BASE EDITORS, COMPOSITIONS, AND METHODS FOR MODIFYING THE MITOCHONDRIAL GENOME
20250011748 ยท 2025-01-09
Assignee
- The Broad Institute, Inc. (Cambridge, MA)
- University Of Washington (Seattle, WA)
- President And Fellows Of Harvard College (Cambridge, MA)
Inventors
- David R. Liu (Cambridge, MA)
- Beverly Mok (Cambridge, MA, US)
- Joseph D. Mougous (Seattle, WA, US)
- Snow Brook Peterson (Seattle, WA, US)
- Marcos de Moraes (Seattle, WA, US)
- Julian Willis (Cambridge, MA, US)
Cpc classification
C12N9/78
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C07K2319/81
CHEMISTRY; METALLURGY
International classification
Abstract
The specification provides programmable base editors that are capable of introducing a nucleotide change and/or which could alter or modify the nucleotide sequence at a target site in mitochondrial DNA (mtDNA) with high specificity and efficiency. Moreover, the disclosure provides fusion proteins and compositions comprising a programmable DNA binding protein (e.g., a mitoTALE, a mitoZFP, or a CRISPR/Casp) and double-stranded DNA deaminase that is capable of being delivered to the mitochondria and carrying out precise installation of nucleotide changes in the mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but also may be used for base editing of any double-stranded target DNA.
Claims
1. A non-naturally occurring polypeptide variant comprising a double-stranded DNA deaminase activity.
2-12. (canceled)
13. A non-naturally occurring polypeptide fragment of a double-stranded DNA deaminase obtained by splitting the deaminase in the deaminase domain at a split site.
14. The non-naturally occurring polypeptide fragment of claim 13, wherein the fragment corresponds to an N-terminal fragment, wherein said fragment comprises an N-terminal portion of a split deaminase domain.
15. The non-naturally occurring polypeptide fragment of claim 13, wherein the fragment corresponds to a C-terminal half fragment, wherein said fragment comprises a C-terminal portion of a split deaminase domain.
16-19. (canceled)
20. The non-naturally occurring polypeptide fragment of claim 14, wherein the N-terminal half fragment comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 349 or 351.
21. The non-naturally occurring polypeptide fragment of claim 15, wherein the C-terminal half fragment comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 350 or 352.
22. A base editor comprising a heterodimer having first and second monomers, said first monomer comprising a first programmable DNA binding protein and an N-terminal fragment of a split double-stranded DNA deaminase, and said second monomer comprising a second programmable DNA binding protein and a C-terminal fragment of a split double-stranded DNA deaminase, wherein dimerization of the first and second monomers reconstitutes the double-stranded DNA deaminase activity.
23-24. (canceled)
25. The base editor of claim 22, wherein the first and/or second programmable DNA binding protein is a nucleic acid programmable DNA binding protein (napDNAbp), a TALE protein, a zinc finger protein, or a mitoTALE protein.
26-36. (canceled)
37. The base editor of claim 22, wherein the N-terminal fragment of the split double-stranded DNA deaminase domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 349 or 351; and wherein the C-terminal fragment of the split double-stranded DNA deaminase domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 350 or 352.
38-46. (canceled)
47. The base editor of claim 22, wherein the first monomer comprises a linker that joins the first programmable DNA binding protein with the N-terminal fragment of the split double-stranded DNA deaminase, and wherein the second monomer comprises a linker that joins the second programmable DNA binding protein with the C-terminal fragment of the split double-stranded DNA deaminase.
48-51. (canceled)
52. The base editor of claim 22, further comprising one or more uracil glycosylase inhibitor (UGI) domains.
53. (canceled)
54. The base editor of claim 22, further comprising one or more targeting sequences.
55. The base editor of claim 54, wherein the one or more targeting sequences is a nuclear localization sequence (NLS) or a mitochondrial targeting sequence (MTS).
56-57. (canceled)
58. The base editor of claim 5557, wherein the MTS comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 13, 14, and 299, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 13, 14, or 299.
59-60. (canceled)
61. The base editor of claim 22, wherein the first and/or second monomers have one of the following structures: (a) [A]-[programmable DNA binding protein]-[N-terminal or C-terminal fragment of a split double-stranded DNA deaminase]-[B]; or (b) [A]-[N-terminal or C-terminal fragment of a split double-stranded DNA deaminase]-[programmable DNA binding protein]-[B], wherein [A] and/or [B] represent optional one or more additional functional domains; and wherein ]-[ is an optional linker.
62-65. (canceled)
66. The base editor of claim 22, wherein the first monomer comprises one of the following structures: [SOD2]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 349 or DddA.sub.tox-C of SEQ ID NO: 350]-[UGI].sub.1-2 or [COX8A]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 351 or DddA.sub.tox-C of SEQ ID NO: 3521-[UGI].sub.1-2; and wherein the second monomer comprises one of the following structures: [SOD21-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 349 or DddA.sub.tox-C of SEQ ID NO: 350]-[UGI].sub.1-2 or [COX8A]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 351 or DddA.sub.tox-C of SEQ ID NO: 352]-[UGI].sub.1-2.
67. The base editor of claim 66, wherein the first monomer comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 360 or 361, and wherein the second monomer comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 360 or 361.
68-73. (canceled)
74. The base editor of claim 22, wherein the first and second monomers bind to first and second nucleotide sequences, respectively, on either side of a target site; and wherein the target site comprises a target base that becomes deaminated by the base editor.
75-84. (canceled)
85. An isolated nucleic acid encoding the first monomer and/or the second monomer of the base editor of claim 22.
86-88. (canceled)
89. A vector comprising the isolated nucleic acid of claim 85.
90. A cell comprising a vector of claim 89.
91. A method of editing a target nucleotide sequence at a target site, comprising contacting a target nucleotide sequence with a base editor of claim 22, wherein the first monomer targets a first nucleotide sequence flanking a target site, and the second monomer targets a second nucleotide sequence flanking the target site, thereby inducing deamination of a target base at the target site.
92-110. (canceled)
111. A method of delivering a base editor of claim 22 to a cell comprising transforming a cell with one or more vectors encoding the first and second monomers of the base editor, wherein once in the cell the first and second monomers are expressed and dimerize, thereby forming a base editor in the cell.
112-114. (canceled)
115. A method of delivering a base editor of claim 22 to a mitochondria comprising transforming a cell with one or more vectors encoding the first and second monomers of the base editor, wherein once in the cell the first and second monomers are expressed, transported to the mitochondria, and dimerize therein, thereby forming a base editor in the mitochondria, wherein the first and second programmable DNA binding proteins of the base editor are mitoTALE domains.
116-121. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0058] All previously described cytidine deaminases, including those used in base editing, operate on single-stranded DNA and thus when used for genome editing require unwinding of double-stranded DNA by macromolecules such as CRISPR-Cas9 complexed with a guide RNA. The difficulty of delivering guide RNAs into the mitochondria has thus far precluded base editing in mitochondrial DNA (mtDNA). The ability of DddA to deaminate double-stranded DNA raises the possibility of RNA-free precision base editing, rather than simple elimination of targeted mtDNA copies following double-strand DNA breaks. Split-DddA halves were engineered that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, TALE array proteins, and uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyze C.Math.G-to-T.Math.A conversions efficiently and with high DNA sequence specificity and product purity at targeted sites within mtDNA in human cells.
[0059] DddA-mediated base editing was used to model a disease-associated mtDNA mutation in human cell lines, resulting in changes in rates of respiration and oxidative phosphorylation. CRISPR-free, DddA-mediated base editing enables precision editing of mtDNA, with important basic science and biomedical implications.
[0060]
[0061] In the context of base editing, all previously described cytidine deaminases utilize single-stranded DNA as a substrate (e.g., the R-loop region of a Cas9-gRNA/dsDNA complex). Base editing in the context of mitochondrial DNA has not heretofore been possible due to the challenges of introducing and/or expressing the gRNA needed for a Cas9-based system into mitochondria. The inventors have recognized for the first time that the catalytic properties of DddA can be leveraged to conduct base editing directly on a double strand DNA substrate by separating the DddA into inactive portions, which when co-localized within a cell will become reconstituted as an active DddA. This avoids or at least minimizes the toxicity associated with delivering and/or expressing a fully active DddA in a cell. For example, a DddA may be divided into two fragments at a split site, i.e., a peptide bond between two adjacent residues in the primary structure or sequence of a DddA. The split site may be positioned anywhere along the length of the DddA amino acid sequence, so long as the resulting fragments do not on their own possess a toxic property (which could be a complete or partial deaminase activity). In certain embodiments, the split site is located in a loop region of the DddA protein. In the embodiment shown in
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DEFINITIONS
[0157] As used herein and in the claims, the singular forms a, an, and the include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to an agent includes a single agent and a plurality of such agents.
AAV
[0158] An adeno-associated virus or AAV is a virus which infects humans and some other primate species. The wild-type AAV genome is a single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The genome comprises two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs): rep and cap between the ITRs. The rep ORF comprises four overlapping genes encoding Rep proteins required for the AAV life cycle. The cap ORF comprises overlapping genes encoding capsid proteins: VP1, VP2 and VP3, which interact together to form the viral capsid. VP1, VP2 and VP3 are translated from one mRNA transcript, which can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two isoforms of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA isoform. The capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a non-enveloped, T-1 icosahedral lattice capable of protecting the AAV genome. The mature capsid is composed of VP1, VP2, and VP3 (molecular masses of approximately 87, 73, and 62 kDa respectively) in a ratio of about 1:1:10.
[0159] rAAV particles may comprise a nucleic acid vector (e.g., a recombinant genome), which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest (e.g., a split Cas9 or split nucleobase) or an RNA of interest (e.g., a gRNA), or one or more nucleic acid regions comprising a sequence encoding a Rep protein; and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g., heterologous nucleic acid regions). In some embodiments, the nucleic acid vector is between 4 kb and 5 kb in size (e.g., 4.2 to 4.7 kb in size). In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein. In some embodiments, the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, a double-stranded nucleic acid vector may be, for example, a self-complimentary vector that contains a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, initiating the formation of the double-strandedness of the nucleic acid vector.
Adenosine Deaminase
[0160] As used herein, the term adenosine deaminase or adenosine deaminase domain refers to a protein or enzyme that catalyzes a deamination reaction of an adenosine (or adenine). The terms are used interchangeably. In certain embodiments, the disclosure provides base editor fusion proteins comprising one or more adenosine deaminase domains. For instance, an adenosine deaminase domain may comprise a heterodimer of a first adenosine deaminase and a second deaminase domain, connected by a linker. Adenosine deaminases (e.g., engineered adenosine deaminases or evolved adenosine deaminases) provided herein may be enzymes that convert adenine (A) to inosine (I) in DNA or RNA. Such adenosine deaminase can lead to an A:T to G:C base pair conversion. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism. In some embodiments, the deaminase does not occur in nature. For example, in some embodiments, the deaminase is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase.
[0161] In some embodiments, the adenosine deaminase is derived from a bacterium, such as, E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. Reference is made to U.S. Patent Publication No. 2018/0073012, published Mar. 15, 2018, which is incorporated herein by reference.
Antisense Strand
[0162] In genetics, the antisense strand of a segment within double-stranded DNA is the template strand, and which is considered to run in the 3 to 5 orientation. By contrast, the sense strand is the segment within double-stranded DNA that runs from 5 to 3, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3 to 5. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense.
Base Editing
[0163] Base editing refers to genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus (e.g., including in a mtDNA). In certain embodiments, this can be achieved without requiring double-stranded DNA breaks (DSB), or single stranded breaks (i.e., nicking). To date, other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g. typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the present inventors previously modified the CRISPR/Cas9 system to directly convert one DNA base into another without DSB formation. See, Komor, A. C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016), the entire contents of which is incorporated by reference herein.
Base Editor
[0164] The term base editor (BE) as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., mtDNA) that converts one base to another (e.g., A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, T to G). In some embodiments, the BE refers to those fusion proteins described herein which are capable of modifying bases directly in mtDNA. Such BEs can also be referred to herein as mtDNA base editors or mtDNA BEs.0 Such BEs can refer to those fusion proteins comprising a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a double-stranded DNA deaminase (DddA) to precisely install nucleotide changes and/or correct pathogenic mutations in mtDNA, rather than destroying the mtDNA with double-strand breaks (DSBs). It should be noted that in some places DddA is referred to as DddE (e.g.,
[0165] In some embodiments, the base editors contemplated herein comprise a nuclease-inactive Cas9 (dCas9) fused to a deaminase which binds a nucleic acid in a guide RNA-programmed manner via the formation of an R-loop, but does not cleave the nucleic acid. For example, the dCas9 domain of the fusion protein may include a D10A and a H840A mutation (which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex), as described in PCT/US2016/058344, which published as WO 2017/070632 on Apr. 27, 2017 and is incorporated herein by reference in its entirety. The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA (the targeted strand, or the strand in which editing or deamination occurs), whereas the RuvC1 subdomain cleaves the non-complementary strand containing the PAM sequence (the non-edited strand). The RuvC1 mutant D10A generates a nick in the targeted strand, while the HNH mutant H840A generates a nick on the non-edited strand (see Jinek et al., Science, 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).
[0166] BEs that convert a C to T, in some embodiments, comprise a cytidine deaminase (e.g., a double-stranded DNA deaminase or DddA). A cytidine deaminase (including those DddAs disclosed herein) refers to an enzyme that catalyzes the chemical reaction cytosine+H.sub.2O.fwdarw.uracil+NH.sub.3 or 5-methyl-cytosine+H.sub.2O.fwdarw.thymine+NH.sub.3. As it may be apparent from the reaction formula, such chemical reactions result in a C to U/T nucleobase change. In the context of a gene, such a nucleotide change, or mutation, may in turn lead to an amino acid change in the protein, which may affect the protein's function, e.g., loss-of-function or gain-of-function. In some embodiments, the C to T nucleobase editor comprises a dCas9 or nCas9 fused to a cytidine deaminase. In some embodiments, the cytidine deaminase domain is fused to the N-terminus of the dCas9 or nCas9.
[0167] In some embodiments, the nucleobase editor further comprises a domain that inhibits uracil glycosylase, and/or a nuclear localization signal.
[0168] Cas9 domains used in base editing have been described in the following references, the contents of which may be applied in the instant disclosure to modify and/or include in BEs described herein, which can target mtDNA, e.g., in Rees & Liu, Nat Rev Genet. 2018; 19(12):770-788 and Koblan et al., Nat Biotechnol. 2018; 36(9):843-846; as well as. U.S. Patent Publication No. 2018/0073012, published Mar. 15, 2018, which issued as U.S. Pat. No. 10,113,163; on Oct. 30, 2018; U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Pat. No. 10,167,457 on Jan. 1, 2019; International Publication No. WO 2017/070633, published Apr. 27, 2017; U.S. Patent Publication No. 2015/0166980, published Jun. 18, 2015; U.S. Pat. No. 9,840,699, issued Dec. 12, 2017; U.S. Pat. No. 10,077,453, issued Sep. 18, 2018; International Publication No. WO 2019/023680, published Jan. 31, 2019; International Publication No. WO 2018/0176009, published Sep. 27, 2018, International Application No PCT/US2019/033848, filed May 23, 2019, International Application No. PCT/US2019/47996, filed Aug. 23, 2019; International Application No. PCT/US2019/049793, filed Sep. 5, 2019; U.S. Provisional Application No. 62/835,490, filed Apr. 17, 2019; International Application No. PCT/US2019/61685, filed Nov. 15, 2019; International Application No. PCT/US2019/57956, filed Oct. 24, 2019; U.S. Provisional Application No. 62/858,958, filed Jun. 7, 2019; International Publication No. PCT/US2019/58678, filed Oct. 29, 2019, the contents of each of which are incorporated herein by reference in their entireties.
[0169] Exemplary adenine and cytosine base editors are also described in Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 2018; 19(12):770-788; as well as U.S. Patent Publication No. 2018/0073012, published Mar. 15, 2018, which issued as U.S. Pat. No. 10,113,163, on Oct. 30, 2018; U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Pat. No. 10,167,457 on Jan. 1, 2019; International Publication No. WO 2017/070633, published Apr. 27, 2017; U.S. Patent Publication No. 2015/0166980, published Jun. 18, 2015; U.S. Pat. No. 9,840,699, issued Dec. 12, 2017; and U.S. Pat. No. 10,077,453, issued Sep. 18, 2018, PCT Application PCT/US2017/045381, filed Aug. 3, 2017, which published as WO 2018/027078, and PCT Application No. PCT/US2019/033848, which published as WO 2019/226953, each of which is herein incorporated by reference. Any of the deaminase components of these adenine or cytidine BEs could be modified using a method of directed evolution (e.g., PACE or PANCE) to obtain a deaminase which may use double-stranded DNA as a substrate, and thus, which could be used in the BEs described herein which are intended for use in conducting base editing directly on mtDNA, i.e., on a double-stranded DNA target.
Cas9
[0170] The term Cas9 or Cas9 nuclease refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 domain as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and/or the gRNA binding domain of Cas9. A Cas9 protein is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3-5 exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (sgRNA, or simply gNRA) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., Complete genome sequence of an M1 strain of Streptococcus pyogenes. Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.
[0171] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a dCas9 protein (for nuclease-dead Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as Cas9 variants. A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 28). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 28). In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 28). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 28).
[0172] As used herein, the term nCas9 or Cas9 nickase refers to a Cas9 or a variant thereof, which cleaves or nicks only one of the strands of a target cut site thereby introducing a nick in a double strand DNA molecule rather than creating a double strand break. This can be achieved by introducing appropriate mutations in a wild-type Cas9 which inactivates one of the two endonuclease activities of the Cas9. Any suitable mutation which inactivates one Cas9 endonuclease activity but leaves the other intact is contemplated, such as one of D10A or H840A mutations in the wild-type S. pyogenes Cas9 amino acid sequence, or a D10A mutation in the wild-type S. aureus Cas9 amino acid sequence, may be used to form the nCas9.
cDNA
[0173] The term cDNA refers to a strand of DNA copied from an RNA template. cDNA is complementary to the RNA template.
Circular Permutant
[0174] As used herein, the term circular permutant refers to a protein or polypeptide comprising a circular permutation, which is change in the protein's structural configuration involving a change in order of amino acids appearing in the protein's amino acid sequence. In other words, circular permutants are proteins that have altered N- and C-termini as compared to a wild-type counterpart, e.g., the wild-type C-terminal half of a protein becomes the new N-terminal half. Circular permutation (or CP) is essentially the topological rearrangement of a protein's primary sequence, connecting its N- and C-terminus, often with a peptide linker, while concurrently splitting its sequence at a different position to create new, adjacent N- and C-termini. The result is a protein structure with different connectivity, but which often can have the same overall similar three-dimensional (3D) shape, and possibly include improved or altered characteristics, including, reduced proteolytic susceptibility, improved catalytic activity, altered substrate or ligand binding, and/or improved thermostability. Circular permutant proteins can occur in nature (e.g., concanavalin A and lectin). In addition, circular permutation can occur as a result of posttranslational modifications or may be engineered using recombinant techniques. Any of the polypeptides contemplated for use in the mtDNA base editors disclosed herein may be converted to circular permutant variants, including any pDNAbp (e.g., Cas9, mitoTALE, or mitoZFP) and any double-stranded DNA deaminase (e.g., DddA).
Circularly Permuted napDNAbp
[0175] In the case of circular permutant Cas9s or other napDNAbps that could be used with the mtDNA base editors contemplated herein, the term circularly permuted napDNAbp refers to any napDNAbp protein, or variant thereof (e.g., SpCas9), that occurs as or engineered as a circular permutant, whereby its N- and C-termini have been topically rearranged. Such circularly permuted proteins (CP-napDNAbp, such as CP-Cas9 in the case of Cas9), or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al., Protein Engineering of Cas9 for enhanced function, Methods Enzymol, 2014, 546: 491-511 and Oakes et al., CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification, Cell, Jan. 10, 2019, 176: 254-267, each of are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA). Such CP variants of Cas9 can be used with the mtDNA base editors described herein.
Cytidine Deaminase
[0176] As used herein, a cytidine deaminase encoded by the CDA gene is an enzyme that catalyzes the removal of an amine group from cytidine (i.e., the base cytosine when attached to a ribose ring) to uridine (C to U) and deoxycytidine to deoxyuridine (C to U). A non-limiting example of a cytidine deaminase is APOBEC1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1). Another example is AID (activation-induced cytidine deaminase). Under standard Watson-Crick hydrogen bond pairing, a cytosine base hydrogen bonds to a guanine base. When cytidine is converted to uridine (or deoxycytidine is converted to deoxyuridine), the uridine (or the uracil base of uridine) undergoes hydrogen bond pairing with the base adenine. Thus, a conversion of C to uridine (U) by cytidine deaminase will cause the insertion of A instead of a G during cellular repair and/or replication processes. Since the adenine A pairs with thymine T, the cytidine deaminase in coordination with DNA replication causes the conversion of an C-G pairing to a T-A pairing in the double-stranded DNA molecule.
CRISPR
[0177] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively compose, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (mc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3-5 exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (sgRNA, or simply gRNA) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA speciesthe guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., Complete genome sequence of an M1 strain of Streptococcus pyogenes. Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
Deaminase
[0178] The term deaminase or deaminase domain refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine (or adenine) deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA) to inosine. In other embodiments, the deaminase is a cytidine (or cytosine) deaminase, which catalyzes the hydrolytic deamination of cytidine or cytosine. In preferred aspects, the deaminase is a double-stranded DNA deaminase, or is modified, evolved, or otherwise altered to be able to utilize double-strand DNA as a substrate for deamination.
[0179] The deaminase embraces the DddA domains described herein, and defined below. The DddA is a type of deaminase, but where the activity of the deaminase is against double-stranded DNA, rather than single-stranded DNA, which is the case for deaminases prior to the present disclosure.
[0180] The deaminases provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase.
DNA Editing Efficiency
[0181] The term DNA editing efficiency, as used herein, refers to the number or proportion of intended base pairs that are edited. For example, if a base editor edits 10% of the base pairs that it is intended to target (e.g., within a cell or within a population of cells), then the base editor can be described as being 10% efficient. Some aspects of editing efficiency embrace the modification (e.g. deamination) of a specific nucleotide within DNA, without generating a large number or percentage of insertions or deletions (i.e., indels). It is generally accepted that editing while generating less than 5% indels (as measured over total target nucleotide substrates) is high editing efficiency. The generation of more than 20% indels is generally accepted as poor or low editing efficiency. Indel formation may be measured by techniques known in the art, including high-throughput screening of sequencing reads.
Downstream
[0182] As used herein, the terms upstream and downstream are relative terms that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5-to-3 direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5 to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5 side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3 to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3 side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the sense or coding strand. In genetics, a sense strand is the segment within double-stranded DNA that runs from 5 to 3, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3 to 5. Thus, as an example, a SNP nucleobase is downstream of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3 side of the promoter on the sense or coding strand.
[0183] In another example, the mtDNA BEs contemplated herein can comprise a pair of fusion proteins wherein a first fusion protein binds upstream of a target nucleobase pair target of deamination, and a second fusion protein binds just downstream of the target nucleobase pair that is being targeted for deamination. The pair of fusion proteins each comprise a pDNAbp (e.g., a Cas9 domain, a mitoTALE, or a mitoZFP) which bind to a target site on either side of the targeted nucleobase pair. Each of the pDNAbps of each fusion protein are each fused to a DddA half portion (e.g., an N-terminal half and a C-terminal half of a DddA which is divided into two inactive fragments at a split site), which become co-localized at the target nucleobase pair upon binding of the pDNAbp domains at their respective upstream and downstream sites.
DddA
[0184] The term double-stranded DNA deaminase domain or DddA (or equivalently, DddE) refers to a protein which catalyzes a deamination of a target nucleotide (e.g., C, A, G, C) in a double-stranded DNA molecule. Reference to DddA and double-stranded DNA deaminase are equivalent. In one embodiment, the DddA deaminates a cytidine. Deamination of cytidine, results in a uracil (or deoxyuracil in the case of deoxycytidine), and through replication and/or repair processes, converts the original C:G base pair to a T:A base pair. This change can also be referred to as a C-to-T edit because the C of the C:G pair is converted to a T of T:A pair. DddA, when expressed naturally, can be toxic to biological systems. While the mechanism of action is not clearly documented, one rationale for the observed toxicity is DddA's activity may cause indiscriminant deamination of cytidine in vivo on double-stranded target DNA (e.g., the cellular genome). Such indiscriminant deaminations may provoke celluar repair responses, including, but not limited to, degradation of genomic DNA.
Effective Amount
[0185] The term effective amount, as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of any of the fusion proteins as described herein, or compositions thereof, may refer to the amount of the fusion proteins sufficient to edit a target nucleotide sequence (e.g., mtDNA). In some embodiments, an effective amount of any of the fusion proteins as described herein, or compositions thereof (e.g., a fusion protein comprising a first mitoTALE or another pDNAbp and a first portion of a DddA, a second fusion protein comprising a second mitoTALE or another pDNAbp and a second portion of a DddA) that is sufficient to induce editing of a target nucleotide, which is proximal to a target nucleic acid sequence specifically bound and edited by the fusion protein (e.g., by the first or second mitoTALE). As will be appreciated by the skilled artisan, the effective amount of an agent (e.g., a fusion protein, a second fusion protein), may vary depending on various factors as, for example, on the desired biological response on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.
Fusion Protein
[0186] The term fusion protein as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins (e.g., a first mitoTALE, a first portion of a DddA, a second mitoTALE, a second portion of a DddA). One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. A protein may comprise different domains, for example, a nucleic acid binding site (e.g., a first or second mitoTALE) and a catalytic domain of a nucleic-acid editing protein (e.g., a first or second portion of a DddA). Another example includes a mitoTALE to a DddA or portion thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4.sup.th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
Guide Nucleic Acid
[0187] In embodiments involving mtDNA base editors that comprise Cas9 domains as the pDNAbp component, the Cas9 domain requires a guide RNA (or more generically, a guide nucleic acid) to program the binding of the Cas9 to a target site. The term guide nucleic acid or napDNAbp-programming nucleic acid molecule or equivalently guide sequence refers the one or more nucleic acid molecules which associate with and direct or otherwise program a napDNAbp protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the napDNAbp protein to bind to the nucleotide sequence at the specific target site. A non-limiting example is a guide RNA of a Cas protein of a CRISPR-Cas genome editing system.
[0188] Guide RNA is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospace sequence of the guide RNA. As used herein, a guide RNA refers to a synthetic fusion of the endogenous bacterial crRNA and tracrRNA that provides both targeting specificity and scaffolding and/or binding ability for Cas9 nuclease to a target DNA. This synthetic fusion does not exist in nature and is also commonly referred to as an sgRNA. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein. In addition, methods for designing appropriate guide RNA sequences are provided herein.
Guide RNA (gRNA)
[0189] In embodiments involving pDNAbp/DddA base editors that comprise Cas9 domains as the pDNAbp component, the Cas9 domain requires a guide RNA (or more generically, a guide nucleic acid) to program the binding of the Cas9 to a target site. As used herein, the term guide RNA is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospace sequence of the guide RNA. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein.
[0190] Guide RNAs may comprise various structural elements that include, but are not limited to (a) a spacer sequencethe sequence in the guide RNA (having 20 nts in length) which binds to a complementary strand of the target DNA (and has the same sequence as the protospacer of the DNA) and (b) a gRNA core (or gRNA scaffold or backbone sequence)-refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer sequence that is used to guide Cas9 to target DNA.
Guide RNA Target Sequence
[0191] As used herein, the guide RNA target sequence refers to the 20 nucleotides that are complementary to the protospacer sequence in the PAM strand. The target sequence is the sequence that anneals to or is targeted by the spacer sequence of the guide RNA. The spacer sequence of the guide RNA and the protospacer have the same sequence (except the spacer sequence is RNA and the protospacer is DNA).
Guide RNA Scaffold Sequence
[0192] As used herein, the guide RNA scaffold sequence refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer/targeting sequence that is used to guide Cas9 to target DNA.
Host Cell
[0193] The term host cell, as used herein, refers to a cell that can host, replicate, and transfer a phage vector useful for a continuous evolution process as provided herein. In embodiments where the vector is a viral vector, a suitable host cell is a cell that may be infected by the viral vector, can replicate it, and can package it into viral particles that can infect fresh host cells. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the disclosure is not limited in this respect. In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is an E. coli cell. Suitable E. coli host strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, Top10F, DH12S, ER2738, ER2267, and XL1-Blue MRF. These strain names are art recognized and the genotype of these strains has been well characterized. It should be understood that the above strains are exemplary only and that the invention is not limited in this respect. The term fresh, as used herein interchangeably with the terms non-infected or uninfected in the context of host cells, refers to a host cell that has not been infected by a viral vector comprising a gene of interest as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.
[0194] In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.
Inteins and Split-Inteins
[0195] In some embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered to include intein and/or split-intein amino acid sequences.
[0196] As used herein, the term intein refers to auto-processing polypeptide domains found in organisms from all domains of life. An intein (intervening protein) carries out a unique auto-processing event known as protein splicing in which it excises itself out from a larger precursor polypeptide through the cleavage of two peptide bonds and, in the process, ligates the flanking extein (external protein) sequences through the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally), as intein genes are found embedded in frame within other protein-coding genes. Furthermore, intein-mediated protein splicing is spontaneous; it requires no external factor or energy source, only the folding of the intein domain. This process is also known as cis-protein splicing, as opposed to the natural process of trans-protein splicing with split inteins.
[0197] Split inteins are a sub-category of inteins. Unlike the more common contiguous inteins, split inteins are transcribed and translated as two separate polypeptides, the N-intein and C-intein, each fused to one extein. Upon translation, the intein fragments spontaneously and non-covalently assemble into the canonical intein structure to carry out protein splicing in trans.
[0198] Inteins and split inteins are the protein equivalent of the self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from a precursor protein with the concomitant fusion of the flanking protein sequences, known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).
[0199] As used herein, the term protein splicing refers to a process in which an interior region of a precursor protein (an intein) is excised and the flanking regions of the protein (exteins) are ligated to form the mature protein. This natural process has been observed in numerous proteins from both prokaryotes and eukaryotes (Perler, F. B., Xu, M. Q., Paulus, H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F. B. Nucleic Acids Research 1999, 27, 346-347). The intein unit contains the necessary components needed to catalyze protein splicing and often contains an endonuclease domain that participates in intein mobility (Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thomer, J., Belfort, M. Nucleic Acids Research 1994, 22, 1127-1127). The resulting proteins are linked, however, not expressed as separate proteins. Protein splicing may also be conducted in trans with split inteins expressed on separate polypeptides spontaneously combine to form a single intein which then undergoes the protein splicing process to join to separate proteins.
[0200] The elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, J. Amer. Chem. Soc., 121:5597-5598 (1999); Chong, et al., Gene, 192:271-281 (1997), Chong, et al., Nucleic Acids Res., 26:5109-5115 (1998); Chong, et al., J. Biol. Chem., 273:10567-10577 (1998); Cotton, et al. J. Am. Chem. Soc., 121:1100-1101 (1999); Evans, et al., J. Biol. Chem., 274:18359-18363 (1999); Evans, et al., J. Biol. Chem., 274:3923-3926 (1999); Evans, et al., Protein Sci., 7:2256-2264 (1998); Evans, et al., J. Biol. Chem., 275:9091-9094 (2000); Iwai and Pluckthun, FEBS Lett. 459:166-172 (1999); Mathys, et al., Gene, 231:1-13 (1999); Mills, et al., Proc. Natl. Acad. Sci. USA 95:3543-3548 (1998); Muir, et al., Proc. Natl. Acad. Sci. USA 95:6705-6710 (1998); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999); Severinov and Muir, J. Biol. Chem., 273:16205-16209 (1998); Shingledecker, et al., Gene, 207:187-195 (1998); Southworth, et al., EMBO J. 17:918-926 (1998); Southworth, et al., Biotechniques, 27:110-120 (1999); Wood, et al., Nat. Biotechnol., 17:889-892 (1999); Wu, et al., Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998a); Wu, et al., Biochim Biophys Acta 1387:422-432 (1998b); Xu, et al., Proc. Natl. Acad. Sci. USA 96:388-393 (1999); Yamazaki, et al., J. Am. Chem. Soc., 120:5591-5592 (1998)). Each reference is incorporated herein by reference.
Lentiviral Vectors
[0201] Lentiviral vectors are derived from human immunodeficiency virus-1 (HIV-1). The lentiviral genome consists of single-stranded RNA that is reverse-transcribed into DNA and then integrated into the host cell genome. Lentiviruses can infect both dividing and non-dividing cells, making them attractive tools for gene therapy.
[0202] The lentiviral genome is around 9 kb in length and contains three major structural genes: gag, pol, and env. The gag gene is translated into three viral core proteins: 1) matrix (MA) proteins, which are necessary for virion assembly and infection of non-dividing cells; 2) capsid (CA) proteins, which form the hydrophobic core of the virion; and 3) nucleocapsid (NC) proteins, which protect the viral genome by coating and associating tightly with the RNA. The pol gene encodes for the viral protease, reverse transcriptase, and integrase enzymes which are essential for viral replication. The env gene encodes for the viral surface glycoproteins, which are essential for virus entry into the host cell by enabling binding to cellular receptors and fusion with cellular membranes. In some embodiments, the viral glycoprotein is derived from vesicular stomatitis virus (VSV-G). The viral genome also contains regulatory genes, including tat and rev. Tat encodes transactivators critical for activating viral transcription, while rev encodes a protein that regulates the splicing and export of viral transcripts. Tat and rev are the first proteins synthesized following viral integration and are required to accelerate production of viral mRNAs.
[0203] To improve the safety of lentivirus, the components necessary for viral production are split across multiple vectors. In some embodiments, the disclosure relates to delivery of a heterologous gene (e.g., transgene) via a recombinant lentiviral transfer vector encoding one or more transgenes of interest flanked by long terminal repeat (LTR) sequences. These LTRs are identical nucleotide sequences that are repeated hundreds or thousands of times and facilitate the integration of the transfer plasmid sequences into the host cell genome. Methods of the current disclosure also describe one or more accessory plasmids. These accessory plasmids may include one or more lentiviral packaging plasmids, which encode the pol and rev genes that are necessary for the replication, splicing, and export of viral particles. The accessory plasmids may also include a lentiviral envelope plasmid, which encodes the genes necessary for producing the viral glycoproteins which will allow the viral particle to fuse with the host cell.
Ligand-Dependent Intein
[0204] In some embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered to include ligand-dependent inteins.
[0205] The term ligand-dependent intein, as used herein refers to an intein that comprises a ligand-binding domain. Typically, the ligand-binding domain is inserted into the amino acid sequence of the intein, resulting in a structure intein (N)ligand-binding domainintein (C). Typically, ligand-dependent inteins exhibit no or only minimal protein splicing activity in the absence of an appropriate ligand, and a marked increase of protein splicing activity in the presence of the ligand. In some embodiments, the ligand-dependent intein does not exhibit observable splicing activity in the absence of ligand but does exhibit splicing activity in the presence of the ligand. In some embodiments, the ligand-dependent intein exhibits an observable protein splicing activity in the absence of the ligand, and a protein splicing activity in the presence of an appropriate ligand that is at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times, at least 2500 times, at least 5000 times, at least 10000 times, at least 20000 times, at least 25000 times, at least 50000 times, at least 100000 times, at least 500000 times, or at least 1000000 times greater than the activity observed in the absence of the ligand. In some embodiments, the increase in activity is dose dependent over at least 1 order of magnitude, at least 2 orders of magnitude, at least 3 orders of magnitude, at least 4 orders of magnitude, or at least 5 orders of magnitude, allowing for fine-tuning of intein activity by adjusting the concentration of the ligand. Suitable ligand-dependent inteins are known in the art, and in include those provided below and those described in published U.S. Patent Application U.S. 2014/0065711 A1; Mootz et al., Protein splicing triggered by a small molecule. J. Am. Chem. Soc. 2002; 124, 9044-9045; Mootz et al., Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., Proc. Natt. Acad. Sci. USA. 2004; 101, 10505-10510); Skretas & Wood, Regulation of protein activity with small-molecule-controlled inteins. Protein Sci. 2005; 14, 523-532; Schwartz, et al., Post-translational enzyme activation in an animal via optimized conditional protein splicing. Nat. Chem. Biol. 2007; 3, 50-54; Peck et al., Chem. Biol. 2011; 18 (5), 619-630; the entire contents of each are hereby incorporated by reference. Exemplary sequences are as follows:
TABLE-US-00001 SEQ NAME SEQUENCEOFLIGAND-DEPENDENTINTEIN IDNO: 2-4 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVI 17 INTEIN: GLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYDPTSPFSEASMMGLLINLADRELVHMINWAKRVPGFVDLTLHD QAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 3-2 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVI 18 INTEIN GLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHD QAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 30R3-1 CLAEGTRIFDPVIGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVI 19 INTEIN GLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPIPYSEYDPTSPFSEASMMGLLINLADRELVHMINWAKRVPGFVDLTLHD QAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSILKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 30R3-2 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVI 20 INTEIN GLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHD QAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 30R3-3 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVI 21 INTEIN GLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHD QAHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 37R3-1 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVI 22 INTEIN GLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYNPTSPFSEASMMGLLINLADRELVHMINWAKRVPGFVDLTLHD QAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 37R3-2 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGILLARPVVSWFDQGTRDVI 23 INTEIN GLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYDPTSPFSEASMMGLLINLADRELVHMINWAKRVPGFVDLILHD QAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSILKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC 37R3-3 CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVI 24 INTEIN GLRIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVS ALLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHD QAHLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSILKSLEEKDHIHRALDKITDTLIH LMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLD AHRLHAGGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEE LHTLVAEGVVVHNC
Linker
[0206] In various embodiments, the herein disclosed fusion proteins (e.g., the mtDNA base editors) or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered to include one or more linker sequences that join two or more polypeptides (e.g., a pDNAbp and a DddA half) to one another.
[0207] The term linker, as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, a first or second mitoTALE can be fused to a first or second portion of a DddA, by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 1-100 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer linkers are also contemplated.
mitoTALE
[0208] In various embodiments, the mtDNA base editors embrace fusion proteins comprising a DddA (or inactive fragment thereof) and a mitoTALE domain. As used herein, a mitoTALE protein or domain refers to a modified TALE protein that can be designed to localize to the mitochondria. In one embodiment, a mitoTALE comprises a TALE domain fused to a mitochondrial targeting sequences (MTS). In another embodiment, a mitoTALE comprises a TALE domain fused to an MTS in place of the endogenous LS (localization signal) of the TALE, or into the repeat variable diresidue (RVD) of the TALE. MTS domains can include, but are not limited to, SOD2, Cox8a, bipartitie nuclear localization signals (BPNLS), zmLOC100282174 MLS), which are disclosed herein.
[0209] Transcription activator-like effector proteins (TALE proteins) are class of naturally occurring DNA binding proteins which bind specific promoter sequences and which can activate the expression of genes. TALE proteins can be engineered to recognize a desired DNA sequence. TALEs have a modular DNA-binding domain (DBD) consisting of repetitive sequences of amino acids with each repeat region comprising of 34 amino acids. The two amino acids at residue positions 12 and 13 of each repeat region determine the nucleotide specificity of the TALE. This pair of residues is referred to as the repeat variable diresidue (RVD). A final region, known as the half-repeat, is typically truncated to 20 amino acids. Using these factors, one of ordinary skill in the art can sythesize sequence-specific synthetic TALEs, which target user defined nucleotide sequences. See Garg A.; Lohmueller J. J.; Silver P. A.; Armel T. Z. (2012), Engineering synthetic TAL effectors with orthogonal target sites, Nucleic Acids Res. 40, 7584-7595, which is incorporated herein by reference. Further reference to designing sequence specific TALEs can be found in Carlson et al., Targeting DNA with fingers and TALENs, Mol. Ther. Nucleic Acids, 2012, 1, e3.10.1038/mtna.2011, which is incorporated herein by reference. For example, the C-terminus typically contains a localization signal (LS), which directs a TALE to the particular cellular component (e.g., mitochondria), as well as a functional domain that modulates transcription, such as an acidic activation domain (AD). The endogenous LS can be replaced by an organism-specific localization signal, such as a specific MLS to localize the TALE to the mitochondria. For example, an LS derived from the simian virus 40 large T-antigen can be used in mammalian cells.
mitoZFP
[0210] In various embodiments, the mtDNA base editors embrace fusion proteins comprising a DddA (or inactive fragment thereof) and a mitoZFP domain.
[0211] A zinc finger DNA binding protein or ZFP is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein can be abbreviated as zinc finger protein or ZFP. A mitoZFP refers to a zinc finger DNA binding protein that has been modified to comprise one or more mitochondral targeting sequences (MTS).
[0212] Zinc finger binding domains can be engineered to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for engineering zinc finger proteins are design and selection. A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261; and 6,785,613; see, also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496; and U.S. Pat. Nos. 6,746,838; 6,866,997; and 7,030,215, each of which are incorporated herein by reference.
[0213] Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes.
[0214] The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. If the zinc finger domains are perfectly specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 basepairs can, in theory, target a single locus in a mammalian genome. The most straightforward method to generate new zinc-finger arrays is to combine smaller zinc-finger modules of known specificity. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger array that can recognize a 9 basepair target site.
Mitochondrial Targeting Sequence (MTS)
[0215] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered to include one or more mitochondrial targeting sequences (MTS) (or mitochondrial localization sequence (MLS)) which facilitate that translocation of a polypeptide into the mitochondria. MTS are known in the art and exemplary sequences are provided herein. In general MTSs are short peptide sequences (about 3-70 amino acids long) that direct a newly synthesized protein to the mitochondria within a cell. It is usually found at the N-terminus and consists of an alternating pattern of hydrophobic and positively charged amino acids to form what is called an amphipathic helix. Mitochondrial localization sequences can contain additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix. One exemplary mitochondrial localization sequence is the mitochondrial localization sequence derived from Cox8, a mitochondrial cytochrome c oxidase subunit VIII. In embodiments, a mitochondrial localization sequence derived from Cox8 includes the amino acid sequence: MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 299). In the embodiments, the mitochondrial localization sequence derived from Cox8 includes an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 299.
Nucleic Acid Molecule
[0216] The term nucleic acid, as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 0(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2-fluororibose, ribose, 2-deoxyribose, 2-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5 N phosphoramidite linkages).
Mutation
[0217] The term mutation, as used herein, refers to a substitution of a residue within a sequence, e.g. a nucleic acid or amino acid sequence, with another residue; a deletion or insertion of one or more residues within a sequence; or a substitution of a residue within a sequence of a genome in a subject to be corrected. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4.sup.th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include loss-of-function mutations which are mutations that reduce or abolish a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. There are some exceptions where a loss-of-function mutation is dominant, one example being haploinsufficiency, where the organism is unable to tolerate the approximately 50% reduction in protein activity suffered by the heterozygote. This is the explanation for a few genetic diseases in humans, including Marfan syndrome, which results from a mutation in the gene for the connective tissue protein called fibrillin. Mutations also embrace gain-of-function mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Alternatively, the mutation could lead to overexpression of one or more genes involved in control of the cell cycle, thus leading to uncontrolled cell division and hence to cancer. Because of their nature, gain-of-function mutations are usually dominant.
napDNAbp
[0218] In various embodiments, the mtDNA base editors may comprise pDNAbps which are nucleic acid programmable. The term napDNAb which stand for nucleic acid programmable DNA binding protein refers to any protein that may associate (e.g., form a complex) with one or more nucleic acid molecules (i.e., which may broadly be referred to as a napDNAbp-programming nucleic acid molecule and includes, for example, guide RNA in the case of Cas systems) which direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. This term napDNAbp embraces CRISPR-Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or modified), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), C2c3 (a type V CRISPR-Cas system), dCas9, GeoCas9, CjCas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12g, Cas12h, Cas12i, Cas13d, Cas14, Argonaute, and nCas9. Further Cas-equivalents are described in Makarova et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 2016; 353 (6299), the contents of which are incorporated herein by reference. However, the nucleic acid programmable DNA binding protein (napDNAbp) that may be used in connection with this invention are not limited to CRISPR-Cas systems. The invention embraces any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo) which may also be used for DNA-guided genome editing. NgAgo-guide DNA system does not require a PAM sequence or guide RNA molecules, which means genome editing can be performed simply by the expression of generic NgAgo protein and introduction of synthetic oligonucleotides on any genomic sequence. See Gao et al., DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nature Biotechnology 2016; 34(7):768-73, which is incorporated herein by reference.
[0219] In some embodiments, the napDNAbp is a RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though gRNA is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 (or equivalent) complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is homologous to a tracrRNA as depicted in
[0220] The napDNAbp nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using napDNAbp nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acid Res. (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).
Nickase
[0221] The term nickase refers to a napDNAbp having only a single nuclease activity that cuts only one strand of a target DNA, rather than both strands. Thus, a nickase type napDNAbp does not leave a double-strand break.
Nuclear Localization Signal
[0222] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be further engineered to include one or more nuclear localization signals.
[0223] A nuclear localization signal or sequence (NLS) is an amino acid sequence that tags, designates, or otherwise marks a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. Thus, a single nuclear localization signal can direct the entity with which it is associated to the nucleus of a cell. Such sequences may be of any size and composition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5, or 4 amino acids, but will preferably comprise at least a four to eight amino acid sequence known to function as a nuclear localization signal (NLS).
Nucleic Acid Molecule
[0224] The term nucleic acid molecule as used herein, refers to RNA as well as single and/or double-stranded DNA. Nucleic acid molecules may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g. a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms nucleic acid, DNA, RNA, and/or similar terms include nucleic acid analogs, e.g. analogs having other than a phosphodiester backbone. Nucleic acids may be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g. in the case of chemically synthesized molecules, nucleic acids may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5 to 3 direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g. 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, inosinedenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g. methylated bases); intercalated bases; modified sugars (e.g. 2-fluororibose, ribose, 2-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g. phosphorothioates and 5-N-phosphoramidite linkages).
PACE
[0225] The term phage-assisted continuous evolution (PACE), as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Application, U.S. Pat. No. 9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference. PACE can be used, for instance, to evolve a deaminase (e.g., a cytidine or adenosine deaminase) which uses single strand DNA as a substrate to obtain a deaminase which is capable of using double-strand DNA as a substrate (e.g., DddA).
Programmable DNA Binding Protein (pDNAbp)
[0226] As used herein, the term programmable DNA binding protein, pDNA binding protein, pDNA binding protein domain or pDNAbp refers to any protein that localizes to and binds a specific target DNA nucleotide sequence (e.g. a gene locus of a genome). This term embraces RNA-programmable proteins, which associate (e.g. form a complex) with one or more nucleic acid molecules (i.e., which includes, for example, guide RNA in the case of Cas systems) that direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., DNA sequence) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein. The term also embraces proteins which bind directly to nucleotide sequence in an amino acid-programmable manner, e.g., zinc finger proteins and TALE proteins. Exemplary RNA-programmable proteins are CRISPR-Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g. engineered or modified), and may include a Cas9 equivalent from any type of CRISPR system (e.g. type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), C2c3 (a type V CRISPR-Cas system), dCas9, GeoCas9, CjCas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12g, Cas12h, Cas12i, Cas13d, Cas14, Argonaute, and nCas9. Further Cas-equivalents are described in Makarova et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 2016; 353(6299), the contents of which are incorporated herein by reference.
Promoter
[0227] The term promoter is recognized in the art as referring to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream (i.e., closer to or toward the 3 end of the nucleic acid strand) gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule inducer for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.
Protein, Peptide, and Polypeptide
[0228] The terms protein, peptide, and polypeptide are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
[0229] The term amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups {e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms non-naturally occurring amino acid and unnatural amino acid refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
[0230] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the njPAC-R7B Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms polypeptide, peptide and protein are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A fusion protein refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.
[0231] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another: [0232] 1) Alanine (A), Glycine (G); [0233] 2) Aspartic acid (D), Glutamic acid (E); [0234] 3) Asparagine (N), Glutamine (Q); [0235] 4) Arginine (R), Lysine (K); [0236] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); [0237] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); [0238] 7) Serine (S), Threonine (T); and [0239] 8) Cysteine (C), Methionine (M).
RNA-Protein Recruitment System
[0240] In various embodiments, two separate protein domains (e.g., a pDNAbp and a DddA or a DddA N-terminal half and a DddA C-terminal half) may be colocalized to one another to form a functional complex (akin to the function of a fusion protein comprising the two separate protein domains) by using an RNA-protein recruitment system, such as the MS2 tagging technique. Such systems generally tag one protein domain with an RNA-protein interaction domain (aka RNA-protein recruitment domain) and the other with an RNA-binding protein that specifically recognizes and binds to the RNA-protein interaction domain, e.g., a specific hairpin structure. These types of systems can be leveraged to colocalize the domains of a base editor, as well as to recruitment additional functionalities to a base editor, such as a UGI domain. In one example, the MS2 tagging technique is based on the natural interaction of the MS2 bacteriophage coat protein (MCP or MS2cp) with a stem-loop or hairpin structure present in the genome of the phage, i.e., the MS2 hairpin. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). Thus, in one exemplarly scenario a deaminase-MS2 fusion can recruit a Cas9-MCP fusion.
[0241] A review of other modular RNA-protein interaction domains are described in the art, for example, in Johansson et al., RNA recognition by the MS2 phage coat protein, Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., Organization of intracellular reactions with rationally designed RNA assemblies, Science, 2011, Vol. 333: 470-474; Mali et al., Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering, Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the com hairpin, which specifically recruits the Com protein. See Zalatan et al.
[0242] The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the MS2 aptamer) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 25).
[0243] The amino acid sequence of the MCP or MS2cp is:
TABLE-US-00002 (SEQIDNO:26) GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT CSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTI PIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY.
Sense Strand
[0244] In genetics, a sense strand is the segment within double-stranded DNA that runs from 5 to 3, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3 to 5. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense.
Split Site (e.g., of a DddA)
[0245] As used herein, the term split site, as in a split site of a DddA, refers to a specific peptide bond between any two immediately adjacent amino acid residues in the amino acid sequence of a DddA at which the complete DddA polypeptide is divided into two half portions, i.e., an N-terminal half portion and a C-terminal half portion. The N-terminal half portion of the DddA may be referred to as DddA-N half and the C-terminal half portion of the DddA may be referred to as the DddA-C half. Alternately, DddA-N half may be referred to as the DddA-N fragment or portion and the DddA-C half may be referred to as the DddA-C fragment of portion. Depending on the location of the split site, the DddA-N half and the DddA-C half may be the same or different size and/or sequence length. The term half does not connote the requirement that the DddA-N and DddA-C portions are identically half of the size and/or sequence length of a complete DddA, or that the split site is required to be at the mid point of the complete DddA polypeptide. To the contrary, and as noted above, the split site can be between any pair of residues in the DddA polypeptide, thereby giving rise to half portions which are unequal in size and/or sequence length. For clarity, as used herein, the term half when used in the context of a split molecule (e.g., protein, intein, delivery molecule, nucleic acid, etc.), shall not be interpreted to require, and shall not imply, that the size of the resulting portions (e.g., as split or broken into smaller portions) of the molecule are one-half (e.g., , 50%) of the original molecule. The term shall be interpreted to be illustriative of idea that they are portion(s) of a larger molecule that has been broken into smaller fragments (e.g., portions), but that when reconstituted may regain the activity of the molecule as a whole. Thus, by way of example, a half (e.g., portion) may be any portion of the molecule from which it is obtained (e.g., is less than 100% of the whole of the molecule), such that there is at least one additional portion formed (e.g., a second half, other half, second portion), which also is less than 100% of the whole of the molecule. It is important to note, that the molecule may be formed into additional portions (e.g., third, fourth, etc., halves (e.g., portions)), which is readily envisioned by using the term definition above, and such additional halves to not constitute a molecule larger than or in addition to the whole from which they were derived. Further, it should be noted that in the event there are more than two halves (e.g., two portions) formed from the splitting of a molecule it may only require two of the portions to reconstitute the activity of the molecule as a whole. By way of example, if an enzyme is split into three halves (e.g., three portions), wherein the catalytic domain of the enzyme possessing the enzymatic activity of interest is only split into two halves (e.g., two portions) only the two portions of the catalytic domain may be necessary to be used to carry out the activity of interest. Thus, when referring to using two halves, it is not necessary that the two halves, together, comprise 100% of the whole of the molecule from which they were derived. In certain embodiments, the split site is within a loop region of the DddA.
[0246] As used herein, reference to splitting a DddA at a split site embraces direct and indirect means for obtaining two half portions of a DddA. In one embodiment, splitting a DddA refers to the direct splitting a DddA polypeptide at a split site in the protein to obtain the DddA-N and DddA-C half portions. For example, the cleaving of a peptide bond between two adjacent amino acid residues at a split site may be achieved by enzymatic or chemical means. In another embodiment, a DddA may be split by engineering separate nucleic acid sequences, each encoding a different half portion of the DddA. Such methods can be used to obtain expression vectors for expressing the DddA half portions in a cell in order to reconstitute the DddA.
Subject
[0247] The term subject, as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
Target Site
[0248] The term target site refers to a sequence within a nucleic acid molecule (e.g., a mtDNA) that is edited by a mtDNA base editor disclosed herein. The target site further refers to the sequence within a nucleic acid molecule to which a complex of the mtDNA base editor binds. In cases wherein the pDNAbp of the mtDNA base editor is a Cas9 domain, typically, the target site is a sequence that includes the unique 20 bp target specified by the gRNA plus the genomic PAM sequence. CRISPR-Cas9 mechanisms recognize DNA targets that are complementary to a short CRISPR sgRNA sequence. The part of the sgRNA sequence that is complementary to the target sequence is known as a protospacer. In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.
Transition
[0249] As used herein, transitions refer to the interchange of purine nucleobases (A.Math.G) or the interchange of pyrimidine nucleobases (C.Math.T). This class of interchanges involves nucleobases of similar shape. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule. These changes involve A.Math.G, G.Math.A, C.Math.T, or T.Math.C. In the context of a double-strand DNA with Watson-Crick paired nucleobases, transitions refer to the following base pair exchanges: A:T.Math.G:C, G:G.Math.A:T, C:G.Math.T:A, or T:A.Math.C:G. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.
Transversion
[0250] As used herein, transversions refer to the interchange of purine nucleobases for pyrimidine nucleobases, or in the reverse and thus, involve the interchange of nucleobases with dissimilar shape. These changes involve T.Math.A, T.Math.G, C.Math.G, C.Math.A, A.Math.T, A.Math.C, G.Math.C, and G.Math.T. In the context of a double-strand DNA with Watson-Crick paired nucleobases, transversions refer to the following base pair exchanges: T:A.Math.A:T, T:A.Math.G:C, C:G.Math.G:C, C:G.Math.A:T, A:T.Math.T:A, A:T.Math.C:G, G:C.Math.C:G, and G:C.Math.T:A. The compositions and methods disclosed herein are capable of inducing one or more transversions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.
Treatment
[0251] The terms treatment, treat, and treating, refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms treatment, treat, and treating refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
Upstream
[0252] As used herein, the terms upstream and downstream are terms of relativety that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5-to-3 direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5 to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5 side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3 to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3 side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the sense or coding strand. In genetics, a sense strand is the segment within double-stranded DNA that runs from 5 to 3, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3 to 5. Thus, as an example, a SNP nucleobase is downstream of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3 side of the promoter on the sense or coding strand.
Uracil Glycosylase Inhibitor
[0253] The term uracil glycosylase inhibitor or UGI, as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 27. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as UGI variants. A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 27. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 27. In some embodiments, the UGI comprises the following amino acid sequence: MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML (SEQ ID NO: 27) (P14739|UNGI_BPPB2 Uracil-DNA glycosylase inhibitor), or the same sequence but without the N-terminal methionine.
[0254] Other UGI proteins may include those described in Example 6, as follows:
TABLE-US-00003 SEQ UGI Sequence IDNO: Canonical TNLSDIIEKETGKQLVIQESILMLPEEVE 341 UGI EVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKML UGI2 MTLELQLKHYITNLFNLPKDEKWHCESIE 445 EIADDILPDQYVRLGALSNKILQTYTYYS DTLHESNIYPFILYYQKQLIAIGYIDENH DMDFLYLHNTIMPLLDQRYLLTGGQ UGI3 MNKNFDEVKADLRTVTGKKIEFKERLKNI 446 LRVQMNQLGFEDSYMIQVQVSSDQEEWVE CHENMSLSDFEVMYGNISGEIKRMTVVKY EEANIEKLVELKFEYEYAKAHQEYIRAYT KLMSNTLYGRKPSL UGI5 MNEEKMHYRDAIKEVELTMMSLDSHFRTH 447 KEFTDSYLLVLILEDVVGETRVEVSEGLT FDEASYIIGGTSDNILNMHMINYCEKNRE EIYKWLKVSRVNTFKSNYAKMLLNTAYGK DLLKGVVK UGI7 MNNHFMSIGRNCSKCNNVRLNEDFSKSEE 556 ICNECFDKEERFVDSYTLIYITEDETGKR FEAILENQTIEETEIIYGNIIDKIIVWNV ILTM UGI12 DGNEHWEVHPGLSLSDFEVVYGNNPHQIV 376 KLRLDKEVGGSGGSMVQNDFIDSYTLCWL LRDDSGGGGSMVQNDFIDSYTLCWLLRDD DGNEHWEVHPGLSLSDFEVVYGNNPHQIV KLRLDKEV
Variant
[0255] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered as variants.
[0256] As used herein, the term variant refers to a protein having characteristics that deviate from what occurs in nature that retains at least one functional i.e. binding, interaction, or enzymatic ability and/or therapeutic property thereof. A variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the wild type protein. For instance, a variant of Cas9 may comprise a Cas9 that has one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. As another example, a variant of a deaminase may comprise a deaminase that has one or more changes in amino acid residues as compared to a wild type deaminase amino acid sequence, e.g. following ancestral sequence reconstruction of the deaminase. These changes include chemical modifications, including substitutions of different amino acid residues truncations, covalent additions (e.g. of a tag), and any other mutations. The term also encompasses circular permutants, mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence. This term also embraces fragments of a wild type protein.
[0257] The level or degree of which the property is retained may be reduced relative to the wild type protein but is typically the same or similar in kind. Generally, variants are overall very similar, and in many regions, identical to the amino acid sequence of the protein described herein. A skilled artisan will appreciate how to make and use variants that maintain all, or at least some, of a functional ability or property.
[0258] The variant proteins may comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, identical to, for example, the amino acid sequence of a wild-type protein, or any protein provided herein (e.g. DddA).
[0259] By a polypeptide having an amino acid sequence at least, for example, 95% identical to a query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[0260] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the amino acid sequence of a protein such as a DddA protein, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
[0261] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
Vector
[0262] The term vector, as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.
Wild Type
[0263] As used herein the term wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
[0264] These and other exemplary embodiments are described in more detail in the Detailed Description, Examples, and claims. The invention is not intended to be limited in any manner by the above exemplary embodiments.
Detailed Description of Certain Embodiments
[0265] Each mammalian cell contains hundreds to thousands of copies of a circular mtDNA.sup.10. Homoplasmy refers to a state in which all mtDNA molecules are identical, while heteroplasmy refers to a state in which a cell contains a mixture of wild-type and mutant mtDNA. Current approaches to engineer mtDNA rely on DNA-binding proteins such as transcription activator-like effectors nucleases (mitoTALENs).sup.11-17 and zinc finger nucleases (mitoZFNs).sup.18-20 fused to mitochondrial targeting sequences to induce double-strand breaks (DSBs). Such proteins do not rely on nucleic acid programmability (e.g., such as with Cas9 domains). Linearized mtDNA is rapidly degraded,.sup.21-23 resulting in heteroplasmic shifts to favor uncut mtDNA genomes. As a candidate therapy however, this approach cannot be applied to homoplasmic mtDNA mutations.sup.24 since destroying all mtDNA copies is presumed to be harmful..sup.22,25 In addition, using DSBs to eliminate heteroplasmic mtDNA mutations, which tend to be functionally recessive,.sup.26 implicitly requires the edited cell to restore its wild-type mtDNA copy number. During this transient period of mtDNA repopulation, the loss of mtDNA copies could result in cellular toxicity.
[0266] The present disclosure relates in part to the inventors' discovery of a double-stranded DNA deaminase, referred to herein as DddA, and to its application in base editing of double-stranded nucleic acid molecules, and in particular, the editing of mitochondrial DNA.
[0267] Accordingly, the disclosure provides a novel platform of precision genome editing using a double-stranded DNA deaminase (DddA) and a programmable DNA binding protein (pDNAbp), such as a TALE domain, zinc finger binding domain, or a napDNAbp (e.g., Cas9), to target the deamination of a target base, which through cellular DNA repair and/or replication, is converted to a new base, thereby installing a base edit at a target site. In some embodiments, the deaminase activity is a cytidine deminase, which deaminates a cytidine, leading to a C-to-T edit at that site. In some other embodiments, that deaminase activity is an adenosine deminase, which deaminates an adenosine, leading to a A-to-G edit at that site. In various embodiments, the disclosure further relates to split-constructs and split-delivery of said constructs whereby to address the toxic nature of fully active DddA in cells (as discovered by the inventors), the DddA protein is split or otherwise divided into two or more DddA fragments which can be separately delivered, expressed, or otherwise provided to cells to avoid the toxicity of fully active DddA. Further, the DddA fragments may be delivered, expressed, or otherwise provided as separate fusion proteins to cells with programmable DNA binding proteins (e.g., zinc finger domains, TALE domains, or Cas9 domains) which are programmed to localize the DddA fragments to a target edit site, through the binding of the DNA binding proteins to DNA sites upstream and downstream of the target edit site. Once co-localized to the target edit site, the separately provided DddA fragments may associate (covalently or non-covalently) to reconstitute an active DddA protein with a double-stranded DNA deaminase activity. In certain embodiments where the objective is to base edit mitochondrial DNA targets, the programmable DNA binding proteins can be modified with one or more mitochondrial localization signals (MLS) so that the DddA-pDNAbp fusions are translocated into the mitochondria, thereby enabling them to act on mtDNA targets.
[0268] The inventors are believed to be the first to identify DddA, initially being discovered as a bacterial toxin. The inventors further conceived of the idea of splitting the DddA into two or more domains, which apart do not have a deaminase activity (and as such, lack toxicity), but which may be reconstituted to restore the deaminase activity of the protein. This allows the separate delivery DddA fragments to cells, or delivery of nucleic acid molecules expressing such DddA fragments to a cell, such that once present or expressed within a cell, DddA fragments may associate with one another By associate it is meant the two or more DddA fragments may come into contact with one another (e.g., in a cell) and form a functional DddA protein within a cell. The association of the two or more fragments may be through covalent interactions or non-covalent interactions. In addition, the DddA domains may be fused or otherwise non-covalently linked to a programmable DNA binding protein, such as a Cas9 domain or other napDNAbp domain, zinc finger domain or protein (ZF, ZFD, or ZFP), or a transcription activator-like effector protein (TALE), which allows for the co-localization of the two or more DddA fragments to a particular desired site in a target nucleic acid molecule which is to be edited, such that when the DddA fragments are co-localized at the desired editing site, they reform a functional DddA that is capable deaminating a target site on a double-stranded DNA molecule. In certain embodiments, the programmable DNA binding proteins can be engineered to comprise one or more mitochondrial localization signals (MLS) such the DddA domains become translocated into the mitochondria, thereby providing a means by which to conduct base editing directly on the mitochondrial genome.
[0269]
[0270] In the context of base editing, all previously described cytidine deaminases utilize single-stranded DNA as a substrate (e.g., the R-loop region of a Cas9-gRNA/dsDNA complex). Base editing in the context of mitochondrial DNA has not heretofore been possible due to the challenges of introducing and/or expressing the gRNA needed for a Cas9-based system into mitochondria. The inventors have recognized for the first time that the catalytic properties of DddA can be leveraged to conduct base editing directly on a double strand DNA substrate by separating the DddA into inactive portions, which when co-localized within a cell will become reconstituted as an active DddA. This avoids or at least minimizes the toxicity associated with delivering and/or expressing a fully active DddA in a cell.
[0271] For example, a DddA may be divided into two fragments at a split site, i.e., a peptide bond between two adjacent residues in the primary structure or sequence of a DddA. The split site may be positioned anywhere along the length of the DddA amino acid sequence, so long as the resulting fragments do not on their own possess a toxic property (which could be a complete or partial deaminase activity). In certain embodiments, the split site is located in a loop region of the DddA protein. In the embodiment shown in
[0272] Accordingly, this disclosure provides compositions, kits, and methods of modifying double-stranded DNA (e.g., mitochondrial DNA or mtDNA) using genome editing strategies that comprise the use of a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a double-stranded DNA deaminase (DddA) to precisely install nucleotide changes and/or correct pathogenic mutations in double-stranded DNA (e.g., mtDNA), rather than destroying the DNA (e.g., mtDNA) with double-strand breaks (DSBs). The present disclosure provides pDNAbp polypeptides, DddA polypeptides, fusion proteins comprising pDNAbp polypeptides and DddA polypeptides, nucleic acid molecules encoding the pDNAbp polypeptides, DddA polypeptides, and fusion proteins described herein, expression vectors comprising the nucleic acid molecules described herein, cells comprising the nucleic acid molecules, expression vectors, pDNAbp polypeptides, DddA polypeptides, and/or fusion proteins described herein, pharmaceutical compositions comprising the polypeptides, fusion proteins, nucleic acid molecules, vectors, or cells described herein, and kits comprising the polypeptides, fusion proteins, nucleic acid molecules, vectors, or cells described herein for modifying double-stranded DNA (e.g., mtDNA) by base editing.
[0273] Mitochondrial diseases (e.g., MELAS/Leigh syndrome and Leber's hereditary optic neuropathy) are diseases often resulting from errors or mutations in the mitochondrial DNA (mtDNA). In many cases, the mutated mtDNA co-exists with the wild-type mtDNA (mtDNA heteroplasmy). In such instances, residual wild type mtDNA can partially compensate for the mutation before biochemical and clinical manifestations occur. Multiple approaches to reduce the levels of mutant mtDNA have been tried. None of these approaches, however, have been successful in treating or correcting these abnormalities. The present disclosure, including the disclosed DddA/pDNAbp fusion proteins, nucleic acid molecules and vectors encoding same can be used to treat one or more mitochondrial diseases, which can include, but are not limited to: Alper's Disease, Autosomal Dominant Optic Atrophy (ADOA), Barth Syndrome, Carnitine Deficiency, Chronic Progressive External Ophthalmoplegia (CPEO), Co-Enzyme Q10 Deficiency, Creatine Deficiency Syndrome, Fatty Acid Oxidation Disorders, Friedreich's Ataxia, Kearns-Sayre Syndrome (KSS), Lactic Acidosis, Leber Hereditary Optic Neuropathy (LHON), Leigh Syndrome, MELAS, Mitochondrial Myopathy, Multiple Mitochondrial Dysfunction Syndrome, Primary Mitochondrial Myopathy, and TK2d, among others.
[0274] The present disclosure addresses many of the shortcomings of the exisiting technologies with a new precision mtDNA editing fusion protein and technique. The proposed technology permits the editing (e.g., deamination) of single, or multiple, nucleotides in the mtDNA allowing for the correction or modification of the nucleotide, and by extension the codon in which it is contained. In various embodiment, however, the present disclosure is not limited to editing mtDNA, but may also be used to target the editing of any double-stranded DNA in the cell, including the genomic DNA in the nucleus.
mtDNA BEs
[0275] Provided herein are base editor fusion proteins, vectors and nucleic acid molecule encoding base editor fusion proteins, kits, and methods of modifying mitochondrial DNA (mtDNA) using genome editing strategies that comprise the use of a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a double-stranded DNA deaminase (DddA) to precisely install nucleotide changes and/or correct pathogenic mutations in mtDNA, rather than destroying the mtDNA with double-strand breaks (DSBs). In various embodiments, these polypeptides may be combined as fusion proteins referred to as mtDNA base editors. In various embodiments, that base editor fusion proteins may be provided as separate components, i.e., not as a fusion protein, but rather as separate pDNAbp and DddA domains which associate in the cell to target the desired edit site.
[0276] Also provided herein are base editor fusion proteins, vectors and nucleic acid molecule encoding base editor fusion proteins, kits, and methods of modifying any double-stranded DNA (e.g., genomic DNA) using genome editing strategies that comprise the use of a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a double-stranded DNA deaminase (DddA) to precisely install nucleotide changes and/or correct pathogenic mutations in double-stranded DNA, rather than destroying the DNA with double-strand breaks (DSBs). In various embodiments, that base editor fusion proteins may be provided as separate components, i.e., not as a fusion protein, but rather as separate pDNAbp and DddA domains which associate in the cell to target the desired edit site.
[0277] The present disclosure provides mtDNA base editors, pDNAbp polypeptides, DddA polypeptides, nucleic acid molecules encoding the pDNAbp polypeptides, DddA polypeptides, and fusion proteins described herein, expression vectors comprising the nucleic acid molecules, cells comprising the nucleic acid molecules, expression vectors, and/or pDNAbp polypeptides, DddA polypeptides, or fusion proteins, pharmaceutical compositions comprising the polypeptides, fusion proteins, nucleic acid molecules, vectors, or the cells described herein, and kits comprising the polypeptides, fusion proteins, nucleic acid molecules, vectors, or the cells described herein for modifying mtDNA by base editing.
[0278] In some embodiments, the mtDNA base editors comprise a pDNAbp (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas) and a DddAs (or inactive fragment there). In other embodiments, the mtDNA base editors comprise separately expressed pDNAbps and DddAs, which may be co-localized at a desired target site through the use of split-intein sequences, RNA-protein recruitment systems, or other elements that facilitate the co-localization of separately expressed elements to a target site. In various other embodiments, the fusion proteins and/or the separately expressed pDNAbps and DddAs become translocated into the mitochondria. To effect translocation, the fusion proteins and/or the separately expressed pDNAbps and DddAs can comprise one or more mitochondrial targeting sequences (MTS).
[0279] In still other embodiments, the mtDNA base editors comprise a DddA domain which has been inactivated. In one embodiment, this inactivation can be achieved by engineering a whole DddA polypeptide into two or more fragments, each alone which is inactive and non-toxic to a cell. When the DddA inactive fragments become co-localization in the cell, e.g., inside the mitochondria, the fragments reconstitute the deaminase activity. The co-localization of the DddA fragments can be effectuated by fusing each DddA fragment to a separate pDNAbp that binds on either one side or the other of a target deamination site. For example, the embodiments depicted in
[0280] In certain embodiments, the reconstituted activity of the co-localized two or more fragments can comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% of the deaminase activity of a wildtype DddA.
[0281] In terms of the spacing between the target site A and target site B from the site of deamination, any suitable spacing may be used, and which may be further dependent on the length of the linkers (if present) between the pDNAbp and the DddA domains, as well as the properties of the DddA domains. If the target nucleobase site (C on the deamination strand or a G:C nucleobase pair if referring to both strands) is assigned an arbitrary value of 0, then 3-most position of target site A, in various embodiments, may be spaced at least 1 nucleotide upstream of the target G:C nucleobase pair, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides upstream of the G:C nucleobase pair (or otherwise the target site of deamination). Likewise, the 3-most position of target site B (i.e., which is on the opposite strand in this instance), may be spaced at least 1 nucleotide upstream of the target G:C nucleobase pair, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides upstream of the G:C nucleobase pair (or otherwise the target site of deamination).
[0282] Looking at
[0283] In certain embodiments, the DddA can be separated into two fragments by dividing the DddA at a split site. A split site refers to a position between two adjacent amino acids (in a wildtype DddA amino acid sequence) that marks a point of division of a DddA. In certain embodiments, the DddA can have a least one split site, such that once divided at that split site, the DddA forms an N-terminal fragment and a C-terminal fragment. The N-terminal and C-terminal fragments can be the same or difference sizes (or lengths), wherein the size and/or polypeptide length depends on the location or position of the split site. As used herein, reference to a fragment of DddA (or any other polypeptide) can be referred equivalently as a portion. Thus, a DddA which is divided at a split site can form an N-terminal portion and a C-terminal portion. Preferably, the N-terminal fragment (or portion) and the C-terminal fragment (or portion) or DddA do not have a deaminase activity.
[0284] In various embodiments, a DddA may be split into two or more inactive fragments by directly cleaving the DddA at one or more split sites. Direct cleaving can be carried out by a protease (e.g., trypsin) or other enzyme or chemical reagent. In certain embodiments, such chemical cleavage reactions can be designed to be site-selective (e.g., Elashal and Raj, Site-selective chemical cleavage of peptide bonds, Chemical Communications, 2016, Vol. 52, pages 6304-6307, the contents of which are incorporated herein by reference.) In other embodiments, chemical cleavage reactions can be designed to be non-selective and/or occur in a random fashion.
[0285] In other embodiments, the two or more inactive DddA fragments can be engineered as separately expressed polypeptides. For instance, for a DddA having one split site, the N-terminal DddA fragment could be engineered from a first nucleotide sequence that encodes the N-terminal DddA fragment (which extends from the N-terminus of the DddA up to and including the residue on the amino-terminal side of the split site). In such an example, the C-terminal DddA fragment could be engineered from a second nucleotide sequence that encodes the C-terminal DddA fragment (which extends from the carboxy-terminus of the split site up to including the natural C-terminus of the DddA protein). The first and second nucleotide sequences could be on the same or different nucleotide molecules (e.g., the same or different expression vectors).
[0286] In various embodiments, that N-terminal portion of the DddA may be referred to as DddA-N half and the C-terminal portion of the DddA may be referred to as the DddA-C half. Reference to the term half does not connote the requirement that the DddA-N and DddA-C portions are identically half of the size and/or sequence length of a complete DddA, or that the split site is required to be at the mid point of the complete DddA polypeptide. To the contrary, and as noted above, the split site can be between any pair of residues in the DddA polypeptide, thereby giving rise to half portions which are unequal in size and/or sequence length. In certain embodiments, the split site is within a loop region of the DddA.
[0287] Accordingly, in one aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins, in some embodiments, can comprise a first fusion protein comprising a first pDNAbp (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a first portion or fragment of a DddA, and a second fusion protein comprising a second pDNAbp (e.g., mitoTALE, mitoZFP, or a CRISPR/Cas9) and a second portion or fragment of a DddA, such that the first and the second portions of the DddA reconstitute a DddA upon co-localization in a cell and/or mitochondria. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a DddA. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0288] [pDNAbp]-[DddA half.sup.A] and [pDNAbp]-[DddA half.sup.B]; [0289] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[pDNAbp]; [0290] [pDNAbp]-[DddA half.sup.A] and [DddA-half.sup.B]-[pDNAbp]; or [0291] [DddA-half.sup.A]-[pDNAbp] and [pDNAbp]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0292] In another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first mitoTALE and a first portion or fragment of a DddA, and a second fusion protein comprising a second mitoTALE and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a Ddda. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0293] [mitoTALE]-[DddA half.sup.A] and [mitoTALE]-[DddA half.sup.B]; [0294] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[mitoTALE]; [0295] [mitoTALE]-[DddA half.sup.A] and [DddA-half.sup.B]-[mitoTALE]; or [0296] [DddA-half.sup.A]-[mitoTALE] and [mitoTALE]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0297] In yet another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first mitoZFP and a first portion or fragment of a DddA, and a second fusion protein comprising a second mitoZFP and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a Ddda. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0298] [mitoZFP]-[DddA half.sup.A] and [mitoZFP]-[DddA half.sup.B]; [0299] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[mitoZFP]; [0300] [mitoZFP]-[DddA half.sup.A] and [DddA-half.sup.B]-[mitoZFP]; or [0301] [DddA-half.sup.A]-[mitoZFP] and [mitoZFP]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0302] In yet another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first Cas9 and a first portion or fragment of a DddA, and a second fusion protein comprising a second Cas9 and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA (i.e., DddA half.sup.A as shown in
[0307] In each instance above of ]-[ can be in reference to a linker sequence.
[0308] In addition, the fusion proteins may have any suitable architecture, include any those depicted in
[0309] In some embodiments, a first fusion protein comprises, a first mitochondrial transcription activator-like effector (mitoTALE) domain and a first portion of a DNA deaminase effector (DddA). In some embodiments, the first portion of the DddA comprises an N-terminal truncated DddA. In some embodiments, the first mitoTALE is configured to bind a first nucleic acid sequence proximal to a target nucleotide. In some embodiments, the first portion of a DddA is linked to the remainder of the first fusion protein by the C-terminus of the first portion of a DddA.
[0310] In one aspect, the present disclosure provides mitochondrial DNA editor fusion proteins for use in editing mitochondrial DNA. As used herein, these mitochondrial DNA editor fusion proteins may be referred to as mtDNA editors or mtDNA editing systems.
[0311] In various embodiments, the mtDNA editors described herein comprise (1) a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE domain, mitoZFP domain, or a CRISPR/Cas9 domain) and a double-stranded DNA deaminase domain, which is capable of carrying out a deamination of a nucleobase at a target site associated with the binding site of the programmable DNA binding protein (pDNAbp).
[0312] In some embodiments, the double-stranded DNA deaminase is split into two inactive half portions, with each half portion being fused to a programmable DNA binding protein that binds to a nucleotide sequence either upstream or downstream of a target edit site, and wherein once in the mitochondria, the two half portions (i.e., the N-terminal half and the C-terminal half) reassociate at the target edit site by the co-localization of the programmable DNA binding proteins to binding sites upstream and downstream of the target edit site to be acted on by the DNA deaminase. The reassociation of the two half portions of the double-stranded DNA deaminase restores the deaminase activity at the target edit site. In other embodiments, the double-stranded DNA deaminase can initially be set in an inactive state which can be induced when in the mitochondria. The double-stranded DNA deaminase is preferably delivered initially in an inactive form in order to avoid toxicity inherent with the protein. Any means to regulate the toxic properties of the double-stranded DNA deaminase until such time as the activity is desired to be activated (e.g., in the mitochondria) is contemplated.
[0313] The mtDNA base editors described herein contemplate fusion proteins comprising a mitoTALE and a DddA domain or fragment or portion thereof (e.g., an N-terminal or C-terminal fragment or portion of a DddA), and optionally the joining of the two by a linker. The application contemplates any suitable mitoTALE and a DddA domain to be combined in a single fusion protein. Examples of mitoTALEs and DddA domains are each defined herein.
[0314] In some embodiments, a first fusion protein comprises a first portion of a DddA fused (e.g., attached) to a first mitoTALE. In some embodiments, a second fusion protein comprises a second portion of a DddA fused (e.g., attached) to a second mitoTALE. In some embodiments, the first fusion protein comprises a first portion of a DddA linked to the remainder of the first fusion protein by the C-terminus of the first portion of a DddA. In some embodiments, a second fusion protein comprises a second portion of a DddA linked to the remainder of the second fusion protein by the C-terminus of the second portion of a DddA.
[0315] In some embodiments, the first fusion protein comprises a first mitoTALE to bind a target nucleic acid sequence proximal (as defined herein above) to the target nucleotide. In some embodiments, the second fusion protein comprises a mitoTALE to bind a target nucleic acid sequence proximal to the nucleotide complementary to the target nucleotide. In some embodiments, the first and second mitoTALEs are configured to bind proximally to the same target nucleotide (or nucleotide complementary thereto, as described herein above). In some embodiments, the first and second fusion proteins comprise mitoTALEs configured to bind first and second target nucleic acid sequences such that the first and second portions of DddA can dimerize (i.e., re-assemble) at or near the target nucleotide, such that re-assembled first and second portions of a DddA regain, at least partially, the native activity (e.g., deamination) of a full-length DddA. In some embodiments, the first and second fusion proteins comprise mitoTALEs configured to bind first and second target nucleic acid sequences such that that the first and second portions of a DddA can dimerize (i.e., re-assemble) at or near the target nucleotide, such that the target nucleotide is affected by activity of a re-assembled first and second portions of a DddA. Any suitable architecture of the fusion proteins comprising mitoTALEs are contemplated, and shows in
[0316] The mtDNA base editors described herein also contemplate fusion proteins comprising a mitoZF and a DddA domain or fragment or portion thereof (e.g., an N-terminal or C-terminal fragment or portion of a DddA), and optionally the joining of the two by a linker. The application contemplates any suitable mitoZF and a DddA domain to be combined in a single fusion protein. Examples of mitoZFs and DddA domains are each defined herein.
[0317] In some embodiments, a first fusion protein comprises a first portion of a DddA fused (e.g., attached) to a first mitoZF. In some embodiments, a second fusion protein comprises a second portion of a DddA fused (e.g., attached) to a second mitoZF. In some embodiments, the first fusion protein comprises a first portion of a DddA linked to the remainder of the first fusion protein by the C-terminus of the first portion of a DddA. In some embodiments, a second fusion protein comprises a second portion of a DddA linked to the remainder of the second fusion protein by the C-terminus of the second portion of a DddA.
[0318] In some embodiments, the first fusion protein comprises a first mitoZF to bind a target nucleic acid sequence proximal (as defined herein above) to the target nucleotide. In some embodiments, the second fusion protein comprises a mitoZF to bind a target nucleic acid sequence proximal to the nucleotide complementary to the target nucleotide. In some embodiments, the first and second mitoZFs are configured to bind proximally to the same target nucleotide (or nucleotide complementary thereto, as described herein above). In some embodiments, the first and second fusion proteins comprise mitoZFs configured to bind first and second target nucleic acid sequences such that the first and second portions of DddA can dimerize (i.e., re-assemble) at or near the target nucleotide, such that re-assembled first and second portions of a DddA regain, at least partially, the native activity (e.g., deamination) of a full-length DddA. In some embodiments, the first and second fusion proteins comprise mitoTALEs configured to bind first and second target nucleic acid sequences such that that the first and second portions of a DddA can dimerize (i.e., re-assemble) at or near the target nucleotide, such that the target nucleotide is affected by activity of a re-assembled first and second portions of a DddA. Any suitable architecture of the fusion proteins comprising mitoZFs are contemplated, and shows in
[0319] In some embodiments, the first fusion protein comprises the amino acid sequence of any one of SEQ ID NOs.: 360-375. In some embodiments, the first fusion protein comprises an amino acid sequence with 75% or greater percent identity (e.g., 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 99.9% or greater percent identity) any one of SEQ ID NOs.: 360-375. In some embodiments, the second fusion protein comprises the amino acid sequence of any one of SEQ ID NOs.: 360-375. In some embodiments, the second fusion protein comprises an amino acid sequence with 75% or greater percent identity (e.g., 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 99.9% or greater percent identity) to any one of SEQ ID NOs.: 360-375.
[0320] In some embodiments, the first and second fusion protein form pairs which result from the targeting of a similar target nucleotide, or which first and second portion of a DddA form a pair of portions which can re-assemble (e.g., dimerize) to form a protein with, at least partially, the activity of a full-length DddA (e.g., deamination). In some embodiments, the pair of fusion proteins comprise a first fusion protein comprising the first fusion protein of any one of and a second fusion protein comprising the second fusion protein wherein the first mitoTALE of the first fusion protein is configured to bind a first nucleic acid sequence proximal to a target nucleotide and the second mitoTALE of the second fusion protein is configured to bind a second nucleic acid sequence proximal to a nucleotide opposite the target nucleotide. In some embodiments, the first nucleic acid sequence is upstream of the target nucleotide and the second nucleic acid sequence is upstream of a nucleic acid of the complementary nucleotide of the target nucleotide. In some embodiments, the re-assembly (i.e., dimerization) of the first and second fusion proteins facilitate deamination of the target nucleotide.
mtDNA BEs Comprising mitoTALES
[0321] The mtDNA base editors described herein contemplate fusion proteins comprising a mitoTALE and a DddA domain or fragment or portion thereof (e.g., an N-terminal or C-terminal fragment or portion of a DddA), and optionally the joining of the two by a linker. The application contemplates any suitable mitoTALE and a DddA domain to be combined in a single fusion protein. Examples of mitoTALEs and DddA domains are each defined herein.
[0322] In some embodiments, the mtDNA base editors comprise DddA domains which are DdCBE, i.e., DddA which deaminates a C. Examples of general architecture of mtDNA base editors comprising DdCBEs and mitoTALEs and their amino acid and nucleotide sequences are as follows:
[0323] All right-side halves of DdCBEs have the general architecture of (from N- to C-terminus): COX8A MTS-3FLAG-mitoTALE-2aa linker-DddA.sub.tox half-4aa linker-1-UGI-ATP5B 3UTR
[0324] All left-side halves of DdCBEs have the general architecture of (from N- to C-terminus): SOD2 MTS-3HA-mitoTALE-2aa linker-DddA.sub.tox half-4aa linker-1-UGI-SOD2 3UTR
TABLE-US-00004 (A)SOD2MTS (SEQIDNO:298) MLSRAVCGTSRQLAPVLGYLGSRQKHSLPD (B)COX8AMTS (SEQIDNO:299) MSVLTPLLLRGLTGSARRLPVPRAKIHSL (C)SOD23UTR (SEQIDNO:300) ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCTGTCT TCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCCTGGATAA TTTTTGTTTGATTATTCATTGAAGAAACATTTATTTTCCAATTGTGTGAAGTTTTTGA CTGTTAATAAAAGAATCTGTCAACCATCAAAAAAAAAAAAAAA (D)ATP5B3UTR (SEQIDNO:301) ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCTGTCT TCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCCTGGATAA TTTTTGTTTGATTATTCATTGAAGAAACATTTATTTTCCAATTGTGTGAAGTTTTTGA CTGTTAATAAAAGAATCTGTCAACCATCAAAAAAAAAAAAAAA (E)ND6-DdCBE:LeftmitoTALE-G1397-DddA.sub.tox-N-1x-UGI (SEQIDNO:302) ATGGCCCTGTCCCGTGCGGTTTGTGGCACCTCCCGTCAACTGGCTCCGGTTCTGGGT TATCTGGGTTCCCGTCAAAAACACTCCCTGCCGGACTACCCGTATGATGTTCCGGAT TACGCTGGCTACCCATACGACGTCCCAGACTACGCTGGCTACCCATACGACGTCCC AGACTACGCTATGGACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGC AGGAGAAGATCAAGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCT GGTGGGCCACGGTTTCACCCACGCTCACATTGTGGCCCTGAGCCAGCACCCAGCCG CGCTGGGCACCGTGGCCGTGAAATATCAGGATATGATTGCTGCCCTGCCAGAGGCC ACCCATGAAGCTATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGA GGCGCTGCTGACCGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGACACCG GTCAGCTGCTGAAAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCAT GCTTGGCGTAATGCTCTGACCGGTGCGCCGCTGAACCTGACCCCGCAGCAGGTGGT GGCTATTGCCAGCAACAACGGCGGTAAACAGGCCCTGGAGACCGTGCAGCGCCTG CTGCCGGTGCTGTGCCAGGCCCATGGTCTGACCCCGGAGCAGGTGGTGGCGATCGC TAGCAACATCGGCGGCAAGCAGGCCCTGGAAACCGTGCAGGCGCTGTTACCGGTGC TGTGCCAGGCTCATGGCCTGACCCCGGAACAAGTIGTGGCTATTGCCAGCCATGAT GGCGGTAAACAGGCTCTGGAAACCGTGCAGCGTCTGTTGCCGGTGCTGTGCCAAGC CCATGGCCTGACCCCGGAGCAAGTTGTGGCTATTGCGAGCCATGATGGCGGCAAGC AGGCGCTGGAAACCGTTCAGCGCCTGTTACCGGTGCTGTGCCAAGCTCATGGTCTG ACCCCGGAACAGGTGGTGGCCATTGCTTCCCATGATGGCGGTAAACAGGCCCTGGA AACCGTTCAGCGTCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAAC AAGTGGTTGCTATTGCCAGCCACGATGGCGGCAAGCAGGCTCTGGAGACCGTTCAG CGCCTGCTTCCGGTGCTGTGCCAGGCCCATGGCTTAACCCCGGAACAAGTTGTTGCT ATTGCTAGTCATGATGGCGGTAAACAGGCGCTGGAGACCGTTCAGCGTCTGTTACC GGTGCTGTGCCAGGCGCATGGCTTAACCCCGGAGCAGGTTGTTGCCATTGCCTCCA ATATCGGCGGCAAGCAGGCTCTGGAAACCGTTCAGGCCCTGTTGCCGGTGCTGTGC CAGGCCCATGGACTGACCCCGCAGCAAGTTGTTGCCATTGCCAGCAATGGCGGTGG CAAACAGGCGCTGGAAACTGTTCAGCGCCTGCTCCCGGTGCTGTGCCAAGCGCATG GTCTGACCCCGCAGCAAGTGGTTGCTATTGCTAGCAATGGTGGCGGTCGTCCGGCG CTGGAAAGCATTGTGGCTCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGAC CAACGATCACCTGGTGGCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCGG TGAAGAAAGGCCTGGGTGGATCCGGCAGCTACGCCCTGGGTCCGTATCAGATTAGC GCCCCGCAGCTGCCAGCTTACAATGGTCAGACCGTGGGTACCTTCTACTATGTGAA CGACGCGGGCGGTCTGGAGAGCAAGGTGTTTAGCAGCGGCGGTCCAACCCCGTACC CAAACTATGCCAATGCCGGTCATGTGGAGGGTCAGAGCGCCCTGTTCATGCGTGAT AACGGCATCAGCGAGGGTCTGGTGTTCCACAACAACCCGGAAGGCACCTGCGGTTT TTGCGTGAACATGACCGAGACCCTGCTGCCGGAAAACGCGAAAATGACCGTGGTGC CGCCGGAAGGTTCTGGCGGCTCAACTAATCTGAGCGACATCATTGAGAAGGAGACT GGGAAACAGCTGGTCATTCAGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGA AGTGATCGGCAACAAGCCAGAGTCTGACATCCTGGTGCACACCGCCTACGACGAGT CCACAGATGAGAATGTGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGG GCCCTGGTCATCCAGGATTCTAACGGCGAGAATAAGATCAAGATGCTGTGATAAAC CACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCTGTCTTC TAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCCTGGATAATT TTTGTTTGATTATTCATTGAAGAAACATTTATTTTCCAATTGTGTGAAGTTTTTGACT GTTAATAAAAGAATCTGTCAACCATCAAA (F)ND6-DdCBE:RightmitoTALE-G1397-DddA.sub.tox-N-1x-UGI (SEQIDNO:303) TCCGTTCTGACCCCGCTGCTGCTGCGTGGCCTGACCGGCTCCGCTCGTCGTCTGCCA GTTCCGCGTGCGAAAATCCATTCCCTGGACTACAAAGACCATGACGGTGATTATAA AGATCATGACATCGATTACAAGGATGACGATGACAAGATGGACATCGCGGATCTGC GTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCAAGCCAAAGGTGCGCAG CACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGGCTTCACCCACGCGCACA TCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCGTGGCCGTGAAATATCAG GACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCGATTGTGGGTGTGGGCAA GAGAGGAGCCGGTGCTCGTGCGCTGGAGGCCCTGCTGACCGTGGCCGGTGAACTGC GTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGAAAATCGCGAAACGTGGC GGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAATGCTCTGACCGGTGCCCC GCTGAACCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCAACAACGGCGGTAAAC AGGCTCTGGAAACCGTGCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCATGGTCTG ACCCCGGAGCAGGTGGTGGCGATCGCTAGCAACATCGGCGGCAAGCAGGCTCTGG AGACCGTTCAGGCCCTGTTACCGGTGCTGTGCCAAGCCCATGGTCTGACCCCGCAG CAAGTTGTGGCTATTGCCAGCAATGGCGGTGGCAAACAGGCGCTGGAGACCGTGCA GCGTCTGTTGCCGGTGCTGTGCCAAGCCCATGGGCTGACCCCGCAGCAAGTGGTTG CCATCGCCAGCAACAACGGTGGCAAGCAGGCCCTGGAGACCGTTCAGCGCCTGTTA CCGGTGCTGTGCCAGGCCCATGGCTTAACCCCGCAGCAAGTTGTGGCCATCGCTAG CAACAACGGTGGCAAACAGGCTCTGGAGACTGTTCAGCGTCTGCTTCCGGTGCTGT GCCAAGCGCATGGCCTGACCCCGGAACAAGTTGTTGCTATTGCCAGCCATGATGGT GGCAAGCAGGCGCTGGAAACCGTTCAGCGCCTGCTTCCGGTGCTGTGCCAGGCGCA TGGATTAACCCCGCAGCAAGTGGTGGCCATCGCCAGCAATGGTGGCGGTAAACAG GCCCTGGAAACCGTTCAGCGTCTGTTACCGGTGCTGTGCCAGGCCCATGGATTAAC CCCGGAACAAGTIGTGGCTATTGCGTCCAATATCGGCGGCAAGCAGGCGCTGGAAA CTGTGCAGGCTCTGCTCCCGGTGCTGTGCCAGGCCCATGGGTTAACCCCGCAGCAG GTTGTTGCCATTGCGAGCAACGGCGGTGGCAAACAGGCTCTGGAGACGGTTCAGCG CCTGCTCCCGGTGCTGTGCCAGGCCCATGGTTTAACCCCGCAGCAGGTGGTTGCTAT TGCTAGCAATGGCGGCGGCAAGCAGGCGCTGGAAACGGTGCAGCGTCTGCTACCG GTGCTGTGCCAGGCACATGGCCTTACCCCGCAGCAAGTTGTGGCCATTGCTAGCAA TGGCGGTGGCCGTCCGGCCCTGGAAAGCATTGTGGCGCAGCTGAGCCGTCCAGACC CGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGCCTGCCTGGGTGGC CGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGCGGATCCGCCATTCCAGTGAA GCGCGGCGCTACCGGTGAAACCAAAGTGTTTACCGGTAACAGCAACAGCCCGAAG AGCCCGACCAAAGGCGGTTGCTCTGGCGGCTCAACTAATCTGAGCGACATCATTGA GAAGGAGACTGGGAAACAGCTGGTCATTCAGGAGTCCATCCTGATGCTGCCTGAGG AGGTGGAGGAAGTGATCGGCAACAAGCCAGAGTCTGACATCCTGGTGCACACCGC CTACGACGAGTCCACAGATGAGAATGTGATGCTGCTGACCTCTGACGCCCCCGAGT ATAAGCCTTGGGCCCTGGTCATCCAGGATTCTAACGGCGAGAATAAGATCAAGATG CTGTGATAAGGGGTCTTTGTCCTCTGTACTGTCTCTCTCCTTGCCCCTAACCCAAAA AGCTTCATTTTTCTGTGTAGGCTGCACAAGAGCCTTGATTGAAGATATATTCTTTCT GAACAGTATTTAAGGTTTCCAATAAAATGTACACCCCTCAGAA (G)ND1-DdCBERightmitoTALErepeat (SEQIDNO:304) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCGGAACAGGTGGTTGCCATCGCATC CAATAATGGTGGTAAACAAGCTCTGGAGACCGTTCAAGCCCTGCTGCCAGTGCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAAGTTGTGGCTATTGCCAGCAACATCGGC GGCAAGCAGGCCCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAGGCCCA TGGCCTGACCCCGCAGCAAGTGGTTGCTATCGCCAGCAACAACGGCGGTAAACAGG CTCTGGAAACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAAGCCCATGGTCTGACC CCGGAGCAGGTGGTGGCGATTGCTAGCAACGGCGGTGGCAAGCAGGCTCTGGAGA CCGTTCAGGCCCTGCTTCCGGTGCTGTGCCAAGCGCATGGCCTGACCCCGGAACAA GTTGTTGCCATTGCCAGCAATGGTGGCGGTAAACAGGCGCTGGAAACCGTGCAGGC TCTGTTACCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAGCAAGTGGTGGCTA TTGCGAGCAATGGCGGTGGCAAGCAGGCCCTGGAAACCGTGCAGGCGCTGTTGCCG GTGCTGTGCCAAGCCCATGGATTAACCCCGGAACAAGTGGTGGCGATCGCTAGCAA CAACGGTGGCAAACAGGCGCTGGAGACCGTTCAGCGTCTGTTACCGGTGCTGTGCC AGGCGCATGGCTTAACCCCGGAACAGGTTGTTGCGATTGCCAGCAACATTGGTGGC AAGCAGGCTCTGGAAACCGTTCAGGCCCTGCTCCCGGTGCTGTGCCAGGCCCATGG TTTAACCCCGGAACAGGTGGTGGCCATTGCCAGCAACGGTGGCGGTAAACAGGCCC TGGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCCCATGGACTGACCCCG GAGCAAGTTGTTGCCATTGCTAGCAACAACGGCGGCAAGCAGGCGCTGGAGACCG TGCAGGCTCTGCTTCCGGTGCTGTGCCAGGCCCATGGGTTAACCCCGGAGCAGGTT GTGGCCATCGCCAGCCACGACGGCGGTAAACAGGCCCTGGAAACCGTTCAGGCGCT GCTACCGGTGCTGTGCCAGGCACATGGCTTAACCCCGGAGCAGGTGGTTGCCATCG CCTCCAATGGCGGTGGCAAGCAGGCTCTGGAAACGGTGCAGGCCCTGCTGCCGGTG CTGTGCCAAGCCCATGGGTTGACCCCGGAACAAGTGGTGGCTATTGCTAGCCACGA CGGTGGCAAACAGGCTCTGGAGACTGTTCAGCGTCTGCTTCCGGTGCTGTGCCAGG CTCATGGCTTAACCCCGCAGCAAGTTGTTGCTATTGCCTCCAATATTGGTGGCAAGC AGGCGCTGGAAACCGTTCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCATGGGCTT ACCCCGGAACAAGTTGTGGCCATTGCCTCCCATGATGGTGGCAAACAGGCGCTGGA AACTGTGCAGGCTCTGCTCCCGGTGCTGTGCCAGGCTCATGGATTAACCCCGCAGC AAGTGGTGGCCATTGCTAGCCACGATGGTGGCAAGCAGGCCCTGGAGACGGTTCAG CGTCTGCTCCCGGTGCTGTGCCAGGCCCATGGGCTAACCCCGCAGCAGGTTGTTGCT ATTGCCAGTCATGATGGTGGCAAACAGGCTCTGGAAACTGTGCAGCGCCTGCTACC GGTGCTGTGCCAGGCTCACGGTCTGACCCCGCAGCAGGTGGTGGCAATCGCAAGCA ACGGTGGTGGTCGTCCGGCACTGGAAAGCATTGTGGCGCAGCTGAGCCGTCCAGAC CCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGCCTGCCTGGGTGG CCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0325] Translated amino acid sequence:
TABLE-US-00005 (SEQIDNO:305) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV QALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAAL TNDHLVALACLGGRPALDAVKKGLG (H)ND1-DdCBELeftmitoTALErepeat (SEQIDNO:306) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGG TTTCACCCACGCTCACATTGTGGCCCTGAGCCAGCACCCAGCCGCGCTGGGCACCG TGGCCGTGAAATATCAGGATATGATTGCTGCCCTGCCAGAGGCCACCCATGAAGCT ATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCGCTGCTGAC CGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGACACCGGTCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAAT GCTCTGACCGGTGCGCCGCTGAACCTGACCCCGGAACAAGTGGTTGCTATCGCATC CCATGACGGCGGTAAACAAGCCCTGGAGACCGTTCAAGCCCTGCTGCCAGTGCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCAATGGCGGT GGCAAGCAGGCGCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAAGCCCA TGGCCTGACCCCGCAGCAAGTTGTGGCTATCGCCAGCAACATTGGTGGCAAACAGG CCCTGGAAACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAGGCCCATGGTCTGACC CCGGAGCAGGTGGTGGCGATCGCTAGCAACAACGGTGGCAAGCAGGCTCTGGAAA CCGTGCAGGCCCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAACAA GTTGTGGCTATTGCCAGCCACGACGGTGGCAAACAGGCGCTGGAAACCGTGCAGGC TCTGTTACCGGTGCTGTGCCAAGCGCATGGCCTGACCCCGGAACAGGTGGTGGCTA TTGCTAGCCACGATGGTGGCAAGCAGGCCCTGGAGACCGTTCAGGCGCTGTTGCCG GTGCTGTGCCAGGCGCATGGCTTAACCCCGGAACAAGTTGTTGCGATTGCTAGCAA CGGTGGCGGTAAACAGGCTCTGGAGACCGTTCAGCGTCTGTTACCGGTGCTGTGCC AGGCACATGGCCTGACCCCGGAGCAAGTTGTTGCCATTGCCAGCAACATCGGCGGC AAGCAGGCTCTGGAGACCGTGCAGGCCCTGCTCCCGGTGCTGTGCCAGGCCCATGG CTTAACCCCGGAGCAAGTIGTGGCCATTGCCAGCAACAACGGCGGTAAACAGGCGC TGGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCTCATGGCTTGACCCCG GAACAGGTTGTTGCGATTGCGAGCCATGATGGCGGCAAGCAGGCGCTGGAAACCG TTCAGGCTCTGCTTCCGGTGCTGTGCCAGGCCCATGGATTAACCCCGGAGCAGGTT GTTGCTATTGCCAGCCATGATGGCGGTAAACAGGCCCTGGAGACCGTGCAGGCGCT GCTACCGGTGCTGTGCCAGGCTCATGGGCTGACCCCGGAGCAAGTGGTTGCTATCG CGAGCAACAATGGCGGCAAGCAGGCTCTGGAAACGGTGCAGGCCCTGCTGCCGGT GCTGTGCCAGGCCCATGGGTTAACCCCGGAACAAGTGGTGGCCATCGCTAGCAACG GCGGTGGCAAACAGGCCCTGGAGACTGTTCAGCGTCTGCTTCCGGTGCTGTGCCAG GCCCATGGGCTAACCCCGCAGCAAGTGGTTGCCATTGCCAGCAATGGCGGCGGCAA GCAGGCTCTGGAAACTGTGCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCACGGTC TGACCCCGCAACAGGTGGTGGCAATCGCAAGCAATGGTGGTGGTCGTCCGGCACTG GAGAGCATTGTGGCTCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAA CGATCACCTGGTGGCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCGGTGA AGAAAGGCCTGGGT
[0326] Translated amino acid sequence:
TABLE-US-00006 (SEQIDNO:307) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (I)ND2-DdCBERightmitoTALErepeat (SEQIDNO:308) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCGGAACAAGTGGTTGCCATCGCATC CAATATCGGTGGTAAACAAGCCCTGGAGACCGTTCAAGCCCTGCTGCCAGTGCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAGGTGGTGGCCATTGCGAGCAACAATGGC GGCAAGCAGGCGCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAAGCCCA TGGCCTGACCCCGCAGCAAGTGGTTGCTATCGCCAGCAACATTGGCGGTAAACAGG CCCTGGAAACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAGGCCCATGGTCTGACC CCGGAGCAGGTGGTGGCGATCGCTAGCAACATCGGCGGCAAGCAGGCTCTGGAAA CCGTGCAGGCCCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAACAA GTTGTGGCTATTGCCAGCCATGATGGCGGTAAACAGGCGCTGGAAACCGTGCAGGC TCTGTTACCGGTGCTGTGCCAAGCGCATGGCCTGACCCCGGAACAGGTGGTGGCTA TTGCGAGCAATGGCGGTGGCAAGCAGGCCCTGGAGACCGTTCAGGCGCTGTTGCCG GTGCTGTGCCAGGCGCATGGCTTAACCCCGGAACAAGTTGTTGCGATCGCTAGCAA CAACGGTGGCAAACAGGCTCTGGAGACCGTTCAGCGTCTGTTACCGGTGCTGTGCC AGGCACATGGCCTGACCCCGGAGCAAGTTGTTGCCATTGCCAGCCACGATGGTGGC AAGCAGGCTCTGGAGACCGTGCAGGCCCTGCTCCCGGTGCTGTGCCAGGCCCATGG CTTAACCCCGGAGCAAGTTGTGGCTATCGCCAGCAACGGTGGCGGTAAACAGGCGC TGGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCTCATGGCTTGACCCCG GAACAGGTTGTTGCCATTGCGTCCAATATCGGCGGCAAGCAGGCGCTGGAAACCGT TCAGGCTCTGCTTCCGGTGCTGTGCCAGGCCCATGGATTAACCCCGGAGCAGGTTG TGGCGATTGCGAGCAACGGCGGTGGCAAACAGGCCCTGGAGACCGTGCAGGCGCT GCTACCGGTGCTGTGCCAGGCTCATGGGCTGACCCCGGAGCAAGTGGTTGCTATTG CTAGCAATGGCGGCGGCAAGCAGGCTCTGGAAACGGTGCAGGCCCTGCTGCCGGT GCTGTGCCAGGCCCATGGGTTAACCCCGGAACAAGTGGTGGCCATCGCTTCCAATA TTGGCGGTAAACAGGCCCTGGAGACTGTTCAGCGTCTGCTTCCGGTGCTGTGCCAG GCCCATGGGCTAACCCCGCAGCAAGTTGTTGCTATTGCCTCCAATGGCGGTGGCAA GCAGGCTCTGGAAACTGTGCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCACGGCC TGACCCCGCAGCAAGTTGTGGCAATCGCAAGCAATGGTGGTGGTCGTCCGGCTCTG GAGAGCATTGTGGCGCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAA TGATCACCTGGTGGCCCTGGCCTGCCTGGGTGGCCGTCCGGCTCTGGATGCCGTGA AGAAAGGTCTGGGC
[0327] Translated amino acid sequence:
TABLE-US-00007 (SEQIDNO:309) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (J)ND2-DdCBELeftmitoTALErepeat (SEQIDNO:310) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCGGAACAAGTGGTGGCTATCGCGTC CCATGATGGTGGTAAACAGGCTCTGGAGACCGTGCAAGCTCTGCTGCCAGTGCTGT GCCAGGCCCATGGTCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCAATGGCGGT GGCAAGCAGGCGCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAGGCTCA TGGCCTGACCCCGCAGCAAGTIGTGGCTATTGCCAGCAACGGTGGCGGTAAACAGG CCCTGGAGACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAAGCCCATGGCCTGACC CCGGAGCAGGTGGTGGCGATCGCTAGCAACATCGGCGGCAAGCAGGCTCTGGAAA CCGTGCAGGCCCTGCTTCCGGTGCTGTGCCAGGCCCATGGCTTAACCCCGGAACAG GTTGTTGCTATTGCCAGCAACAACGGCGGTAAACAGGCGCTGGAAACCGTGCAGGC TCTGTTACCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAACAAGTTGTGGCTA TTGCGAGCCATGATGGCGGCAAGCAGGCCCTGGAAACCGTGCAGGCGCTGTTGCCG GTGCTGTGCCAAGCCCATGGATTAACCCCGGAACAAGTTGTTGCGATCGCTAGCAA CATTGGCGGTAAACAGGCTCTGGAAACCGTTCAGCGTCTGTTACCGGTGCTGTGCC AGGCGCATGGTCTGACCCCGGAACAGGTTGTGGCCATTGCCTCCAATGGCGGTGGC AAGCAGGCTCTGGAGACCGTTCAGGCCCTGCTCCCGGTGCTGTGCCAAGCGCATGG CCTGACCCCGGAACAGGTGGTGGCTATCGCCAGCAACATTGGTGGCAAACAGGCGC TGGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCACATGGTCTGACCCCG GAGCAAGTTGTGGCCATTGCTAGCCACGATGGTGGCAAGCAGGCGCTGGAAACCGT TCAGGCTCTGCTTCCGGTGCTGTGCCAGGCCCATGGTTTAACCCCGGAACAAGTGG TTGCCATTGCGTCCAATGGTGGCGGTAAACAGGCCCTGGAAACCGTTCAGGCGCTG CTACCGGTGCTGTGCCAGGCTCATGGGCTGACCCCGGAGCAAGTGGTTGCTATTGC TTCCCATGATGGCGGCAAGCAGGCTCTGGAAACGGTGCAGGCCCTGCTGCCGGTGC TGTGCCAAGCCCATGGGTTAACCCCGGAACAGGTGGTTGCGATTGCTAGCCACGAC GGCGGTAAACAGGCCCTGGAAACGGTTCAGCGTCTGCTTCCGGTGCTGTGCCAGGC CCATGGACTTACCCCGCAGCAGGTTGTGGCGATTGCCTCCAATGGCGGTGGCAAGC AGGCTCTGGAAACTGTGCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCATGGTTTA ACCCCGGAGCAGGTTGTTGCCATCGCCAGCCACGACGGTGGCAAACAGGCGCTGG AAACTGTGCAGGCTCTGCTCCCGGTGCTGTGCCAGGCTCATGGACTTACCCCGGAG CAGGTGGTTGCCATTGCTAGCAACATTGGTGGCAAGCAGGCCCTGGAGACTGTTCA GGCGCTGTTACCGGTGCTGTGCCAGGCCCATGGGTTAACCCCGGAGCAAGTTGTTG CCATTGCCTCCAATATTGGTGGCAAACAGGCTCTGGAGACTGTTCAGGCCCTGCTG CCGGTGCTGTGCCAGGCTCACGGTCTGACCCCGCAGCAAGTGGTGGCAATCGCAAG CAATGGTGGTGGTCGTCCGGCTCTGGAGAGCATTGTGGCGCAGCTGAGCCGTCCAG ACCCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGCCTGCCTGGGT GGCCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0328] Translated amino acid sequence:
TABLE-US-00008 (SEQIDNO:311) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (K)ND4-DdCBERightmitoTALErepeat (SEQIDNO:312) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCGGAACAAGTGGTTGCGATTGCGTC CCATGATGGTGGTAAACAAGCCCTGGAGACCGTTCAAGCTCTGCTGCCAGTGCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCCATGATGGC GGCAAGCAGGCTCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAAGCCCA TGGCCTGACCCCGCAGCAAGTTGTGGCTATTGCCAGCAACGGCGGTGGCAAACAGG CGCTGGAAACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAAGCGCATGGTCTGACC CCGGAGCAGGTGGTGGCCATCGCTAGCAACAACGGTGGCAAGCAGGCCCTGGAAA CCGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCTTAACCCCGGAACAA GTGGTGGCCATTGCGAGCAATGGTGGCGGTAAACAGGCTCTGGAAACCGTGCAGG CCCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGGAACAAGTTGTGGCT ATCGCCAGCAACATCGGCGGCAAGCAGGCGCTGGAGACCGTTCAGGCTCTGTTACC GGTGCTGTGCCAGGCGCATGGCCTGACCCCGGAACAGGTGGTGGCGATCGCTAGCA ACATTGGCGGTAAACAGGCCCTGGAGACCGTTCAGCGCCTGCTCCCGGTGCTGTGC CAGGCCCATGGTCTGACCCCGGAACAGGTTGTTGCTATTGCCAGCAACAACGGCGG CAAGCAGGCCCTGGAGACCGTGCAGGCGCTGCTACCGGTGCTGTGCCAGGCCCATG GACTGACCCCGGAGCAGGTTGTGGCCATCGCGTCCAATGGCGGTGGCAAACAGGCT CTGGAGACCGTTCAGCGTCTGTTACCGGTGCTGTGCCAGGCACATGGCCTGACCCC GGAGCAAGTTGTTGCCATCGCTAGCAACATTGGTGGCAAGCAGGCGCTGGAAACCG TTCAGCGCCTGCTACCGGTGCTGTGCCAGGCTCATGGCTTAACCCCGGAGCAGGTT GTCGCCATTGCCAGCAACAATGGTGGCAAACAGGCTCTGGAAACTGTGCAGGCCCT GCTACCGGTGCTGTGCCAGGCCCATGGGTTAACCCCGGAACAGGTTGTGGCCATTG CCTCCAATAACGGTGGCAAGCAGGCGCTGGAAACGGTGCAGGCTCTGCTTCCGGTG CTGTGCCAGGCTCATGGGCTGACCCCGGAGCAAGTGGTTGCTATTGCGTCCAACAT TGGTGGCAAACAGGCCCTGGAAACCGTTCAGGCGCTGCTCCCGGTGCTGTGCCAGG CCCATGGGCTAACCCCGGAACAGGTGGTTGCCATTGCCTCCAACAATGGTGGCAAG CAGGCCCTGGAAACGGTTCAGCGTCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCT TACCCCGCAGCAAGTTGTTGCTATCGCCAGCAATATTGGTGGCAAACAGGCTCTGG AAACGGTGCAGCGCCTGCTACCGGTGCTGTGCCAGGCTCATGGTTTAACCCCGCAG CAGGTGGTTGCGATTGCCTCCAACAACGGTGGCAAGCAGGCGCTGGAAACTGTTCA GCGTCTGCTCCCGGTGCTGTGCCAGGCTCACGGCCTGACCCCGCAGCAAGTGGTGG CTATCGCCTCCAACGGTGGTGGTCGCCCGGCTCTGGAAAGCATTGTGGCGCAGCTG AGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGC CTGCCTGGGTGGCCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0329] Translated amino acid sequence:
TABLE-US-00009 (SEQIDNO:313) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG GKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQA HGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (L)ND4-DdCBELeftmitoTALErepeat (SEQIDNO:314) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCGGAACAGGTGGTGGCAATCGCAAG CAATAATGGTGGTAAACAGGCTCTGGAAACCGTGCAAGCTCTGCTGCCAGTTCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCCATGATGGC GGCAAGCAGGCCCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGTGCCAGGCCCA TGGCCTGACCCCGCAGCAAGTIGTGGCTATTGCCAGCAACGGCGGTGGCAAACAGG CTCTGGAGACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAAGCCCATGGTCTGACC CCGGAGCAGGTGGTGGCGATCGCTAGCAACATTGGTGGCAAGCAGGCCCTGGAAA CCGTGCAGGCGCTGTTGCCGGTGCTGTGCCAAGCCCATGGGCTGACCCCGGAACAA GTTGTTGCCATTGCCAGCAACAATGGTGGCAAACAGGCTCTGGAAACTGTGCAGGC CCTGCTTCCGGTGCTGTGCCAGGCCCATGGATTAACCCCGGAACAAGTTGTGGCTA TTGCGAGCAATGGCGGCGGCAAGCAGGCGCTGGAAACCGTGCAGGCTCTGTTACCG GTGCTGTGCCAGGCGCATGGCCTGACCCCGGAGCAAGTGGTGGCCATCGCTAGCAA CATTGGCGGTAAACAGGCGCTGGAGACCGTTCAGCGTCTGTTACCGGTGCTGTGCC AGGCACATGGCCTTACCCCGGAACAAGTTGTGGCCATTGCCAGCAACATCGGCGGC AAGCAGGCCCTGGAAACGGTGCAGGCGCTGCTCCCGGTGCTGTGCCAGGCCCATGG GTTAACCCCGGAACAAGTGGTTGCTATTGCTAGCCATGATGGCGGTAAACAGGCCC TGGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCCCATGGTTTAACCCCG GAACAGGTTGTTGCGATTGCTAGCCACGATGGCGGCAAGCAGGCTCTGGAGACCGT TCAGGCCCTGCTCCCGGTGCTGTGCCAGGCCCATGGGCTTACCCCGGAGCAAGTTG TTGCTATTGCCTCCAATATTGGCGGTAAACAGGCGCTGGAAACCGTTCAGGCTCTG CTTCCGGTGCTGTGCCAGGCTCATGGCCTCACCCCGGAACAAGTTGTGGCGATTGC GTCCCATGATGGCGGCAAGCAGGCCCTGGAAACTGTGCAGGCGCTGCTACCGGTGC TGTGCCAGGCCCATGGGCTAACCCCGGAACAGGTGGTTGCGATTGCTAGCAACAAC GGCGGTAAACAGGCTCTGGAGACTGTTCAGCGTCTGCTTCCGGTGCTGTGCCAGGC TCATGGGCTGACCCCGCAGCAAGTGGTTGCTATTGCCAGCAATGGCGGTGGCAAGC AGGCGCTGGAGACTGTTCAGCGCCTGCTCCCGGTGCTGTGCCAGGCTCATGGTTTA ACCCCGGAGCAGGTTGTGGCGATCGCCAGCAATGGTGGCGGTAAACAGGCTCTGG AAACGGTGCAGGCCCTGCTCCCGGTGCTGTGCCAGGCTCATGGACTGACCCCGGAG CAAGTTGTTGCCATTGCGTCCCACGACGGCGGCAAGCAGGCGCTGGAGACGGTGCA GGCTCTGCTCCCGGTGCTGTGCCAGGCTCACGGTCTGACCCCGCAACAGGTGGTGG CAATCGCAAGCAACGGTGGTGGTCGTCCGGCACTGGAGAGCATTGTGGCGCAGCTG AGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGC CTGCCTGGGTGGCCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0330] Translated amino acid sequence:
TABLE-US-00010 (SEQIDNO:315) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV QALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (M)ND5.1-DdCBERightmitoTALErepeat (SEQIDNO:316) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCACAGCAGGTGGTGGCAATCGCAAG CCACGACGGAGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCCGTGCTG TGCCAGGCACACGGACTGACACCTGAACAGGTCGTGGCAATCGCATCCAACGGAG GCGGCAAGCAGGCCCTGGAAACCGTGCAGCGCCTGTTACCCGTGCTGTGCCAGGCC CACGGCCTGACACCCCAGCAGGTGGTGGCCATCGCCTCTAATGGAGGGGGCAAGC AGGCCCTGGAGACGGTGCAGCGGCTGCTGCCTGTGCTGTGCCAGGCTCATGGACTG ACACCAGAACAGGTGGTCGCAATCGCAAGCAACGGAGGTGGCAAGCAGGCCCTGG AGACTGTGCAGGCCCTGCTTCCCGTGCTGTGCCAGGCTCACGGACTGACACCTCAG CAGGTCGTCGCCATCGCCTCCAACAATGGTGGCAAGCAGGCCCTGGAGACAGTGCA GAGACTGCTGCCAGTGCTGTGCCAAGCCCATGGACTGACACCACAGCAGGTCGTCG CTATCGCCTCTAATAACGGCGGCAAGCAGGCCCTGGAGACGGTACAGAGGCTGTTA CCCGTGCTGTGCCAAGCACACGGACTGACACCAGAGCAGGTCGTCGCAATCGCCAG CAATATCGGTGGCAAGCAGGCCCTGGAGACGGTCCAGCGCCTGCTCCCCGTGCTGT GCCAAGCCCACGGCCTGACCCCTCAGCAGGTCGTGGCTATTGCTAGCAATAACGGG GGCAAGCAGGCCCTGGAGACGGTTCAGCGGCTGTTGCCCGTGCTGTGCCAAGCCCA CGGTCTGACCCCTCAGCAGGTGGTCGCTATTGCTTCTAATGGAGGAGGCAAGCAGG CCCTGGAGACGGTACAGAGACTGTTACCTGTGCTGTGCCAGGCACATGGCCTGACA CCAGAGCAGGTGGTCGCTATCGCCAGCAACATAGGTGGCAAGCAGGCCCTGGAGA CGGTACAGAGGCTGCTTCCCGTGCTGTGCCAAGCTCATGGCCTGACACCTGAACAG GTGGTCGCCATTGCTAGCAATAACGGTGGCAAGCAGGCCCTGGAGACGGTACAGC GGCTGTTACCAGTGCTGTGCCAAGCACATGGCTTAACCCCTCAACAGGTCGTCGCA ATTGCCTCTAATATCGGAGGCAAGCAGGCCCTGGAGACGGTACAGCGGCTGCTCCC CGTGCTGTGCCAGGCGCACGGCCTGACTCCTCAGCAGGTCGTGGCAATCGCCAGCA ACATCGGCGGCAGACCTGCCCTGGAGAGCATTGTGGCGCAGCTGAGCCGTCCAGAC CCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGCCTGCCTGGGTGG CCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0331] Translated amino acid sequence:
TABLE-US-00011 (SEQIDNO:317) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLG (N)ND5.1-DdCBELeftmitoTALErepeat (SEQIDNO:318) CTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAATATCGGAGGCAAGCAGGCCCT GGAGACCGTGCAGGCCCTGCTGCCCGTGCTGTGCCAGGCACACGGACTGACACCTC AGCAGGTCGTCGCCATCGCCTCCAACAATGGCGGCAAGCAGGCCCTGGAAACCGTG CAGAGGCTGTTACCCGTGCTGTGCCAGGCCCACGGCCTGACACCCCAGCAGGTGGT GGCAATCGCATCTCACGATGGGGGCAAGCAGGCCCTGGAGACGGTGCAGCGCCTG CTGCCTGTGCTGTGCCAGGCTCATGGACTGACACCAGAACAGGTCGTGGCCATCGC CAGCAACATTGGCGGCAAGCAGGCCCTGGAGACTGTCCAGGCCCTGTTACCCGTGC TGTGCCAAGCCCATGGACTGACACCTGAACAGGTCGTGGCAATCGCATCCAATGGA GGTGGCAAGCAGGCCCTGGAGACAGTGCAGGCCCTGCTGCCAGTGCTGTGCCAGGC TCACGGCCTGACACCAGAACAGGTGGTCGCAATCGCATCTAATGGAGGAGGCAAG CAGGCCCTGGAGACGGTACAGGCCCTGTTGCCCGTGCTGTGCCAAGCCCACGGACT GACACCAGAGCAGGTCGTCGCTATTGCTTCCAACATTGGAGGCAAGCAGGCCCTGG AGACGGTCCAGCGGCTGCTTCCCGTGCTGTGCCAAGCTCATGGCCTGACACCAGAG CAGGTGGTCGCTATTGCCTCCAACAATGGAGGCAAGCAGGCCCTGGAGACGGTTCA GGCCCTGCTTCCCGTGCTGTGCCAGGCTCATGGTCTGACACCCGAACAGGTGGTCG CTATCGCCTCTCACGATGGAGGCAAGCAGGCCCTGGAGACGGTACAGAGGCTGTTA CCTGTGCTGTGCCAGGCCCATGGGCTGACCCCAGAACAGGTGGTCGCCATCGCCAG CAACATCGGCGGCAAGCAGGCCCTGGAGACGGTACAGGCCCTGCTCCCCGTGCTGT GCCAAGCACATGGCCTGACACCCGAGCAGGTCGTGGCTATTGCTAGCAACAACGGG GGCAAGCAGGCCCTGGAGACGGTACAGGCCCTGCTACCAGTGCTGTGCCAAGCGC ACGGGCTGACCCCAGAGCAGGTCGTCGCAATCGCCTCTAACAACGGTGGCAAGCA GGCCCTGGAGACGGTACAGGCCCTGCTGCCCGTGCTGTGCCAAGCGCATGGGCTGA CTCCAGAACAGGTGGTGGCTATCGCCAGCAACATTGGAGGCAAGCAGGCCCTGGA GACGGTACAGCGGCTGCTACCCGTGCTGTGCCAAGCGCACGGTCTGACACCTCAGC AGGTGGTCGCTATCGCTTCTAACATAGGGGGCAAGCAGGCCCTGGAGACGGTACAG CGGCTGCTGCCCGTGCTGTGCCAAGCGCACGGACTGACCCCACAGCAGGTCGTCGC TATCGCCTCTAACGGAGGAGGCAGACCCGCCCTGGAG
[0332] Translated amino acid sequence:
TABLE-US-00012 (SEQIDNO:319) LTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQR LLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVV AIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQ AHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (O)ND5.2-DdCBERightmitoTALErepeat (SEQIDNO:320) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGG TTTCACCCACGCTCACATCGTGGCCCTGAGCCAGCACCCAGCCGCGCTGGGCACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCCACCCATGAAGCT ATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCGCTGCTGAC CGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGATACCGGTCAGCTGCTGA AAATTGCCAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAAT GCTCTGACCGGTGCGCCGCTGAACCTGACCCCGCAGCAGGTGGTGGCTATTGCCAG CAACAACGGCGGTAAACAGGCTCTGGAGACCGTGCAGCGTCTGCTGCCGGTGCTGT GCCAGGCTCATGGTCTGACCCCGGAGCAGGTGGTGGCCATTGCTAGCCATGATGGC GGCAAGCAGGCGCTGGAAACCGTGCAGCGCCTGTTACCGGTGCTGTGCCAAGCCCA TGGTCTGACCCCGCAGCAAGTIGTGGCTATTGCGAGCAACGGCGGTGGCAAACAGG CCCTGGAAACCGTTCAGCGTCTGTTACCGGTGCTGTGCCAGGCCCATGGCCTGACC CCGGAACAAGTGGTGGCTATCGCCAGCAACATTGGTGGCAAGCAGGCCCTGGAAA CCGTGCAGGCGCTGTTGCCGGTGCTGTGCCAAGCCCATGGGCTGACCCCGCAGCAA GTGGTTGCGATCGCTAGCAACAACGGTGGCAAACAGGCTCTGGAAACCGTTCAGCG CCTGCTTCCGGTGCTGTGCCAAGCGCATGGCTTAACCCCGCAGCAAGTTGTGGCCA TTGCGAGCAACAACGGTGGCAAGCAGGCGCTGGAGACCGTTCAGCGTCTGCTTCCG GTGCTGTGCCAGGCGCATGGCCTGACCCCGGAGCAAGTGGTGGCTATTGCTAGCCA CGATGGTGGCAAACAGGCCCTGGAGACCGTGCAGCGCCTGCTCCCGGTGCTGTGCC AGGCCCATGGATTAACCCCGCAGCAAGTGGTGGCCATCGCCAGCAATGGCGGCGG CAAGCAGGCTCTGGAAACTGTGCAGCGTCTGTTACCGGTGCTGTGCCAGGCCCATG GGTTAACCCCGCAGCAGGTTGTTGCCATTGCCTCCAATAATGGCGGTAAACAGGCG CTGGAGACTGTGCAGCGCCTGCTACCGGTGCTGTGCCAGGCACATGGTCTGACCCC GGAACAAGTTGTTGCCATTGCGTCCCATGATGGCGGCAAGCAGGCCCTGGAGACTG TTCAGCGTCTGCTCCCGGTGCTGTGCCAGGCCCATGGTTTAACCCCGGAACAAGTTG TGGCCATTGCTAGCCACGATGGCGGTAAACAGGCTCTGGAAACTGTTCAGCGCCTG CTGCCGGTGCTGTGCCAAGCACATGGCTTAACCCCGGAACAGGTTGTTGCTATTGC CAGCAACATCGGCGGCAAGCAGGCTCTGGAGACCGTTCAGGCCCTGTTGCCGGTGC TGTGCCAGGCCCATGGGCTTACCCCGGAACAAGTGGTTGCCATCGCCAGCAACATT GGCGGTAAACAGGCGCTGGAAACCGTTCAGGCTCTGTTGCCGGTGCTGTGCCAGGC TCATGGCCTTACCCCGCAGCAAGTTGTGGCGATTGCTAGCAATGGCGGTGGCAAGC AGGCGCTGGAGACGGTTCAGCGTCTGCTACCGGTGCTGTGCCAGGCTCATGGATTG ACCCCGCAGCAGGTCGTGGCCATTGCCTCCAATAACGGTGGCAAACAGGCGCTGGA GACAGTTCAGCGCCTGCTGCCGGTGCTGTGCCAGGCTCATGGGTTGACCCCGCAGC AGGTAGTTGCTATTGCTAGCAATGGTGGCGGTCGTCCGGCCCTGGAGAGCATTGTG GCGCAGCTGAGCCGTCCAGACCCGGCGCTGGCGGCTCTGACCAACGATCACCTGGT GGCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCCGTGAAGAAAGGCCTGG GT
[0333] Translated amino acid sequence:
TABLE-US-00013 (SEQIDNO:321) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALAC LGGRPALDAVKKGLG (P)ND5.2-DdCBELeftmitoTALErepeat (SEQIDNO:322) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGG TTTCACCCACGCTCACATTGTGGCCCTGAGCCAGCACCCAGCCGCGCTGGGCACCG TGGCCGTGAAATATCAGGATATGATTGCTGCCCTGCCAGAGGCCACCCATGAAGCT ATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCGCTGCTGAC CGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGACACCGGTCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAAT GCTCTGACCGGTGCGCCGCTGAACCTGACCCCTGAGCAGGTGGTGGCAATCGCAAG CCACGACGGAGGCAAGCAGGCCCTGGAGACAGTGCAGGCCCTGCTGCCCGTGCTGT GCCAGGCACACGGCCTGACACCTGAGCAGGTGGTGGCCATCGCCTCCAACATCGGC GGCAAGCAGGCCCTGGAGACAGTACAGAGGCTGTTACCCGTGCTGTGCCAGGCCCA CGGCCTGACACCCCAGCAGGTCGTCGCCATCGCCTCTAATATTGGAGGCAAGCAGG CCCTGGAGACAGTCCAGCGCCTGCTGCCTGTGCTGTGCCAGGCTCATGGCCTGACA CCAGAACAGGTCGTGGCCATCGCCAGTAATATTGGGGGCAAGCAGGCCCTGGAGA CAGTTCAGGCCCTGTTACCCGTGCTGTGCCAAGCCCATGGCCTGACACCTGAACAG GTGGTCGCCATCGCCTCCAATATTGGTGGCAAGCAGGCCCTGGAGACAGTACAGGC CCTGCTGCCAGTGCTGTGCCAGGCTCACGGCCTGACACCAGAGCAGGTCGTCGCAA TCGCATCTCATGATGGCGGCAAGCAGGCCCTGGAGACAGTACAGGCCCTGTTACCC GTGCTGTGCCAAGCGCACGGCCTGACCCCTGAACAGGTCGTGGCTATTGCAAGCCA CGATGGTGGCAAGCAGGCCCTGGAGACAGTACAGCGGCTGCTTCCCGTGCTGTGCC AAGCTCATGGCCTGACACCTGAGCAGGTCGTCGCTATTGCTAGCAATATTGGCGGC AAGCAGGCCCTGGAGACAGTACAGGCCCTGCTCCCCGTGCTGTGCCAAGCACACGG CCTGACACCCGAACAGGTGGTGGCTATCGCCTCTAATGGAGGTGGCAAGCAGGCCC TGGAGACAGTACAGAGGCTGCTTCCTGTGCTGTGCCAGGCCCATGGCCTGACCCCT GAGCAGGTCGTGGCTATTGCCAGTAATATAGGAGGCAAGCAGGCCCTGGAGACAG TACAGGCCCTGCTACCCGTGCTGTGCCAAGCGCATGGCCTGACCCCAGAACAGGTC GTGGCAATCGCATCTCATGACGGCGGCAAGCAGGCCCTGGAGACAGTACAGGCCCT GCTACCAGTGCTGTGCCAAGCACATGGCCTGACCCCCGAACAGGTGGTGGCAATCG CCTCTCACGACGGGGGCAAGCAGGCCCTGGAGACAGTACAGGCCCTGCTACCCGTG CTGTGCCAAGCGCACGGCCTGACGCCAGAACAGGTGGTCGCTATCGCAAGCAACG GCGGTGGCAAGCAGGCCCTGGAGACAGTACAGCGGCTGCTACCCGTGCTGTGCCAA GCGCACGGCCTGACTCCTCAGCAGGTCGTCGCTATCGCATCTCATGATGGTGGCAA GCAGGCCCTGGAGACAGTACAGCGGCTGCTACCCGTGCTGTGCCAAGCGCACGGCC TGACACCACAGCAGGTCGTCGCAATTGCATCTAACGGAGGAGGCAGACCCGCCCTG GAGAGCATTGTGGCTCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAA CGATCACCTGGTGGCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCGGTGA AGAAAGGCCTGGGT
[0334] Translated amino acid sequence:
TABLE-US-00014 (SEQIDNO:323) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQ ALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALET VQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVA QLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (Q)ND5.3-DdCBERightmitoTALErepeat (SEQIDNO:324) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACACCACAGCAGGTCGTCGCTATCGCTTC AAACATTGGGGGGAAACAGGCACTGGAAACCGTCCAGAGACTGCTGCCCGTCCTGT GCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAATATCGGA GGCAAGCAGGCCCTGGAGACCGTGCAGCGGCTGCTGCCCGTGCTGTGCCAAGCCCA CGGCTTAACACCTCAGCAGGTCGTGGCTATCGCCTCCAACAATGGCGGCAAGCAGG CCCTGGAGACGGTGCAGAGACTGCTGCCAGTGCTGTGCCAGGCCCACGGCTTAACA CCAGAACAGGTCGTGGCCATCGCCTCTAACATTGGCGGCAAGCAGGCCCTGGAGAC TGTGCAGGCCCTGCTGCCCGTGCTGTGCCAGGCCCACGGCCTTACACCACAGCAGG TGGTGGCAATCGCCAGCAATGGAGGGGGCAAGCAGGCCCTGGAGACAGTGCAGAG GCTGCTGCCCGTGCTGTGCCAAGCCCACGGCCTGACACCTCAGCAGGTGGTCGCCA TCGCCTCCAACGGAGGTGGCAAGCAGGCCCTGGAGACGGTACAGCGCCTGCTGCCC GTGCTGTGCCAAGCCCACGGCCTAACACCCGAACAGGTCGTCGCCATCGCCTCTAA CATCGGCGGCAAGCAGGCCCTGGAGACGGTCCAGCGGCTGCTGCCTGTGCTGTGCC AAGCCCACGGCCTTACCCCTCAGCAGGTCGTGGCAATCGCCAGCAACAATGGTGGC AAGCAGGCCCTGGAGACGGTTCAGAGACTGCTGCCCGTGCTGTGCCAAGCCCACGG CCTCACACCTCAGCAGGTGGTGGCCATTGCCTCCAACGGAGGAGGCAAGCAGGCCC TGGAGACGGTACAGAGGCTGCTGCCAGTGCTGTGCCAGGCCCACGGCCTAACACCA GAACAGGTGGTCGCTATTGCCTCTAACATTGGTGGCAAGCAGGCCCTGGAGACGGT ACAGCGCCTGCTGCCCGTGCTGTGCCAAGCCCACGGCCTAACGCCAGAACAGGTCG TCGCTATCGCCAGCAACGGAGGAGGCAAGCAGGCCCTGGAGACGGTACAGCGGCT GCTGCCCGTGCTGTGCCAAGCCCACGGCCTAACCCCACAGCAGGTCGTGGCCATTG CCTCCAATAACGGCGGCAAGCAGGCCCTGGAGACGGTACAGCGGCTGCTGCCCGTG CTGTGCCAAGCCCACGGCCTAACTCCCCAGCAAGTCGTCGCTATTGCCTCTAATAAC GGGGGCAAGCAGGCCCTGGAGACGGTACAGAGACTGCTGCCCGTGCTGTGCCAAG CCCACGGCCTGACACCACAGCAGGTCGTCGCCATCGCAAGCAACGGAGGAGGGAG GCCCGCACTGGAGAGCATTGTGGCGCAGCTGAGCCGTCCAGACCCGGCCCTGGCGG CTCTGACCAATGATCACCTGGTGGCCCTGGCCTGCCTGGGTGGCCGTCCGGCTCTGG ATGCCGTGAAGAAAGGTCTGGGC
[0335] Translated amino acid sequence:
TABLE-US-00015 (SEQIDNO:325) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV KKGLG (R)ND5.3-DdCBELeftmitoTALErepeat (SEQIDNO:326) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGG TTTCACCCACGCTCACATTGTGGCCCTGAGCCAGCACCCAGCCGCGCTGGGCACCG TGGCCGTGAAATATCAGGATATGATTGCTGCCCTGCCAGAGGCCACCCATGAAGCT ATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCGCTGCTGAC CGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGACACCGGTCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAAT GCTCTGACCGGTGCGCCGCTGAACCTGACTCCCGAACAGGTGGTCGCTATCGCTTCT CATGATGGCGGAAAACAGGCTCTGGAAACCGTCCAGGCTCTGCTGCCCGTGCTGTG CCAGGCCCACGGCCTGACCCCACAGCAGGTCGTCGCAATCGCCAGCAATATCGGAG GCAAGCAGGCCCTGGAGACCGTGCAGCGGCTGCTGCCCGTGCTGTGCCAAGCCCAC GGCTTAACACCTCAGCAGGTGGTGGCCATCGCCTCCAACAATGGCGGCAAGCAGGC CCTGGAGACGGTGCAGAGACTGCTGCCAGTGCTGTGCCAGGCCCACGGCTTAACAC CAGAACAGGTCGTGGCAATCGCCTCTAACGGAGGGGGCAAGCAGGCCCTGGAGAC TGTGCAGGCCCTGCTGCCCGTGCTGTGCCAGGCCCACGGCCTTACACCAGAACAGG TGGTCGCCATTGCCAGCAATGGAGGTGGCAAGCAGGCCCTGGAGACAGTCCAGGC CCTGCTGCCCGTGCTGTGCCAAGCCCACGGCCTGACACCTGAACAGGTGGTCGCAA TCGCCTCCCACGATGGGGGCAAGCAGGCCCTGGAGACGGTACAGGCCCTGCTGCCC GTGCTGTGCCAAGCCCACGGCCTAACACCCGAACAGGTGGTGGCCATTGCCTCTAA CGGAGGAGGCAAGCAGGCCCTGGAGACGGTCCAGCGGCTGCTGCCTGTGCTGTGCC AAGCCCACGGCCTTACCCCTGAACAAGTCGTGGCCATCGCCAGCAATGGAGGAGGC AAGCAGGCCCTGGAGACGGTTCAGGCCCTGCTGCCCGTGCTGTGCCAAGCCCACGG CCTCACACCTGAACAAGTIGTGGCCATCGCCTCCCACGATGGTGGCAAGCAGGCCC TGGAGACGGTACAGAGGCTGCTGCCAGTGCTGTGCCAGGCCCACGGCCTAACACCA GAACAGGTGGTGGCTATCGCCTCTAACATTGGCGGCAAGCAGGCCCTGGAGACGGT ACAGGCCCTGCTGCCCGTGCTGTGCCAAGCCCACGGCCTAACGCCAGAACAGGTCG TCGCTATTGCCAGCAACATTGGGGGCAAGCAGGCCCTGGAGACGGTACAGGCCCTG CTGCCCGTGCTGTGCCAAGCCCACGGCCTAACCCCTGAACAGGTGGTGGCAATCGC CTCCAACATTGGTGGCAAGCAGGCCCTGGAGACGGTACAGGCCCTGCTGCCCGTGC TGTGCCAAGCCCACGGCCTAACTCCCGAGCAGGTCGTCGCCATCGCCTCTAATGGC GGCGGCAAGCAGGCCCTGGAGACGGTACAGAGGCTGCTGCCTGTGCTGTGCCAAG CCCACGGCCTAACGCCGCAGCAAGTCGTCGCTATTGCCAGCAATATTGGCGGCAAG CAGGCCCTGGAGACGGTACAGCGCCTGCTGCCCGTGCTGTGCCAAGCCCACGGCCT GACCCCCCAGCAGGTGGTGGCAATCGCTTCAAACGGAGGAGGGAGACCCGCTCTG GAAAGCATTGTGGCTCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAA CGATCACCTGGTGGCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCGGTGA AGAAAGGCCTGGGT
[0336] Translated amino acid sequence:
TABLE-US-00016 (SEQIDNO:327) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQ LSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (S)ATP8-DdCBERightmitoTALErepeat (SEQIDNO:328) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCAAAGGTGCGCAGCACCGTGGCCCAGCACCATGAAGCTCTGGTGGGTCACGG CTTCACCCACGCGCACATCGTGGCTCTGAGCCAGCACCCAGCCGCGCTGGGTACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCTACCCATGAAGCG ATTGTGGGTGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCCCTGCTGAC CGTGGCCGGTGAACTGCGTGGCCCGCCGCTGCAGCTGGATACCGGCCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCTGTGGAAGCTGTGCATGCCTGGCGTAAT GCTCTGACCGGTGCCCCGCTGAACCTGACCCCTCAGCAGGTGGTGGCCATCGCCAG CAACATCGGCGGCAAGCAGGCCCTGGAGACAGTGCAGAGGCTGCTGCCCGTGCTGT GCCAGGCACACGGCCTGACACCTGAGCAGGTGGTGGCAATCGCATCCAATGGAGG AGGCAAGCAGGCCCTGGAGACAGTACAGCGCCTGTTACCCGTGCTGTGCCAGGCCC ACGGCCTGACACCCCAGCAGGTCGTCGCCATCGCCTCTAACAATGGGGGCAAGCAG GCCCTGGAGACAGTCCAGCGGCTGCTGCCTGTGCTGTGCCAGGCTCATGGCCTGAC ACCAGAACAGGTCGTGGCTATTGCCAGCAACAATGGTGGCAAGCAGGCCCTGGAG ACAGTTCAGGCCCTGCTTCCCGTGCTGTGCCAGGCTCACGGCCTGACACCACAGCA GGTCGTGGCCATCGCCTCCAACAATGGCGGCAAGCAGGCCCTGGAGACAGTACAG AGACTGCTGCCAGTGCTGTGCCAAGCCCATGGCCTGACCCCTCAGCAGGTCGTGGC AATCGCATCTCACGACGGTGGCAAGCAGGCCCTGGAGACAGTACAGAGGCTGTTAC CCGTGCTGTGCCAAGCACACGGCCTGACACCAGAGCAGGTCGTCGCAATCGCAAGC AACGGCGGCGGCAAGCAGGCCCTGGAGACAGTACAGCGCCTGCTCCCCGTGCTGTG CCAAGCCCACGGCCTGACACCTCAGCAGGTGGTCGCCATTGCCAGCAACGGCGGGG GCAAGCAGGCCCTGGAGACAGTACAGCGGCTGTTGCCCGTGCTGTGCCAAGCCCAC GGCCTGACGCCCCAGCAGGTGGTCGCCATCGCATCTAACGGCGGTGGCAAGCAGGC CCTGGAGACAGTACAGCGGCTGCTTCCTGTGCTGTGCCAGGCCCATGGCCTGACCC CCGAACAGGTCGTGGCTATCGCTAGCAACAATGGCGGCAAGCAGGCCCTGGAGAC AGTACAGAGACTGTTACCCGTGCTGTGCCAAGCGCATGGCCTGACCCCTGAACAGG TCGTGGCAATTGCCTCCAATAACGGTGGCAAGCAGGCCCTGGAGACAGTACAGCGG CTGCTACCAGTGCTGTGCCAAGCACATGGCCTGACCCCCCAGCAGGTCGTGGCTAT TGCATCTAATGGAGGAGGCAGACCCGCCCTGGAGAGCATTGTGGCGCAGCTGAGCC GTCCAGACCCGGCCCTGGCGGCTCTGACCAATGATCACCTGGTGGCCCTGGCCTGC CTGGGTGGCCGTCCGGCTCTGGATGCCGTGAAGAAAGGTCTGGGC
[0337] Translated amino acid sequence:
TABLE-US-00017 (SEQIDNO:329) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG (T)ATP8-DdCBELeftmitoTALErepeat (SEQIDNO:330) GACATCGCGGATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCA AGCCGAAGGTGCGCAGCACCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGG TTTCACCCACGCTCACATCGTGGCCCTGAGCCAGCACCCAGCCGCGCTGGGCACCG TGGCCGTGAAATATCAGGACATGATTGCTGCCCTGCCAGAGGCCACCCATGAAGCT ATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGGAGGCGCTGCTGAC CGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGATACCGGTCAGCTGCTGA AAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAAT GCTCTGACCGGTGCGCCGCTGAACCTGACCCCGGAGCAGGTGGTGGCTATCGCCAG CAACATTGGCGGTAAACAGGCCCTGGAAACCGTGCAGGCGCTGCTGCCGGTGCTGT GCCAGGCTCATGGTCTGACCCCGCAGCAGGTGGTGGCGATCGCTAGCAACGGCGGT GGCAAGCAGGCTCTGGAGACCGTGCAGCGTCTGTTACCGGTGCTGTGCCAAGCCCA TGGCCTGACCCCGCAGCAAGTTGTGGCCATTGCGAGCAATGGTGGCGGTAAACAGG CGCTGGAAACCGTGCAGCGCCTGTTGCCGGTGCTGTGCCAAGCCCATGGGCTGACC CCGGAACAAGTTGTTGCTATCGCCAGCAACATCGGCGGCAAGCAGGCTCTGGAAAC CGTGCAGGCCCTGCTTCCGGTGCTGTGCCAAGCGCATGGTCTGACCCCGGAACAAG TGGTGGCCATCGCTTCCAATATTGGCGGTAAACAGGCGCTGGAGACCGTGCAGGCT CTGCTCCCGGTGCTGTGCCAAGCACATGGTCTGACCCCGGAGCAAGTTGTGGCTAT TGCCTCCAATATCGGCGGCAAGCAGGCCCTGGAGACCGTTCAGGCGCTGTTACCGG TGCTGTGCCAGGCCCATGGATTAACCCCGGAGCAAGTGGTGGCTATTGCTAGCCAT GATGGCGGTAAACAGGCCCTGGAGACTGTTCAGCGTCTGCTACCGGTGCTGTGCCA GGCCCATGGTTTAACCCCGGAACAGGTTGTTGCCATCGCTTCCAACATCGGCGGCA AGCAGGCTCTGGAAACGGTGCAGGCCCTGTTACCGGTGCTGTGCCAGGCCCATGGG TTAACCCCGGAACAAGTTGTGGCCATTGCCTCCCATGACGGCGGTAAACAGGCTCT GGAGACCGTTCAGCGCCTGCTACCGGTGCTGTGCCAGGCGCATGGCTTAACCCCGG AACAAGTGGTTGCCATTGCGTCCAATATCGGCGGCAAGCAGGCGCTGGAGACCGTT CAGGCTCTGCTTCCGGTGCTGTGCCAGGCACATGGCCTTACCCCGGAACAAGTGGT CGCGATCGCTTCCAACATTGGCGGTAAACAGGCCCTGGAAACGGTTCAGGCGCTGC TTCCGGTGCTGTGCCAGGCCCATGGGCTTACCCCGGAACAGGTTGTGGCTATTGCC AGTAATATCGGCGGCAAGCAGGCTCTGGAAACTGTGCAGGCCCTGCTACCGGTGCT GTGCCAGGCTCATGGGCTGACCCCGGAGCAAGTGGTTGCCATTGCCTCCCATGATG GCGGTAAACAGGCGCTGGAAACGGTGCAGCGTCTGCTTCCGGTGCTGTGCCAGGCT CATGGCTTAACCCCGCAGCAAGTTGTTGCGATTGCTAGCAATGGCGGTGGCAAGCA GGCCCTGGAAACTGTTCAGCGCCTGTTACCGGTGCTGTGCCAGGCCCATGGGCTAA CCCCGGAACAGGTGGTTGCTATTGCCAGCAACATTGGTGGCAAACAGGCGCTGGAA ACTGTGCAGGCTCTGCTTCCGGTGCTGTGCCAGGCCCATGGGCTGACCCCGCAGCA AGTGGTTGCTATTGCTAGCAATGGTGGCGGTCGTCCGGCCCTGGAGAGCATTGTGG CGCAGCTGAGCCGTCCAGACCCGGCCCTGGCGGCTCTGACCAACGATCACCTGGTG GCGCTGGCTTGCCTGGGCGGTCGTCCGGCCCTGGATGCGGTGAAGAAAGGCCTGGG T
[0338] Translated amino acid sequence:
TABLE-US-00018 (SEQIDNO:331) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHP AALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETV QALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLT PQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPA LDAVKKGLG
[0339] Other exemplary mtDNA base editors may comprise DdCBE/mitoTALE fusion proteins, as follows:
[0340] All right-side halves of DdCBEs have the general architecture of (from N- to C-terminus): COX8A MTS-3FLAG-mitoTALE-2aa linker-DddAtox half-4aa linker-1-UGI-ATP5B 3UTR
[0341] All left-side halves of DdCBEs have the general architecture of (from N- to C-terminus): SOD2 MTS-3HA-mitoTALE-2aa linker-DddAtox half-4aa linker-1-UGI-SOD2 3UTR
[0342] mitoTALE domains are annotated as: bold for N-terminal domain, underlined for RVD and bolded italics for C-terminal domain.
TABLE-US-00019 ND6-DdCBE:LeftmitoTALE-G1397-DddAtox-N-1x-UGI(Note:TerminalNG RVDrecognizesamismatchedTinsteadofaGinthereferencegenome) (SEQIDNO:360) MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVP DYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAAL GTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQL LKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETV QALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGP YQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFM RDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETG KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQ DSNGENKIKML** ND6-DdCBE:RightmitoTALE-G1397-DddAtox-N-1x-UGI(Note:TerminalNG RVDrecognizesamismatchedTinsteadofaGinthereferencegenome.TheNTDwasalso engineeredtobepermissiveforA,T,CandGnucleotidesattheN0position) (SEQIDNO:361) MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIAD LRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQ DMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGV TAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVAL ACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDI IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKP WALVIQDSNGENKIKML** ND1-DdCBERightmitoTALErepeat (SEQIDNO:362) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV QALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAAL TNDHLVALACLGGRPALDAVKKGLG ND1-DdCBELeftmitoTALErepeat (SEQIDNO:363) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND2-DdCBERightmitoTALErepeat (SEQIDNO:364) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND2-DdCBELeftmitoTALErepeat (SEQIDNO:365) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNG GKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPV LCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVA IASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV QALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNI GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALT NDHLVALACLGGRPALDAVKKGLG ND4-DdCBERightmitoTALErepeat (SEQIDNO:366) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG GKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQA HGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND4-DdCBELeftmitoTALErepeat (SEQIDNO:367) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV QALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.1-DdCBERightmitoTALErepeat (SEQIDNO:368) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLG ND5.1-DdCBELeftmitoTALErepeat (SEQIDNO:369) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.2-DdCBERightmitoTALErepeat(Note:TerminalNGRVDrecognizesa mismatchedTinsteadofaGinthereferencegenome) (SEQIDNO:370) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALG TVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLL KIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL PVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTND HLVALACLGGRPALDAVKKGLG ND5.2-DdCBELeftmitoTALErepeat (SEQIDNO:371) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQ ALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALET VQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVA QLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.3-DdCBERightmitoTALErepeat (SEQIDNO:372) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV KKGLG ND5.3-DdCBELeftmitoTALErepeat (SEQIDNO:373) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQ LSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ATP8-DdCBERightmitoTALErepeat(Note:TerminalNGRVDrecognizesa mismatchedTinsteadofaCinthereferencegenome) (SEQIDNO:374) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ATP8-DdCBELeftmitoTALErepeat (SEQIDNO:375) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGG KQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA LLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLG GRPALDAVKKGLG
mtDNA BEs Comprising mitoZFs
[0343] The mtDNA base editors described herein contemplate fusion proteins comprising a mitoZF and a DddA domain or fragment or portion thereof (e.g., an N-terminal or C-terminal fragment or portion of a DddA), and optionally the joining of the two by a linker. The application contemplates any suitable mitoZF and a DddA domain to be combined in a single fusion protein. Examples of mitoZFs and DddA domains are each defined herein.
[0344] In some embodiments, the mtDNA base editors comprise DddA domains which are DdCBE, i.e., DddA which deaminates a C. Examples of general architecture of mtDNA base editors comprising DdCBEs and mitoZFs and their amino acid and nucleotide sequences are as follows:
TABLE-US-00020 R8v6 MLGFVGRVAAAPASGALRRLTPS MTS ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSTSGSLSR FLAGtag HIRTHTGEKPFQCDICMRNFSQSG DYKDDDDK(SEQIDNO:395) SLTRHIRTHTGSEKPFQCDICMRN NES FSRSDALSQHIRTHTGEKPFQCDI VDEMTKKFGTLTIHDTEK(SEQIDNO: CMRNFSRNDNRITHIRTHTGEKPF 396) QCDICMRNFSRSDHLTQHTKIHL Linker RGSGGGGSGGSGGSGSYALGPY GS QISAPQLPAYNGQTVGTFYYVND NES2 AGGLESKVFSSGGPTPYPNYANA LQKKLEELELD(SEQIDNO:397) GHVEGQSALFMRDNGISEGLVFH Linker NNPEGTCGFCVNMTETLLPENAK AA MTVVPPEGSGGSTNLSDIIEKETG ZF(R8) KQLVIQESILMLPEEVEEVIGNKP MAERPFQCDICMRNFSTSGSLSRHIRTH ESDILVHTAYDESTDENVMLLTS TGEKPFQCDICMRNFSQSGSLTRHIRTH DAPEYKPWALVIQDSNGENKIKM TGSEKPFQCDICMRNFSRSDALSQHIRT LGSGATNFSLLKQAGDVEENPGP HTGEKPFQCDICMRNFSRNDNRITHIRT MASVLTPLLLRGLTGSARRLPVP HTGEKPFQCDICMRNFSRSDHLTQHTKI RAKIHSLGSTNLSDIIEKETGKQL HLR(SEQIDNO:398) VIQESILMLPEEVEEVIGNKPESDI Linker LVHTAYDESTDENVMLLTSDAPE GSGGGGSGGSGGS(SEQIDNO:399) YKPWALVIQDSNGENKIKML SplitDddA(DddA-G1397N) (SEQIDNO:382) GSYALGPYQISAPQLPAYNGQTVGTFY YVNDAGGLESKVFSSGGPTPYPNYANA GHVEGQSALFMRDNGISEGLVFHNNPE GTCGFCVNMTETLLPENAKMTVVPPE G(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 4-R8v6 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSQASNLIS FLAGtag HIRTHTGEKPFQCDICMRNFSTSH DYKDDDDK(SEQIDNO:395) SLTEHIRTHTGSEKPFQCDICMRN NES FSERSHLREHIRTHTGEKPFQCDI VDEMTKKFGTLTIHDTEK(SEQIDNO: CMRNFSQSGNLTEHIRTHTGEKP 396) FQCDICMRNFSSKKALTEHTKIHL Linker RGSGGGGSGGSGGSAIPVKRGAT GS GETKVFTGNSNSPKSPTKGGCSG NES2 GSTNLSDIIEKETGKQLVIQESILM LQKKLEELELD(SEQIDNO:397) LPEEVEEVIGNKPESDILVHTAYD Linker ESTDENVMLLTSDAPEYKPWAL AA VIQDSNGENKIKMLGSGATNESL ZF(5xZnF-4-R8) LKQAGDVEENPGPMASVLTPLLL MAERPFQCDICMRNFSQASNLISHIRTH RGLTGSARRLPVPRAKIHSLGSTN TGEKPFQCDICMRNFSTSHSLTEHIRTH LSDIIEKETGKQLVIQESILMLPEE TGSEKPFQCDICMRNFSERSHLREHIRT VEEVIGNKPESDILVHTAYDESTD HTGEKPFQCDICMRNFSQSGNLTEHIRT ENVMLLTSDAPEYKPWALVIQDS HTGEKPFQCDICMRNFSSKKALTEHTKI NGENKIKML(SEQIDNO:383) HLR(SEQIDNO:402) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 10-R8 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v6 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSQASNLIS FLAGtag HIRTHTGEKPFQCDICMRNFSQR DYKDDDDK(SEQIDNO:395) ANLRAHIRTHTGSEKPFQCDICM NES RNFSQASNLISHIRTHTGEKPFQC VDEMTKKFGTLTIHDTEK(SEQIDNO: DICMRNFSTSHSLTEHIRTHTGEK 396) PFQCDICMRNFSERSHLREHTKIH Linker LRGSGGGGSGGSGGSAIPVKRGA GS TGETKVFTGNSNSPKSPTKGGCS NES2 GGSTNLSDIIEKETGKQLVIQESIL Linker MLPEEVEEVIGNKPESDILVHTAY LQKKLEELELD(SEQIDNO:397) DESTDENVMLLTSDAPEYKPWA AA LVIQDSNGENKIKMLGSGATNFS ZF(5xZnF-10-R8) LLKQAGDVEENPGPMASVLTPLL MAERPFQCDICMRNFSQASNLISHIRTH LRGLTGSARRLPVPRAKIHSLGST TGEKPFQCDICMRNFSQRANLRAHIRT NLSDIIEKETGKQLVIQESILMLPE HTGSEKPFQCDICMRNFSQASNLISHIR EVEEVIGNKPESDILVHTAYDEST THTGEKPFQCDICMRNFSTSHSLTEHIR DENVMLLTSDAPEYKPWALVIQ THTGEKPFQCDICMRNFSERSHLREHT DSNGENKIKML(SEQIDNO:384) KIHLR(SEQIDNO:403) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) R13-1 MLGFVGRVAAAPASGALRRLTPS MTS v6 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSRSDNLST FLAGtag HIRTHTGEKPFQCDICMRNFSDRS DYKDDDDK(SEQIDNO:395) DLSRHIRTHTGEKPFQCDICMRNF NES SQSGDLTRHIRTHTGSEKPFQCDI VDEMTKKFGTLTIHDTEK(SEQIDNO: CMRNFSRSDSLSAHIRTHTGEKPF 396) QCDICMRNFSQKATRITHTKIHLR Linker GSGGGGSGGSGGSGSYALGPYQI GS SAPQLPAYNGQTVGTFYYVNDA NES2 GGLESKVFSSGGPTPYPNYANAG LQKKLEELELD(SEQIDNO:397) HVEGQSALFMRDNGISEGLVFHN Linker NPEGTCGFCVNMTETLLPENAK AA MTVVPPEGSGGSTNLSDIIEKETG ZF(R13-1) KQLVIQESILMLPEEVEEVIGNKP MAERPFQCDICMRNFSRSDNLSTHIRTH ESDILVHTAYDESTDENVMLLTS TGEKPFQCDICMRNFSDRSDLSRHIRTH DAPEYKPWALVIQDSNGENKIKM TGEKPFQCDICMRNFSQSGDLTRHIRTH LGSGATNFSLLKQAGDVEENPGP TGSEKPFQCDICMRNFSRSDSLSAHIRT MASVLTPLLLRGLTGSARRLPVP HTGEKPFQCDICMRNFSQKATRITHTKI RAKIHSLGSTNLSDIIEKETGKQL HLR(SEQIDNO:404) VIQESILMLPEEVEEVIGNKPESDI Linker LVHTAYDESTDENVMLLTSDAPE GSGGGGSGGSGGS(SEQIDNO:399) YKPWALVIQDSNGENKIKML SplitDddA(DddA-G1397N) (SEQIDNO:385) GSYALGPYQISAPQLPAYNGQTVGTFY YVNDAGGLESKVFSSGGPTPYPNYANA GHVEGQSALFMRDNGISEGLVFHNNPE GTCGFCVNMTETLLPENAKMTVVPPE G(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 9-R13 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v6 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSQSSSLVR FLAGtag HIRTHTGEKPFQCDICMRNFSRSD DYKDDDDK(SEQIDNO:395) NLVRHIRTHTGSEKPFQCDICMR NES NFSQAGHLASHIRTHTGEKPFQC VDEMTKKFGTLTIHDTEK(SEQIDNO: DICMRNFSRKDNLKNHIRTHTGE 396) KPFQCDICMRNFSRKDALRGHTK Linker IHLRGSGGGGSGGSGGSAIPVKR GS GATGETKVFTGNSNSPKSPTKGG NES2 CSGGSTNLSDIIEKETGKQLVIQES LQKKLEELELD(SEQIDNO:397) ILMLPEEVEEVIGNKPESDILVHT Linker AYDESTDENVMLLTSDAPEYKP AA WALVIQDSNGENKIKMLGSGAT ZF(5xZnF-9-R13) NFSLLKQAGDVEENPGPMASVLT MAERPFQCDICMRNFSQSSSLVRHIRTH PLLLRGLTGSARRLPVPRAKIHSL TGEKPFQCDICMRNFSRSDNLVRHIRTH GSTNLSDIIEKETGKQLVIQESILM TGSEKPFQCDICMRNFSQAGHLASHIRT LPEEVEEVIGNKPESDILVHTAYD HTGEKPFQCDICMRNFSRKDNLKNHIR ESTDENVMLLTSDAPEYKPWAL THTGEKPFQCDICMRNFSRKDALRGHT VIQDSNGENKIKML(SEQIDNO: KIHLR(SEQIDNO:405) 386) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 12-R13 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v6 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKGSLQKKLEELELDAA IDNO:394) MAERPFQCDICMRNFSRSDHLTT FLAGtag HIRTHTGEKPFQCDICMRNFSQSS DYKDDDDK(SEQIDNO:395) SLVRHIRTHTGSEKPFQCDICMRN NES FSRSDNLVRHIRTHTGEKPFQCDI VDEMTKKFGTLTIHDTEK(SEQIDNO: CMRNFSQAGHLASHIRTHTGEKP 396) FQCDICMRNFSRKDNLKNHTKIH Linker LRGSGGGGSGGSGGSAIPVKRGA GS TGETKVFTGNSNSPKSPTKGGCS NES2 GGSTNLSDIIEKETGKQLVIQESIL LQKKLEELELD(SEQIDNO:397) MLPEEVEEVIGNKPESDILVHTAY Linker DESTDENVMLLTSDAPEYKPWA AA LVIQDSNGENKIKMLGSGATNFS ZF(5xZnF-12-R13) LLKQAGDVEENPGPMASVLTPLL MAERPFQCDICMRNFSRSDHLTTHIRT LRGLTGSARRLPVPRAKIHSLGST HTGEKPFQCDICMRNFSQSSSLVRHIRT NLSDIIEKETGKQLVIQESILMLPE HTGSEKPFQCDICMRNFSRSDNLVRHIR EVEEVIGNKPESDILVHTAYDEST THTGEKPFQCDICMRNFSQAGHLASHI DENVMLLTSDAPEYKPWALVIQ RTHTGEKPFQCDICMRNFSRKDNLKNH DSNGENKIKML(SEQIDNO:387) TKIHLR(SEQIDNO:406) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQID NO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKI HSL(SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) R8v3 MLGFVGRVAAAPASGALRRLTPS MTS ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSTSGSLSRHIRTHTGEKPFACDI FLAGtag CGRKFAQSGSLTRHTKIHTGGQR DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSRSDALSQHIRTH NES TGEKPFACDICGRKFARNDNRIT ZF(R8) HTKIHTGEKPFQCRICMRKFARS VDEMTKKFGTLTIHDTEK(SEQIDNO: DHLTQHTKIHLRGSGGGGSGGSG 396) GSGSYALGPYQISAPQLPAYNGQ Linker TVGTFYYVNDAGGLESKVFSSGG AA PTPYPNYANAGHVEGQSALFMR MAERPFQCRICMRNFSTSGSLSRHIRTH DNGISEGLVFHNNPEGTCGFCVN TGEKPFACDICGRKFAQSGSLTRHTKIH MTETLLPENAKMTVVPPEGSGGS TGGQRPFQCRICMRNFSRSDALSQHIRT TNLSDIIEKETGKQLVIQESILMLP HTGEKPFACDICGRKFARNDNRITHTKI EEVEEVIGNKPESDILVHTAYDES HTGEKPFQCRICMRKFARSDHLTQHTK TDENVMLLTSDAPEYKPWALVIQ IHLR(SEQIDNO:407) DSNGENKIKML(SEQIDNO:388) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFY YVNDAGGLESKVFSSGGPTPYPNYANA GHVEGQSALFMRDNGISEGLVFHNNPE GTCGFCVNMTETLLPENAKMTVVPPE G(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 4-R8v3 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSQASNLISHIRTHTGEKPFACDI FLAGtag CGRKFATSHSLTEHTKIHTGSQKP DYKDDDDK(SEQIDNO:395) FQCRICMRNFSERSHLREHIRTHT NES GEKPFACDICGRKFAQSGNLTEH VDEMTKKFGTLTIHDTEK(SEQIDNO: TKIHTGEKPFQCRICMRKFASKK 396) ALTEHTKIHLRGSGGGGSGGSGG Linker SAIPVKRGATGETKVFTGNSNSP AA KSPTKGGCSGGSTNLSDIIEKETG ZF(5xZnF-4-R8) KQLVIQESILMLPEEVEEVIGNKP MAERPFQCRICMRNFSQASNLISHIRTH ESDILVHTAYDESTDENVMLLTS TGEKPFACDICGRKFATSHSLTEHTKIH DAPEYKPWALVIQDSNGENKIKM TGSQKPFQCRICMRNFSERSHLREHIRT L(SEQIDNO:389) HTGEKPFACDICGRKFAQSGNLTEHTKI HTGEKPFQCRICMRKFASKKALTEHTK IHLR(SEQIDNO:408) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 10-R8 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v3 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSQASNLISHIRTHTGEKPFACDI FLAGtag CGRKFAQRANLRAHTKIHTGSQK DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSQASNLISHIRTH NES TGEKPFACDICGRKFATSHSLTEH VDEMTKKFGTLTIHDTEK(SEQIDNO: TKIHTGEKPFQCRICMRKFAERSH 396)Linker LREHTKIHLRGSGGGGSGGSGGS AA AIPVKRGATGETKVFTGNSNSPK ZF(5xZnF-10-R8) SPTKGGCSGGSTNLSDIIEKETGK MAERPFQCRICMRNFSQASNLISHIRTH QLVIQESILMLPEEVEEVIGNKPES TGEKPFACDICGRKFAQRANLRAHTKI DILVHTAYDESTDENVMLLTSDA HTGSQKPFQCRICMRNFSQASNLISHIR PEYKPWALVIQDSNGENKIKML THTGEKPFACDICGRKFATSHSLTEHTK (SEQIDNO:390) IHTGEKPFQCRICMRKFAERSHLREHTK IHLR(SEQIDNO:409) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) R13-1 MLGFVGRVAAAPASGALRRLTPS MTS v3 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSRSDNLSTHIRTHTGEKPFACD FLAGtag ICGRKFADRSDLSRHTKIHTGEKP DYKDDDDK(SEQIDNO:395) FQCRICMRKFAQSGDLTRHTKIH NES TGSQKPFQCRICMRNFSRSDSLSA VDEMTKKFGTLTIHDTEK(SEQIDNO: HIRTHTGEKPFACDICGRKFAQK 396) ATRITHTKIHLRGSGGGGSGGSG Linker GSGSYALGPYQISAPQLPAYNGQ AA TVGTFYYVNDAGGLESKVFSSGG ZF(R13-1) PTPYPNYANAGHVEGQSALFMR MAERPFQCRICMRNFSRSDNLSTHIRTH DNGISEGLVFHNNPEGTCGFCVN TGEKPFACDICGRKFADRSDLSRHTKIH MTETLLPENAKMTVVPPEGSGGS TGEKPFQCRICMRKFAQSGDLTRHTKI TNLSDIIEKETGKQLVIQESILMLP HTGSQKPFQCRICMRNFSRSDSLSAHIR EEVEEVIGNKPESDILVHTAYDES THTGEKPFACDICGRKFAQKATRITHT TDENVMLLTSDAPEYKPWALVIQ KIHLR(SEQIDNO:410) DSNGENKIKML(SEQIDNO:391) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFY YVNDAGGLESKVFSSGGPTPYPNYANA GHVEGQSALFMRDNGISEGLVFHNNPE GTCGFCVNMTETLLPENAKMTVVPPE G(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 9-R13 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v3 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSQSSSLVRHIRTHTGEKPFACD FLAGtag ICGRKFARSDNLVRHTKIHTGSQ DYKDDDDK(SEQIDNO:395) KPFQCRICMRNFSQAGHLASHIR NES THTGEKPFACDICGRKFARKDNL VDEMTKKFGTLTIHDTEK(SEQIDNO: KNHTKIHTGEKPFQCRICMRKFA 396) RKDALRGHTKIHLRGSGGGGSGG Linker SGGSAIPVKRGATGETKVFTGNS AA NSPKSPTKGGCSGGSTNLSDIIEK ZF(5xZnF-9-R13) ETGKQLVIQESILMLPEEVEEVIG MAERPFQCRICMRNFSQSSSLVRHIRTH NKPESDILVHTAYDESTDENVML TGEKPFACDICGRKFARSDNLVRHTKI LTSDAPEYKPWALVIQDSNGENK HTGSQKPFQCRICMRNFSQAGHLASHI IKML(SEQIDNO:392) RTHTGEKPFACDICGRKFARKDNLKNH TKIHTGEKPFQCRICMRKFARKDALRG HTKIHLR(SEQIDNO:411) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341) 5xZnF- MLGFVGRVAAAPASGALRRLTPS MTS 12-R13 ASLPPAQLLLRAAPTAVHPVRDY MLGFVGRVAAAPASGALRRLTPSASLP v3 AAQDYKDDDDKVDEMTKKFGT PAQLLLRAAPTAVHPVRDYAAQ(SEQ LTIHDTEKAAMAERPFQCRICMR IDNO:394) NFSRSDHLTTHIRTHTGEKPFACD FLAGtag ICGRKFAQSSSLVRHTKIHTGSQK DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSRSDNLVRHIRTH NES TGEKPFACDICGRKFAQAGHLAS VDEMTKKFGTLTIHDTEK(SEQIDNO: HTKIHTGEKPFQCRICMRKFARK 396) DNLKNHTKIHLRGSGGGGSGGSG Linker GSAIPVKRGATGETKVFTGNSNS AA PKSPTKGGCSGGSTNLSDIIEKET ZF(5xZnF-12-R13) GKQLVIQESILMLPEEVEEVIGNK MAERPFQCRICMRNFSRSDHLTTHIRTH PESDILVHTAYDESTDENVMLLT TGEKPFACDICGRKFAQSSSLVRHTKIH SDAPEYKPWALVIQDSNGENKIK TGSQKPFQCRICMRNFSRSDNLVRHIRT ML(SEQIDNO:393) HTGEKPFACDICGRKFAQAGHLASHTK IHTGEKPFQCRICMRKFARKDNLKNHT KIHLR(SEQIDNO:412) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTK GGC(SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQIDNO:341)
DddAs
[0345] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may be engineered to include a DddA, or an inactive fragment thereof.
[0346] In various embodiments, the DddA protein has the following amino acid sequence:
[0347] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYAN AGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIP VKRGATGETKVFTGNSNSPKSPTKGGC (SEQ IN NO: 338), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 338, or a fragment thereof.
[0348] This full length DddA may also be referred to as DddAtox since it is toxic to cells, as described in Example 1.
[0349] In other embodiments, the DddA has the following amino acid sequence: XGSSHHHHHHSQDPIGLNGG ANVYHYAPNP VGWVDPWGLA GSYALGPYQI SAPQLPAYNGQTVGTFYYVN DAGGLESKVF SSGGPTPYPN YANAGHVEGQ SALFXRDNGI SEGLVFHNNPEGTCGFCVNX TETLLPENAK XTVVPPEGAI PVKRGATGET KVFTGNSNSPKSPTKGGC (SEQ ID NO: 413) (which corresponds to PDB Accession No. 6U08_A of Burkholderia cenocepacia), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of 6U08_A (SEQ ID NO: 413).
[0350] In various other embodiments, a split DddA can have the following sequences: [0351] G1333 DddAtox-NGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG (SEQ ID NO: 349), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 349.
[0352] G1333 DddAtox-C PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMT VVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO: 350), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 350.
[0353] G1397 DddAtox-NGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVE GQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG (SEQ ID NO: 351), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 351.
[0354] G1397 DddAtox-C AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO: 352), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 352.
[0355] Split DddA (DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVE GQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG (SEQ ID NO: 351), and can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of SEQ ID NO: 351.
TABLE-US-00021 SplitDddA(DddA-G1397C) (SEQIDNO:352) AIPVKRGATGETKVFTGNSNSPKSPTKGGC.
[0356] The disclosure also contemplates the use of any variant of DddAtox, or proteins comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA-G1397C, or a biologically active fragment of DddA-G1397C.
[0357] As shown in
[0358] In preferred embodiments, as depicted in
[0359] In some embodiments, the DddA comprises a first portion and a second portion. In some embodiments, the first portion and the second portion together comprise a full length DddA. In some embodiments, the first and second portion comprise less than the full length DddA portion. In some embodiments, the first and second portion independently do not have any, or have minimal, native DddA activity (e.g., deamination activity). In some embodiment, the first and second portion can re-assemble (i.e., dimerize) into a DddA protein with, at least partial, native DddA activity (e.g., deamination activity).
[0360] In some embodiments, the first and second portion of the DddA are formed by truncating (i.e., dividing or splitting the DddA protein) at specified amino acid residues. In some embodiments, the first portion of a DddA comprises a full-length DddA truncated at its N-terminus. In some embodiments, the second portion of a DddA comprises a full-length DddA truncated at its C-terminus. In some embodiments, additional truncations are performed to either the full-length DddA or to the first or second portions of the DddA. In some embodiments, the first and second portions of a DddA may comprise additional truncations, but which the first and second portion can dimerize or re-assemble, to restore, at least partially, native DddA activity (e.g., deamination). In some embodiments, the first and second portions comprise full-length DddA truncated at, or around, a residue in DddA selected from the group comprising: 62, 71, 73, 84, 94, 108, 110, 122, 135, 138, 148, and 155. In some embodiments, the truncation of DddA occurs at residue 148.
[0361] In certain embodiments, the DddA can be separated into two fragments by dividing the DddA at a split site. A split site refers to a position between two adjacent amino acids (in a wildtype DddA amino acid sequence) that marks a point of division of a DddA. In certain embodiments, the DddA can have a least one split site, such that once divided at that split site, the DddA forms an N-terminal fragment and a C-terminal fragment. The N-terminal and C-terminal fragments can be the same or difference sizes (or lengths), wherein the size and/or polypeptide length depends on the location or position of the split site. As used herein, reference to a fragment of DddA (or any other polypeptide) can be referred equivalently as a portion. Thus, a DddA which is divided at a split site can form an N-terminal portion and a C-terminal portion. Preferably, the N-terminal fragment (or portion) and the C-terminal fragment (or portion) or DddA do not have a deaminase activity.
[0362] In various embodiments, a DddA may be split into two or more inactive fragments by directly cleaving the DddA at one or more split sites. Direct cleaving can be carried out by a protease (e.g., trypsin) or other enzyme or chemical reagent. In certain embodiments, such chemical cleavage reactions can be designed to be site-selective (e.g., Elashal and Raj, Site-selective chemical cleavage of peptide bonds, Chemical Communications, 2016, Vol. 52, pages 6304-6307, the contents of which are incorporated herein by reference.) In other embodiments, chemical cleavage reactions can be designed to be non-selective and/or occur in a random fashion.
[0363] In other embodiments, the two or more inactive DddA fragments can be engineered as separately expressed polypeptides. For instance, for a DddA having one split site, the N-terminal DddA fragment could be engineered from a first nucleotide sequence that encodes the N-terminal DddA fragment (which extends from the N-terminus of the DddA up to and including the residue on the amino-terminal side of the split site). In such an example, the C-terminal DddA fragment could be engineered from a second nucleotide sequence that encodes the C-terminal DddA fragment (which extends from the carboxy-terminus of the split site up to including the natural C-terminus of the DddA protein). The first and second nucleotide sequences could be on the same or different nucleotide molecules (e.g., the same or different expression vectors).
[0364] In various embodiments, that N-terminal portion of the DddA may be referred to as DddA-N half and the C-terminal portion of the DddA may be referred to as the DddA-C half. Reference to the term half does not connote the requirement that the DddA-N and DddA-C portions are identically half of the size and/or sequence length of a complete DddA, or that the split site is required to be at the mid point of the complete DddA polypeptide. To the contrary, and as noted above, the split site can be between any pair of residues in the DddA polypeptide, thereby giving rise to half portions which are unequal in size and/or sequence length. In certain embodiments, the split site is within a loop region of the DddA.
[0365] Accordingly, in one aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins, in some embodiments, can comprise a first fusion protein comprising a first pDNAbp (e.g., a mitoTALE, mitoZFP, or a CRISPR/Cas9) and a first portion or fragment of a DddA, and a second fusion protein comprising a second pDNAbp (e.g., mitoTALE, mitoZFP, or a CRISPR/Cas9) and a second portion or fragment of a DddA, such that the first and the second portions of the DddA reconstitute a DddA upon co-localization in a cell and/or mitochondria. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a DddA. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0366] [pDNAbp]-[DddA half.sup.A] and [pDNAbp]-[DddA half.sup.B]; [0367] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[pDNAbp]; [0368] [pDNAbp]-[DddA half.sup.A] and [DddA-half.sup.B]-[pDNAbp]; or [0369] [DddA-half.sup.A]-[pDNAbp] and [pDNAbp]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0370] In another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first mitoTALE and a first portion or fragment of a DddA, and a second fusion protein comprising a second mitoTALE and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a Ddda. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0371] [mitoTALE]-[DddA half.sup.A] and [mitoTALE]-[DddA half.sup.B]; [0372] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[mitoTALE]; [0373] [mitoTALE]-[DddA half.sup.A] and [DddA-half.sup.B]-[mitoTALE]; or [0374] [DddA-half.sup.A]-[mitoTALE] and [mitoTALE]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0375] In yet another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first mitoZFP and a first portion or fragment of a DddA, and a second fusion protein comprising a second mitoZFP and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA and the second portion of the DddA is C-terminal fragment of a Ddda. In other embodiments, the first portion of the DddA is a C-terminal fragment of a DddA and the second portion of the DddA is an N-terminal fragment of a DddA. In this aspect, the structure of the pair of fusion proteins can be, for example: [0376] [mitoZFP]-[DddA half.sup.A] and [mitoZFP]-[DddA half.sup.B]; [0377] [DddA-half.sup.A]-[pDNAbp] and [DddA-half.sup.B]-[mitoZFP]; [0378] [mitoZFP]-[DddA half.sup.A] and [DddA-half.sup.B]-[mitoZFP]; or [0379] [DddA-half.sup.A]-[mitoZFP] and [mitoZFP]-[DddA half.sup.B], wherein A or B can be the N-terminal or C-terminal half of DddA.
[0380] In yet another aspect, the disclosure relates to a pair of fusion proteins useful for making modifications to the sequence of mitochondrial DNA (e.g., mtDNA). The pair of fusion proteins can comprise a first fusion protein comprising a first Cas9 and a first portion or fragment of a DddA, and a second fusion protein comprising a second Cas9 and a second portion or fragment of a DddA, such that the first and the second portions of the DddA, upon co-localization in a cell and/or mitochondria, are reconstituted an active DddA. In certain embodiments, that first portion of the DddA is an N-terminal fragment of a DddA (i.e., DddA half.sup.A as shown in
[0385] In each instance above of ]-[ can be in reference to a linker sequence.
[0386] In some embodiments, a first fusion protein comprises, a first mitochondrial transcription activator-like effector (mitoTALE) domain and a first portion of a DNA deaminase effector (DddA). In some embodiments, the first portion of the DddA comprises an N-terminal truncated DddA. In some embodiments, the first mitoTALE is configured to bind a first nucleic acid sequence proximal to a target nucleotide. In some embodiments, the first portion of a DddA is linked to the remainder of the first fusion protein by the C-terminus of the first portion of a DddA.
[0387] In one aspect, the present disclosure provides mitochondrial DNA editor fusion proteins for use in editing mitochondrial DNA. As used herein, these mitochondrial DNA editor fusion proteins may be referred to as mtDNA editors or mtDNA editing systems.
[0388] In various embodiments, the mtDNA editors described herein comprise (1) a programmable DNA binding protein (pDNAbp) (e.g., a mitoTALE domain, mitoZFP domain, or a CRISPR/Cas9 domain) and a double-stranded DNA deaminase domain, which is capable of carrying out a deamination of a nucleobase at a target site associated with the binding site of the programmable DNA binding protein (pDNAbp).
[0389] In some embodiments, the double-stranded DNA deaminase is split into two inactive half portions, with each half portion being fused to a programmable DNA binding protein that binds to a nucleotide sequence either upstream or downstream of a target edit site, and wherein once in the mitochondria, the two half portions (i.e., the N-terminal half and the C-terminal half) reassociate at the target edit site by the co-localization of the programmable DNA binding proteins to binding sites upstream and downstream of the target edit site to be acted on by the DNA deaminase. The reassociation of the two half portions of the double-stranded DNA deaminase restores the deaminase activity at the target edit site. In other embodiments, the double-stranded DNA deaminase can initially be set in an inactive state which can be induced when in the mitochondria. The double-stranded DNA deaminase is preferably delivered initially in an inactive form in order to avoid toxicity inherent with the protein. Any means to regulate the toxic properties of the double-stranded DNA deaminase until such time as the activity is desired to be activated (e.g., in the mitochondria) is contemplated.
[0390] In various embodiments, the following exemplary DddA enzymes can be used with the mtDNA base editors described herein, or a sequence (amino acid or nucleotide as the case may be) having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the following mitoTALE sequences:
TABLE-US-00022 DddA Description DddAaminoacidand/ornucleotidsequence DddA >ATF83755.1hypotheticalproteinCO712_00910[Burkholderiagladiolipv. homologin gladioli] Burkholderia MYEAARVTDPIEHTSALAGFLVGAVLGIALIAAVAFATFTCGFGVALL gladioli AGMAAGIGAQVLLSLGESIGKMFSSQSGAITLGSPNVYVNGKQAAYA PROTEIN TLSSVTCSKHNPTPLVAQGSTNIFINGKPAARKDDKITCGAAISDGSHD TYFHGGIQTCLPIDDEVPPWLRTATDWAFALAGLVGGLGGLLKEAGG LSHAVMPCAAKFIGGYVLGEAASRYVIGPAINSAIGGMFGNPVDVTT GRKILPAESETDYVVPSPMPVAIRRFYSSDLDYVGTLGRGWVLPWEL RLHARDGRLWYTDAQGRESGFPILKPGQAAFSEADQRYLTCTPDGRY ILHDVGETYYDFGRYEPGSGRIGWVRRIEDQAGQWCQFERDSRGRVR EIQTCGGLLAVLDYEPEHERLAEVSLVSGDQRRLVVAYGYDENGQM ASVTDANGAVVRRFTYADGRMTSHSNALGFTSGYTWKVIDGTPRVV ATHTSEGEAWAFEYDIEGRRTHVRHADGRHAQWRYDAQFQIVEYLD FDGRRYGLKYNAAGMPVMLTLPGERTVMFEYDDAGRIVAETDPLGR TTKTRYDGNSMRPVEIILPDGSAWHAEYDRQGRLLVTRDPLDRENRY EYPEALSALPVAHVDALGGRKTFEWNRLGELVAYTDCSGKTTRNFF DAFGLPLARENALGHRVSFDLRPTGETRRVTYPDGSSESYEYDAAGL MIRHIGLGGRMQTLQRNARGQLVEAVDPAGRRTRYHYDAEGRLREL QQAHARYAFAYSAGGRLVSETRPDGVLRRFEYGEAGDLAALEIVGT ADDCAPNDRPVRAIRFERDRMGNLCVQHTPTEVTRYERDAGGRLLE VASVPTAAGLALGIAPDTLTFEYDKAGRLSAEHGANGSVQYTLDALD NVLKLALPHEQTLQMLRYGSGHVHQIRHGDQVVSDFERDDLHRELT RTQGPLTERTAYDLLGRKIWQSAGFQPDALARGQGQLWRNYGYDA AGELVESHDSLRGSTQFSYDPAGYLTQRVNTADRQLESFAWDAAGN LLDDAQRSSRGYVEGNRLRMWQNLRFDYDAFGNLATKLRGANQRQ QFTYDGQDRLVAVRTQGARGVVETRFAYDPLGRRIAKTDRTLDVRG VTLREETKRFVWEGLRLAQEVRDTGVSSYVYSPDAPYMPAARVDAV KAEALANAAIDKARQATRIYHFHTDVSGAPQEATNEAGDIVWAGQY SAWGKVAPNQHAPARIDQPLRYAGQYADDSTELHYNTFRFYDPDVG RFINQDPIGLMGGLNLYQYAPNSIAWTDWWGLAGSYTLGSYQISAPQ LPAYNGQTVGTFYYVNDAGGLESRTFSSGGPTPYPNYANAGHVEGQ SALFMRDNGISDGLVFHNNPEGTCGFCVNMTETLLPENSKLTVVPPE GSIPVKRGATGETRTFTGNSKSPKSPVKGGC[SEQIDNO:130] DddA >CO712_00910NZ_CP023522.1:185368-189645Burkholderiagladiolipv. homologin gladiolistrainFDAARGOS_389chromosome1,completesequence Burkholderia GTGTACGAAGCGGCCCGCGTCACGGATCCGATCGAGCACACCAGC gladioli GCGCTGGCCGGCTTCCTGGTGGGCGCCGTGCTCGGTATCGCCCTGA DNA TTGCTGCCGTGGCGTTCGCCACGTTCACCTGCGGCTTCGGCGTGGC ACTGCTGGCCGGCATGGCGGCCGGCATCGGCGCGCAGGTGCTGTT GTCGTTAGGGGAATCGATCGGGAAGATGTTCAGTTCGCAATCCGG CGCGATCACGCTCGGCTCGCCGAACGTCTACGTGAACGGCAAGCA GGCCGCCTACGCCACGCTCAGCAGCGTGACGTGCAGCAAGCACAA CCCGACGCCGCTCGTCGCGCAGGGCTCCACCAACATCTTCATCAAC GGCAAGCCGGCCGCGCGCAAGGACGACAAGATCACCTGCGGCGC GGCCATCTCGGACGGCTCGCACGACACCTACTTCCACGGAGGCAT CCAGACCTGCCTGCCGATCGACGACGAAGTGCCGCCGTGGCTGCG CACCGCCACCGACTGGGCGTTCGCGCTGGCCGGGCTGGTGGGCGG GCTCGGCGGCCTACTCAAGGAAGCGGGCGGGCTGTCGCACGCGGT GATGCCGTGCGCGGCGAAGTTCATCGGCGGCTACGTGCTCGGCGA GGCGGCGAGCCGCTACGTGATCGGCCCGGCCATCAACAGCGCGAT CGGCGGGATGTTCGGCAACCCGGTAGACGTCACCACTGGGCGCAA GATCCTCCCTGCCGAATCGGAAACCGATTACGTCGTGCCCAGCCC GATGCCGGTGGCGATCCGGCGCTTCTATTCGAGCGACCTCGATTAC GTCGGCACGCTTGGGCGCGGCTGGGTGCTGCCGTGGGAGCTGCGC CTGCACGCGCGTGACGGTCGGCTCTGGTACACCGACGCGCAGGGG CGCGAGAGCGGCTTCCCGATCCTGAAACCGGGCCAGGCCGCGTTC AGCGAGGCCGATCAGCGCTATCTGACCTGCACGCCGGATGGCCGC TACATCCTCCACGACGTCGGCGAAACCTATTACGACTTCGGCCGCT ACGAGCCGGGCTCGGGCCGCATCGGCTGGGTGCGCCGGATCGAGG ATCAGGCCGGCCAGTGGTGCCAGTTCGAGCGCGACAGCCGTGGCC GCGTGCGTGAAATCCAGACCTGCGGCGGCTTGCTGGCCGTGCTCG ATTACGAGCCGGAGCACGAGCGGCTCGCCGAGGTGTCGCTCGTCA GCGGCGATCAGCGCCGCCTCGTCGTGGCCTACGGCTACGACGAAA ACGGCCAGATGGCCTCCGTGACCGACGCGAACGGCGCGGTGGTGC GCCGCTTCACCTATGCCGACGGGCGCATGACGAGCCATTCGAACG CGCTCGGTTTCACGTCGGGCTATACGTGGAAGGTCATCGACGGCA CGCCGCGAGTGGTCGCCACCCACACCAGCGAGGGCGAGGCCTGGG CGTTCGAGTACGACATCGAAGGCCGCCGCACCCATGTGCGGCATG CCGACGGCCGCCACGCGCAATGGCGCTACGACGCGCAATTCCAGA TCGTCGAGTACCTCGATTTCGACGGCCGTCGCTACGGGCTCAAGTA CAACGCTGCCGGCATGCCCGTGATGCTGACGCTGCCCGGCGAACG AACCGTGATGTTCGAGTACGACGACGCCGGCCGCATCGTCGCCGA AACCGATCCCCTCGGCCGCACCACGAAAACGCGCTACGACGGCAA CAGCATGCGGCCCGTCGAGATCATCTTGCCCGACGGCAGCGCCTG GCACGCCGAATACGACCGGCAGGGCCGGCTGCTCGTCACCCGTGA TCCGCTCGACCGGGAGAATCGCTACGAATATCCGGAGGCACTGAG CGCGCTCCCGGTGGCGCATGTCGATGCGCTGGGCGGGCGCAAGAC GTTCGAGTGGAACCGGCTCGGCGAGCTGGTGGCCTACACCGATTG CTCGGGCAAGACCACGCGCAATTTTTTCGATGCATTCGGCCTGCCG CTCGCGCGCGAGAACGCGCTCGGGCACCGCGTGTCGTTCGATCTG CGCCCGACCGGCGAGACGCGCCGCGTCACCTATCCCGACGGCAGT TCCGAAAGCTACGAATACGACGCCGCCGGGCTGATGATCCGGCAC ATCGGGCTGGGCGGCCGGATGCAGACGTTGCAGCGCAATGCGCGC GGGCAACTCGTCGAGGCGGTCGATCCGGCCGGGCGGCGAACCCGC TACCACTACGACGCCGAAGGGCGGCTGCGCGAGCTGCAACAGGCC CACGCGCGCTACGCATTCGCGTACAGCGCAGGCGGGCGGCTTGTC AGCGAAACGCGGCCCGACGGCGTGCTGCGCCGCTTCGAATACGGC GAGGCCGGCGATCTGGCGGCGCTCGAGATCGTCGGAACGGCCGAT GATTGCGCTCCAAACGATCGCCCGGTTCGCGCGATCCGCTTCGAGC GCGACCGGATGGGTAACCTGTGCGTGCAGCACACGCCTACCGAGG TGACGCGCTACGAGCGCGACGCCGGCGGCCGCCTGCTCGAAGTCG CGAGCGTGCCGACCGCGGCCGGACTGGCGCTCGGCATCGCGCCCG ACACGCTGACCTTCGAATACGACAAGGCCGGGCGGCTGAGCGCCG AACACGGCGCGAACGGCAGCGTCCAGTACACGCTCGACGCGCTCG ACAACGTGTTGAAGCTCGCCTTGCCGCACGAACAGACGCTGCAGA TGCTGCGCTACGGCTCGGGGCACGTGCACCAGATTCGCCACGGCG ACCAGGTCGTCAGCGATTTCGAGCGCGACGACCTGCATCGCGAGT TGACGCGCACGCAGGGCCCCCTGACCGAGCGGACCGCCTACGACC TGCTGGGCCGCAAGATCTGGCAATCAGCCGGCTTCCAGCCCGACG CGCTTGCGCGTGGGCAGGGCCAGCTGTGGCGCAACTACGGCTACG ACGCCGCCGGGGAACTGGTCGAGAGCCACGACAGCCTGCGCGGCA GCACGCAGTTCAGCTACGATCCGGCCGGCTATCTGACGCAGCGCG TGAACACCGCCGACCGGCAGCTCGAATCGTTCGCCTGGGACGCCG CCGGCAACCTGCTCGACGATGCGCAACGCAGCAGCCGCGGCTATG TCGAGGGCAACCGGCTGCGCATGTGGCAGAACCTGCGCTTCGACT ACGACGCGTTCGGCAATCTCGCGACCAAGCTGCGCGGCGCGAATC AGCGCCAGCAGTTCACGTACGATGGGCAGGATCGGCTCGTGGCCG TGCGCACGCAGGGCGCGCGCGGCGTGGTGGAGACGCGTTTCGCCT ACGATCCGCTCGGGCGGCGCATCGCCAAGACCGATAGGACACTCG ACGTGCGCGGCGTAACGCTGCGCGAGGAAACGAAGCGGTTCGTAT GGGAAGGGCTGCGGCTCGCGCAGGAGGTGCGCGACACCGGCGTG AGCAGCTACGTGTACAGCCCGGATGCGCCTTACATGCCCGCGGCG CGGGTCGATGCGGTGAAAGCCGAAGCGCTCGCAAACGCCGCGATC GACAAGGCCAGACAGGCGACGCGGATCTATCACTTTCATACCGAT GTGTCGGGCGCACCGCAAGAAGCGACGAACGAGGCCGGCGACAT TGTTTGGGCCGGCCAATACTCAGCCTGGGGCAAGGTGGCGCCGAA CCAGCATGCCCCAGCCCGGATCGATCAGCCGCTCCGCTACGCCGG ACAATATGCCGATGACAGTACCGAGCTGCACTACAACACGTTTCG TTTCTACGATCCGGATGTCGGCCGGTTTATCAATCAGGATCCAATC GGGTTGATGGGGGGGCTGAATCTTTACCAATATGCACCCAACTCA ATCGCGTGGACCGACTGGTGGGGGCTGGCCGGCAGCTATACGCTC GGTTCCTATCAAATTTCTGCTCCTCAACTTCCCGCCTACAATGGGC AGACTGTTGGGACCTTCTACTATGTAAACGACGCGGGCGGGCTCG AATCGAGGACATTCTCTTCTGGAGGGCCGACCCCTTATCCAAATTA TGCCAATGCCGGGCACGTGGAAGGCCAGTCCGCACTGTTCATGAG GGATAACGGAATTTCAGACGGACTGGTTTTCCACAACAACCCTGA GGGTACTTGCGGATTCTGCGTCAATATGACCGAAACGCTTTTGCCT GAAAATTCCAAACTTACCGTCGTTCCGCCCGAGGGCTCGATTCCGG TCAAGCGGGGCGCGACGGGCGAAACGAGAACATTTACAGGGAAC AGCAAGTCTCCGAAGTCCCCTGTCAAAGGAGGATGTTGA(SEQID NO:131) DddA >AJY63123.1RHSrepeat-associatedcoredomainprotein[Burkholderia homologin glumaeLMG2196=ATCC33617] Burkholderia MYEAARVTDPIEHTSALTGFLVGAVLGIALIAAVAFATFTCGFGVALL glumaeLMG AGMAAGIGAQVLLSLGESIGKMFSSQSGAITLGSPNVYVNGKPTAYA 2196 MLSSVTCSKHNPTPLVAQGSTNIFINGKPAARKDDKITCGATISDGSH PROTEIN DTYFHGGTQTCLPIDDEVPPWLRTATDWAFALAGLVGGLGGLLKEA GGLSRAVMPCAAKFIGGYVLGEAASRYVVGPAINSAIGGMFGNPVDV TTGRKILLAESETDYVVPSPMPVAIRRFYSSDLDYVGTLGRGWVLPW ELRLHARDGRLWYTDAQGRESGFPMLQPGHAAFSEADQRYLTCTPD GRYILHDLGETYYDFGHYEPGSGRIGWVRRIEDQAGQWCQFERDSRG RVREIQTCGGLLAVLDYEPEHGRLAGVSLVSGDQRRLVVAYGYDEH GQMASVTDANGALVRRFTYADGRMTSHSNALGFTSGYTWQAVGGA PRVVATHTSEGEAWAFEYDIEGRRTHVRHADGRHAQWRYDAQFQIV EYLDFDGRRYGLKYNDAGMPVMLTLPGERTVTFEYDDAGRIVAETD PLGRTTKTRYDGNSRRPVEIIAPDGSAWHAEYDRQGRLLATRDPLDR ENRYEYPKALSALPIAHVDALGGRKTFEWNRLGELVAYTDCSGKTTR NFYDAFGLPLARENALGHRVTFDLRPTGEARRVTYPDGSTESYEYDA AGLMIRHVGLGGRTQIALRNARGQIVEAVDPAGRRTCYRYDAEGRL RELQQGHARYAFTYSAGGRLTSETRPDGVRRRFEYGEAGDLAALDIV GAADDATANDRPVRTIRFERDRMGNLCAQHTPTEVTRYTRDTGGRL LEVACVPTAAGLALGIAPDTLTFEYDKAGRLSAEHGANGSVRYTLDA LDNVMKLALPHEQTLQMLRYGSGHVHQIRCGDQVVSDFERDDLHRE LTRTQGRLTERTAYDLLGRKIWQSAGFQPDALARGQGQVWRNYGY DAAGELAESHDSLRGSTQFSYDPAGYLTQRVNTADRQLESFAWDAA GNLLDDAQRRSRGYVEGNRLRMWQNLRFEYDPFGNLATKLRGANQ RQQFTYDGQDRLVAVRTQDARGVVETRFAYDPLGRRIAKTDIVRDA RGVALREETKRFVWEGLRLAQEVRDTGVSSYVYSPDAPYTPAARVD AVLAEAMAAAAIEQARQATRIYHFHTDVSGAPQEATNEAGDIVWAG QYSAWGKVAPNQHAPARIDQPLRYAGQYADDSTELHYNTFRFYDPD VGRFINQDPIGLMGGLNLYQYAPNSIAWTDWWGLAGSYTLGSYQISA PQLPAYNGQTVGTFYYVNGAGGLESRTFSSGGPTPYPNYANAGHVE GQSALFMRDNGISDGLVFHNNPEGTCGFCVNMTETLLPENSKLTVVP PEGAIPVKRGATGETRTFTGNSKSPKSPVKGEC[SEQIDNO:132] DddA >KS03_3390CP009434.1:65330-69607BurkholderiaglumaeLMG2196= homologin ATCC33617chromosomeII,completesequence Burkholderia GTGTACGAAGCGGCCCGCGTCACCGACCCGATCGAACACACCAGC GCGCTGACCGGCTTTCTGGTGGGCGCCGTGCTCGGCATTGCCCTGA glumaeLMG TCGCCGCGGTGGCGTTCGCCACCTTCACCTGCGGCTTCGGCGTGGC 2196 GCTGCTGGCCGGCATGGCCGCCGGCATCGGCGCGCAGGTGCTGTT DNA GTCGTTAGGAGAATCGATCGGGAAGATGTTCAGTTCGCAATCCGG CGCGATCACGCTCGGCTCGCCGAACGTCTATGTGAACGGCAAGCC GACCGCCTACGCCATGCTCAGCAGCGTGACGTGCAGCAAGCACAA CCCGACGCCGCTCGTCGCGCAGGGGTCCACCAACATCTTCATCAA CGGCAAGCCGGCCGCCCGCAAGGACGACAAGATCACCTGCGGCGC GACCATCTCCGACGGCTCGCACGACACCTATTTCCACGGCGGCAC CCAGACCTGCCTGCCGATCGACGACGAAGTGCCGCCGTGGCTGCG CACCGCCACCGACTGGGCGTTCGCGCTGGCCGGGCTGGTGGGCGG GCTCGGCGGCCTGCTCAAGGAAGCGGGGGGCTGTCGCGCGCGGT GATGCCGTGCGCGGCGAAGTTCATCGGCGGCTACGTGCTCGGCGA GGCGGCGAGCCGCTACGTGGTCGGCCCGGCCATCAACAGCGCGAT CGGCGGGATGTTCGGCAACCCGGTGGACGTCACCACCGGGCGCAA GATCCTGCTGGCGGAATCGGAAACCGATTACGTGGTGCCCAGCCC GATGCCGGTGGCGATCCGGCGCTTCTATTCGAGCGACCTCGACTAC GTCGGCACGCTCGGGCGCGGCTGGGTGCTGCCGTGGGAACTGCGG CTGCACGCGCGCGACGGGCGGCTCTGGTACACCGACGCGCAGGGG CGCGAGAGCGGCTTCCCGATGCTCCAGCCGGGCCATGCCGCGTTC AGCGAGGCCGACCAGCGCTATCTGACCTGCACCCCGGATGGCCGC TACATCCTGCACGACCTCGGCGAAACCTATTACGACTTCGGCCACT ACGAGCCGGGCTCGGGCCGCATCGGCTGGGTGCGCCGCATCGAGG ATCAGGCCGGCCAGTGGTGCCAGTTCGAGCGCGACAGCCGCGGCC GCGTGCGCGAAATCCAGACCTGCGGCGGCTTGCTGGCCGTGCTCG ATTACGAGCCGGAACACGGGCGGCTCGCCGGGGTGTCGCTCGTCA GCGGGGATCAGCGCCGCCTCGTGGTGGCTTACGGCTATGACGAGC ACGGCCAGATGGCGTCCGTGACCGATGCGAACGGCGCGCTGGTGC GCCGCTTCACCTATGCCGACGGGCGCATGACGAGCCATTCGAACG CGCTCGGCTTCACGTCGGGCTATACGTGGCAAGCCGTCGGCGGCG CGCCGCGGGTGGTTGCCACCCACACCAGCGAGGGCGAGGCCTGGG CCTTCGAGTACGACATTGAAGGACGCCGCACCCACGTGCGTCACG CCGACGGCCGCCACGCGCAATGGCGCTACGACGCGCAATTCCAGA TCGTCGAGTACCTCGATTTCGACGGCCGGCGCTACGGGCTCAAGT ACAACGACGCCGGCATGCCCGTGATGCTGACGCTGCCCGGCGAAC GGACCGTGACGTTCGAGTACGACGATGCCGGCCGCATCGTCGCCG AAACCGATCCACTCGGCCGCACCACGAAAACGCGCTACGACGGCA ACAGCAGGCGGCCCGTCGAGATCATCGCGCCCGACGGCAGCGCCT GGCACGCCGAATACGACCGGCAAGGCCGGCTGCTCGCCACCCGCG ATCCGCTCGACCGGGAAAACCGCTACGAATACCCGAAGGCGCTCA GCGCGCTGCCGATCGCGCACGTCGATGCGCTGGGCGGGCGCAAGA CGTTCGAGTGGAACCGGCTCGGCGAGCTGGTGGCCTATACCGATT GCTCGGGCAAGACCACACGCAATTTTTACGACGCATTCGGTCTGCC GCTCGCGCGCGAGAACGCGCTCGGCCACCGCGTGACGTTCGACCT GCGCCCGACCGGCGAGGCGCGGCGCGTCACCTATCCCGACGGCAG TACAGAAAGCTACGAATACGACGCCGCCGGGCTGATGATCCGGCA CGTCGGGCTGGGCGGCCGGACGCAGATTGCGCTGCGCAACGCGCG TGGGCAGATCGTGGAGGCGGTCGATCCGGCCGGACGGCGCACCTG CTACCGCTACGACGCCGAGGGGCGGCTGCGCGAGCTGCAACAGGG GCACGCGCGTTACGCGTTCACCTACAGCGCGGGCGGGCGGCTCAC CAGCGAAACCCGGCCCGACGGCGTGCGGCGCCGCTTCGAATACGG CGAGGCCGGCGATCTGGCGGCGCTCGACATCGTCGGCGCGGCCGA CGACGCCACGGCGAACGATCGTCCGGTTCGCACCATCCGCTTCGA GCGCGACCGCATGGGCAATCTGTGCGCGCAGCACACGCCCACCGA GGTGACGCGCTACACGCGCGACACCGGCGGCCGCCTGCTCGAAGT CGCATGCGTGCCGACCGCGGCCGGGCTGGCGCTCGGCATCGCGCC CGACACGCTGACCTTCGAATACGACAAGGCCGGGCGGCTGAGTGC CGAACACGGCGCGAACGGCAGCGTCCGATACACGCTCGACGCGCT CGACAACGTGATGAAGCTCGCCCTGCCGCACGAGCAGACGCTGCA GATGCTGCGCTACGGCTCGGGGCACGTGCATCAGATCCGCTGCGG CGACCAGGTGGTCAGCGATTTCGAGCGCGACGACCTGCATCGCGA GCTGACGCGCACTCAGGGCCGCCTGACCGAGCGTACCGCCTACGA CCTGCTGGGCCGCAAGATCTGGCAATCGGCCGGCTTCCAGCCCGA CGCGCTTGCGCGCGGGCAGGGCCAGGTGTGGCGCAACTACGGCTA CGACGCCGCCGGCGAACTGGCCGAGAGCCACGATAGCCTGCGCGG CAGCACGCAGTTCAGCTACGATCCGGCCGGCTATCTGACGCAGCG CGTCAATACCGCCGACCGGCAGCTCGAATCGTTCGCCTGGGATGC CGCCGGCAACCTGCTCGACGATGCGCAGCGCCGCAGCCGCGGTTA TGTCGAGGGCAACCGGCTGCGCATGTGGCAGAACCTGCGCTTCGA ATACGACCCGTTCGGCAATCTCGCGACCAAGCTGCGCGGCGCGAA CCAGCGCCAGCAGTTCACTTACGACGGGCAGGATCGGCTCGTGGC GGTGCGCACGCAGGACGCGCGCGGCGTGGTGGAGACGCGTTTCGC CTACGATCCGCTGGGGCGGCGCATCGCCAAGACGGATATTGTGCG CGACGCGCGCGGCGTAGCGCTGCGCGAGGAAACGAAGCGGTTCGT GTGGGAGGGGCTGCGGCTCGCGCAGGAGGTGCGCGACACGGGCG TGAGCAGCTACGTGTACAGCCCGGACGCGCCCTATACGCCCGCGG CGCGCGTGGATGCCGTGCTGGCCGAGGCCATGGCCGCCGCTGCCA TCGAGCAGGCCAGACAGGCGACGCGGATCTATCACTTTCATACCG ATGTGTCGGGCGCACCGCAAGAAGCGACGAACGAGGCTGGCGAC ATTGTTTGGGCCGGCCAATACTCAGCCTGGGGCAAGGTGGCGCCG AACCAGCATGCCCCCGCCCGGATCGATCAGCCGCTCCGCTACGCC GGACAATATGCCGACGACAGTACCGAGCTGCACTACAACACGTTT CGTTTCTACGATCCGGACGTCGGCCGGTTTATCAATCAGGATCCAA TCGGGTTGATGGGGGGGCTGAATCTTTACCAATATGCACCCAACTC GATCGCATGGACCGACTGGTGGGGGCTGGCCGGCAGCTATACGCT CGGTTCCTATCAAATTTCTGCGCCTCAACTTCCGGCCTACAATGGA CAGACTGTTGGGACCTTCTACTACGTGAACGGCGCGGGCGGGCTC GAATCGAGGACATTCTCTTCCGGAGGGCCGACCCCTTATCCAAATT ATGCCAATGCCGGGCACGTGGAGGGCCAGTCCGCGCTGTTCATGA GGGATAACGGAATTTCAGACGGACTGGTTTTCCACAACAACCCTG AGGGCACTTGCGGATTCTGCGTTAATATGACCGAAACGCTTTTGCC TGAAAATTCCAAACTTACCGTCGTTCCGCCCGAGGGCGCGATCCC GGTCAAGCGGGGCGCGACGGGCGAAACGAGAACATTTACGGGGA ACAGCAAGTCTCCGAAGTCCCCTGTCAAAGGAGAATGTTGA[SEQ IDNO:133] DddA >ACR30728.1Rhsfamilyprotein[BurkholderiaglumaeBGR1] homologin MYEAARVTDPIEHTSALTGFLVGAVLGIALIAAVAFATFTCGFGVALL Burkholderia AGMAAGIGAQVLLSLGESIGKMFSSQSGAITLGSPNVYVNGKPTAYA glumaeBGR1 MLSSVTCSKHNPTPLVAQGSTNIFINGKPAARKDDKITCGATISDGSH PROTEIN DTYFHGGTQTCLPIDDEVPPWLRTATDWAFALAGLVGGLGGLLKEA GGLSRAVMPCAAKFIGGYVLGEAASRYVVGPAINSAIGGMFGNPVDV TTGRKILLAESETDYVVPSPMPVAIRRFYSSDLDYVGTLGRGWVLPW ELRLHARDGRLWYTDAQGRESGFPMLQPGHAAFSEADQRYLTCTPD GRYILHDLGETYYDFGHYEPGSGRIGWVRRIEDQAGQWCQFERDSRG RVREIQTCGGLLAVLDYEPEHGRLAGVSLVSGDQRRLVVAYGYDEH GQMASVTDANGALVRRFTYADGRMTSHSNALGFTSGYTWQAVGGA PRVVATHTSEGEAWAFEYDIEGRRTHVRHADGRHAQWRYDAQFQIV EYLDFDGRRYGLKYNDAGMPVMLTLPGERTVTFEYDDAGRIVAETD PLGRTTKTRYDGNSRRPVEIIAPDGSAWHAEYDRQGRLLATRDPLDR ENRYEYPKALSALPIAHVDALGGRKTFEWNRLGELVAYTDCSGKTTR NFYDAFGLPLARENALGHRVTFDLRPTGEARRVTYPDGSTESYEYDA AGLMIRHVGLGGRTQIALRNARGQIVEAVDPAGRRTCYRYDAEGRL RELQQGHARYAFTYSAGGRLTSETRPDGVRRRFEYGEAGDLAALDIV GAADDATANDRPVRTIRFERDRMGNLCAQHTPTEVTRYTRDTGGRL LEVACVPTAAGLALGIAPDTLTFEYDKAGRLSAEHGANGSVRYTLDA LDNVMKLALPHEQTLQMLRYGSGHVHQIRCGDQVVSDFERDDLHRE LTRTQGRLTERTAYDLLGRKIWQSAGFQPDALARGQGQVWRNYGY DAAGELAESHDSLRGSTQFSYDPAGYLTQRVNTADRQLESFAWDAA GNLLDDAQRRSRGYVEGNRLRMWQNLRFEYDPFGNLATKLRGANQ RQQFTYDGQDRLVAVRTQDARGVVETRFAYDPLGRRIAKTDIVRDA RGVALREETKRFVWEGLRLAQEVRDTGVSSYVYSPDAPYTPAARVD AVLAEAMAAAAIEQARQATRIYHFHTDVSGAPQEATNEAGDIVWAG QYSAWGKVAPNQHAPARIDQPLRYAGQYADDSTELHYNTFRFYDPD VGRFINQDPIGLMGGLNLYQYAPNSIAWTDWWGLAGSYTLGSYQISA PQLPAYNGQTVGTFYYVNGAGGLESRTFSSGGPTPYPNYANAGHVE GQSALFMRDNGISDGLVFHNNPEGTCGFCVNMTETLLPENSKLTVVP PEGAIPVKRGATGETRTFTGNSKSPKSPVKGEC[SEQIDNO:134] DddA >bglu_2g02600NC_012721.2:303868-308145BurkholderiaglumaeBGR1 homologin chromosome2,completesequence Burkholderia GTGTACGAAGCGGCCCGCGTCACCGACCCGATCGAACACACCAGC glumaeBGR1 GCGCTGACCGGCTTTCTGGTGGGCGCCGTGCTCGGCATTGCCCTGA DNA TCGCCGCGGTGGCGTTCGCCACCTTCACCTGCGGCTTCGGCGTGGC GCTGCTGGCCGGCATGGCCGCCGGCATCGGCGCGCAGGTGCTGTT GTCGTTAGGAGAATCGATCGGGAAGATGTTCAGTTCGCAATCCGG CGCGATCACGCTCGGCTCGCCGAACGTCTATGTGAACGGCAAGCC GACCGCCTACGCCATGCTCAGCAGCGTGACGTGCAGCAAGCACAA CCCGACGCCGCTCGTCGCGCAGGGGTCCACCAACATCTTCATCAA CGGCAAGCCGGCCGCCCGCAAGGACGACAAGATCACCTGCGGCGC GACCATCTCCGACGGCTCGCACGACACCTATTTCCACGGCGGCAC CCAGACCTGCCTGCCGATCGACGACGAAGTGCCGCCGTGGCTGCG CACCGCCACCGACTGGGCGTTCGCGCTGGCCGGGCTGGTGGGCGG GCTCGGCGGCCTGCTCAAGGAAGCGGGCGGGCTGTCGCGCGCGGT GATGCCGTGCGCGGCGAAGTTCATCGGCGGCTACGTGCTCGGCGA GGCGGCGAGCCGCTACGTGGTCGGCCCGGCCATCAACAGCGCGAT CGGCGGGATGTTCGGCAACCCGGTGGACGTCACCACCGGGCGCAA GATCCTGCTGGCGGAATCGGAAACCGATTACGTGGTGCCCAGCCC GATGCCGGTGGCGATCCGGCGCTTCTATTCGAGCGACCTCGACTAC GTCGGCACGCTCGGGCGCGGCTGGGTGCTGCCGTGGGAACTGCGG CTGCACGCGCGCGACGGGCGGCTCTGGTACACCGACGCGCAGGGG CGCGAGAGCGGCTTCCCGATGCTCCAGCCGGGCCATGCCGCGTTC AGCGAGGCCGACCAGCGCTATCTGACCTGCACCCCGGATGGCCGC TACATCCTGCACGACCTCGGCGAAACCTATTACGACTTCGGCCACT ACGAGCCGGGCTCGGGCCGCATCGGCTGGGTGCGCCGCATCGAGG ATCAGGCCGGCCAGTGGTGCCAGTTCGAGCGCGACAGCCGCGGCC GCGTGCGCGAAATCCAGACCTGCGGCGGCTTGCTGGCCGTGCTCG ATTACGAGCCGGAACACGGGCGGCTCGCCGGGGTGTCGCTCGTCA GCGGGGATCAGCGCCGCCTCGTGGTGGCTTACGGCTATGACGAGC ACGGCCAGATGGCGTCCGTGACCGATGCGAACGGCGCGCTGGTGC GCCGCTTCACCTATGCCGACGGGCGCATGACGAGCCATTCGAACG CGCTCGGCTTCACGTCGGGCTATACGTGGCAAGCCGTCGGCGGCG CGCCGCGGGTGGTTGCCACCCACACCAGCGAGGGCGAGGCCTGGG CCTTCGAGTACGACATTGAAGGACGCCGCACCCACGTGCGTCACG CCGACGGCCGCCACGCGCAATGGCGCTACGACGCGCAATTCCAGA TCGTCGAGTACCTCGATTTCGACGGCCGGCGCTACGGGCTCAAGT ACAACGACGCCGGCATGCCCGTGATGCTGACGCTGCCCGGCGAAC GGACCGTGACGTTCGAGTACGACGATGCCGGCCGCATCGTCGCCG AAACCGATCCACTCGGCCGCACCACGAAAACGCGCTACGACGGCA ACAGCAGGCGGCCCGTCGAGATCATCGCGCCCGACGGCAGCGCCT GGCACGCCGAATACGACCGGCAAGGCCGGCTGCTCGCCACCCGCG ATCCGCTCGACCGGGAAAACCGCTACGAATACCCGAAGGCGCTCA GCGCGCTGCCGATCGCGCACGTCGATGCGCTGGGCGGGCGCAAGA CGTTCGAGTGGAACCGGCTCGGCGAGCTGGTGGCCTATACCGATT GCTCGGGCAAGACCACACGCAATTTTTACGACGCATTCGGTCTGCC GCTCGCGCGCGAGAACGCGCTCGGCCACCGCGTGACGTTCGACCT GCGCCCGACCGGCGAGGCGCGGCGCGTCACCTATCCCGACGGCAG TACAGAAAGCTACGAATACGACGCCGCCGGGCTGATGATCCGGCA CGTCGGGCTGGGCGGCCGGACGCAGATTGCGCTGCGCAACGCGCG TGGGCAGATCGTGGAGGCGGTCGATCCGGCCGGACGGCGCACCTG CTACCGCTACGACGCCGAGGGGCGGCTGCGCGAGCTGCAACAGGG GCACGCGCGTTACGCGTTCACCTACAGCGCGGGCGGGCGGCTCAC CAGCGAAACCCGGCCCGACGGCGTGCGGCGCCGCTTCGAATACGG CGAGGCCGGCGATCTGGCGGCGCTCGACATCGTCGGCGCGGCCGA CGACGCCACGGCGAACGATCGTCCGGTTCGCACCATCCGCTTCGA GCGCGACCGCATGGGCAATCTGTGCGCGCAGCACACGCCCACCGA GGTGACGCGCTACACGCGCGACACCGGCGGCCGCCTGCTCGAAGT CGCATGCGTGCCGACCGCGGCCGGGCTGGCGCTCGGCATCGCGCC CGACACGCTGACCTTCGAATACGACAAGGCCGGGCGGCTGAGTGC CGAACACGGCGCGAACGGCAGCGTCCGATACACGCTCGACGCGCT CGACAACGTGATGAAGCTCGCCCTGCCGCACGAGCAGACGCTGCA GATGCTGCGCTACGGCTCGGGGCACGTGCATCAGATCCGCTGCGG CGACCAGGTGGTCAGCGATTTCGAGCGCGACGACCTGCATCGCGA GCTGACGCGCACTCAGGGCCGCCTGACCGAGCGTACCGCCTACGA CCTGCTGGGCCGCAAGATCTGGCAATCGGCCGGCTTCCAGCCCGA CGCGCTTGCGCGCGGGCAGGGCCAGGTGTGGCGCAACTACGGCTA CGACGCCGCCGGCGAACTGGCCGAGAGCCACGATAGCCTGCGCGG CAGCACGCAGTTCAGCTACGATCCGGCCGGCTATCTGACGCAGCG CGTCAATACCGCCGACCGGCAGCTCGAATCGTTCGCCTGGGATGC CGCCGGCAACCTGCTCGACGATGCGCAGCGCCGCAGCCGCGGTTA TGTCGAGGGCAACCGGCTGCGCATGTGGCAGAACCTGCGCTTCGA ATACGACCCGTTCGGCAATCTCGCGACCAAGCTGCGCGGCGCGAA CCAGCGCCAGCAGTTCACTTACGACGGGCAGGATCGGCTCGTGGC GGTGCGCACGCAGGACGCGCGCGGCGTGGTGGAGACGCGTTTCGC CTACGATCCGCTGGGGCGGCGCATCGCCAAGACGGATATTGTGCG CGACGCGCGCGGCGTAGCGCTGCGCGAGGAAACGAAGCGGTTCGT GTGGGAGGGGCTGCGGCTCGCGCAGGAGGTGCGCGACACGGGCG TGAGCAGCTACGTGTACAGCCCGGACGCGCCCTATACGCCCGCGG CGCGCGTGGATGCCGTGCTGGCCGAGGCCATGGCCGCCGCTGCCA TCGAGCAGGCCAGACAGGCGACGCGGATCTATCACTTTCATACCG ATGTGTCGGGCGCACCGCAAGAAGCGACGAACGAGGCTGGCGAC ATTGTTTGGGCCGGCCAATACTCAGCCTGGGGCAAGGTGGCGCCG AACCAGCATGCCCCCGCCCGGATCGATCAGCCGCTCCGCTACGCC GGACAATATGCCGACGACAGTACCGAGCTGCACTACAACACGTTT CGTTTCTACGATCCGGACGTCGGCCGGTTTATCAATCAGGATCCAA TCGGGTTGATGGGGGGGCTGAATCTTTACCAATATGCACCCAACTC GATCGCATGGACCGACTGGTGGGGGCTGGCCGGCAGCTATACGCT CGGTTCCTATCAAATTTCTGCGCCTCAACTTCCGGCCTACAATGGA CAGACTGTTGGGACCTTCTACTACGTGAACGGCGCGGGCGGGCTC GAATCGAGGACATTCTCTTCCGGAGGGCCGACCCCTTATCCAAATT ATGCCAATGCCGGGCACGTGGAGGGCCAGTCCGCGCTGTTCATGA GGGATAACGGAATTTCAGACGGACTGGTTTTCCACAACAACCCTG AGGGCACTTGCGGATTCTGCGTTAATATGACCGAAACGCTTTTGCC TGAAAATTCCAAACTTACCGTCGTTCCGCCCGAGGGCGCGATCCC GGTCAAGCGGGGCGCGACGGGCGAAACGAGAACATTTACGGGGA ACAGCAAGTCTCCGAAGTCCCCTGTCAAAGGAGAATGTTGA[SEQ IDNO:135] DddA >AOT60363.1tRNAnucleaseWapAprecursor[Streptomyces homologin rubrolavendulae] Streptomyces MSSSDAGRAFGVPENVLARFTRYPGGARRRAGRTARARRLGIVLSAV rubrolavendulae LSATLLPAEAWAIAPPAPRTGPTLDALQQEEEVDPDPAAMEELDDWD PROTEIN GGPVEPPADYTPTEVTPPTGGTAPVPLDSAGEELVPAGTLPVRIGQAS PTEEDPAPPAPSGTWDVTVEPRATTEAAAVDGAIIKLTPPASGSTPVD VELDYGRFEDLFGTEWSSRLKLTQLPECFLTTPELEECGTPITIPTSNDP ATGTVRATVDPADGQPQGLAAQSGGGPAVLAATDSASGAGGTYKAT SLSATGSWTAGGSGGGFSWSYPLTIPDTPAGPAPKISLSYSSQSVDGRT SVANGQASWIGDGWDYHPGFVERRYRSCNDDRSGTPNNDNSADKE KSDLCWASDNVVMSLGGSTTELVRDDTTGTWVAQNDTGARIEYKD KDGGALAAQTAGYDGEHWVVTTRDGTRYWFGRNTLPGRGAPTNSA LTVPVFGNHTGEPCHAATYAASSCTQAWRWNLDYVEDVHGNAMVV DWKKEQNRYAKNEKFKAAVSYDRDAYPTQILYGLRADDLAGPPAG KVVFHAAPRCLESAATCSEAKFESKNYADKQPWWDTPATLHCKAGD ENCYVTSPTFWSRVRLSAIETQGQRTPGSTALSTVDRWTLHQSFPKQR TDTHPPLWLESITRVGFGRPDASGNQSSKALPAVTFLPNKVDMPNRV LKSTTDQTPDFDRLRVEVIRTETGGETHVTYSAPCPVGGTRPTPASNG TRCFPVHWSPDPAAFSDENLDKSGYEPPLEWFNKYVVTKVTEMDLV AEQPSVETVYTYEGDAAWAKNTDEYGKPALRTYDQWRGYASVVTR TGTTANTGAADATEQSQTRTRYFRGMSGDAGRAKVHVTLTDVTGTA TTVEDLLPYQGMAAETLTYTKAGGDVAARELAFPYSRKTASRARPG LPALEAYRTGTTRTDSIQHISGDRTRAAQNHTTYDDAYGLPTQTYSLT LSPNDSGTLVAGDERCTVTTYVHNTAAHIIGLPDRVRATTGDCAAAP NATTGQIVSDSRTAYDALGAFGTAPVKGLPVQVDTISGGGTSWITSAR TEYDALGRATKVTDAAGNSTTTTYSPATGPAFEVTVTNAAGHATTTT LDPGRGSALTVTDQNGRKTTSTYDELGRATGVWTPSRPVNQDASVR FVYQIEDSKVPAVHTRVLRDAGTYEESIELYDGFLRPRQTQREALGGG RIVTETLYNANGSAKEVRDGYLAEGEPARELFVPLSLDQVPSATRTA YDGLGRPVRTTTLHRGVPRHSATTAYGGDWELSRTGMSPDGTTPLSG SRAVKATTDALGRPARIQHFTTQNVSAESVDTTYTYDPRGPLAQVTD AQQNTWTYTYDARGRKTSSTDPDAGAAYFGYNALDQQVWSKDNQ GRLQYTTYDVLGRQTELRDDSASGPLVAKWTFDTLPGAKGHPVAST RYNDGAAFTSEVTGYDTEYRPTGNKVTIPSTPMTTGLAGTYTYASTY TPTGKVQSVDLPATPGGLAAEKVITRYDGEDSPTTMSGLAWYTADTF LGPYGEVLRTASGEAPRRVWTTNVYDEDTRRLTRTTAHRETAPHPVS TTTYGYDTVGNITSIADQQPAGTEEQCFSYDPMGRLVHAWTDGNSA VCPRTSTAPGAGPARADVSAGVDGGGYWHSYAFDAIGNRTKLTVHD RTDAALDDTYTYTYGKTLPGNPQPVQPHTLTQVDAVLNEPGSRVEPR STYAYDTSGNTTQRVIGGDTQTLAWDRRNKLTSVDTNNDGTPDVKY LYDASGNRLVEDDGTTRTLFLGEAEIVVNTAGQAVDARRYYSSPGAP TTIRTTGGKTTGHKLTVMLSDHHSTATTAVELTDTQPVTRRRFDPYG NPRGTEPTTWPDRRTYLGVGIDDPATGLTHIGAREYDASTGRFISVDP VMDLTDPLQMNGYTYANADPINNSDPTGLLLDARGGGTQKCVGTCV KDVTNRKGIPLPPGEEWKHEGEAQTDFNGDGFITVFPTVNVPAKWKK AKKYTEAFYKAVDTACFYGRESCADPEYPSRAHSINNWKGKACKAV GGKCPERLSWGEGPAFAGGFAIAAEEYAGRGGYRGGGARRGSPCKC FLAGTEVLMADGSTKSIEDIKLGDEVVATDPVTGEAGAHPVSALIATE NDKRFNELVIITSEGVERLTATHEHPFWSPSEGEWLEAGELRTGMTLR SDSGETLVVAGNRAFTQRARTYNLTVADLHTYYVLAGQTPVLVHNA NCGPHLKDLQKDYPRRTVGILDVGTDQLPMISGPGGQSGLLKNLPGR TKANGEHVETHAAAFLRMNPGVRKAVLYIDYPTGTCGTCRSTLPDM LPEGVQLWVISPRRTEKFTGLPD[SEQIDNO:136] DddA >A4G23_03234CP017316.1:3756245-3763321Streptomyces homologin rubrolavendulaestrainMJM4426,completegenome Streptomyces rubrolavendulae ATGTCCTCGTCCGATGCGGGACGCGCCTTCGGCGTGCCCGAAAAC DNA GTCCTGGCGCGTTTCACGCGGTATCCCGGCGGGGCGCGACGCCGT GCCGGGCGCACGGCGCGCGCCCGGCGCCTGGGCATCGTGCTGTCC GCCGTCCTCTCGGCGACCCTGCTGCCCGCCGAGGCATGGGCCATC GCGCCCCCGGCGCCGCGCACCGGTCCGACCCTGGACGCCCTCCAG CAGGAGGAGGAGGTCGATCCGGACCCGGCCGCCATGGAAGAGCT GGACGACTGGGACGGTGGGCCGGTCGAGCCCCCGGCCGACTACAC CCCCACCGAGGTCACGCCTCCCACCGGCGGCACCGCCCCGGTGCC GCTGGACAGCGCGGGCGAGGAACTGGTCCCGGCCGGGACCCTGCC CGTGCGCATCGGCCAGGCGTCCCCCACCGAGGAGGACCCGGCACC CCCGGCACCCAGCGGCACGTGGGACGTCACCGTGGAGCCCCGCGC CACCACCGAGGCGGCCGCCGTGGACGGCGCCATCATCAAGCTCAC CCCGCCCGCCAGCGGCTCCACACCGGTCGACGTGGAACTCGACTA CGGCCGGTTCGAGGACCTGTTCGGCACCGAGTGGTCCTCCCGGCTC AAGCTGACGCAGCTCCCGGAGTGCTTCCTCACGACGCCCGAGCTG GAGGAGTGCGGCACCCCCATCACCATCCCGACGAGCAACGACCCG GCCACCGGGACGGTCCGGGCCACCGTCGACCCGGCCGACGGGCAG CCGCAGGGCCTGGCCGCGCAGTCGGGCGGCGGTCCCGCCGTCCTC GCCGCGACCGACTCGGCGTCCGGCGCCGGCGGCACGTACAAGGCG ACCTCCCTCTCGGCCACCGGCTCCTGGACGGCCGGCGGCAGCGGC GGCGGCTTCTCCTGGTCGTATCCGCTCACCATCCCGGACACCCCGG CCGGCCCCGCGCCGAAGATCTCCCTGTCGTACTCCTCCCAGTCCGT CGACGGCCGCACCTCCGTCGCCAACGGCCAGGCGTCGTGGATAGG CGACGGCTGGGACTACCACCCCGGCTTCGTCGAGCGCCGCTACCG CTCCTGCAACGACGACCGCTCCGGCACCCCGAACAACGACAACAG TGCGGACAAGGAGAAGTCCGACCTGTGCTGGGCGAGCGACAACGT CGTGATGTCGCTCGGCGGCTCCACCACCGAACTCGTCCGCGACGA CACGACCGGCACGTGGGTCGCGCAGAACGACACCGGTGCCCGGAT CGAGTACAAGGACAAGGACGGCGGAGCCCTGGCCGCCCAGACCG CCGGCTACGACGGCGAGCACTGGGTCGTCACCACCCGCGACGGAA CCCGCTACTGGTTCGGCCGCAACACCCTCCCCGGCCGCGGCGCCCC CACGAACTCCGCCCTCACCGTCCCCGTCTTCGGCAACCACACCGGC GAGCCCTGCCACGCCGCCACCTACGCCGCCTCCTCCTGCACCCAGG CGTGGCGCTGGAACCTCGACTACGTCGAGGACGTCCACGGCAACG CGATGGTCGTCGACTGGAAGAAGGAGCAGAACCGGTACGCGAAG AACGAGAAGTTCAAGGCGGCTGTCTCCTACGACCGCGACGCGTAT CCGACGCAGATCCTCTACGGCCTGCGCGCCGACGACCTGGCGGGC CCGCCCGCCGGCAAGGTCGTCTTCCACGCCGCCCCGCGCTGCCTCG AAAGCGCGGCCACCTGCTCCGAAGCCAAGTTCGAGTCCAAGAACT ACGCGGACAAGCAGCCCTGGTGGGACACACCGGCCACCCTGCACT GCAAGGCCGGTGACGAGAACTGCTACGTCACCTCGCCGACGTTCT GGAGCCGCGTCCGCCTGTCGGCGATCGAGACGCAGGGTCAGCGCA CGCCCGGCTCGACGGCGCTGTCCACGGTCGACCGCTGGACCCTGC ACCAGTCGTTCCCGAAGCAGCGCACCGACACCCACCCGCCGCTCT GGCTGGAGTCGATCACCCGCGTGGGCTTCGGCCGGCCGGACGCCT CCGGCAACCAGTCGAGCAAGGCCCTCCCGGCGGTGACCTTCCTGC CCAACAAGGTCGACATGCCGAACCGCGTGCTGAAGAGCACGACGG ACCAGACGCCCGATTTCGACCGCCTGCGCGTCGAGGTCATCCGCA CGGAGACCGGCGGCGAGACCCATGTGACGTACTCCGCCCCCTGCC CCGTCGGCGGCACCCGCCCCACCCCGGCCTCCAACGGCACCCGCT GCTTCCCGGTCCACTGGTCCCCCGACCCGGCGGCCTTCTCCGACGA GAACCTGGACAAGAGCGGCTACGAGCCGCCCCTCGAGTGGTTCAA CAAGTACGTCGTCACCAAGGTCACCGAGATGGACCTCGTGGCGGA GCAGCCCAGCGTCGAGACCGTCTACACCTACGAGGGCGACGCCGC CTGGGCGAAGAACACCGACGAGTACGGCAAGCCCGCCCTGCGCAC CTACGACCAGTGGCGCGGCTACGCGAGCGTCGTCACCCGCACGGG CACCACGGCCAACACCGGCGCCGCCGACGCCACCGAGCAGTCCCA GACCCGCACCCGGTACTTCCGCGGCATGTCCGGCGACGCGGGCCG CGCCAAGGTGCACGTCACGCTCACGGACGTGACCGGCACCGCGAC CACCGTCGAGGACCTGCTCCCGTACCAGGGCATGGCCGCCGAGAC CCTTACCTACACCAAGGCGGGCGGCGACGTCGCCGCCCGCGAGCT GGCCTTCCCCTACAGCAGGAAGACCGCCTCCCGCGCCCGCCCCGG CCTCCCCGCCCTGGAGGCGTACCGCACGGGCACGACGCGCACGGA CTCCATCCAGCACATCAGCGGCGACCGGACGCGCGCCGCTCAGAA CCACACCACATACGACGACGCGTACGGCCTGCCCACCCAGACCTA CTCGCTGACACTCTCGCCGAACGACTCCGGCACCCTTGTCGCCGGT GACGAGCGGTGCACCGTCACGACGTACGTCCACAACACCGCCGCG CACATCATCGGCCTCCCCGACCGCGTCCGCGCCACGACGGGCGAC TGCGCCGCCGCGCCGAACGCCACCACCGGCCAGATCGTCTCCGAC AGCCGCACCGCGTACGACGCGCTCGGCGCCTTCGGCACGGCCCCG GTCAAGGGCCTGCCGGTCCAGGTGGACACGATCTCCGGAGGCGGC ACGAGCTGGATCACCTCGGCGCGCACGGAGTACGACGCGCTGGGC CGTGCGACCAAGGTCACCGACGCGGCGGGCAACTCCACCACGACC ACGTACAGCCCGGCGACCGGCCCCGCGTTCGAGGTCACCGTGACC AACGCGGCTGGTCATGCCACGACCACCACCCTCGACCCCGGTCGC GGCTCGGCGCTGACCGTCACCGACCAGAACGGCCGCAAGACCACC AGCACGTACGACGAACTCGGCCGGGCCACCGGCGTGTGGACGCCC TCCCGCCCGGTGAACCAGGACGCGTCCGTGCGCTTCGTCTACCAG ATCGAGGACAGCAAGGTCCCGGCGGTGCACACTCGGGTCCTGCGC GACGCCGGTACGTACGAGGAGTCGATCGAGCTCTACGACGGCTTC CTCCGCCCCCGTCAGACCCAGCGCGAGGCGCTGGGCGGCGGCCGA ATCGTCACCGAGACCCTCTACAACGCCAACGGCTCTGCGAAGGAA GTGCGCGACGGCTACCTGGCGGAGGGCGAGCCCGCGCGGGAACTG TTCGTCCCGCTCTCCCTCGACCAGGTGCCGAGCGCGACGAGGACG GCCTATGACGGCCTGGGCCGGCCCGTCCGGACGACGACCCTCCAC AGGGGAGTCCCCCGGCACTCCGCCACCACGGCGTACGGCGGCGAC TGGGAACTGAGCCGCACCGGCATGTCGCCCGACGGAACGACGCCG CTCTCTGGCAGCCGCGCCGTGAAGGCGACGACGGACGCGCTCGGC CGCCCGGCCCGCATCCAGCACTTCACCACCCAGAACGTGTCGGCC GAGAGCGTCGACACCACGTACACCTACGACCCCCGCGGCCCCCTT GCCCAGGTCACCGACGCCCAGCAGAACACCTGGACGTACACGTAC GACGCCCGTGGGCGCAAGACGTCCTCCACCGACCCGGACGCGGGC GCCGCCTACTTCGGCTACAACGCGCTGGACCAGCAGGTCTGGTCG AAGGACAACCAGGGCCGCCTGCAGTACACGACGTACGACGTCCTG GGCCGCCAGACCGAGCTGCGCGACGACTCCGCGTCCGGCCCGCTG GTGGCGAAGTGGACCTTCGACACCCTGCCGGGCGCCAAGGGCCAC CCGGTCGCGTCGACCCGCTACAACGACGGCGCCGCGTTCACCAGC GAGGTGACCGGTTACGACACCGAGTACCGTCCGACCGGCAACAAG GTCACCATCCCCAGCACCCCGATGACCACGGGCCTCGCCGGCACG TACACGTACGCCAGCACGTACACCCCGACCGGCAAGGTCCAGTCC GTCGACCTGCCCGCGACGCCCGGCGGGCTCGCCGCGGAGAAGGTG ATCACCCGCTACGACGGCGAGGACTCGCCCACCACGATGTCGGGC CTGGCCTGGTACACGGCCGACACCTTCCTCGGCCCGTACGGGGAA GTGCTGCGCACGGCGTCGGGCGAGGCCCCGCGCCGCGTGTGGACG ACCAACGTCTACGACGAGGACACCCGCCGCCTCACCAGGACCACC GCGCACCGGGAGACGGCTCCCCACCCGGTCAGCACGACCACCTAC GGCTACGACACGGTCGGCAACATCACGTCCATCGCCGACCAGCAG CCGGCGGGTACCGAGGAGCAGTGCTTCTCGTACGACCCGATGGGG CGCCTCGTCCACGCCTGGACGGACGGCAACAGCGCCGTCTGCCCC AGGACGTCCACGGCACCGGGCGCCGGCCCGGCCCGCGCCGACGTC TCGGCCGGTGTCGACGGCGGCGGATACTGGCACTCGTACGCGTTC GACGCGATCGGCAACCGGACGAAGCTGACCGTCCACGACCGCACC GACGCGGCCCTGGACGACACGTACACCTACACCTACGGCAAGACC CTGCCGGGTAACCCGCAGCCGGTCCAGCCGCACACCCTCACCCAG GTCGACGCGGTGCTCAACGAGCCCGGATCGAGAGTCGAACCGCGC TCCACATACGCCTACGACACCTCCGGCAACACCACCCAGCGCGTC ATCGGCGGCGACACCCAGACCCTGGCCTGGGACCGCCGCAACAAG CTGACGTCCGTCGACACGAACAACGACGGCACACCGGACGTGAAG TACCTGTACGACGCGTCGGGCAACCGCCTGGTCGAGGACGACGGC ACCACGCGCACCCTCTTCCTCGGCGAGGCCGAGATCGTCGTCAAC ACGGCCGGCCAGGCCGTGGACGCGCGCCGCTACTACAGCAGCCCC GGCGCCCCGACGACGATCCGCACGACCGGCGGCAAGACCACGGG CCACAAGCTGACCGTCATGCTGTCGGACCACCACAGCACGGCGAC GACCGCGGTCGAGCTGACCGACACCCAGCCGGTCACCCGCCGCCG CTTCGACCCGTACGGCAACCCCCGCGGCACCGAGCCGACCACCTG GCCCGACCGCCGCACCTACCTGGGCGTCGGCATCGACGACCCCGC CACGGGCCTGACCCACATCGGCGCCCGCGAATACGACGCATCGAC GGGCCGCTTCATCTCCGTCGATCCGGTCATGGACCTCACGGACCCG CTCCAGATGAACGGGTACACCTACGCCAACGCGGACCCGATCAAC AACAGCGACCCCACCGGACTGTTGCTCGACGCCCGAGGCGGCGGC ACTCAGAAGTGCGTGGGAACCTGCGTCAAGGACGTCACGAACCGA AAGGGAATTCCGCTCCCGCCTGGCGAGGAGTGGAAGCATGAAGGG GAGGCGCAAACCGATTTCAACGGTGACGGCTTCATCACCGTCTTCC CGACCGTGAATGTTCCGGCGAAGTGGAAGAAGGCGAAGAAGTAC ACGGAGGCTTTCTACAAGGCGGTTGATACTGCTTGCTTCTATGGAC GCGAAAGCTGTGCGGATCCGGAGTACCCTTCGCGGGCGCATAGCA TCAACAACTGGAAGGGAAAGGCATGCAAAGCCGTAGGGGGAAAA TGCCCTGAGAGGTTGTCGTGGGGGGAGGGTCCGGCGTTCGCTGGT GGCTTCGCGATAGCAGCGGAAGAGTATGCGGGGAGAGGGGGCTA CCGGGGCGGTGGGGCGAGGAGGGGGTCGCCCTGTAAGTGCTTCCT TGCCGGCACCGAGGTGCTCATGGCGGATGGCAGCACTAAAAGTAT CGAGGACATCAAGCTCGGTGACGAAGTGGTTGCGACTGATCCGGT AACCGGTGAGGCCGGTGCGCACCCTGTCTCGGCGCTGATCGCCAC CGAGAACGACAAGCGTTTCAACGAGCTGGTCATTATCACCAGCGA GGGTGTAGAGCGTCTTACCGCAACGCATGAGCACCCCTTCTGGTC GCCATCCGAAGGGGAGTGGTTGGAGGCGGGTGAGCTGCGCACTGG CATGACGCTGCGCTCCGACTCTGGCGAAACTCTCGTAGTCGCAGG AAACCGCGCCTTCACCCAGCGAGCCCGGACCTACAACCTCACGGT TGCAGACCTCCACACGTACTATGTGCTGGCGGGCCAGACTCCGGT ACTGGTTCACAATGCAAACTGTGGACCTCACCTGAAGGACCTGCA AAAGGACTACCCCCGGCGCACTGTGGGCATCCTTGACGTCGGAAC TGATCAGCTCCCGATGATTAGCGGCCCAGGTGGCCAGTCGGGACT TCTCAAGAACCTCCCAGGTCGTACGAAGGCCAACGGGGAGCACGT GGAGACTCACGCAGCAGCGTTCTTGCGTATGAACCCGGGTGTCAG AAAGGCCGTGCTCTACATCGACTACCCGACGGGGACCTGCGGAAC ATGTAGAAGTACATTGCCTGACATGCTGCCCGAGGGTGTTCAGTTG TGGGTGATCTCGCCGCGTAGGACTGAAAAATTCACGGGACTTCCT GACTGA[SEQIDNO:137] DddA >AVT32940.1hypotheticalproteinC6361_29650[Plantactinosporasp.BC1] homologin MGDRLPAFVDGGDTLGIFSRGGIERDLASGVAGPASSLPKGTPGFNGL Plantactinospora VKSHVEGHAAALMRQNGIPNAELYINRVPCGSGNGCAAMLPHMLPE sp.BC1 GATLRVYGPNGYDRTFTGLPD[SEQIDNO:138] PROTEIN DddA >C6361_29650CP028158.1:6764267-6764614Plantactinosporasp.BC1 homologin chromosome,completegenome Plantactinospora CTGGGTGACCGGCTCCCTGCCTTCGTGGACGGTGGAGACACGTTG sp.BC1 GGCATCTTTTCTCGCGGAGGTATTGAGCGGGACCTCGCCAGCGGA DNA GTTGCGGGTCCTGCAAGTAGCCTTCCTAAAGGCACGCCTGGCTTCA ATGGTCTTGTAAAGAGTCATGTTGAAGGGCATGCGGCTGCGCTAA TGAGACAAAATGGAATTCCGAACGCTGAGCTGTATATCAACAGAG TGCCGTGCGGTTCAGGTAATGGCTGCGCAGCGATGTTGCCGCATAT GCTTCCGGAAGGTGCCACCCTCCGCGTATATGGGCCGAACGGGTA CGATAGAACCTTCACTGGACTTCCGGACTGA[SEQIDNO:139] DddA >BAJ27137.1hypotheticalproteinKSE_13070[KitasatosporasetaeKM- homologin 6054] Kitasatospora MAAVPSAEALAAKRARDTIWTPPNTPLGSQTKSVDGENLVPGRLPGP setaeKM- LEPEPADWTPGGPASVPAPGSADVTLGFDSAEAAAARKATGGAAPAS 6054 DGAALRAGSLPVVIGAAKDAKSGAHRIRVELVDQAKSRAAHLDSPLI PROTEIN ALTDTEPDTPPSGRTTKVSLDLKGIGAQTWADRARLVALPACALETP DRPECQQQTPVQSSVDLRSGLLTAEVILPAATEGTAPPTKSSLGSGTAS GVVQAGLTTAAPAKAAPTVLAATAGASGSGGSFSATSLSPSAAWGA GSNVGNFTYSYPIQTPPSLGGTAPSVGLGYDSSAVDGKTSAQNSQSS WLGEGWGYEAGFIERGYKSCNTAGIANSSDMCWGGQNATLSLAGHS GTLVRDDTTGVWHLQSDDGTKIEQLTGAPNGLQNGEHWRITTTDGT QFYFGRNHLPGGDGTDPASNSAFKEPVYSPKSGDPCYNSSTATGSWC TMGWRWNLDYAVDVHGNLITYTYAQETNYYSRGAGQNSGSGTLTD YTRAGYLTQIAYGQRLSEQVTAKGAAKAAALITFTAAERCVPSGSITC TEAQRTTANASYWPDTPLDQVCASTGTCTRAGPTFFTTKRLASLTTQ VLVSGAYRTVDTWTLTHSFKDPGDGNAKSLWLDSIQRTGTNGQTAV TMPPVTFTAVMKPNRVDGDLTLKDGTKVTVTPFNRPRLQQVTTETG GQINVVYTTSSDAAHPACSRLAGTMPAAADGNTLACAPVKWYLPGS SSPDPVDDWFNKYLISAVTEQDAISGTTLIKATNYTYNGDAAWHRND AEFTDAKTRTWDGFRGYQSVTSTTGSAYPGEAPRTQQTATYLRGMD GDVKADGSTRSVQVANPLGGPALTDSPWLAGSSFATQTYDQAGGTV ISANGSVAGGQQVTATHAQSGGMPALVARYPASQVTTTSKSKLSDGT WRTNTTVSTSDPAHANRPLSSDDKGDGTPGAELCSTNGYATGTNPM MLNILAERTVTKGACGTPVTSANTVSSARTLYDGKPYGQAGDLAEST SALTLDHYDTGGNPVYVHTAASTFDAYGRLTSVSEANGATYDAAGN QLTAPNLTPATTRTAYTPATGAIATTVTQTTPTGWTTTLTQDPGRAEA LVSTDANGRATTQQYDGLGRLTAAWSPERATNLTPSQKFSYAVNGT TGPSVVTSQWLKEAGGYAYKNELYDGLGRLRQVQRTSDTYSGRLIT DTVYDSHGWPVKTASPYYEKTTAPNSTVYLPQDSQVPAQTWVTFDG IGRTTRSAFVSYGQQQWATTTAYPGADRTDVTPPNGKYPTSTFTDGR NQVSALWQYRTATPTGNPADATVTTYTYDAANRPATRKDAAGNTW SYGYDLRGRQTTVTDPDTGTTTTAYDVNSRAVSTTDGKGNTLVVSY DLIGRKTGLYQGSIAPANQLAGWTYDTLPGGKGKPTSSTRYVGGAGG SAYTQAVTGYDAGYRPTGTSVTIPASEGKLAGTYTTGLTYNPVLGTL KQTDLPAIGAAPAESVMYTYNISGVLQKSYSDTYYVYDVQYDAFGRP VRTTTGDAGTQVVSTQLDKTDYTYNQAGDVTSVTDVQNGTATDAQ CFTYDHLGRLTQAWTDTAGSTSTTSGTWTDTSGTVHNSGSSQSVPAL GACANANGPASTGSPAKLSVGGPSPYWQSYGYDSTGNRTTLVQHDT TGNTTKDTTTTQTFGPAGSVNTATGAPNTGGGTGGPHALLTSSTTGP TGTQVTSYQYDQLGNTTAVTETSGTTTLAWNGEDKLASVTKTGQAQ ATSYLYDADGNQLIRRNPGKTTLNLGSDEVTLDTAANSLTDTRYYSA PGGISIARTTGPTGASALAYQASDPHGTANVQINVDAAQTTTRRPTDP FGNPRGTQPAPNTWAGDKGFVGGTKDDTTGLTNLGAREYQPTTGRF LNPDPLLDAGNPQQWNGYAYSDNDPVNSSDPSGLITNALADGDTYV ARPAAFCVTMSCVEQTSGPGFWEDKRVGDAVFAAVVQATTQSNGN GSSQTKKEKGIWGQAWDWTKKNGGAILGALVEGAVESTCFIGAGFA APATGGITVIAGAAACGAVAGEAGALTTNILTPDADHSVDGITNDMV VGEITGAAVSAASEGASSLAKPAVRKLLGMEAEEGLEAAGRAATGPC NSFPAGVTVLLADGTTKPIEQIAQGDQVTATDPQTGTTQAEPVTDTIIG HDDTEFTDLTLTNDADPRAPPSEITSTTHHPYWNATTSRWTDAGDLK PGDHVRTPDGTELTVNTVYSYTTQPRTARNLTVADLHTYYVLAGNTP VLVHNTGPGCGEPGFVSDAANSLSGRRITTGQIFDASGNPIGPEITSGG GSLADRAQSYLADSPNIRNLPAKARYASADHVEAQYAVWMRENGV TDASVVINQNYVCGLPLGCQAAVPAILPRGSTMTVWYPGSGSPIVLR GVG[SEQIDNO:140] DddA >KSE_13070NC_016109.1:1451556-1458878KitasatosporasetaeKM-6054 homologin DNA,completegenome Kitasatospora GTGCTGGGGACAGCGGCCGCGCTCGCGGTCATGATGTCCATGGCG setaeKM- GCGGTGCCGTCCGCCGAGGCACTGGCCGCGAAGCGGGCACGCGAC 6054 ACCATCTGGACGCCGCCCAACACCCCGCTGGGCAGCCAGACCAAG DNA TCCGTCGACGGCGAGAACCTCGTCCCGGGCCGCCTGCCCGGCCCC CTGGAGCCGGAACCGGCCGACTGGACACCCGGCGGACCGGCATCC GTGCCCGCTCCGGGCAGCGCGGACGTCACCCTCGGCTTCGACTCC GCGGAGGCCGCCGCCGCCCGCAAGGCCACCGGCGGCGCCGCCCCC GCCTCCGACGGCGCGGCCCTCCGCGCGGGCTCCCTCCCCGTCGTCA TCGGCGCGGCGAAGGACGCCAAGAGCGGCGCCCACCGGATCCGC GTCGAGCTCGTGGACCAGGCCAAGAGCCGTGCCGCACACCTCGAC AGCCCGCTGATCGCACTCACCGACACCGAGCCGGACACCCCGCCC TCCGGTCGGACCACGAAGGTGTCCCTCGACCTGAAGGGCATCGGC GCCCAGACCTGGGCGGACCGCGCGCGACTCGTCGCCCTGCCCGCC TGCGCCCTGGAGACGCCCGACAGGCCCGAGTGCCAGCAGCAGACC CCCGTGCAGAGCTCCGTCGACCTGCGCTCCGGACTGCTGACGGCC GAGGTCATTCTGCCCGCCGCCACCGAGGGCACCGCCCCGCCCACC AAGAGCTCCCTCGGCTCGGGCACCGCCTCCGGCGTCGTCCAGGCC GGCCTCACCACGGCGGCGCCCGCCAAGGCCGCGCCCACGGTGCTC GCCGCGACCGCCGGCGCGTCCGGCTCGGGCGGCAGCTTCTCGGCG ACCTCGCTGTCGCCCTCCGCGGCCTGGGGCGCCGGCTCCAACGTCG GCAACTTCACCTACTCGTACCCGATCCAGACGCCTCCCTCGCTCGG CGGGACCGCCCCCTCCGTGGGCCTCGGGTACGACTCGTCCGCCGTC GACGGGAAGACCTCCGCGCAGAACTCCCAGTCCTCCTGGCTCGGC GAGGGCTGGGGCTACGAGGCCGGGTTCATCGAGCGCGGCTACAAG TCCTGCAACACGGCCGGCATCGCGAACTCCTCGGACATGTGCTGG GGCGGGCAGAACGCCACCCTCTCGCTGGCCGGCCACTCCGGCACC CTGGTGCGCGACGACACCACCGGCGTCTGGCACCTGCAGAGCGAC GACGGCACGAAGATCGAACAGCTCACCGGCGCGCCCAACGGCCTG CAGAACGGCGAGCACTGGCGGATCACCACGACCGACGGCACGCA GTTCTACTTCGGCCGCAACCACCTGCCCGGCGGCGACGGCACCGA CCCGGCGAGCAACTCCGCCTTCAAGGAACCGGTGTACTCGCCCAA GAGCGGCGACCCCTGCTACAACTCCTCCACCGCCACCGGCTCCTG GTGCACGATGGGCTGGCGCTGGAACCTCGACTACGCCGTCGACGT CCACGGCAACCTGATCACCTACACCTACGCCCAGGAGACCAACTA CTACAGCCGAGGCGCCGGCCAGAACAGCGGCAGCGGCACCCTGAC CGACTACACCCGCGCCGGCTACCTCACCCAGATCGCCTACGGCCA GCGCCTGAGCGAGCAGGTCACCGCCAAGGGCGCGGCCAAGGCCG CTGCCCTCATCACCTTCACCGCCGCGGAACGCTGCGTCCCGTCCGG CTCGATCACCTGCACCGAGGCACAGCGCACGACCGCGAACGCCTC GTACTGGCCGGACACCCCGCTCGACCAGGTCTGCGCCTCCACCGG CACCTGCACCCGGGCCGGCCCGACGTTCTTCACCACCAAGCGCCTC GCCTCCCTCACCACCCAGGTCCTGGTCTCCGGCGCCTACCGCACCG TCGACACCTGGACGCTCACCCATTCCTTCAAGGACCCGGGCGACG GCAACGCCAAGTCGCTGTGGCTCGACTCGATCCAGCGCACCGGCA CCAACGGGCAGACCGCGGTCACCATGCCGCCCGTCACCTTCACGG CGGTGATGAAGCCGAACCGGGTGGACGGGGACCTCACCCTCAAGG ACGGCACCAAGGTCACCGTCACCCCGTTCAACCGGCCCCGCCTCC AGCAGGTCACCACGGAGACCGGCGGCCAGATCAACGTCGTCTACA CCACCTCCTCCGACGCCGCGCACCCCGCCTGCTCGCGCCTGGCCGG CACCATGCCCGCCGCGGCGGACGGCAACACCCTCGCCTGCGCCCC CGTCAAGTGGTACCTGCCCGGATCCAGCTCCCCGGACCCGGTCGA CGACTGGTTCAACAAGTACCTGATCAGCGCCGTCACCGAACAGGA CGCGATCAGCGGCACCACCCTGATCAAGGCCACCAACTACACCTA CAACGGCGACGCCGCCTGGCACCGCAACGACGCCGAGTTCACCGA CGCCAAGACCCGCACCTGGGACGGCTTCCGCGGCTACCAGTCCGT CACCAGCACCACCGGCAGCGCCTACCCGGGCGAGGCCCCCAGGAC CCAGCAGACCGCGACCTACCTGCGCGGCATGGACGGCGACGTCAA GGCCGACGGCTCCACCCGCAGCGTCCAGGTCGCCAACCCGCTCGG CGGCCCGGCCCTCACCGACAGCCCGTGGCTGGCCGGCTCCAGCTT CGCCACCCAGACCTACGACCAGGCCGGCGGCACCGTCATCTCCGC CAACGGCTCCGTCGCCGGCGGCCAGCAGGTCACCGCCACCCACGC CCAGAGCGGCGGCATGCCGGCCCTGGTCGCCCGCTACCCCGCCTC CCAGGTCACCACCACCTCCAAGTCCAAGCTCTCCGACGGGACCTG GCGCACCAACACCACCGTCAGCACCAGCGACCCCGCGCACGCCAA CCGCCCCCTCAGCAGCGACGACAAGGGCGACGGCACCCCCGGCGC CGAACTGTGCAGCACCAACGGCTACGCCACCGGCACCAACCCGAT GATGCTGAACATCCTCGCCGAGCGGACGGTCACCAAGGGCGCCTG CGGCACCCCCGTGACCTCGGCCAACACCGTCTCCTCCGCCCGCACC CTCTACGACGGCAAGCCCTACGGCCAGGCCGGCGACCTCGCCGAG TCCACCAGCGCCCTGACCCTGGACCACTACGACACCGGCGGCAAC CCCGTCTACGTCCACACCGCCGCCTCCACCTTCGACGCCTACGGCC GGCTTACCAGCGTCAGCGAGGCCAACGGCGCCACCTACGACGCCG CGGGCAACCAGCTCACCGCGCCCAACCTCACCCCCGCCACCACCC GCACCGCCTACACCCCGGCCACCGGCGCCATCGCCACCACCGTCA CCCAGACCACGCCCACCGGCTGGACCACCACCCTCACCCAGGACC CGGGCCGCGCCGAAGCTCTGGTCTCCACCGACGCCAACGGCCGCG CCACCACCCAGCAGTACGACGGCCTCGGCCGCCTGACCGCCGCCT GGTCACCGGAGCGCGCGACCAACCTCACCCCCAGCCAGAAGTTCT CCTACGCGGTCAACGGCACCACCGGCCCCTCCGTCGTCACCTCCCA GTGGCTCAAGGAAGCCGGCGGCTACGCGTACAAGAACGAGCTGTA CGACGGCCTCGGCCGCCTGCGCCAGGTCCAGCGCACCAGCGACAC CTACTCCGGGCGGCTGATCACCGACACCGTCTACGACTCGCACGG CTGGCCCGTCAAGACCGCCAGCCCGTACTACGAGAAGACCACCGC GCCCAACAGCACCGTCTACCTGCCGCAGGACTCCCAGGTGCCCGC CCAGACCTGGGTCACCTTCGACGGCATCGGCCGGACCACCCGCTC CGCGTTCGTCTCCTACGGACAGCAGCAGTGGGCCACCACCACCGC CTACCCCGGCGCCGACCGCACCGACGTCACCCCGCCCAACGGCAA ATACCCGACCAGCACCTTCACCGACGGCCGCAACCAGGTCAGCGC CCTGTGGCAGTACCGCACCGCCACCCCCACCGGCAACCCGGCCGA CGCGACCGTCACCACCTACACCTACGACGCCGCCAACCGGCCCGC CACCCGCAAGGACGCCGCCGGGAACACCTGGAGCTACGGCTACGA CCTGCGCGGCCGCCAGACCACCGTCACCGACCCCGACACCGGCAC CACCACCACCGCCTACGACGTCAACTCGCGCGCCGTCTCCACCACC GACGGCAAGGGCAACACCCTCGTCGTCAGCTACGACCTGATCGGC CGCAAGACCGGCCTCTACCAGGGCAGCATCGCCCCGGCCAACCAG CTCGCCGGCTGGACGTACGACACCCTGCCGGGCGGAAAGGGCAAG CCCACCTCCTCCACCCGCTACGTCGGGGGCGCCGGCGGCTCGGCCT ACACCCAGGCCGTCACCGGCTACGACGCCGGCTACCGGCCCACCG GCACCTCGGTGACGATCCCCGCCAGCGAAGGCAAGCTCGCCGGTA CCTACACCACCGGCCTGACGTACAACCCGGTCCTCGGCACGCTCA AGCAGACCGACCTGCCGGCCATCGGCGCGGCGCCCGCCGAGAGCG TCATGTACACCTACAACATCTCCGGCGTCCTGCAGAAGTCCTACAG CGACACCTACTACGTCTACGACGTGCAGTACGACGCCTTCGGCCG CCCGGTCCGCACGACCACCGGCGACGCCGGAACCCAGGTCGTCTC CACCCAGCTCGACAAGACCGACTACACCTACAACCAGGCCGGCGA CGTCACCTCGGTCACCGACGTCCAGAACGGCACCGCCACCGACGC CCAGTGCTTCACCTACGACCACCTCGGGCGCCTCACCCAGGCCTGG ACCGACACCGCGGGCTCCACCAGCACCACCAGCGGCACCTGGACC GACACCTCCGGCACCGTCCACAACAGCGGCTCCTCCCAGTCCGTCC CCGCACTCGGCGCCTGCGCCAACGCCAACGGCCCCGCCAGCACCG GCAGCCCCGCCAAGCTCTCCGTCGGCGGCCCCTCCCCGTACTGGCA GAGCTACGGCTACGACAGCACCGGCAACCGCACCACCCTCGTCCA GCACGACACCACCGGCAACACCACCAAGGACACCACCACCACCCA GACCTTCGGCCCCGCCGGATCGGTCAACACCGCCACCGGCGCCCC CAACACCGGCGGCGGCACCGGCGGCCCGCACGCCCTGCTCACCAG CAGCACCACCGGACCCACCGGGACCCAGGTCACCAGCTACCAGTA CGACCAGCTCGGCAACACCACCGCGGTCACCGAGACGTCCGGAAC CACCACCCTCGCCTGGAACGGCGAGGACAAGCTCGCCTCCGTCAC CAAGACCGGCCAGGCCCAGGCCACCAGCTACCTCTACGACGCCGA CGGCAACCAGCTCATCCGCCGCAACCCCGGCAAGACCACCCTCAA CCTCGGCAGCGACGAGGTCACCCTCGACACCGCCGCCAACTCCCT CACCGACACCCGCTACTACAGCGCCCCCGGCGGCATCAGCATCGC CCGCACCACCGGACCCACCGGCGCAAGCGCCCTCGCCTACCAGGC CTCCGACCCCCACGGCACCGCCAACGTCCAGATCAACGTCGACGC CGCCCAGACCACCACCCGCCGCCCCACCGACCCCTTCGGCAACCC CCGCGGCACCCAGCCCGCCCCCAACACCTGGGCCGGCGACAAGGG CTTCGTCGGCGGCACCAAGGACGACACCACCGGACTCACCAACCT CGGCGCCCGCGAATACCAACCCACCACCGGCCGCTTCCTCAACCC CGACCCACTCCTCGACGCCGGCAACCCCCAGCAGTGGAACGGCTA CGCCTACAGCGACAACGACCCCGTCAACAGCTCCGACCCCAGCGG ACTCATCACCAACGCCCTGGCCGACGGCGACACCTACGTCGCCCG CCCCGCCGCCTTCTGCGTCACCATGTCGTGCGTCGAGCAGACCAGC GGCCCCGGTTTCTGGGAGGACAAGCGCGTCGGTGACGCCGTCTTC GCCGCCGTCGTCCAGGCCACCACGCAGAGCAACGGCAACGGGTCA TCCCAGACCAAGAAAGAGAAGGGCATCTGGGGCCAGGCCTGGGA CTGGACCAAGAAGAACGGCGGCGCCATCCTCGGAGCGCTGGTAGA GGGAGCGGTCTTCAGCACATGCTTCATCGGAGCTGGATTCGCCGC ACCTGCAACGGGAGGAATCACCGTCATCGCCGGTGCTGCGGCCTG CGGGGCTGTGGCCGGCGAGGCAGGGGCACTGACCACCAATATCCT CACCCCAGATGCCGACCACTCCGTCGACGGCATCACCAACGACAT GGTCGTTGGTGAAATCACCGGGGCGGCTGTCAGCGCAGCGAGCGA GGGCGCAAGCTCCCTCGCCAAGCCGGCGGTCCGCAAACTCCTGGG CATGGAAGCCGAGGAAGGACTCGAGGCAGCAGGCCGCGCCGCCA CCGGACCTTGCAACAGTTTCCCGGCCGGCGTCACCGTCCTCCTCGC CGACGGCACCACCAAGCCCATCGAACAGATCGCCCAGGGCGACCA GGTAACCGCCACCGACCCGCAGACAGGCACCACCCAGGCAGAACC CGTCACCGACACGATCATCGGCCACGACGACACGGAATTCACCGA CCTCACCCTCACCAACGACGCAGACCCCCGCGCCCCGCCCAGCGA GATCACCTCCACCACCCACCACCCCTACTGGAACGCCACCACCAG CCGCTGGACCGATGCCGGCGACCTCAAGCCCGGCGACCACGTCCG CACCCCCGACGGCACCGAACTGACCGTCAACACCGTCTACAGCTA CACCACACAACCCCGGACCGCGCGCAACCTCACCGTCGCAGACCT CCACACGTACTATGTGCTCGCTGGAAATACGCCGGTCCTAGTGCAT AACACCGGCCCGGGATGTGGTGAGCCGGGATTCGTTAGTGACGCT GCTAATTCTCTCTCGGGCAGGCGCATCACCACGGGACAAATATTTG ATGCGAGCGGGAATCCGATCGGGCCTGAGATCACGAGCGGCGGCG GCAGTCTGGCAGATAGGGCGCAGAGTTATCTTGCCGACTCCCCTA ATATTCGAAATCTGCCCGCTAAGGCGAGATATGCGTCGGCTGACC ACGTTGAGGCGCAATATGCAGTGTGGATGCGAGAAAATGGAGTGA CCGACGCCAGTGTGGTCATCAATCAAAACTATGTATGTGGGCTGC CCCTAGGCTGCCAGGCGGCGGTGCCCGCTATCCTCCCTCGCGGCTC GACCATGACGGTATGGTATCCAGGGTCAGGAAGTCCCATCGTATT GCGGGGAGTGGGTTAA[SEQIDNO:141] DddA >ATE59819.1typeIVsecretionproteinRhs[Thauerasp.K11] homologin MRAFRLIACLLAFSAAAAPAAADTSSMLGRLPEASARQLKERLAPRG Thauerasp. LASAAALRQYLDASQRELDTAPEADDVPARSQRFAARAGELTALREQ K11 ARRDLASLEDAAKASGSAEATQRIGRIRGQVDARFDRLEGLFTTWRN PROTEIN APQGSERRQARRELRAALATLRHAGTPAPAAIPVPTLGPLQPAGEPAA NPPAARLPAYAQADDATGDPFTPGGFRLMKVAALPPAVAAEAATDC SATSADLADDGKDVRLTQPIRDLAASLDYSPARILRWTQQNVAFEPY WGALKGAEGVLQTRAGNSTDQASLLIALLRASNIPARYVRGTVQLND TAAQDDAGGRAQRWLGTKRYRASAAVLAGGGTSAGLQSIDGTVRGI RFSHVWVQACVPHGAYRGARAEAGGYRWLALDAAVKDHDYQQGI AVDVPLTDAAFYTPYLAARSDQLPHEHFAQKVAEAARATDANAALA DVPYAGTPRPLRYDVLPGSLPYEVEAFTNWPGLGSSETASLPDAHRH TFTVTVRNGATTLASAALPYPQNAFKRVTLSYQPTAASQAAWNAWT GDLPAAADGSIQVVPQIKADGTVLAAGAPANALPLAGVHNVILKVSQ GERSGAACINDSGNPADPKDTDGTCLNKTVYTNIKAGAYHALGLNA LHTSNAFLGQRLEALAAGVQAYPVAPTPAAGAGYEATVGELLHLVL QDYLHQTEQADQRNAALRGFKSVGPYDLGLTASDLETDYLFDIPVAI KPAGVFVDFKGGLYGFVKLDTTAETAAARAAENVDLAKLSIYSGSAL EHHVWQQALRTDAVSTVRGLQFAAEQGIPLVTFTAANIGQYDSLMQ MSGATSMAAYKSAIQNAVKGSDNGNHGVVTVPRAQIAYADPVDPAS KWTGAVYMSQNPVTGEYGAIINGTIAGGFPLLNSTPFSNLYNFDSFVP NTLLGTNGGAGAVQTLPGGTQGESSWITKAGDPVNMLTGNYTLQAR DFTIKGRGGLPIVLERWFNAQNATDGPFGFGWTHSFNHQLRFYGIESG QSKVGWVDGTGAQRFYAVAAAGSIAPGTTLAAQAGVFTTLSRLADG RFQVRETNGLTYSFESLTSPTTPPAAGSEPRARLLAIADRHGNTLTLNY SGSQLASVSDSLGRTVLSFTWNGNRIGKVKDVSGREVNYAYEDGNG NLTRVTDPLGQATRYSYYTSADGAKLDHALRRHTLPRGNGMEFEYY AGGQVFRHTPFDTSGNLIPESALTFHYNSYRRESWTVDGRGAEERFLF DTHGNVIQQTAANGATHTYAYADPNDPHLRTRMTDPVGRVTQYSYT AEGYLQTLTLPSGAVQAWRDYDAFGQPRRVKDARGNWTLHHYDTA GTRTDSIRVKSGVVPTVGTAPAAANVVSWIKYQGDSVGNLTGVKRL RDWTGATLGNFASGSGPVVTTTFDAARLNVASVGRSGNRNGSQISET SPIFSHDALGRLTGGVDGRWYPVAFDYDVLDRVTRATDATGQPRRY AFDVNGNRIGTELIAGGSRIDSSVAAFDVQDRVAHVLDHAGNRVAYA YDAVGNRVSVESPDGYAIGFDYDLAGRPYSAYDEDGNRVFSAFDVA GRVRAVIDPNGAATLYDYHGDEQDGRLARVEQPAIPGQNAGRAAET DYDAGGLPIRVRQVSAGGEAREGYRFHDELGRVVRSVSAPDDVGQR LQVCYSYDALSNLTQVRAGATTDTTSAACAGSPAVQLTQSWDDFGN LLTRTDALGRVWKFEYDAHGNLVASQTPEQAKVSTRSTYRYDPALH GLLAGRSVPGSGSAGQSVSYARNALGQVIRAETRDGAGNLVVAYDY QYDAAHRVVRIVDSRGGKALDYAWTPAGRLASITLDGHVWRFQYD GVGRLAAIVAPNGATIAMARDAAGRLTERRWPDGAKSAFDWLPEGS LAAIEHSAGGSALAQFAYAYDAWGNRTSATETLAGTSRSLAYGYDA LDRLKTVTTDGATETHAFDLFGNRTSKTTGGVTTDYLFDAAHQLTQV QIAGTPTERLAYDDNGNLRKHCVGSPSGSTSDCTGTTVLSLAWNGLD QLIQAARTGLPAESYAYDDAGRRVTKAVGSSATHFAYDGPDILAEYA SPAGSPTAVYAHGAGIDEPLLRLTGATSTPAASAHHYAQDGLGSIVA AYGEIGASGPVSAASVSATHSYSAGSYPPAKLIDGETTGSTGFWAGSS GNFAADPAVITLELGAEKSVSRVRLHRVASYLPDYVVKDAEVQVRKP DNSWQTVGTLTNNTSEDSPEIVLTGAPGSALRVLVKGVRNGSLVLMA EVTMSADGGAASVATARYDAWGNVTQASGSIPAFGYTGREPDATGL VYYRARYYHPALGRFASRDPLGLAAGINPYAYAGGNPILYNDPDGLL AQLAWNTAASYWGQPIVQETVATIRNGAAVAAGNFVPDTVNGATG WFEQFLHQESGSFGRMDSWVDVRNPVAQDVAQDLRGVAAVGLMM TPLRYGRASNASFNPPVANLPLNTGGKTSGMLHIPGQESLSLTSGIAGP SQVVRGQGLPGFNGNQLTHVEGHAAAYMRTHKVSEAVLDINKAPCT AGSGGGCNGLLPRMLPEGAHLTIRHPNGVQVYIGTPD[SEQIDNO: 142] DddA >CCZ27_07525NZ_CP023439.1:1708666-1716450Thauerasp.K11 homologin chromosome,completegenome Thauerasp. ATGCGTGCCTTCCGCCTGATCGCCTGCCTTCTCGCCTTTTCGGCGG K11 CAGCCGCACCTGCTGCGGCTGACACGTCGTCGATGCTGGGGCGTC DNA TGCCTGAAGCAAGCGCCCGCCAGCTCAAGGAGCGGTTGGCGCCGC GTGGCCTTGCCTCCGCTGCCGCCTTGCGCCAGTACCTGGACGCCTC GCAACGCGAGCTGGACACCGCACCGGAAGCGGACGACGTACCCG CCCGCAGCCAACGCTTTGCCGCAAGGGCGGGCGAACTCACCGCGC TGCGCGAACAGGCGCGCCGGGATCTCGCCAGTCTGGAGGACGCCG CGAAGGCGAGCGGCTCGGCCGAGGCGACGCAGCGCATCGGTCGA ATCCGCGGGCAGGTGGACGCACGCTTCGACCGGCTCGAAGGGCTT TTTACCACTTGGCGCAATGCGCCCCAGGGCAGCGAACGCCGCCAG GCCCGCCGCGAACTGCGTGCCGCGCTCGCCACGCTCCGCCATGCC GGCACCCCGGCTCCGGCTGCGATTCCTGTTCCTACCCTCGGCCCCC TGCAACCGGCCGGCGAGCCGGCTGCCAACCCACCGGCCGCGCGCT TGCCAGCCTATGCGCAAGCGGATGACGCGACTGGCGACCCCTTTA CCCCCGGTGGCTTCCGGCTGATGAAGGTCGCCGCACTGCCGCCGG CGGTCGCGGCCGAGGCGGCAACGGACTGCTCCGCCACCAGCGCCG ACCTGGCCGACGACGGCAAGGACGTGCGCCTGACCCAGCCGATCC GCGACCTCGCGGCATCGCTCGACTACTCACCGGCACGCATCCTGC GCTGGACGCAGCAGAACGTCGCCTTCGAACCCTACTGGGGGGCAC TCAAGGGGGCGGAAGGCGTGCTGCAGACGCGCGCCGGCAACAGC ACCGACCAGGCCAGCCTGCTGATCGCACTCTTGCGGGCCTCCAAC ATTCCCGCCCGCTACGTACGCGGCACCGTGCAGCTCAACGACACT GCCGCGCAGGACGACGCAGGCGGGCGGGCGCAGCGCTGGCTGGG CACCAAGCGCTACCGTGCATCGGCCGCGGTACTCGCCGGCGGCGG AACTTCCGCCGGCCTGCAGTCGATCGACGGCACCGTCCGCGGCAT CCGCTTCAGCCATGTCTGGGTCCAGGCCTGCGTTCCCCATGGCGCT TACCGCGGTGCCCGCGCGGAAGCCGGCGGCTATCGCTGGCTGGCG CTGGACGCGGCGGTGAAGGACCATGACTACCAGCAGGGCATCGCG GTCGATGTGCCGCTCACCGATGCCGCGTTCTACACGCCCTATCTGG CGGCGCGCAGCGACCAGTTGCCGCACGAGCATTTCGCACAGAAGG TGGCGGAGGCGGCGCGTGCGACCGACGCCAATGCGGCGCTGGCCG ACGTGCCCTACGCCGGTACGCCGCGGCCGCTGCGCTACGACGTGC TGCCCGGTTCGCTGCCCTACGAGGTCGAAGCCTTCACCAACTGGCC CGGCCTCGGTTCGTCCGAAACCGCAAGCCTGCCGGACGCACACCG CCACACCTTCACCGTGACGGTCAGGAACGGCGCCACCACGTTGGC GAGCGCCGCGCTGCCCTATCCGCAGAACGCCTTCAAGCGCGTCAC GCTGTCCTATCAGCCGACTGCCGCCTCGCAGGCGGCCTGGAACGC CTGGACGGGCGATCTGCCCGCCGCGGCCGACGGCAGCATCCAGGT CGTGCCGCAGATCAAGGCCGACGGTACCGTGCTCGCCGCAGGTGC GCCCGCCAACGCGCTGCCGCTCGCCGGCGTGCACAACGTCATCCT CAAGGTCTCGCAGGGCGAGCGCAGCGGTGCCGCGTGCATCAACGA CAGCGGCAACCCCGCCGACCCGAAGGACACCGACGGCACCTGCCT CAACAAGACCGTCTACACCAACATCAAGGCCGGCGCCTACCACGC CCTGGGCCTGAATGCGCTGCACACCTCGAATGCCTTCCTCGGCCAG CGGCTCGAAGCGCTGGCGGCCGGCGTGCAGGCCTATCCCGTCGCG CCCACGCCGGCCGCGGGTGCCGGCTACGAGGCCACGGTCGGTGAA TTGCTGCATCTGGTGCTGCAGGACTACCTGCACCAGACCGAGCAG GCCGACCAGCGCAACGCCGCGTTGCGCGGCTTCAAGAGCGTGGGG CCGTACGACCTCGGGCTGACCGCGTCCGACCTCGAAACCGACTAC CTCTTCGACATCCCGGTCGCGATCAAGCCGGCCGGCGTGTTCGTGG ACTTCAAGGGCGGCCTCTACGGTTTCGTCAAACTCGATACCACGGC CGAGACGGCCGCGGCACGCGCCGCCGAAAACGTGGATCTGGCCAA GCTCTCGATCTACTCCGGCTCCGCGCTCGAACACCACGTCTGGCAG CAGGCGCTGCGCACCGATGCGGTGTCCACCGTGCGTGGGCTGCAG TTCGCCGCCGAGCAGGGCATTCCGCTCGTCACCTTCACCGCGGCCA ACATCGGCCAGTACGACAGCCTCATGCAGATGAGCGGCGCCACCA GCATGGCCGCTTACAAGAGCGCGATCCAGAACGCGGTGAAGGGCT CGGACAACGGCAACCACGGCGTCGTCACCGTGCCGCGCGCCCAGA TCGCCTACGCCGACCCCGTCGATCCGGCGAGCAAATGGACCGGCG CGGTCTACATGTCTCAGAACCCCGTCACCGGAGAGTACGGGGCGA TCATCAACGGCACCATCGCCGGCGGCTTCCCGCTGCTCAACAGCA CGCCCTTCAGCAATCTCTACAACTTCGATTCCTTCGTGCCCAACAC CCTCCTTGGCACGAACGGGGGTGCCGGTGCGGTGCAGACCCTGCC CGGCGGCACCCAGGGCGAGAGTTCCTGGATCACCAAGGCCGGCGA CCCGGTGAACATGCTCACCGGCAACTACACGCTGCAGGCACGCGA CTTCACCATCAAGGGCCGGGGCGGACTGCCGATCGTGCTGGAGCG CTGGTTCAACGCGCAGAACGCCACCGACGGGCCGTTCGGCTTCGG CTGGACGCACAGCTTCAACCATCAGTTGCGTTTCTACGGCATCGAG AGCGGCCAGTCCAAGGTCGGCTGGGTGGACGGCACTGGCGCCCAG CGCTTCTACGCCGTGGCCGCCGCCGGCAGCATTGCGCCGGGCACG ACGCTGGCCGCGCAGGCCGGGGTGTTCACGACGCTGTCGCGTCTG GCCGACGGCCGCTTCCAGGTGCGCGAGACCAACGGCCTCACCTAC AGCTTCGAATCGCTCACGAGCCCGACCACCCCGCCGGCCGCGGGC AGCGAACCGCGCGCAAGACTGCTGGCCATCGCCGACCGCCACGGC AACACCCTGACGCTCAACTACAGCGGCAGCCAGCTTGCCTCGGTG AGCGACAGCCTCGGCCGCACGGTGCTCAGCTTCACCTGGAACGGC AACCGCATCGGCAAGGTGAAGGACGTCAGCGGACGGGAAGTGAA CTACGCCTACGAGGACGGCAACGGCAACCTCACGCGCGTCACCGA TCCGCTGGGTCAAGCCACGCGCTACAGCTACTACACCAGTGCCGA CGGTGCCAAGCTCGACCACGCCCTGCGCCGCCACACCCTGCCGCG CGGCAACGGCATGGAGTTCGAGTACTACGCCGGTGGCCAGGTCTT CCGCCACACGCCGTTCGACACCAGCGGCAACCTCATTCCCGAATC GGCGCTGACCTTCCACTACAACAGTTATCGGCGCGAGAGCTGGAC GGTCGATGGCCGCGGTGCCGAGGAGCGCTTCCTGTTCGACACGCA CGGCAACGTGATCCAGCAGACCGCCGCCAACGGTGCCACCCACAC CTACGCGTACGCCGACCCGAACGATCCGCATCTGCGCACGCGCAT GACAGACCCGGTCGGCCGCGTCACCCAGTACAGCTATACCGCCGA AGGCTATCTGCAGACCCTGACGCTGCCGTCGGGCGCCGTGCAGGC GTGGCGCGACTACGACGCCTTCGGCCAGCCCCGCCGCGTCAAGGA CGCGCGCGGCAACTGGACGCTCCACCACTACGACACCGCCGGGAC ACGGACCGACTCCATCCGGGTCAAATCGGGCGTGGTCCCCACCGT CGGCACCGCGCCTGCCGCGGCCAACGTCGTTTCCTGGATCAAGTA CCAGGGCGACAGCGTGGGCAACCTCACCGGCGTCAAGCGCCTGCG CGACTGGACGGGCGCGACCCTGGGCAATTTCGCCAGCGGCAGCGG CCCCGTCGTCACCACCACCTTCGATGCGGCCAGGCTCAACGTCGCC AGCGTCGGCCGTAGCGGCAACCGCAACGGCAGCCAGATCAGCGA GACCAGCCCGATCTTCTCCCACGACGCGCTGGGGCGCCTCACCGG CGGGGTGGACGGGCGCTGGTATCCGGTCGCCTTCGATTACGACGT GCTCGACCGCGTCACCCGCGCCACCGACGCCACGGGCCAGCCGCG CCGCTACGCGTTCGACGTCAACGGCAACCGCATCGGTACGGAGCT GATTGCCGGCGGCAGCCGTATCGATTCCTCGGTGGCCGCCTTCGAC GTGCAGGACCGCGTCGCCCACGTCCTCGATCACGCCGGCAACCGC GTGGCCTACGCCTACGATGCGGTGGGCAACCGGGTGAGCGTGGAA AGCCCCGACGGCTACGCCATCGGCTTCGACTACGACCTCGCCGGA CGGCCCTATTCGGCCTACGACGAAGACGGCAACCGCGTCTTCTCC GCCTTCGACGTGGCCGGGCGCGTGCGAGCGGTCATCGACCCCAAC GGCGCCGCGACGCTCTACGACTATCACGGCGACGAGCAGGACGGG CGTCTCGCGCGCGTGGAGCAGCCCGCCATCCCGGGCCAGAACGCG GGCCGCGCCGCCGAGACCGACTACGATGCGGGTGGGTTGCCCATC CGCGTGCGCCAGGTCTCGGCCGGCGGCGAAGCGCGCGAAGGCTAC CGTTTCCACGACGAGCTTGGCCGCGTGGTGCGCAGCGTCTCCGCGC CGGACGACGTCGGCCAGCGGCTGCAGGTCTGCTACAGCTACGATG CACTCTCGAACCTCACCCAGGTGCGCGCCGGCGCCACCACCGACA CCACCAGTGCCGCCTGCGCCGGCAGCCCCGCGGTGCAGCTCACCC AGAGCTGGGACGACTTTGGCAACCTGCTGACGCGCACCGACGCGC TGGGCCGGGTGTGGAAGTTCGAGTACGACGCCCACGGCAACCTCG TCGCCAGCCAGACGCCCGAGCAGGCCAAGGTCTCGACGCGCAGCA CCTACCGCTACGATCCGGCGCTGCACGGCTTGCTGGCCGGGCGCA GCGTGCCGGGCAGCGGCAGTGCGGGCCAGAGCGTGAGCTATGCGC GCAACGCGCTCGGCCAGGTCATCCGCGCCGAGACGCGCGACGGCG CGGGCAACCTCGTCGTCGCCTACGACTACCAGTACGACGCCGCCC ACCGTGTGGTGCGCATCGTCGACAGCCGCGGCGGCAAGGCGCTCG ACTACGCCTGGACGCCCGCCGGGCGGCTGGCGAGCATTACCCTGG ACGGCCATGTCTGGCGCTTCCAGTACGACGGCGTCGGCCGGCTCG CCGCGATCGTCGCGCCCAACGGCGCCACCATAGCGATGGCACGCG ATGCCGCCGGGCGGCTCACCGAGCGGCGCTGGCCCGACGGCGCGA AGAGCGCCTTCGACTGGCTGCCCGAAGGCAGCCTCGCCGCCATCG AGCACAGCGCGGGCGGCAGCGCGCTCGCACAGTTCGCCTATGCCT ACGATGCCTGGGGCAACCGCACGAGCGCCACCGAGACCCTCGCGG GCACCAGCCGCAGCCTCGCCTACGGCTACGACGCGCTCGACCGCC TGAAGACCGTCACCACCGACGGTGCGACCGAAACCCATGCCTTCG ATCTCTTCGGCAATCGCACCAGCAAGACCACGGGCGGGGTGACCA CCGACTATCTCTTCGACGCGGCGCACCAGCTCACCCAGGTGCAGA TCGCCGGCACCCCCACCGAGCGGCTCGCCTACGACGACAACGGTA ATCTCCGCAAGCACTGCGTCGGCAGTCCGAGTGGCAGCACCAGCG ATTGCACCGGCACCACCGTGCTGAGCCTCGCCTGGAACGGCCTCG ACCAGTTGATCCAGGCCGCCAGGACGGGCCTGCCCGCCGAGTCCT ACGCCTACGACGATGCCGGGCGGCGTGTCACCAAGGCGGTGGGCA GCAGCGCCACCCACTTCGCCTACGACGGTCCCGACATCCTGGCCG AGTACGCCAGCCCGGCCGGCAGCCCCACCGCCGTCTATGCCCACG GTGCCGGCATCGACGAACCGCTGCTGCGCCTCACCGGCGCGACGA GCACGCCGGCCGCTTCCGCGCACCACTACGCGCAGGACGGGCTGG GCAGCATCGTCGCGGCCTATGGCGAGATCGGCGCCAGCGGTCCGG TCAGTGCCGCGAGCGTATCGGCCACCCACAGTTACAGCGCCGGCA GCTACCCGCCGGCAAAGCTGATCGACGGCGAGACGACCGGAAGC ACCGGGTTCTGGGCTGGCAGCTCGGGCAACTTCGCTGCCGATCCA GCCGTGATCACGCTGGAACTGGGTGCGGAGAAAAGCGTGAGCCGC GTGAGGCTGCACCGGGTGGCCAGCTACCTGCCCGACTACGTGGTC AAGGATGCCGAGGTGCAGGTCCGAAAACCGGACAATTCGTGGCAG ACGGTCGGCACGCTGACAAACAACACCAGCGAAGACAGTCCCGA GATCGTGCTCACCGGCGCCCCCGGCAGCGCGCTGCGCGTGCTCGT CAAGGGCGTGCGCAACGGCAGCCTGGTGCTGATGGCCGAGGTGAC GATGAGTGCGGACGGTGGCGCGGCCAGCGTGGCCACCGCCCGCTA CGACGCCTGGGGCAACGTCACGCAGGCGAGCGGCAGCATCCCGGC CTTCGGCTACACCGGACGCGAGCCCGATGCCACGGGCCTGGTCTA CTACCGCGCCCGCTACTACCACCCCGCGCTCGGCCGCTTCGCCAGC CGCGACCCGCTGGGGCTGGCGGCGGGGATCAATCCCTACGCCTAC GCGGGCGGCAATCCCATCCTCTACAACGATCCGGATGGCTTGCTG GCGCAACTGGCGTGGAATACGGCGGCCAGCTACTGGGGACAGCCG ATAGTTCAAGAAACGGTCGCCACGATTCGAAATGGGGCCGCAGTG GCCGCTGGCAACTTCGTTCCAGACACGGTCAACGGTGCAACAGGT TGGTTTGAGCAGTTCCTGCACCAAGAATCGGGCTCGTTCGGGCGC ATGGACTCGTGGGTGGATGTGCGAAACCCCGTTGCGCAGGACGTA GCCCAGGACCTGCGCGGTGTCGCAGCCGTTGGGTTAATGATGACG CCGCTGCGGTATGGTCGTGCCTCCAACGCGTCTTTCAATCCGCCAG TAGCCAATCTTCCGCTCAACACTGGAGGAAAAACATCTGGCATGT TGCACATTCCAGGGCAAGAATCACTGTCGCTCACGAGCGGAATTG CGGGGCCGTCTCAAGTCGTTAGAGGTCAAGGTTTGCCAGGATTCA ACGGTAATCAGTTGACCCATGTGGAAGGTCATGCTGCTGCTTACAT GCGGACTCACAAGGTCTCTGAGGCTGTTCTGGACATAAACAAAGC ACCTTGCACCGCTGGTAGTGGTGGTGGATGTAATGGGTTGCTTCCC CGAATGCTGCCGGAGGGGGCTCATTTAACAATTCGACACCCAAAT GGTGTTCAAGTTTATATTGGCACTCCTGACTAA[SEQIDNO:143] Chondromyces >AKT41505.1typeIVsecretionproteinRhs[Chondromycescrocatus] crocatus MSMSASRSQPAFPFVSASSPRPRRRPPFPRALLLLIAVLLVGACGDAG PROTEIN GPLLWSSSSQALWEPSPIPPLPPLLCLGPGDGPSPFPPDLTQGTTTAAG TLPGSFSVTSTGEATYTIPVPTLPGRAGIEPSLAITYDSAQGEGLLGIGF HLQGLSSVDRCPRNVAQDGHIAPVRDAEDDALCLDGQRLVPVDPQP GRAPREYRTFPDSFTRVEADFAESEGWPAERGPKRLRAHGKAGLIYE YGGESSGRVLAQGEAVRSWLLTRLSDRDGNTMAVVYRNDLHAKGY TVEHAPQRITYTRHPTVPASRMVEFTYGPLEAADVRVHYARGMELRR SLSLRSIQMFGPGHVLARELRFGYGHGPATGRLRLEAVRECAGDGTC KPPTRFTWHTAGAAGYTQQQTLVEVPLSERGTLMTMDVSGDGLDDL VTSDMVVEAGTEEPITRWSVALNRSQELTPGFFEAAVTGQEQPHFIDA EPPYQPELGTPLDYDHDGRMDLFLHDVHGQSMTWEVLLSNGDGRFT RRDTGVPRPFTMGMTPAGLRSPDASTHLVDVDGDGMVDLLQCYLSA HEQLWYLHRWTAAAGGFAPHGDRVHALSSYPCHAELHAVDVDADG RVDLVMQELILVGSQVRAGWQYVAFSYELSDGSWTRALTGLRLTPP GDRVFFLDVNGDGLPDAVQSSRDDEQLYTSMNIGAGFAAPVPSLATP TLGAARFVRFASVLDHNADGRQDLLLAMSDGGSESLPAWKVLQATG EVGPGTFEIVHPGLPMGIVLQQDELPTPDHPLTPRVTDVNGDGAQDLL YAFNNQVHVFENVLGQEDLLAAVTDGMNAHAPEDAEYLPNVQIRYD HLIDRARTTEGFEDAPGIPSPEQRTYRPLEQSDEEPCRYPVRCVVGHR RVVSGYVLNNGADRPRTFQVAYRNGRHHRLGRGFLGFGTRIVRDLD TGAGTAEFYDNVTFDGAFQAFPFRGQVQRSWRWSPSLPLDAHSAEPA SLELLTTRSYAVVIPTQAGTYFTLSLLEGKSRHQGTFSPGSGKTLEEAV RALEGDLASRMSDTLRTVSDFDLYGNILAEQTQTEGVDLDLSVTRSF DNDPLSWRLGELTRETTCSKAGGETQCRVMHRSYDGRGHVRLERVG GEPFDPEMQLDVWFSRDALGNIHSTRSRDGTGQVRASCTSYDALGL MPYAHRNLEGHQSYTRYDPAVGVLRASVDPNGLVSRWAYDGFGRV TLESLPGRMPTVIRRTWTKDGGAAGNAWNLKIRTASVGGQDETVQL DGLGREVRWWWQALDVGEEQAPRMMQEVAFDARGEHLAWRSLPI VDPAPPGSVQVRETWQYDGMGRVLRHVTPWGAATTHEYIGRDEVIT APGQAVTRIASDPLGRPTAVGDPEGGVSRYTYGPFGGLREVTTPAGA VTLTERDAFGRVRRQVSPDRGVSTAHYDGYGQKISSLDAAGRAVTTR YDTLGRIFRQVDEDGVTEFRWDDAQHGVGQLALVVSPDGHRLRYGF DHLGRPATTTLEIGGESFTSRLSYDLSGRLERIEYPSAPGIGSFAIEREY DPHGRLRALKDAGSGAEFWRATAIDAGNRITGERFGGGTATTLRTFD AARERVSRIETQTAGGPVQQLSYLWNDRRKLVERSDGLHANVERFR YDLLDRLTCAQFGLINAALCERPFTYGPDGNLLQKPGVGAYEYDPAQ PHAVVRAGSAFYGYDAVGNQTSRPGATIAYTAFDLPKRIALTSGDTV DFAYDGLQQRVRKTTATQEIASFGEVYERVTDVVTGAVEHRYHVRN DERVVTLVRRSVAQGTRTLHVHVDHLGSIDVLTDGVTGSVAERRSY DAFGAPRHPDWGSGQPPSPHELSSLGFTGHEADLDLGLVNMKGRIYD PKLGRFLTPDPLVPRPLFGQSWNSYSYVLNSPLSLVDPSGFQEQPPATE DGCSQGCTIWVFGPPREPKPPAPPKVVEGNLEDAAGTGSTQAPVDVG TSGVRSGWSPQLPATLQTLGRGDAIARRIMDGVRIGMARMLLESAKL GILGGTSRVYVAYTNLTAAWNGYKESGLPGALDAVNPASQMVQAG VEAYEAAAAEDWEAAGASLFKAGSIGMSILATAVGVGGAITATVGST AGAAGRAAARAPSLPAYAGGKTSGVLRTTAGDTALLSGYKGPSASM PRGTPGMNGRIKSHVEAHAAAVMREQGMKEGTLYINRVPCSGATGC DAMLPRMLPPDAHLRVVGPNGYDQVFVGLPD[SEQIDNO:144] Chondromyces >CMC5_057130NZ_CP012159.1:7808731-7815414Chondromyces crocatus crocatusstrainCmc5,completegenome DNA ATGTCCATGTCGGCCTCACGGAGTCAGCCCGCATTCCCCTTCGTGT CGGCCTCCTCTCCGCGTCCGCGCCGGCGCCCTCCCTTTCCCCGAGC GCTGCTCCTCCTCATCGCCGTGCTCCTCGTCGGCGCATGCGGCGAC GCTGGCGGCCCGCTTCTCTGGTCGAGCAGCTCCCAGGCCCTCTGGG AACCCTCCCCGATCCCGCCGCTCCCCCCGCTCCTGTGCCTCGGCCC CGGCGACGGTCCCTCCCCCTTTCCGCCTGACCTTACGCAGGGGACC ACCACCGCGGCGGGGACCCTGCCAGGGAGCTTTTCGGTCACGAGC ACGGGCGAGGCGACGTACACGATCCCGGTCCCCACGCTGCCTGGC CGTGCCGGCATCGAGCCCTCGCTGGCGATCACCTACGACAGTGCG CAGGGTGAAGGGCTGCTCGGGATCGGCTTCCACTTGCAGGGCCTC TCGTCGGTCGATCGCTGCCCCCGGAACGTCGCGCAGGATGGTCAC ATCGCGCCGGTCCGGGATGCCGAGGACGACGCCTTGTGCCTCGAT GGGCAGCGGCTCGTCCCCGTGGACCCGCAGCCAGGGCGTGCGCCG CGGGAATACCGCACGTTCCCGGACAGCTTCACGCGCGTCGAGGCC GACTTCGCGGAGAGCGAGGGGTGGCCGGCGGAGCGTGGGCCGAA GCGGCTGCGGGCGCATGGCAAAGCGGGGCTGATCTACGAATACGG TGGAGAATCATCGGGCCGGGTGCTCGCGCAAGGGGAGGCGGTGCG GTCCTGGTTGCTGACGCGGCTCAGCGACCGGGATGGCAACACGAT GGCGGTGGTCTACCGGAATGACCTCCACGCGAAGGGCTACACCGT CGAGCACGCGCCGCAGCGGATCACCTACACCAGGCACCCGACTGT GCCGGCCTCGCGCATGGTGGAGTTCACGTACGGGCCGCTGGAGGC GGCGGACGTGCGCGTACACTATGCCCGCGGGATGGAGCTGCGCCG CTCGCTGAGCTTGCGCTCGATCCAGATGTTCGGGCCGGGACACGT GCTCGCGAGGGAGCTGCGCTTCGGTTACGGGCATGGGCCGGCGAC GGGTCGCTTGCGACTGGAGGCGGTTCGGGAGTGCGCAGGTGACGG GACGTGCAAGCCGCCGACACGCTTCACCTGGCACACGGCCGGAGC GGCTGGATACACGCAGCAGCAGACACTGGTGGAGGTGCCGCTGTC GGAGCGCGGCACGTTGATGACGATGGACGTCAGCGGCGATGGCCT CGACGACCTGGTGACGTCCGACATGGTGGTGGAGGCCGGCACGGA AGAGCCGATCACCCGCTGGTCGGTCGCGCTCAACCGGAGCCAGGA GCTGACGCCGGGGTTCTTCGAGGCGGCCGTCACTGGGCAGGAGCA GCCGCATTTCATCGACGCAGAGCCGCCGTACCAGCCGGAGCTGGG GACGCCGCTCGACTACGACCACGATGGCCGGATGGACCTGTTTCT GCACGATGTGCACGGGCAGTCGATGACGTGGGAGGTGCTGCTGTC GAATGGAGATGGGCGGTTCACGCGGCGGGATACGGGGGTGCCGC GGCCGTTCACGATGGGCATGACGCCGGCGGGATTGCGCAGCCCGG ATGCGTCGACCCATCTGGTGGATGTTGACGGTGACGGGATGGTGG ACCTGCTGCAGTGCTACCTGAGCGCGCACGAGCAGCTCTGGTACTT GCACCGCTGGACGGCAGCGGCGGGGGGCTTCGCGCCGCACGGCGA TCGGGTGCATGCGCTGAGCTCCTACCCGTGCCACGCCGAGCTGCA CGCGGTCGATGTCGACGCGGATGGGCGGGTGGACCTGGTGATGCA GGAGCTGATCCTCGTCGGGAGCCAGGTGCGGGCGGGGTGGCAGTA CGTGGCGTTCTCGTACGAGCTGTCCGATGGATCGTGGACGCGCGC GCTGACGGGGCTGCGGCTCACGCCGCCTGGGGACCGGGTGTTCTT CCTCGACGTCAACGGCGATGGGCTGCCCGATGCGGTGCAGAGCAG CCGGGACGATGAGCAGCTGTACACGTCGATGAATATCGGCGCGGG ATTCGCGGCGCCGGTACCGAGCCTGGCGACGCCGACGCTCGGGGC TGCGAGGTTCGTTCGGTTTGCGTCGGTGCTCGATCACAACGCGGAT GGGCGACAAGACCTGCTGCTGGCCATGAGCGATGGGGGATCGGAG TCGCTGCCCGCGTGGAAGGTGCTCCAGGCGACGGGGGAGGTCGGT CCGGGGACGTTCGAGATCGTCCATCCCGGGCTGCCGATGGGCATC GTGCTCCAGCAGGACGAGCTGCCCACGCCCGACCATCCGCTCACG CCGCGGGTCACTGACGTGAATGGGGATGGGGCGCAGGATCTGCTC TATGCGTTCAACAACCAGGTCCATGTGTTCGAGAACGTGCTCGGCC AGGAGGACCTGCTCGCGGCCGTGACCGACGGCATGAATGCGCACG CTCCGGAGGACGCCGAGTACCTGCCCAACGTGCAGATCCGGTACG ACCACCTGATCGATCGTGCGCGGACGACGGAGGGCTTCGAGGATG CTCCAGGGATCCCGTCACCCGAGCAGCGCACCTACCGGCCTCTGG AGCAAAGCGATGAGGAGCCCTGCCGCTATCCGGTGCGGTGCGTGG TCGGGCATCGGCGGGTGGTGAGCGGCTATGTGCTCAACAATGGCG CGGATCGGCCGCGCACCTTCCAGGTGGCCTACCGCAATGGCCGTC ACCATCGCCTGGGCCGAGGGTTTCTGGGGTTCGGGACGCGGATCG TGCGTGACCTCGATACCGGCGCGGGGACGGCCGAGTTCTACGACA ACGTCACGTTTGATGGCGCCTTCCAGGCCTTCCCTTTCCGAGGGCA GGTACAGCGCTCGTGGCGCTGGAGTCCGAGCTTGCCGCTGGACGC GCATAGCGCGGAGCCGGCGTCCCTCGAGCTGCTGACGACGCGGAG CTACGCGGTGGTGATCCCCACGCAAGCGGGGACGTACTTCACCCT CTCGCTGCTGGAGGGCAAGAGCCGTCATCAGGGCACGTTCTCACC GGGGAGTGGGAAAACGCTCGAAGAAGCCGTGCGCGCTCTGGAAG GAGATCTCGCCTCGCGAATGAGCGACACGCTCCGCACCGTCAGCG ACTTCGACCTCTACGGGAACATCCTCGCCGAGCAAACGCAGACGG AGGGCGTCGACCTCGACCTCTCGGTGACGCGCAGCTTCGACAACG ACCCGCTCTCCTGGCGCCTTGGCGAGCTGACGCGAGAGACGACGT GCAGCAAAGCGGGCGGTGAGACGCAGTGCCGGGTGATGCACCGG AGCTATGACGGGCGCGGCCACGTTCGCCTGGAGCGCGTCGGGGGA GAGCCCTTCGACCCGGAGATGCAGCTCGATGTCTGGTTCTCGCGG GACGCGCTGGGCAACATCCACAGCACCCGGTCACGTGATGGGACG GGGCAGGTGCGCGCGAGCTGCACCAGCTACGACGCGCTGGGCTTG ATGCCTTATGCCCACCGCAACCTGGAGGGCCACCAGAGCTATACG CGCTACGACCCGGCCGTGGGCGTGCTGCGGGCGTCGGTGGATCCC AACGGCCTGGTGAGCCGCTGGGCCTACGATGGCTTCGGGCGGGTG ACGCTGGAGAGCCTCCCCGGGCGCATGCCCACCGTCATCCGGCGG ACCTGGACGAAGGACGGCGGAGCGGCTGGCAACGCCTGGAACCT GAAGATCCGCACCGCCTCGGTGGGGGGCCAGGACGAGACCGTGCA GCTCGATGGTCTCGGGCGGGAGGTGCGCTGGTGGTGGCAAGCGCT CGACGTGGGGGAAGAGCAAGCGCCGCGGATGATGCAGGAGGTCG CCTTCGATGCGCGGGGCGAGCACCTCGCGTGGCGCTCGCTGCCGA TCGTGGATCCCGCGCCACCAGGCTCGGTGCAGGTGCGAGAGACGT GGCAATACGACGGGATGGGGGGGGTGCTCCGGCACGTCACGCCGT GGGGGGCGGCGACGACGCACGAGTACATCGGGCGGGACGAGGTC ATCACCGCGCCTGGGCAGGCCGTCACCCGAATCGCCAGCGATCCG CTCGGGAGGCCCACGGCAGTGGGTGATCCCGAAGGTGGCGTCAGC CGGTACACCTACGGTCCCTTCGGGGGGCTGCGCGAGGTGACCACG CCCGCTGGTGCCGTGACGCTGACCGAGCGGGATGCGTTTGGCCGC GTGCGACGGCAGGTGAGCCCGGACCGGGGAGTCTCTACTGCGCAC TACGACGGTTACGGGCAGAAGATCTCATCGCTCGACGCGGCAGGA CGCGCGGTCACGACCCGCTACGACACGCTGGGTCGGATTTTCAGG CAGGTCGACGAAGACGGCGTCACCGAGTTCCGTTGGGATGACGCG CAGCATGGAGTGGGTCAGCTCGCGCTGGTGGTCAGCCCCGATGGG CATCGGCTGCGCTACGGCTTCGACCACCTCGGGCGACCAGCGACG ACGACGCTGGAGATCGGAGGGGAAAGCTTCACCAGCCGGCTGTCT TATGATCTGAGCGGCCGGCTCGAGCGGATCGAGTACCCGAGCGCG CCGGGGATTGGCAGCTTCGCCATCGAGCGGGAGTACGATCCTCAC GGGCGGCTGCGGGCGCTGAAGGATGCGGGGTCGGGGGCGGAGTT CTGGCGAGCCACCGCGATCGATGCGGGGAATCGCATCACGGGGGA GCGCTTCGGTGGGGGGACCGCCACCACGCTCCGCACGTTCGACGC GGCACGGGAGCGGGTGAGTCGGATCGAGACGCAGACGGCAGGTG GGCCCGTCCAGCAGCTCTCCTACCTCTGGAACGATCGCCGCAAGCT CGTCGAGCGCTCCGATGGCCTCCACGCCAACGTCGAGCGCTTTCGT TACGACCTGCTGGACCGGCTGACGTGCGCGCAGTTCGGGCTGATC AATGCTGCCCTCTGCGAGCGACCGTTCACCTACGGACCCGACGGC AACCTGCTCCAGAAGCCCGGCGTCGGTGCCTACGAGTACGACCCC GCGCAGCCCCACGCCGTCGTCCGAGCTGGTAGCGCGTTCTACGGC TACGACGCCGTCGGCAACCAGACCTCACGACCCGGCGCGACCATC GCCTACACCGCGTTCGACCTACCGAAGCGAATCGCGCTCACCAGC GGCGACACCGTCGACTTCGCGTACGACGGCCTCCAGCAGCGGGTG CGCAAGACCACGGCGACGCAGGAGATCGCCTCCTTCGGCGAGGTG TACGAGCGCGTGACCGATGTCGTCACGGGAGCCGTCGAGCATCGC TACCACGTGCGCAACGACGAGCGCGTCGTCACGCTGGTGCGGCGC TCGGTCGCGCAAGGCACGCGCACGCTGCATGTCCATGTCGACCAC CTCGGGTCGATCGATGTGCTCACCGACGGTGTGACCGGCAGCGTC GCCGAGCGCCGCAGCTACGATGCCTTCGGCGCACCGCGCCATCCC GACTGGGGTTCGGGTCAGCCTCCGTCACCCCACGAGCTGTCGTCGC TTGGCTTCACCGGGCACGAGGCCGACCTCGACCTCGGCCTCGTGA ACATGAAGGGGCGCATCTACGACCCCAAGCTCGGACGGTTCCTCA CGCCCGATCCGCTCGTGCCGCGGCCTCTCTTCGGGCAGAGCTGGA ATAGCTATTCGTACGTGCTAAACAGCCCGCTGTCGCTGGTCGATCC CAGTGGGTTTCAAGAGCAGCCACCTGCGACAGAGGACGGATGCTC GCAGGGCTGCACCATCTGGGTGTTCGGTCCTCCCCGCGAGCCGAA GCCACCTGCGCCGCCCAAGGTCGTCGAGGGCAACCTGGAGGACGC CGCTGGCACTGGTTCGACCCAGGCGCCGGTCGATGTCGGGACCTC CGGGGTCCGTAGCGGATGGAGTCCGCAGCTCCCGGCCACGTTGCA GACCTTGGGCCGTGGTGACGCCATCGCCAGGCGCATCATGGACGG CGTCCGCATCGGGATGGCCAGGATGCTGCTGGAGTCCGCAAAGCT CGGCATCCTGGGCGGCACCAGCCGCGTCTACGTCGCCTACACCAA CCTCACCGCCGCCTGGAATGGCTACAAAGAGAGCGGGCTCCCCGG CGCTCTCGACGCCGTCAATCCCGCCAGCCAGATGGTCCAAGCCGG CGTGGAGGCCTACGAGGCTGCCGCCGCAGAGGACTGGGAGGCCGC CGGCGCCAGCTTGTTCAAGGCCGGGTCGATCGGGATGTCGATCCT GGCGACGGCTGTTGGCGTCGGGGGAGCGATCACTGCGACAGTGGG CTCGACGGCAGGAGCGGCGGGGAGGGCAGCCGCAAGAGCCCCCT CACTCCCTGCATATGCTGGCGGAAAAACGTCGGGAGTACTACGGA CCACCGCAGGCGATACAGCACTGCTGAGCGGCTACAAGGGGCCGT CCGCATCGATGCCTCGAGGAACGCCAGGCATGAACGGACGCATCA AGTCGCATGTAGAAGCTCATGCGGCTGCCGTGATGCGAGAGCAAG GGATGAAGGAAGGAACCCTGTACATCAATCGAGTCCCCTGCTCTG GCGCCACCGGATGCGACGCGATGCTCCCAAGAATGCTCCCACCAG ATGCACACCTTCGCGTGGTCGGTCCGAATGGTTACGATCAAGTTTT TGTCGGGCTGCCCGACTGA[SEQIDNO:145]
[0391] In addition, the disclosure contemplates the use any variant of any DddA amino acid sequence, including:
TABLE-US-00023 Sequence(relativetoDddAtoxorwildtypeof SEQID Mutation(s) SEQIDNO:338) NO: DddA GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKV 338 (residues1290- FSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHN 1427)or NPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGAT (DddAtox) GETKVFTGNSNSPKSPTKGGC T1380I GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKV 377 FSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHN NPEGTCGFCVNMIETLLPENAKMTVVPPEGAIPVKRGAT GETKVFTGNSNSPKSPTKGGC T1314A/T1380I GSYALGPYQISAPQLPAYNGQTVGAFYYVNDAGGLESK 378 VFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFH NNPEGTCGFCVNMIETLLPENAKMTVVPPEGAIPVKRGA TGETKVFTGNSNSPKSPTKGGC Q1310R/ GSYALGPYQISAPQLPAYNGRTVGTFYYVNDAGGLESKV 379 S1330I/ FISGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHN T1380I NPEGTCGFCVNMIETLLPENAKMTVVPPEGAIPVKRGAT GETKVFTGNSNSPKSPTKGGC T1380I/T1413I GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKV 380 FSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHN NPEGTCGFCVNMIETLLPENAKMTVVPPEGAIPVKRGAT GETKVFIGNSNSPKSPTKGGC T1314A/T1380I/ GSYALGPYQISAPQLPAYNGQTVGAFYYVNDAGGLESK 381 E1396K VFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFH NNPEGTCGFCVNMIETLLPENAKMTVVPPKGAIPVKRGA TGETKVFTGNSNSPKSPTKGGC
mitoTALEs and mitoZFs
[0392] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may include a mitoTALE as the pDNAbp component.
[0393] MitoTALEs and mitoZFP are known in the art. Each of the proteins may comprise a mitochondrial targeting sequence (MTS) in order to facilitate the translocation of the protein into the mitochondria.
[0394] In one aspect, the methods and compositions described herein involve a TALE protein programmed (e.g., engineered through manipulation of the localization signal in the C-terminus) to localize to the mitochondria (mitoTALE). In some embodiments, the localization signal comprises a sequence to target SOD2. In some embodiments, the LS comprises SEQ ID NO.: 13. In some embodiments, the LS comprises a sequence to target Cox8a. In some embodiments, the LS comprises SEQ ID NO.: 14. In some embodiments, the LS comprises a sequence with 75% or greater percent identity (e.g., 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 99.9% or greater percent identity) to SEQ ID NOs.: 13 or 14.
[0395] The mitoTALE is also used to guide the fusion protein to the appropriate target nucleotide in the mtDNA. By using the RVD in the mitoTALE specific sequences can be targeted, which will place the attached DddA proximal to the target nucleotide. As used herein, proximal or proximally with respect to a target nucleotide shall mean a range of nucleic acids which are arranged consecutively upstream or downstream of the target nucleotide, on either the strand containing the target nucleotide or the strand complementary to the strand containing the target nucleotide, which when targeted and bound by a mitoTALE allow for the dimerization or re-assembly of portions of a DddA to regain, at least partially, the native activity of a full length DddA. Accordingly, the sequence should be selected from a range of nucleotides at or near the target nucleotide, or the nucleotide complementary thereto. In some embodiments, the target nucleic acid sequence is located upstream of the target nucleotide. In some embodiments, the target nucleic acid sequence is between 1 and 40 nucleotides upstream of the target nucleotide. In some embodiments, the target nucleic acid sequence is between 5 and 20 nucleotides upstream of the target nucleotide.
[0396] In some embodiments, a second mitoTALE is used. A second mitoTALE can be used to deliver additional components (e.g., additional DddA, a second portion of a DddA, additional enzymes). In some embodiments, the second mitoTALE is configured to bind a second target nucleic acid sequence. In some embodiments, the second mitoTALE is configured to bind a second target nucleic acid sequence on the nucleic acid strand complementary to the strand containing the target nucleotide. In some embodiments, the second mitoTALE is configured to bind a second target nucleic acid sequence upstream of the nucleotide complementary to the target nucleotide, which complementary nucleotide is on the nucleic acid strand complementary to the strand containing the target nucleotide. In some embodiments, the second target nucleic acid sequence is between 1 and 40 nucleotides upstream of the nucleotide complementarty to the target nucleotide, which is on the strand complementary to the strand containing the target nucleotide. In some embodiments, the second target nucleic acid sequence is between 5 and 20 nucleotides upstream of the nucleotide complementarty to the target nucleotide, which is on the strand complementary to the strand containing the target nucleotide.
[0397] In some embodiments, a mitoTALE comprises an amino acid sequence selected from any one of the following amino acid sequences, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the following mitoTALE sequences:
TABLE-US-00024 SEQID NO. Sequence Description 1 MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDY Mito24 AGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHG FTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGAR ALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGA PLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNG QTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALEMRDNG ISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG 2 MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDY Mito24a AGYPYDVPDYATNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPES DILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS GGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVH TAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSMDIAD LRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAAL GTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP LQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNN GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPV LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPQQVVAIASNGGGRPALESIVAQLSRPDPALAALINDHLVALACLGGR PALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLE SKVFSSGGPTPYPNYANAGHVEGQSALEMRDNGISEGLVFHNNPEGTCG FCVNMTETLLPENAKMTVVPPEG 3 MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDY Mito24b AGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHG FTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGAR ALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGA PLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNG QTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALEMRDNG ISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSINLSDI IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM LLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKET GKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML 4 MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDY Mito24c AGYPYDVPDYATNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPES DILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS MDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGE LRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVA IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ ALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGG KQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC QAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALINDHLVALA CLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGIFYYVND AGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNP EGTCGFCVNMTETLLPENAKMTVVPPEG 5 MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDY Mito24d AGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHG FTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGAR ALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGA PLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNG QTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALEMRDNG ISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDI IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM LLTSDAPEYKPWALVIQDSNGENKIKML 6 MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDD Mito28 DDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTV AGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVK KGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGC 7 MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDD Mito28a DDKTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAY DESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTN LSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTD ENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSMDIADLRTLGYSQ QQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQ DMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQL LKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLT PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPV LCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVA QLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATG ETKVFTGNSNSPKSPTKGGC 8 MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDD Mito28b DDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTV AGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALINDHLVALACLGGRPALDAVK KGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEK ETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQ LVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPE YKPWALVIQDSNGENKIKML 9 MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDD Mito28c DDKTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAY DESTDENVMLLISDAPEYKPWALVIQDSNGENKIKMLSGGSMDIADLRT LGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQL DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ ALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQ QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPAL ESIVAQLSRPDPALAALINDHLVALACLGGRPALDAVKKGLGGSAIPVK RGATGETKVFTGNSNSPKSPTKGGC 10 MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDD Mito28d DDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTV AGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALINDHLVALACLGGRPALDAVK KGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEK ETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKML 11 DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQH Modified PAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL m.13513- RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAI RightTALE ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLP VLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLV ALACLGGRPALDAVKKGLG 12 DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQH m.8490- PAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL RightTALE RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLS RPDPALAALTNDHLVALACLGGRPALDAVKKGLG 13 MLSRAVCGTSRQLAPVLGYLGSRQKHSLPD SOD2MLS 14 MSVLTPLLLRGLTGSARRLPVPRAKIHSL COX8aMLS 15 ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATG SOD23UTR TCCTGTCTTCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTA TAAGGTCCTGGATAATTTTTGTTTGATTATTCATTGAAGAAACATTTAT TTTCCAATTGTGTGAAGTTTTTGACTGTTAATAAAAGAATCTGTCAACC ATCAAAAAAAAAAAAAAA 16 ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATG ATP5b3UTR TCCTGTCTTCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTA TAAGGTCCTGGATAATTTTTGTTTGATTATTCATTGAAGAAACATTTAT TTTCCAATTGTGTGAAGTTTTTGACTGTTAATAAAAGAATCTGTCAACC ATCAAAAAAAAAAAAAAA
[0398] In addition, the mitoTALE and/or mitoZFP may comprising one of the following mitochondrial targeting sequences which help promote mitochondrial localization, or an amino acid or nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the following sequences:
TABLE-US-00025 13 MLSRAVCGTSRQLAPVLGYLGSRQKHSLPD SOD2 (mitochondrial superoxide dismutase) MTS 14 MSVLTPLLLRGLIGSARRLPVPRAKIHSL COX8a (mitochondrial cytochrome Coxidase subunit8A) MTS 15 ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCT SOD23UTR GTCTTCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCC TGGATAATTTTTGTTTGATTATTCATTGAAGAAACATTTATTTTCCAATTGTG TGAAGTTTTTGACTGTTAATAAAAGAATCTGTCAACCATCAAAAAAAAAAAAA AA 16 ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCT ATP5b3UTR GTCTTCTAAGATGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCC TGGATAATTTTTGTTTGATTATTCATTGAAGAAACATTTATTTTCCAATTGTG TGAAGTTTTTGACTGTTAATAAAAGAATCTGTCAACCATCAAAAAAAAAAAAA AA
[0399] In various embodiments, the mtDNA base editors may comprises a mitoZF. A mitoZF may be a ZF protein comprising one or more mitochondrial localization sequences (MLS). A zinc finger is a small, functional, independently folded domain that coordinates one or more zinc ions to stabilize its structure through cysteine and/or histidine residues. Zinc fingers are structurally diverse and exhibit a wide range of functions, from DNA- or RNA-binding to protein-protein interactions and membrane association. There are more than 40 types of zinc fingers annotated in UniProtKB. The most frequent are the C21H2-type, the CCHC-type, the PHD-type and the RING-type. Examples include Accession Nos. Q7Z42, P55197, Q9P2R3, Q9P2G1, Q9P2S6, Q81UH5, P19811, Q92793, P36406, 095081, and Q9ULV3, some of which have the following sequences:
Zinc Finger Protein: Q7Z142-1:
[0400] MPDFTIIQPD RKFDAAAVAG IFVRSSTSSS FPSASSYIAA KKRKNVDNTS TRKPYSYKDR KRKNTEEIRN IKKKLFMDLG IVRTNCGIDN EKQDREKAMK RKVTETIVTT YCELCEQNFS SSKMLLLHRG KVHNTPYIEC HLCMKLFSQT IQFNRHMKTH YGPNAKIYVQ CELCDRQFKD KQSLRTHWDV SHGSGDNQAV LA (SEQ ID NO: 414), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc finger protein: P55197-4 (isoform-4):
[0401] MVSSDRPVSL EDEVSHSMKE MIGGCCVCSD ERGWAENPLV YCDGHGCSVA VHQACYGIVQ VPTGPWFCRK CESQERAARV RCELCPHKDG ALKRTDNGGW AHVVCALYIP EVQFANVSTM EPIVLQSVPH DRYNKTCYIC DEQGRESKAA TGACMTCNKH GCRQAFHVTC AQFAGLLCEE EGNGADNVQY CGYCKYHFSK LKKSKRGSNR SYDQSLSDSS SHSQDKHHEK EKKKYKEKDK HKQKHKKQPE PSPALVPSLT VTTEKTYTST SNNSISGSLK RLEDTTARFT NANFQEVSAH TSSGKDVSET RGSEGKGKKS SAHSSGQRGR KPGGGRNPGT TVSAASPFPQ GSFSGTPGSV KSSSGSSVQS PQDFLSFTDS DLRNDSYSHS QQSSATKDVH KGESGSQEGG VNSFSTLIGL PSTSAVTSQP KSFENSPGDL GNSSLPTAGY KRAQTSGIEE ETVKEKKRKG NKQSKHGPGR PKGNKNQENV SHLSVSSASP TSSVASAAGS ITSSSLQKSP TLLRNGSLQS LSVGSSPVGS EISMQYRHDG ACPTTTFSEL LNAIHNGIYN SNDVAVSFPN VVSGSGSSTP VSSSHLPQQS SGHLQQVGAL SPSAVSSAAP AVATTQANTL SGSSLSQAPS HMYGNRSNSS MAALIAQSEN NQTDQDLGDN SRNLVGRGSS PRGSLSPRSP VSSLQIRYDQ PGNSSLENLP PVAASIEQLL ERQWSEGQQF LLEQGTPSDI LGMLKSLHQL QVENRRLEEQ IKNLTAKKER LQLLNAQLSV PFPTITANPS PSHQIHTFSA QTAPTTDSLN SSKSPHIGNS FLPDNSLPVL NQDLTSSGQS TSSSSALSTP PPAGQSPAQQ GSGVSGVQQV NGVTVGALAS GMQPVTSTIP AVSAVGGIIG ALPGNQLAIN GIVGALNGVM QTPVTMSQNP TPLTHTTVPP NATHPMPATL TNSASGLGLL SDQQRQILIH QQQFQQLLNS QQLTPEQHQA FLYQLMQHHH QQHHQPELQQ LQIPGPTQIP INNLLAGTQA PPLHTATTNP FLTIHGDNAS QKVARLSDKT GPVAQEKS (SEQ ID NO: 415), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc Finger Protein: Q9P2R3-1 (Isoform 1):
[0402] MAEEEVAKLE KHLMLLRQEY VKLQKKLAET EKRCALLAAQ ANKESSSESF ISRLLAIVAD LYEQEQYSDL KIKVGDRHIS AHKFVLAARS DSWSLANLSS TKELDLSDAN PEVTMTMLRW IYTDELEFRE DDVFLTELMK LANRFQLQLL RERCEKGVMS LVNVRNCIRF YQTAEELNAS TLMNYCAEII ASHWDDLRKE DFSSMSAQLL YKMIKSKTEY PLHKAIKVER EDVVFLYLIE MDSQLPGKLN EADHNGDLAL DLALSRRLES IATTLVSHKA DVDMVDKSGW SLLHKGIQRG DLFAATFLIK NGAFVNAATL GAQETPLHLV ALYSSKKHSA DVMSEMAQIA EALLQAGANP NMQDSKGRTP LHVSIMAGNE YVFSQLLQCK QLDLELKDHE GSTALWLAVQ HITVSSDQSV NPFEDVPVVN GTSFDENSFA ARLIQRGSHT DAPDTATGNC LLQRAAGAGN EAAALFLATN GAHVNHRNKW GETPLHTACR HGLANLTAEL LQQGANPNLQ TEEALPLPKE AASLTSLADS VHLQTPLHMA IAYNHPDVVS VILEQKANAL HATNNLQIIP DFSLKDSRDQ TVLGLALWTG MHTIAAQLLG SGAAINDTMS DGQTLLHMAI QRQDSKSALF LLEHQADINV RTQDGETALQ LAIRNQLPLV VDAICTRGAD MSVPDEKGNP PLWLALANNL EDIASTLVRH GCDATCWGPG PGGCLQTLLH RAIDENNEPT ACFLIRSGCD VNSPRQPGAN GEGEEEARDG QTPLHLAASW GLEETVQCLL EFGANVNAQD AEGRTPIHVA ISSQHGVIIQ LLVSHPDIHL NVRDRQGLTP FACAMTFKNN KSAEAILKRE SGAAEQVDNK GRNFLHVAVQ NSDIESVLFL ISVHANVNSR VQDASKLTPL HLAVQAGSEI IVRNLLLAGA KVNELTKHRQ TALHLAAQQD LPTICSVLLE NGVDFAAVDE NGNNALHLAV MHGRLNNIRV LLTECTVDAE AFNLRGQSPL HILGQYGKEN AAAIFDLFLE CMPGYPLDKP DADGSTVLLL AYMKGNANLC RAIVRSGARL GVNNNQGVNI FNYQVATKQL LFRLLDMLSK EPPWCDGSYC YECTARFGVT TRKHHCRHCG RLLCHKCSTK EIPIIKFDLN KPVRVCNICF DVLTLGGVS (SEQ ID NO: 416), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc Finger Protein: Q9P2G1-1:
[0403] MGNTTTKFRK ALINGDENLA CQIYENNPQL KESLDPNTSY GEPYQHNTPL HYAARHGMNK ILGTFLGRDG NPNKRNVHNE TSMHLLCMGP QIMISEGALH PRLARPTEDD FRRADCLQMI LKWKGAKLDQ GEYERAAIDA VDNKKNTPLH YAAASGMKAC VELLVKHGGD LFAENENKDT PCDCAEKQHH KDLALNLESQ MVFSRDPEAE EIEAEYAALD KREPYEGLRP QDLRRLKDML IVETADMLQA PLFTAEALLR AHDWDREKLL EAWMSNPENC CQRSGVQMPT PPPSGYNAWD TLPSPRTPRT TRSSVTSPDE ISLSPGDLDT SLCDICMCSI SVFEDPVDMP CGHDFCRGCW ESFLNLKIQE GEAHNIFCPA YDCFQLVPVD IIESVVSKEM DKRYLQFDIK AFVENNPAIK WCPTPGCDRA VRLTKQGSNT SGSDTLSFPL LRAPAVDCGK GHLFCWECLG EAHEPCDCQT WKNWLQKITE MKPEELVGVS EAYEDAANCL WLLTNSKPCA NCKSPIQKNE GCNHMQCAKC KYDFCWICLE EWKKHSSSTG GYYRCTRYEV IQHVEEQSKE MTVEAEKKHK RFQELDRFMH YYTRFKNHEH SYQLEQRLLK TAKEKMEQLS RALKETEGGC PDTTFIEDAV HVLLKTRRIL KCSYPYGFFL EPKSTKKEIF ELMQTDLEMV TEDLAQKVNR PYLRTPRHKI IKAACLVQQK RQEFLASVAR GVAPADSPEA PRRSFAGGTW DWEYLGFASP EEYAEFQYRR RHRQRRRGDV HSLLSNPPDP DEPSESTLDI PEGGSSSRRP GTSVVSSASM SVLHSSSLRD YTPASRSENQ DSLQALSSLD EDDPNILLAI QLSLQESGLA LDEETRDFLS NEASLGAIGT SLPSRLDSVP RNTDSPRAAL SSSELLELGD SLMRLGAEND PFSTDTLSSH PLSEARSDFC PSSSDPDSAG QDPNINDNLL GNIMAWFHDM NPQSIALIPP ATTEISADSQ LPCIKDGSEG VKDVELVLPE DSMFEDASVS EGRGTQIEEN PLEENILAGE AASQAGDSGN EAANRGDGSD VSSQTPQTSS DWLEQVHLV (SEQ ID NO: 417), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc Finger Protein: Q8IUH5-1 (Isoform 1):
[0404] MGNTTTKFRK ALINGDENLA CQIYENNPQL KESLDPNTSY GEPYQHNTPL HYAARHGMNK ILGTFLGRDG NPNKRNVHNE TSMHLLCMGP QIMISEGALH PRLARPTEDD FRRADCLQMI LKWKGAKLDQ GEYERAAIDA VDNKKNTPLH YAAASGMKAC VELLVKHGGD LFAENENKDT PCDCAEKQHH KDLALNLESQ MVFSRDPEAE EIEAEYAALD KREPYEGLRP QDLRRLKDML IVETADMLQA PLFTAEALLR AHDWDREKLL EAWMSNPENC CQRSGVQMPT PPPSGYNAWD TLPSPRTPRT TRSSVTSPDE ISLSPGDLDT SLCDICMCSI SVFEDPVDMP CGHDFCRGCW ESFLNLKIQE GEAHNIFCPA YDCFQLVPVD IIESVVSKEM DKRYLQFDIK AFVENNPAIK WCPTPGCDRA VRLTKQGSNT SGSDTLSFPL LRAPAVDCGK GHLFCWECLG EAHEPCDCQT WKNWLQKITE MKPEELVGVS EAYEDAANCL WLLTNSKPCA NCKSPIQKNE GCNHMQCAKC KYDFCWICLE EWKKHSSSTG GYYRCTRYEV IQHVEEQSKE MTVEAEKKHK RFQELDRFMH YYTRFKNHEH SYQLEQRLLK TAKEKMEQLS RALKETEGGC PDTTFIEDAV HVLLKTRRIL KCSYPYGFFL EPKSTKKEIF ELMQTDLEMV TEDLAQKVNR PYLRTPRHKI IKAACLVQQK RQEFLASVAR GVAPADSPEA PRRSFAGGTW DWEYLGFASP EEYAEFQYRR RHRQRRRGDV HSLLSNPPDP DEPSESTLDI PEGGSSSRRP GTSVVSSASM SVLHSSSLRD YTPASRSENQ DSLQALSSLD EDDPNILLAI QLSLQESGLA LDEETRDFLS NEASLGAIGT SLPSRLDSVP RNTDSPRAAL SSSELLELGD SLMRLGAEND PFSTDTLSSH PLSEARSDFC PSSSDPDSAG QDPNINDNLL GNIMAWFHDM NPQSIALIPP ATTEISADSQ LPCIKDGSEG VKDVELVLPE DSMFEDASVS EGRGTQIEEN PLEENILAGE AASQAGDSGN EAANRGDGSD VSSQTPQTSS DWLEQVHLV (SEQ ID NO: 417), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc Finger Protein: P36406-1 (Isoform Alpha):
[0405] MATLVVNKLG AGVDSGRQGS RGTAVVKVLE CGVCEDVFSL QGDKVPRLLL CGHTVCHDCL TRLPLHGRAI RCPFDRQVTD LGDSGVWGLK KNFALLELLE RLQNGPIGQY GAAEESIGIS GESIIRCDED EAHLASVYCT VCATHLCSEC SQVTHSTKTL AKHRRVPLAD KPHEKTMCSQ HQVHAIEFVC LEEGCQTSPL MCCVCKEYGK HQGHKHSVLE PEANQIRASI LDMAHCIRTF TEEISDYSRK LVGIVQHIEG GEQIVEDGIG MAHTEHVPGT AENARSCIRA YFYDLHETLC RQEEMALSVV DAHVREKLIW LRQQQEDMTI LLSEVSAACL HCEKTLQQDD CRVVLAKQEI TRLLETLQKQ QQQFTEVADH IQLDASIPVT FTKDNRVHIG PKMEIRVVTL GLDGAGKTTI LFKLKQDEFM QPIPTIGFNV ETVEYKNLKF TIWDVGGKHK LRPLWKHYYL NTQAVVFVVD SSHRDRISEA HSELAKLLTE KELRDALLLI FANKQDVAGA LSVEEITELL SLHKLCCGRS WYIQGCDARS GMGLYEGLDW LSRQLVAAGV LDVA (SEQ ID NO: 418), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
Zinc Finger Protein: Q9ULV3-1 (Isoform-1):
[0406] MFSQQQQQQL QQQQQQLQQL QQQQLQQQQL QQQQLLQLQQ LLQQSPPQAP LPMAVSRGLP PQQPQQPLLN LQGTNSASLL NGSMLQRALL LQQLQGLDQF AMPPATYDTA GLTMPTATLG NLRGYGMASP GLAAPSLTPP QLATPNLQQF FPQATRQSLL GPPPVGVPMN PSQFNLSGRN PQKQARTSSS TTPNRKDSSS QTMPVEDKSD PPEGSEEAAE PRMDTPEDQD LPPCPEDIAK EKRTPAPEPE PCEASELPAK RLRSSEEPTE KEPPGQLQVK AQPQARMTVP KQTQTPDLLP EALEAQVLPR FQPRVLQVQA QVQSQTQPRI PSTDTQVQPK LQKQAQTQTS PEHLVLQQKQ VQPQLQQEAE PQKQVQPQVQ PQAHSQGPRQ VQLQQEAEPL KQVQPQVQPQ AHSQPPRQVQ LQLQKQVQTQ TYPQVHTQAQ PSVQPQEHPP AQVSVQPPEQ THEQPHTQPQ VSLLAPEQTP VVVHVCGLEM PPDAVEAGGG MEKTLPEPVG TQVSMEEIQN ESACGLDVGE CENRAREMPG VWGAGGSLKV TILQSSDSRA FSTVPLTPVP RPSDSVSSTP AATSTPSKQA LQFFCYICKA SCSSQQEFQD HMSEPQHQQR LGEIQHMSQA CLLSLLPVPR DVLETEDEEP PPRRWCNTCQ LYYMGDLIQH RRTQDHKIAK QSLRPFCTVC NRYFKTPRKF VEHVKSQGHK DKAKELKSLE KEIAGQDEDH FITVDAVGCF EGDEEEEEDD EDEEEIEVEE ELCKQVRSRD ISREEWKGSE TYSPNTAYGV DFLVPVMGYI CRICHKFYHS NSGAQLSHCK SLGHFENLQK YKAAKNPSPT TRPVSRRCAI NARNALTALF TSSGRPPSQP NTQDKTPSKV TARPSQPPLP RRSTRLKT (SEQ ID NO: 419), or an amino acid having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, or fragment thereof.
[0407] The present disclosure may use any known or available zinc finger protein, or variant or functional fragment thereof. In some embodiments, a mitoZF comprises an amino acid sequence selected from any one of the following amino acid sequences, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the following mitoZF sequences:
TABLE-US-00026 ZF(R8) (SEQIDNO:398) MAERPFQCDICMRNFSTSGSLSRHIRTHTGEKPFQCDICMRNFSQSGSLT RHIRTHTGSEKPFQCDICMRNFSRSDALSQHIRTHTGEKPFQCDICMRNF SRNDNRITHIRTHTGEKPFQCDICMRNFSRSDHLTQHTKIHLR ZF(5xZnF-4-R8) (SEQIDNO:402) MAERPFQCDICMRNFSQASNLISHIRTHTGEKPFQCDICMRNFSTSHSLT EHIRTHTGSEKPFQCDICMRNFSERSHLREHIRTHTGEKPFQCDICMRNF SQSGNLTEHIRTHTGEKPFQCDICMRNESSKKALTEHTKIHLR ZF(5xZnF-10-R8) (SEQIDNO:403) MAERPFQCDICMRNFSQASNLISHIRTHTGEKPFQCDICMRNFSQRANLR AHIRTHTGSEKPFQCDICMRNFSQASNLISHIRTHTGEKPFQCDICMRNF STSHSLTEHIRTHTGEKPFQCDICMRNFSERSHLREHTKIHLR ZF(R13-1) (SEQIDNO:404) MAERPFQCDICMRNFSRSDNLSTHIRTHTGEKPFQCDICMRNFSDRSDLS RHIRTHTGEKPFQCDICMRNFSQSGDLTRHIRTHTGSEKPFQCDICMRNF SRSDSLSAHIRTHTGEKPFQCDICMRNFSQKATRITHTKIHLR ZF(5xZnF-9-R13) (SEQIDNO:405) MAERPFQCDICMRNFSQSSSLVRHIRTHTGEKPFQCDICMRNFSRSDNLV RHIRTHTGSEKPFQCDICMRNFSQAGHLASHIRTHTGEKPFQCDICMRNF SRKDNLKNHIRTHTGEKPFQCDICMRNFSRKDALRGHTKIHLR ZF(5xZnF-12-R13) (SEQIDNO:406) MAERPFQCDICMRNFSRSDHLTTHIRTHTGEKPFQCDICMRNFSQSSSLV RHIRTHTGSEKPFQCDICMRNFSRSDNLVRHIRTHTGEKPFQCDICMRNF SQAGHLASHIRTHTGEKPFQCDICMRNFSRKDNLKNHTKIHLR
napDNAbp
[0408] In various embodiments, the mtDNA base editors or the polypeptides that comprise the mtDNA base editors (e.g., the pDNAbps and DddA) may include a napDNAbp as the pDNAbp component.
[0409] In one aspect, the methods and base editor compositions described herein involve a nucleic acid programmable DNA binding protein (napDNAbp). Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid programs the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to a complementary sequence. In various embodiments, the napDNAbp can be fused to a herein disclosed adenosine deaminase or cytidine deaminase.
[0410] Without being bound by theory, the binding mechanism of a napDNAbpguide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guideRNA protospacer then hybridizes to the target strand. This displaces a non-target strand that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and/or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a double-stranded break whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is nicked on one strand. Exemplary napDNAbp with different nuclease activities include Cas9 nickase (nCas9) and a deactivated Cas9 having no nuclease activities (dead Cas9 or dCas9).
[0411] The below description of various napDNAbps which can be used in connection with the presently disclose base editors is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein-including any naturally occurring variant, mutant, or otherwise engineered version of Cas9 that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are dead Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). The base editors described herein may also comprise Cas9 equivalents, including Cas12a/Cpf1 and Cas12b proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Cas12a/Cpf1).
[0412] The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3-5 exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (sgRNA, or simply gNRA) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.
[0413] In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a napDNAbp that is mutated to with respect to a corresponding wild-type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.
[0414] As used herein, the term Cas protein refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science 2016; 353(6299), the contents of which are incorporated herein by reference.
[0415] The terms Cas9 or Cas9 nuclease or Cas9 moiety or Cas9 domain embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a Cas9 or equivalent. Exemplary Cas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the base editor (PE) of the invention.
[0416] As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., Complete genome sequence of an M1 strain of Streptococcus pyogenes. Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference).
[0417] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The base editor fusions of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.
(1) Wild Type SpCas9
[0418] In one embodiment, the base editor constructs described herein may comprise the canonical SpCas9 nuclease from S. pyogenes, which has been widely used as a tool for genome engineering. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. As used herein, the canonical SpCas9 protein refers to the wild type protein from Streptococcus pyogenes having the following amino acid sequence:
TABLE-US-00027 Description Sequence SEQIDNO: SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLEDS 28 Streptococcus GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEED pyogenesM1 KKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKER SwissProt GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR Accession RLENLIAQLPGEKKNGLFGNLIALSLGLIPNEKSNEDLAEDAKLQLSKDTYDDDL No.Q99ZW2 DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ Wildtype DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNE FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SpCas9 ATGGATAAAAAATATAGCATTGGCCTGGATATTGGCACCAACAGCGTGGGCTGGG 29 Reverse CGGTGATTACCGATGAATATAAAGTGCCGAGCAAAAAATTTAAAGTGCTGGGCAA translation CACCGATCGCCATAGCATTAAAAAAAACCTGATTGGCGCGCTGCTGTTTGATAGC of GGCGAAACCGCGGAAGCGACCCGCCTGAAACGCACCGCGCGCCGCCGCTATACCC SwissProt GCCGCAAAAACCGCATTTGCTATCTGCAGGAAATTTTTAGCAACGAAATGGCGAA Accession AGTGGATGATAGCTTTTTTCATCGCCTGGAAGAAAGCTTTCTGGTGGAAGAAGAT No.Q99ZW2 AAAAAACATGAACGCCATCCGATTTTTGGCAACATTGTGGATGAAGTGGCGTATC Streptococcus ATGAAAAATATCCGACCATTTATCATCTGCGCAAAAAACTGGTGGATAGCACCGA pyogenes TAAAGCGGATCTGCGCCTGATTTATCTGGCGCTGGCGCATATGATTAAATTTCGC GGCCATTTTCTGATTGAAGGCGATCTGAACCCGGATAACAGCGATGTGGATAAAC TGTTTATTCAGCTGGTGCAGACCTATAACCAGCTGTTTGAAGAAAACCCGATTAA CGCGAGCGGCGTGGATGCGAAAGCGATTCTGAGCGCGCGCCTGAGCAAAAGCCGC CGCCTGGAAAACCTGATTGCGCAGCTGCCGGGCGAAAAAAAAAACGGCCTGTTTG GCAACCTGATTGCGCTGAGCCTGGGCCTGACCCCGAACTTTAAAAGCAACTTTGA TCTGGCGGAAGATGCGAAACTGCAGCTGAGCAAAGATACCTATGATGATGATCTG GATAACCTGCTGGCGCAGATTGGCGATCAGTATGCGGATCTGTTTCTGGCGGCGA AAAACCTGAGCGATGCGATTCTGCTGAGCGATATTCTGCGCGTGAACACCGAAAT TACCAAAGCGCCGCTGAGCGCGAGCATGATTAAACGCTATGATGAACATCATCAG GATCTGACCCTGCTGAAAGCGCTGGTGCGCCAGCAGCTGCCGGAAAAATATAAAG AAATTTTTTTTGATCAGAGCAAAAACGGCTATGCGGGCTATATTGATGGCGGCGC GAGCCAGGAAGAATTTTATAAATTTATTAAACCGATTCTGGAAAAAATGGATGGC ACCGAAGAACTGCTGGTGAAACTGAACCGCGAAGATCTGCTGCGCAAACAGCGCA CCTTTGATAACGGCAGCATTCCGCATCAGATTCATCTGGGCGAACTGCATGCGAT TCTGCGCCGCCAGGAAGATTTTTATCCGTTTCTGAAAGATAACCGCGAAAAAATT GAAAAAATTCTGACCTTTCGCATTCCGTATTATGTGGGCCCGCTGGCGCGCGGCA ACAGCCGCTTTGCGTGGATGACCCGCAAAAGCGAAGAAACCATTACCCCGTGGAA CTTTGAAGAAGTGGTGGATAAAGGCGCGAGCGCGCAGAGCTTTATTGAACGCATG ACCAACTTTGATAAAAACCTGCCGAACGAAAAAGTGCTGCCGAAACATAGCCTGC TGTATGAATATTTTACCGTGTATAACGAACTGACCAAAGTGAAATATGTGACCGA AGGCATGCGCAAACCGGCGTTTCTGAGCGGCGAACAGAAAAAAGCGATTGTGGAT CTGCTGTTTAAAACCAACCGCAAAGTGACCGTGAAACAGCTGAAAGAAGATTATT TTAAAAAAATTGAATGCTTTGATAGCGTGGAAATTAGCGGCGTGGAAGATCGCTT TAACGCGAGCCTGGGCACCTATCATGATCTGCTGAAAATTATTAAAGATAAAGAT TTTCTGGATAACGAAGAAAACGAAGATATTCTGGAAGATATTGTGCTGACCCTGA CCCTGITTGAAGATCGCGAAATGATTGAAGAACGCCTGAAAACCTATGCGCATCT GTTTGATGATAAAGTGATGAAACAGCTGAAACGCCGCCGCTATACCGGCTGGGGC CGCCTGAGCCGCAAACTGATTAACGGCATTCGCGATAAACAGAGCGGCAAAACCA TTCTGGATTTTCTGAAAAGCGATGGCTTTGCGAACCGCAACTTTATGCAGCTGAT TCATGATGATAGCCTGACCTTTAAAGAAGATATTCAGAAAGCGCAGGTGAGCGGC CAGGGCGATAGCCTGCATGAACATATTGCGAACCTGGCGGGCAGCCCGGCGATTA AAAAAGGCATTCTGCAGACCGTGAAAGTGGTGGATGAACTGGTGAAAGTGATGGG CCGCCATAAACCGGAAAACATTGTGATTGAAATGGCGCGCGAAAACCAGACCACC CAGAAAGGCCAGAAAAACAGCCGCGAACGCATGAAACGCATTGAAGAAGGCATTA AAGAACTGGGCAGCCAGATTCTGAAAGAACATCCGGTGGAAAACACCCAGCTGCA GAACGAAAAACTGTATCTGTATTATCTGCAGAACGGCCGCGATATGTATGTGGAT CAGGAACTGGATATTAACCGCCTGAGCGATTATGATGTGGATCATATTGTGCCGC AGAGCTTTCTGAAAGATGATAGCATTGATAACAAAGTGCTGACCCGCAGCGATAA AAACCGCGGCAAAAGCGATAACGTGCCGAGCGAAGAAGTGGTGAAAAAAATGAAA AACTATTGGCGCCAGCTGCTGAACGCGAAACTGATTACCCAGCGCAAATTTGATA ACCTGACCAAAGCGGAACGCGGCGGCCTGAGCGAACTGGATAAAGCGGGCTTTAT TAAACGCCAGCTGGTGGAAACCCGCCAGATTACCAAACATGTGGCGCAGATTCTG GATAGCCGCATGAACACCAAATATGATGAAAACGATAAACTGATTCGCGAAGTGA AAGTGATTACCCTGAAAAGCAAACTGGTGAGCGATTTTCGCAAAGATTTTCAGIT TTATAAAGTGCGCGAAATTAACAACTATCATCATGCGCATGATGCGTATCTGAAC GCGGTGGTGGGCACCGCGCTGATTAAAAAATATCCGAAACTGGAAAGCGAATTTG TGTATGGCGATTATAAAGTGTATGATGTGCGCAAAATGATTGCGAAAAGCGAACA GGAAATTGGCAAAGCGACCGCGAAATATTTTTTTTATAGCAACATTATGAACTTT TTTAAAACCGAAATTACCCTGGCGAACGGCGAAATTCGCAAACGCCCGCTGATTG AAACCAACGGCGAAACCGGCGAAATTGTGTGGGATAAAGGCCGCGATTTTGCGAC CGTGCGCAAAGTGCTGAGCATGCCGCAGGTGAACATTGTGAAAAAAACCGAAGTG CAGACCGGCGGCTTTAGCAAAGAAAGCATTCTGCCGAAACGCAACAGCGATAAAC TGATTGCGCGCAAAAAAGATTGGGATCCGAAAAAATATGGCGGCTTTGATAGCCC GACCGTGGCGTATAGCGTGCTGGTGGTGGCGAAAGTGGAAAAAGGCAAAAGCAAA AAACTGAAAAGCGTGAAAGAACTGCTGGGCATTACCATTATGGAACGCAGCAGCT TTGAAAAAAACCCGATTGATTTTCTGGAAGCGAAAGGCTATAAAGAAGTGAAAAA AGATCTGATTATTAAACTGCCGAAATATAGCCTGTTTGAACTGGAAAACGGCCGC AAACGCATGCTGGCGAGCGCGGGCGAACTGCAGAAAGGCAACGAACTGGCGCTGC CGAGCAAATATGTGAACTTTCTGTATCTGGCGAGCCATTATGAAAAACTGAAAGG CAGCCCGGAAGATAACGAACAGAAACAGCTGTTTGTGGAACAGCATAAACATTAT CTGGATGAAATTATTGAACAGATTAGCGAATTTAGCAAACGCGTGATTCTGGCGG ATGCGAACCTGGATAAAGTGCTGAGCGCGTATAACAAACATCGCGATAAACCGAT TCGCGAACAGGCGGAAAACATTATTCATCTGTTTACCCTGACCAACCTGGGCGCG CCGGCGGCGTTTAAATATTTTGATACCACCATTGATCGCAAACGCTATACCAGCA CCAAAGAAGTGCTGGATGCGACCCTGATTCATCAGAGCATTACCGGCCTGTATGA AACCCGCATTGATCTGAGCCAGCTGGGCGGCGAT
[0419] The base editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any known mutation reported with the SwissProt Accession No. Q99ZW2 entry, which include:
TABLE-US-00028 SpCas9 mutation (relative Function/Characteristic (as reported) to the amino acid sequence (see UniProtKB - Q99ZW2 of the canonical SpCas9 (CAS9_STRPT1) entry - incorporated sequence, SEQ ID NO: 28) herein by reference) D10A Nickase mutant which cleaves the protospacer strand (but no cleavage of non-protospacer strand) S15A Decreased DNA cleavage activity R66A Decreased DNA cleavage activity R70A No DNA cleavage R74A Decreased DNA cleavage R78A Decreased DNA cleavage 97-150 deletion No nuclease activity R165A Decreased DNA cleavage 175-307 deletion About 50% decreased DNA cleavage 312-409 deletion No nuclease activity E762A Nickase H840A Nickase mutant which cleaves the non-protospacer strand but does not cleave the protospacer strand N854A Nickase N863A Nickase H982A Decreased DNA cleavage D986A Nickase 1099-1368 deletion No nuclease activity R1333A Reduced DNA binding
[0420] Other wild type SpCas9 sequences that may be used in the present disclosure, include:
TABLE-US-00029 Description Sequence SEQIDNO: SpCas9 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGT 30 Streptococcus GATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACC pyogenes GCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCG MGAS1882 GAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTAT wildtype TTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTC NC_017053.1 ATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATT TTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCT GCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCT TAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGAT AATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGA AGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGA GTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGC TTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTT TGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAG ATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAAT TTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGC TCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTT TAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAA TCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAA ATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAA ATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAA ATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTT AAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTG GTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACA ATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTAT TGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATA GTTTGCTTTATGAGTATTTTACGGITTATAACGAATTGACAAAGGTCAAATATGTTACT GAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTT ACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAA AAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCA TTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGA AGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGG GGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAA CAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGG TATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTG CCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATT CAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGC TGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGG TCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAG ACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTAT CAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAA ATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAA TTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCAT TAAAGACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAAT CGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTT CTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGG AGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCC AAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAA AATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGA CTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC ATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTT GAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAA GTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGA ACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATC GAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGT GCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAG GCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGT AAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTC AGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAG AGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTT TTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATA TAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTAC AAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGT CATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGA GCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTG TTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGAC AAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGG AGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTA CAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACA CGCATTGATTTGAGTCAGCTAGGAGGTGACTGA SpCas9 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETA 31 Streptococcus EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPI pyogenes FGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKERGHFLIEGDLNPD MGAS1882 NSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNG wildtype LFGNLIALSLGLTPNFKSNFDLAEDAKLILSKDTYDDDLDNLLAQIGDQYADLFLAAKN NC_017053.1 LSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLILLKALVRIILPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKIRTEDNGSIPHQ IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKILKEDYFKKIECFDSVEISGVEDRENAS LGAYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLEDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLINGRDMYVDQE LDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRIITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS HYEKLKGSPEDNEQKILFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYET RIDLSQLGGD SpCas9 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGT 32 Streptococcus CATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACC pyogenes GTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCA wildtype GAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAAT SWBC2D7W014 ATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTC ACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATC TTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCT CAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTC TTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGAC AACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGITTGA AGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCT CTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGG TTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTT CGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCG ACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAAC CTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGC GCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTC TCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAG TCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAA GTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCA ATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAA ATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCT CAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGG GACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACG ATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCAT CGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACA GTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGITAAGTATGTCACT GAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCT GTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGA AAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCA CTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGA AGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGG AAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAA CAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGG GATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCG CCAATAGGAACTTTATGCAGCTGATCCATGATGACTCITTAACCTTCAAAGAGGATATA CAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGC TGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAG TTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAAT CAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGG TATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGC AGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAG GAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTT TTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGA AAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAG CTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAG GGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCC GCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGAC GAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTC GGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATG CGCACGACGCTTATCITAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAG CTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGC GAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTA TGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTA ATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGAC GGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGA CCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCT CGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTA TTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCA AAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGAC TTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAA GTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGC TTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCG TCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGT TGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGA GAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGG GATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCT CGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTT CTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAA ACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGT CTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGG ATGACGATGACAAGGCTGCAGGA SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 33 Streptococcus EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPI pyogenes FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD wildtype NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAILPGEKKNG Encoded LFGNLIALSLGLIPNFKSNEDLAEDAKLILSKDTYDDDLDNLLAQIGDQYADLFLAAKN productof LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQ SWBC2D7W014 SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKIRTEDNGSIPHQ IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKILKEDYFKKIECFDSVEISGVEDRENAS LGTYHDLLKIIKDKDELDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLEDDKVMK QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLINGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITIRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLA SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAG SpCas9 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGT 34 Streptococcus GATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACC pyogenes GCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCG M1GASwild GAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTAT type TTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTC NC_002737.2 ATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATT TTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCT GCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCT TAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGAT AATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGA AGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCITTCTGCACGATTGA GTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGC TTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTT TGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAG ATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAAT TTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGC TCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTT TAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAA TCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAA ATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAA ATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAA ATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTT AAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTG GTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACA ATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTAT TGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATA GTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACT GAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTT ACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAA AAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCA TTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGA AGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGG AGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAA CAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGG TATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTG CCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATT CAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGC TGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGG TCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAAT CAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGG TATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGC AAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAA GAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTT CCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTA AATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAA CTTCTAAACGCCAAGTTAATCACTCAACGTAAGITTGATAATTTAACGAAAGCTGAACG TGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTC GCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGAT GAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTC TGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATG CCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAA CTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGC TAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCA TGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTA ATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCAC AGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGA CAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCT CGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTA TTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTA AAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGAC TTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAA ATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCT AGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGT GGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGC GTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGA GACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCT TGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGT CTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAA ACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 35 Streptococcus EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPI pyogenes FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD M1GASwild NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG type LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN Encoded LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRIQLPEKYKEIFFDQ productof SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKIRTEDNGSIPHQ NC_002737.2 IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET (100% ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT identicalto EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKILKEDYFKKIECFDSVEISGVEDRENAS thecanonical LGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMK Q99ZW2 QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI wildtype) QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLINGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITIRKFDNLTKAERGGLSELDKAGFIKRQLVETRIITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLA SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGD
[0421] The base editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
(2) Wild Type Cas9 Orthologs
[0422] In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species. For example, the following Cas9 orthologs can be used in connection with the base editor constructs described in this specification. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the present base editors.
TABLE-US-00030 Description Sequence LfCas9 MKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERRTFRTTRRRLKRRKWRLHYLDEIFAPHLQEVD Lactobacillus ENFLRRLKQSNIHPEDPTKNQAFIGKLLFPDLLKKNERGYPTLIKMRDELPVEQRAHYPVMNIYKLREAMINEDRQFDLRE fermentum VYLAVHHIVKYRGHFLNNASVDKFKVGRIDFDKSFNVLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQKAVA wildtype KLLEVKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSSETSEDEIEKFREELSDAQNDILTEITSLFS GenBank: QIMLNEIVPNGMSISESMMDRYWTHERQLAEVKEYLATQPASARKEFDQVYNKYIGQAPKERGFDLEKGLKKILSKKENWK SNX31424.11 EIDELLKAGDFLPKQRTSANGVIPHQMHQQELDRIIEKQAKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLV TPEVQKATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLLNEDVLPANSLLYQKYNVLNELNNVRVN GRRLSVGIKQDIYTELFKKKKTVKASDVASLVMAKTRGVNKPSVEGLSDPKKFNSNLATYLDLKSIVGDKVDDNRYQTDLE NIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALVKKRYKGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFKEIVDQPVFK EQIDQLNQKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVGNAPKSISIEFARNEGNKGEITRSRRTQ LQKLFEDQAHELVKDTSLTEELEKAPDLSDRYYFYFTQGGKDMYTGDPINFDEISTKYDIDHILPQSFVKDNSLDNRVLTS RKENNKKSDQVPAKLYAAKMKPYWNQLLKQGLITQRKFENLTKDVDQNIKYRSLGFVKRQLVETRQVIKLTANILGSMYQE AGTEIIETRAGLTKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSFFVYGEYMKFKHGSDLKLRNFNFFH ELMEGDKSQGKVVDQQTGELITTRDEVAKSFDRLLNMKYMLVSKEVHDRSDQLYGATIVTAKESGKLTSPIEIKKNRLVDL YGAYTNGTSAFMTIIKFTGNKPKYKVIGIPTTSAASLKRAGKPGSESYNQELHRIIKSNPKVKKGFEIVVPHVSYGQLIVD GDCKFTLASPTVQHPATQLVLSKKSLETISSGYKILKDKPAIANERLIRVFDEVVGQMNRYFTIFDQRSNRQKVADARDKF LSLPTESKYEGAKKVQVGKTEVITNLLMGLHANATQGDLKVLGLATFGFFQSTTGLSLSEDTMIVYQSPTGLFERRICLKD I(SEQIDNO:36) SaCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY Staphylococcus LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI aureus KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIA wildtype LSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR GenBank: YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF AYD60528.1 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGA SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQIDNO:37) SaCas9 MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH Staphylococcus SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD aureus GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF TNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLIL DELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSK DAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFD NSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLV DTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVM ENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLY DKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDL IKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK (SEQIDNO:38) StCas9 MLFNKCIIISINLDFSNKEKCMTKPYSIGLDIGTNSVGWAVITDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAE Streptococcus GRRLKRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKVYHDEFPTIYHLRKY thermophilus LADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKK UniProtKB/ DRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLS Swiss-Prot: GFLTVTDNETEAPLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKNLLAEFE G3ECR1.2 GADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWS Wildtype IRKRNEKITPWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQKK DIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFED REMIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQ KAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSL KELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASN RGKSDDFPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERGGLLPEDKAGFIQRQLVETRQITKHVARLLDEKENNKKD ENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKSA TEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGL FNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLL EKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHRKYVENHKK EFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKIPRYRDY TPSSLLKDATLIHQSVTGLYETRIDLAKLGEG(SEQIDNO:39) LcCas9 MKIKNYNLALTPSTSAVGHVEVDDDLNILEPVHHQKAIGVAKFGEGETAEARRLARSARRTTKRRANRINHYFNEIMKPEI Lactobacillus DKVDPLMFDRIKQAGLSPLDERKEFRTVIFDRPNIASYYHNQFPTIWHLQKYLMITDEKADIRLIYWALHSLLKHRGHFFN crispatus TTPMSQFKPGKLNLKDDMLALDDYNDLEGLSFAVANSPEIEKVIKDRSMHKKEKIAELKKLIVNDVPDKDLAKRNNKIITQ NCBI IVNAIMGNSFHLNFIFDMDLDKLTSKAWSFKLDDPELDTKFDAISGSMTDNQIGIFETLQKIYSAISLLDILNGSSNVVDA Reference KNALYDKHKRDLNLYFKFLNTLPDEIAKTLKAGYTLYIGNRKKDLLAARKLLKVNVAKNFSQDDFYKLINKELKSIDKQGL Sequence: QTRFSEKVGELVAQNNFLPVQRSSDNVFIPYQLNAITFNKILENQGKYYDFLVKPNPAKKDRKNAPYELSQLMQFTIPYYV WP_133478044.1 GPLVTPEEQVKSGIPKTSRFAWMVRKDNGAITPWNFYDKVDIEATADKFIKRSIAKDSYLLSELVLPKHSLLYEKYEVENE Wildtype LSNVSLDGKKLSGGVKQILFNEVFKKTNKVNTSRILKALAKHNIPGSKITGLSNPEEFTSSLQTYNAWKKYFPNQIDNFAY QQDLEKMIEWSTVFEDHKILAKKLDEIEWLDDDQKKFVANTRLRGWGRLSKRLLTGLKDNYGKSIMQRLETTKANFQQIVY KPEFREQIDKISQAAAKNQSLEDILANSYTSPSNRKAIRKTMSVVDEYIKLNHGKEPDKIFLMFQRSEQEKGKQTEARSKQ LNRILSQLKADKSANKLFSKQLADEFSNAIKKSKYKLNDKQYFYFQQLGRDALTGEVIDYDELYKYTVLHIIPRSKLTDDS QNNKVLTKYKIVDGSVALKFGNSYSDALGMPIKAFWTELNRLKLIPKGKLLNLTTDFSTLNKYQRDGYIARQLVETQQIVK LLATIMQSRFKHTKIIEVRNSQVANIRYQFDYFRIKNLNEYYRGFDAYLAAVVGTYLYKVYPKARRLFVYGQYLKPKKTNQ ENQDMHLDSEKKSQGFNFLWNLLYGKQDQIFVNGTDVIAFNRKDLITKMNTVYNYKSQKISLAIDYHNGAMFKATLFPRND RDTAKTRKLIPKKKDYDTDIYGGYTSNVDGYMLLAEIIKRDGNKQYGFYGVPSRLVSELDTLKKTRYTEYEEKLKEIIKPE LGVDLKKIKKIKILKNKVPFNQVIIDKGSKFFITSTSYRWNYRQLILSAESQQTLMDLVVDPDFSNHKARKDARKNADERL IKVYEEILYQVKNYMPMFVELHRCYEKLVDAQKTFKSLKISDKAMVLNQILILLHSNATSPVLEKLGYHTRFTLGKKHNLI SENAVLVTQSITGLKENHVSIKQML(SEQIDNO:40) PdCas9 MTNEKYSIGLDIGTSSIGFAVVNDNNRVIRVKGKNAIGVRLFDEGKAAADRRSFRTTRRSFRTTRRRLSRRRWRLKLLREI Pedicoccus FDAYITPVDEAFFIRLKESNLSPKDSKKQYSGDILFNDRSDKDFYEKYPTIYHLRNALMTEHRKFDVREIYLAIHHIMKFR damnosus GHFLNATPANNFKVGRLNLEEKFEELNDIYQRVFPDESIEFRTDNLEQIKEVLLDNKRSRADRQRTLVSDIYQSSEDKDIE NCBI KRNKAVATEILKASLGNKAKLNVITNVEVDKEAAKEWSITFDSESIDDDLAKIEGQMTDDGHEIIEVLRSLYSGITLSAIV Reference PENHTLSQSMVAKYDLHKDHLKLFKKLINGMTDTKKAKNLRAAYDGYIDGVKGKVLPQEDFYKQVQVNLDDSAEANEIQTY Sequence: IDQDIFMPKQRTKANGSIPHQLQQQELDQIIENQKAYYPWLAELNPNPDKKRQQLAKYKLDELVTFRVPYYVGPMITAKDQ WP_062913273.1 KNQSGAEFAWMIRKEPGNITPWNFDQKVDRMATANQFIKRMTTTDTYLLGEDVLPAQSLLYQKFEVLNELNKIRIDHKPIS Wildtype IEQKQQIFNDLFKQFKNVTIKHLQDYLVSQGQYSKRPLIEGLADEKRFNSSLSTYSDLCGIFGAKLVEENDRQEDLEKIIE WSTIFEDKKIYRAKLNDLTWLTDDQKEKLATKRYQGWGRLSRKLLVGLKNSEHRNIMDILWITNENFMQIQAEPDFAKLVT DANKGMLEKTDSQDVINDLYTSPQNKKAIRQILLVVHDIQNAMHGQAPAKIHVEFARGEERNPRRSVQRQRQVEAAYEKVS NELVSAKVRQEFKEAINNKRDFKDRLFLYFMQGGIDIYTGKQLNIDQLSSYQIDHILPQAFVKDDSLTNRVLTNENQVKAD SVPIDIFGKKMLSVWGRMKDQGLISKGKYRNLTMNPENISAHTENGFINRQLVETRQVIKLAVNILADEYGDSTQIISVKA DLSHQMREDFELLKNRDVNDYHHAFDAYLAAFIGNYLLKRYPKLESYFVYGDFKKFTQKETKMRRFNFIYDLKHCDQVVNK ETGEILWTKDEDIKYIRHLFAYKKILVSHEVREKRGALYNQTIYKAKDDKGSGQESKKLIRIKDDKETKIYGGYSGKSLAY MTIVQITKKNKVSYRVIGIPTLALARLNKLENDSTENNGELYKIIKPQFTHYKVDKKNGEIIETTDDFKIVVSKVRFQQLI DDAGQFFMLASDTYKNNAQQLVISNNALKAINNTNITDCPRDDLERLDNLRLDSAFDEIVKKMDKYFSAYDANNFREKIRN SNLIFYQLPVEDQWENNKITELGKRTVLTRILQGLHANATTTDMSIFKIKTPFGQLRQRSGISLSENAQLIYQSPTGLFER RVQLNKIK(SEQIDNO:41) FnCas9 MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFEEAKTAAERRVQRNSRRRLKRRKWRLNLLEEIFSNE Fusobaterium ILKIDSNFFRRLKESSLWLEDKSSKEKFTLENDDNYKDYDFYKQYPTIFHLRNELIKNPEKKDIRLVYLAIHSIFKSRGHF nucleatum LFEGQNLKEIKNFETLYNNLIAFLEDNGINKIIDKNNIEKLEKIVCDSKKGLKDKEKEFKEIFNSDKQLVAIFKLSVGSSV NCBI SLNDLFDTDEYKKGEVEKEKISFREQIYEDDKPIYYSILGEKIELLDIAKTFYDFMVLNNILADSQYISEAKVKLYEEHKK Reference DLKNLKYIIRKYNKGNYDKLFKDKNENNYSAYIGLNKEKSKKEVIEKSRLKIDDLIKNIKGYLPKVEEIEEKDKAIFNKIL Sequence: NKIELKTILPKQRISDNGTLPYQIHEAELEKILENQSKYYDFLNYEENGIITKDKLLMTFKFRIPYYVGPLNSYHKDKGGN WP_060798984.1 SWIVRKEEGKILPWNFEQKVDIEKSAEEFIKRMTNKCTYLNGEDVIPKDTFLYSEYVILNELNKVQVNDEFLNEENKRKII DELFKENKKVSEKKFKEYLLVKQIVDGTIELKGVKDSFNSNYISYIRFKDIFGEKLNLDIYKEISEKSILWKCLYGDDKKI FEKKIKNEYGDILTKDEIKKINTFKFNNWGRLSEKLLTGIEFINLETGECYSSVMDALRRTNYNLMELLSSKFTLQESINN ENKEMNEASYRDLIEESYVSPSLKRAIFQTLKIYEEIRKITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYDSCGN DIANFSIDIKEMKNSLISYDNNSLRQKKLYLYYLQFGKCMYTGREIDLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLV LKNENAEKSNEYPVKKEIQEKMKSFWRFLKEKNFISDEKYKRLTGKDDFELRGFMARQLVNVRQTTKEVGKILQQIEPEIK IVYSKAEIASSFREMFDFIKVRELNDTHHAKDAYLNIVAGNVYNTKFTEKPYRYLQEIKENYDVKKIYNYDIKNAWDKENS LEIVKKNMEKNTVNITRFIKEKKGQLFDLNPIKKGETSNEIISIKPKVYNGKDDKLNEKYGYYKSLNPAYFLYVEHKEKNK RIKSFERVNLVDVNNIKDEKSLVKYLIENKKLVEPRVIKKVYKRQVILINDYPYSIVTLDSNKLMDFENLKPLFLENKYEK ILKNVIKFLEDNQGKSEENYKFIYLKKKDRYEKNETLESVKDRYNLEFNEMYDKFLEKLDSKDYKNYMNNKKYQELLDVKE KFIKLNLFDKAFTLKSFLDLFNRKTMADFSKVGLTKYLGKIQKISSNVLSKNELYLLEESVTGLFVKKIKI(SEQID NO:42) EcCas9 MNKYYLGLDMGSASVGWAVTDENYHLVRRKGKDLWGVRTFDVAQTAKERRITRGNRRRQDRRKQRIQILQELLGEEVLKTD Enterococcus PGFFHRMKESRYVVEDKRTLDGKQVELPYALFVDKDYTDKEYYKQFPTINHLIVYLMTTSDTPDIRLVYLALHYYMKNRGN cecorum FLHSGDINNVKDINDILEQLDNVLETFLDGWNLKLKSYVEDIKNIYNRDLGRGERKKAFVNTLGAKTKAEKAFCSLISGGS NCBI TNLAELFDDSSLKEIETPKIEFASSSLEDKIDGIQEALEDRFAVIEAAKRLYDWKTLTDILGDSSSLAEARVNSYQMHHEQ Reference LLELKSLVKEYLDRKVFQEVFVSLNVANNYPAYIGHTKINGKKKELEVKRTKRNDFYSYVKKQVIEPIKKKVSDEAVLTKL Sequence: SEIESLIEVDKYLPLQVNSDNGVIPYQVKLNELTRIFDNLENRIPVLRENRDKIIKTFKFRIPYYVGSLNGVVKNGKCTNW WP_047338501.1 MVRKEEGKIYPWNFEDKVDLEASAEQFIRRMTNKCTYLVNEDVLPKYSLLYSKYLVLSELNNLRIDGRPLDVKIKQDIYEN Wildtype VFKKNRKVTLKKIKKYLLKEGIITDDDALSGLADDVKSSLTAYRDFKEKLGHLDLSEAQMENIILNITLFGDDKKLLKKRL AALYPFIDDKSLNRIATLNYRDWGRLSERFLSGITSVDQETGELRTIIQCMYETQANLMQLLAEPYHFVEAIEKENPKVDL ESISYRIVNDLYVSPAVKRQIWQTLLVIKDIKQVMKHDPERIFIEMAREKQESKKTKSRKQVLSEVYKKAKEYEHLFEKLN SLTEEQLRSKKIYLYFTQLGKCMYSGEPIDFENLVSANSNYDIDHIYPQSKTIDDSFNNIVLVKKSLNAYKSNHYPIDKNI RDNEKVKTLWNTLVSKGLITKEKYERLIRSTPFSDEELAGFIARQLVETRQSTKAVAEILSNWFPESEIVYSKAKNVSNER QDFEILKVRELNDCHHAHDAYLNIVVGNAYHTKFTNSPYRFIKNKANQEYNLRKLLQKVNKIESNGVVAWVGQSENNPGTI ATVKKVIRRNTVLISRMVKEVDGQLFDLTLMKKGKGQVPIKSSDERLTDISKYGGYNKATGAYFTFVKSKKRGKVVRSFEY VPLHLSKQFENNNELLKEYIEKDRGLTDVEILIPKVLINSLFRYNGSLVRITGRGDTRLLLVHEQPLYVSNSFVQQLKSVS SYKLKKSENDNAKLTKTATEKLSNIDELYDGLLRKLDLPIYSYWFSSIKEYLVESRTKYIKLSIEEKALVIFEILHLFQSD AQVPNLKILGLSTKPSRIRIQKNLKDTDKMSIIHQSPSGIFEHEIELTSL(SEQIDNO:444) AhCas9 MQNGFLGITVSSEQVGWAVTNPKYELERASRKDLWGVRLFDKAETAEDRRMFRTNRRLNQRKKNRIHYLRDIFHEEVNQKD Anaerostipes PNFFQQLDESNFCEDDRIVEFNFDTNLYKNQFPTVYHLRKYLMETKDKPDIRLVYLAFSKFMKNRGHFLYKGNLGEVMDFE hadrus NSMKGFCESLEKFNIDFPTLSDEQVKEVRDILCDHKIAKTVKKKNIITITKVKSKTAKAWIGLFCGCSVPVKVLFQDIDEE NCBI IVTDPEKISFEDASYDDYIANIEKGVGIYYEAIVSAKMLFDWSILNEILGDHQLLSDAMIAEYNKHHDDLKRLQKIIKGTG Reference SRELYQDIFINDVSGNYVCYVGHAKTMSSADQKQFYTFLKNRLKNVNGISSEDAEWIDTEIKNGTLLPKQTKRDNSVIPHQ Sequence: LQLREFELILDNMQEMYPFLKENREKLLKIFNFVIPYYVGPLKGVVRKGESTNWMVPKKDGVIHPWNFDEMVDKEASAECF WP_044924278.1 ISRMTGNCSYLFNEKVLPKNSLLYETFEVLNELNPLKINGEPISVELKQRIYEQLFLTGKKVTKKSLTKYLIKNGYDKDIE Wildtype LSGIDNEFHSNLKSHIDFEDYDNLSDEEVEQIILRITVFEDKQLLKDYLNREFVKLSEDERKQICSLSYKGWGNLSEMLLN GITVTDSNGVEVSVMDMLWNTNLNLMQILSKKYGYKAEIEHYNKEHEKTIYNREDLMDYLNIPPAQRRKVNQLITIVKSLK KTYGVPNKIFFKISREHQDDPKRTSSRKEQLKYLYKSLKSEDEKHLMKELDELNDHELSNDKVYLYFLQKGRCIYSGKKLN LSRLRKSNYQNDIDYIYPLSAVNDRSMNNKVLTGIQENRADKYTYFPVDSEIQKKMKGFWMELVLQGFMTKEKYFRLSREN DFSKSELVSFIEREISDNQQSGRMIASVLQYYFPESKIVFVKEKLISSFKRDFHLISSYGHNHLQAAKDAYITIVVGNVYH TKFTMDPAIYFKNHKRKDYDLNRLFLENISRDGQIAWESGPYGSIQTVRKEYAQNHIAVTKRVVEVKGGLFKQMPLKKGHG EYPLKTNDPRFGNIAQYGGYTNVTGSYFVLVESMEKGKKRISLEYVPVYLHERLEDDPGHKLLKEYLVDHRKLNHPKILLA KVRKNSLLKIDGFYYRLNGRSGNALILTNAVELIMDDWQTKTANKISGYMKRRAIDKKARVYQNEFHIQELEQLYDFYLDK LKNGVYKNRKNNQAELIHNEKEQFMELKTEDQCVLLTEIKKLFVCSPMQADLTLIGGSKHTGMIAMSSNVTKADFAVIAED PLGLRNKVIYSHKGEK(SEQIDNO:44) KvCas9 MSQNNNKIYNIGLDIGDASVGWAVVDEHYNLLKRHGKHMWGSRLFTQANTAVERRSSRSTRRRYNKRRERIRLLREIMEDM Kandleria VLDVDPTFFIRLANVSFLDQEDKKDYLKENYHSNYNLFIDKDFNDKTYYDKYPTIYHLRKHLCESKEKEDPRLIYLALHHI vitulina VKYRGNFLYEGQKFSMDVSNIEDKMIDVLRQFNEINLFEYVEDRKKIDEVLNVLKEPLSKKHKAEKAFALFDTTKDNKAAY NCBI KELCAALAGNKFNVTKMLKEAELHDEDEKDISFKFSDATFDDAFVEKQPLLGDCVEFIDLLHDIYSWVELQNILGSAHTSE Reference PSISAAMIQRYEDHKNDLKLLKDVIRKYLPKKYFEVFRDEKSKKNNYCNYINHPSKTPVDEFYKYIKKLIEKIDDPDVKTI Sequence: LNKIELESFMLKQNSRINGAVPYQMQLDELNKILENQSVYYSDLKDNEDKIRSILTFRIPYYFGPLNITKDRQFDWIIKKE WP_031589969.1 GKENERILPWNANEIVDVDKTADEFIKRMRNFCTYFPDEPVMAKNSLTVSKYEVLNEINKLRINDHLIKRDMKDKMLHTLF Wildtype MDHKSISANAMKKWLVKNQYFSNTDDIKIEGFQKENACSTSLTPWIDFTKIFGKINESNYDFIEKIIYDVTVFEDKKILRR RLKKEYDLDEEKIKKILKLKYSGWSRLSKKLLSGIKTKYKDSTRTPETVLEVMERTNMNLMQVINDEKLGFKKTIDDANST SVSGKFSYAEVQELAGSPAIKRGIWQALLIVDEIKKIMKHEPAHVYIEFARNEDEKERKDSFVNQMLKLYKDYDFEDETEK EANKHLKGEDAKSKIRSERLKLYYTQMGKCMYTGKSLDIDRLDTYQVDHIVPQSLLKDDSIDNKVLVLSSENQRKLDDLVI PSSIRNKMYGFWEKLFNNKIISPKKFYSLIKTEFNEKDQERFINRQIVETRQITKHVAQIIDNHYENTKVVTVRADLSHQF RERYHIYKNRDINDFHHAHDAYIATILGTYIGHRFESLDAKYIYGEYKRIFRNQKNKGKEMKKNNDGFILNSMRNIYADKD TGEIVWDPNYIDRIKKCFYYKDCFVTKKLEENNGTFFNVTVLPNDTNSDKDNTLATVPVNKYRSNVNKYGGFSGVNSFIVA IKGKKKKGKKVIEVNKLTGIPLMYKNADEEIKINYLKQAEDLEEVQIGKEILKNQLIEKDGGLYYIVAPTEIINAKQLILN ESQTKLVCEIYKAMKYKNYDNLDSEKIIDLYRLLINKMELYYPEYRKQLVKKFEDRYEQLKVISIEEKCNIIKQILATLHC NSSIGKIMYSDFKISTTIGRINGRTISLDDISFIAESPTGMYSKKYKL(SEQIDNO:45) EfCas9 MRLFEEGHTAEDRRLKRTARRRISRRRNRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDEVAYHE Enterococcus TYPTIYHLRKKLADSSEQADLRLIYLALAHIVKYRGHFLIEGKLSTENTSVKDQFQQFMVIYNQTFVNGESRLVSAPLPES faecalis VLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQFLKLMVGNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEY NCBI SDVFLAAKNVYDAVELSTILADSDKKSHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLFKNEQKDGYAGYIAHAG Reference KVSQLKFYQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFLKENQEKIEQLVTF Sequence: RIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERMTNFDTYLPSEKVLPKHSLLYEKFMVFNELTK WP_016631044.1 ISYTDDRGIKANFSGKEKEKIFDYLFKTRRKVKKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELD Wildtype HPDNAEKLEDIIKILTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILDYLVKDDGV SKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIVDELVAIMGYAPKRIVVEMAREN QTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRDTRLFLYYMQNGKDMYTGDELSLHRLSHYDIDHIIPQSFMK DDSLDNLVLVGSTENRGKSDDVPSKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITK NVAGILDQRYNAKSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYPNLAPEFVYGEYPK FQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKELNYHQMNIVKKVEVQKGGFSKESIKPKGPSNK LIPVKNGLDPQKYGGFDSPVVAYTVLFTHEKGKKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYE FPEGRRRLLASAKEAQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLAYVEQHQPEFQEILERVVDFAEVHTLAKSKVQQIV KLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQSPTGLYETRRKVVD (SEQIDNO:46) Staphylococcus KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSE aureus LSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE Cas9 VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEEL RSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTN LKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE LWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDA QKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSEDNS FNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDT RYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMEN QMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDK DNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLD ITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIK INGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQIDNO:47) Geobacillus MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPRRLARSARRRLRRRKHRLERIRRLFVREGILT thermodenitri- KEELNKLFEKKHEIDVWQLRVEALDRKLNNDELARILLHLAKRRGFRSNRKSERTNKENSTMLKHIEENQSILSSYRTVAE ficans MVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAKQREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTFE Cas9 PKEKRAPKATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFHDVRTLLNLPDDTRFKGLLYDRNTT LKENEKVRFLELGAYHKIRKAIDSVYGKGAAKSFRPIDFDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLADKVYDEE LIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYSTACERAGYTFTGPKKKQKTVLLPNIPPIANPVVMRALTQARKVVNA IIKKYGSPVSIHIELARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTLNPTGLDIVKFKLWSEQNGKCAYSLQPI EIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLVLTKENREKGNRTPAEYLGLGSERWQQFETFVLINKQFSKKKRDRLLRL HYDENEENEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVNGRITAHLRSRWNENKNREESNLHHAVDAAIVAC TTPSDIARVTAFYQRREQNKELSKKTDPQFPQPWPHFADELQARLSKNPKESIKALNLGNYDNEKLESLQPVFVSRMPKRS ITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQLDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKK NGELGPIIRTIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTIDMMKGILPNKAIEPNKPYSEWKEMTE DYTFRFSLYPNDLIRIEFPREKTIKTAVGEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSRTLKRFEKYQVDVL GNIYKVRGEKRVGVASSSHSKAGETIRPL(SEQIDNO:48) ScCas9 MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIRY S.canis LQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAHII 1375AA KFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIA 159.2kDa LALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKR YDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEELLAKLNR DDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSEEAITPW NFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEIIGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQGDS LHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNY WRQLLNAKLITQRKFDNLTKAERGGLSEADKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLV SDFRKDFQLYKVRDINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFYSNIMN FFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESILSKRESAKLIPRKKG WDTRKYGGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIKKELIFKLPKYSLFEL ENGRRRMLASATELQKANELVLPQHLVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKVNSNLKS SFDEQFAVSDSILLSNSFVSLLKYTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD (SEQIDNO:49)
[0423] The base editors described herein may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
[0424] The napDNAbp may include any suitable homologs and/or orthologs or naturally occurring enzymes, such as, Cas9. Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Preferably, the Cas moiety is configured (e.g, mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase, i.e., capable of cleaving only a single strand of the target doubpdditional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the above tables.
(3) Dead Cas9 Variant
[0425] In certain embodiments, the base editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactive both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
[0426] As used herein, the term dCas9 refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a dCas9 or equivalent. Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.
[0427] In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogneous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1)) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1) are provided having amino acid sequences which are shorter, or longer than NC_017053.1 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
[0428] In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10A and an H8 10A substitutions (underlined and bolded), or a variant be variant of SEQ ID NO: 27 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto:
TABLE-US-00031 Description Sequence SEQIDNO: deadCas9or MDKKYSIGLXIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 50 dCas9 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withD10X LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG andH810X ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQE WhereXis DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA anyamino QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV acid DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDXIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD deadCas9or MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 51 dCas9 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withD10A LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG andH810A ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV ILDATLIHQSITGLYETRIDLSQLGGD
(4) Cas9 Nickase Variant
[0429] In one embodiment, the base editors described herein comprise a Cas9 nickase. The term Cas9 nickase of nCas9 refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., Crystal structure of Cas9 in complex with guide RNA and target DNA, Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain could include D10X, H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be D10A, of H983A, or D986A, or E762A, or a combination thereof.
[0430] In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
TABLE-US-00032 SEQ ID Description Sequence NO: Cas9nickase MDKKYSIGLXIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 52 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withD10X, SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI whereinXis LRVNTEITKAPLSASMIKRYDEHHQDLTILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG any ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE alternate DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA aminoacid QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYEDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 53 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withE762X, SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI whereinXis LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG any ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE alternate DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA aminoacid QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIXMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLEDSGETAEA 54 nickase TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withH983X, LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG whereinXis ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQE any DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA alternate QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV aminoacid DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHXAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 55 nickase TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withD986X, LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG whereinXis ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE any DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA alternate QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV aminoacid DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHXAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 56 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withD10A SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLILTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLEDSGETAEA 57 nickase TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withE762A LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKIIKDKDFLDNE ENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIAMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYEDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 58 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withH983A SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLILILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHAAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 59 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withD986A SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHAAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD
[0431] In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. For example, mutations in histidine (H) 840 or asparagine (R) 863 have been reported as loss-of-function mutations of the HNH nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., Crystal structure of Cas9 in complex with guide RNA and target DNA, Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain could include H840X and R863X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be H840A or R863A or a combination thereof.
[0432] In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
TABLE-US-00033 SEQ ID Description Sequence NO: Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60 nickase TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withH840X, LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG whereinXis ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE any DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA alternate QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV aminoacid DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNE ENEDILEDIVLILTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDXIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 61 nickase TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI Streptococcus VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSDVDK pyogenes LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL Q99ZW2Cas9 SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI withH840A, LRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG whereinXis ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE any DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA alternate QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV aminoacid DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNE ENEDILEDIVLILILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 62 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withR863X, SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI whereinXis LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG any ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE alternate DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA aminoacid QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNXGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD Cas9nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 63 Streptococcus TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI pyogenes VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK Q99ZW2Cas9 LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL withR863A, SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI whereinXis LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG any ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE alternate DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA aminoacid QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNE ENEDILEDIVLTLILFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNAGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGD
[0433] In some embodiments, the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus Cas9 nickases include the following sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
TABLE-US-00034 Description Sequence Cas9nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT (Metminus) RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS Streptococcus TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS pyogenes KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL Q99ZW2Cas9 FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAG withH840X, YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL whereinXis KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINEDKNLPNE any KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS alternate VEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMK aminoacid QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDXIVPQSFLKDDSIDNKVLIRSDKNRG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFA TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQIDNO:64) Cas9 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT nickase RRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS (Metminus) TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS Streptococcus KSRRLENLIAQLPGEKKNGLFGNLIALSLGLIPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL pyogenes FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAG Q99ZW2Cas9 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPEL with KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE H840A, KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS whereinXis VEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK any QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH alternate EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS aminoacid QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQIDNO:65) Cas9 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT nickase RRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS (Metminus) TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS Streptococcus KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL pyogenes FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAG Q99ZW2Cas9 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKRTFDNGSIPHQIHLGELHAILRRQEDFYPFL with KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINEDKNLPNE R863X, KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS whereinXis VEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMK any QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH alternate EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS aminoacid QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNXG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFA TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQIDNO:66) Cas9 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT nickase RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS (Metminus) TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS Streptococcus KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL pyogenes FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAG Q99ZW2Cas9 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL withR863A, KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINEDKNLPNE whereinXis KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS any VEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMK alternate QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH aminoacid EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNAG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFA TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQIDNO:67)
(5) Other Cas9 Variants
[0434] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other Cas9 variants having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SEQ ID NO: 28).
[0435] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
[0436] In various embodiments, the base editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.
(6) Small-Sized Cas9 Variants
[0437] In some embodiments, the base editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery.
[0438] The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term small-sized Cas9 variant, as used herein, refers to any Cas9 variant-naturally occurring, engineered, or otherwisethat is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein.
[0439] In various embodiments, the base editors disclosed herein may comprise one of the small-sized Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference small-sized Cas9 protein.
TABLE-US-00035 SEQ ID Description Sequence NO: SaCas9 MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRR 68 Staphylococcus HRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNE aureus VEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLK 1053AA VQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSV 123kDa KYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEED IKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNL NSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHINDNQIAIFNRLKLVPKKVDLSQQ KEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQ KRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH IIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRIS KTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFT SFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPE IETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNL NGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTK YSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVK NLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNR IEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK NmeCas9 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMAR 69 N. RLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDRKLTP meningitidis LEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPAELALNKFE 1083AA KESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSG 124.5kDa DAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLIKLNNLRILEQGSERPLTDTERATLMDEPY RKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPL NLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPL MEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYG SPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRL YEQQHGKCLYSGKEINLGRLNEKGYVEIDAALPESRTWDDSFNNKVLVLGSENQNKGNQTPY EYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVA DRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRF VRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKL RTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLK LKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLID DSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKT ALSFQKYQIDELGKEIRPCRLKKRPPVR CjCas9 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRK 70 C.jejuni ARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILH 984AA IAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRN 114.9kDa KKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSF FTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTK KLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKAL AKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDF LPAFNETYYKDEVINPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEK EQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEID HIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRIL DKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAK SGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKI SELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKE GVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKINKFYAVPIYTMDFALKVLPNKAVARSK KGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK GeoCas9 MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRR 71 G. RKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKR stearothermo- RGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIA philus RDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKR 1087AA APKATYTFQSFIAWEHINKLRLISPSGARGLIDEERRLLYEQAFQKNKITYHDIRTLLHLPD 127kDa DTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDEDTFGYALTL FKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYME QGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVS IHIELARDLSQTFDERRKIKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNG RCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVG TERWQQFETFVLINKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLK FAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAVIVACTTPSDIAKVTAFYQR REQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRM PKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIR QRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVD VFEKDGKYYCVPVYIMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPR EKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLG NIYKVRGEKRVGLASSAHSKPGKTIRPLQSTRD LbaCas12a MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI 72 L.bacterium NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII 1228AA ETILPEFLDDKDEIALVNSENGFTTAFTGFFDNRENMFSEEAKSISIAFRCINENLTRYISN 143.9kDa MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVERNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSESLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDENFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKITTLSYDVYKDKRFSEDQYELHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNENGIRI KTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLISKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVEDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH BhCas12b MATRSFILKIEPNEEVKKGLWKTHEVINHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSK 73 B.hisashii AEIQAELWDFVLKMQKCNSFTHEVDKDEVENILRELYEELVPSSVEKKGEANQLSNKFLYPL 1108AA VDPNSQSGKGTASSGRKPRWYNIKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPL 130.4kDa FIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEY KILEERIKEDIQALKALEQYEKERQEQLLRDILNINEYRLSKRGLRGWREIIQKWLKMDENE PSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAK QQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGW EEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLK SGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASENIKL PGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVP LVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNI DEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGY CYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGL QVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLILDKIAVLKEGDLYP DKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWIRTHGFYKVYCKAYQVDGQTVYIPESKD QKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGE KLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM
(7) Cas9 Equivalents
[0440] In some embodiments, the base editors described herein can include any Cas9 equivalent. As used herein, the term Cas9 equivalent is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 in the present base editors despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and/or three dimensional structure. The base editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution.
[0441] For example, CasX is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the CasX protein described in Liu et al., CasX enzymes comprises a distinct family of RNA-guided genome editors, Nature, 2019, Vol. 566: 218-223, is contemplated to be used with the base editors described herein. In addition, any variant or modification of CasX is conceivable and within the scope of the present disclosure.
[0442] Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.
[0443] In some embodiments, Cas9 equivalents may refer to CasX or CasY, which have been described in, for example, Burstein et al., New CRISPR-Cas systems from uncultivated microbes. Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. Also see Liu et al., CasX enzymes comprises a distinct family of RNA-guided genome editors, Nature, 2019, Vol. 566: 218-223. Any of these Cas9 equivalents are contemplated.
[0444] In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.
[0445] In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, Argonaute, Cas12a, and Cas12b. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. The state of the art may also now refer to Cpf1 enzymes as Casl2a.
[0446] In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation (e.g., a mutation corresponding to the D10A mutation of the wild type Cas9 polypeptide of SEQ ID NO: 28).
[0447] In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cpf1, a CasX, a CasY, a C2c1, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Cas12a, a Cas12b, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.
[0448] Exemplary Cas9 equivalent protein sequences can include the following:
TABLE-US-00036 Description Sequence AsCas12a MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDW (previously ENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQL knownas GTVTTTEHENALLRSFDKFTTYFSGFYENRKNVESAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLR Cpf1) EHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETA Acidaminococcus HIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKILLRNENVLETAEALFNELNSIDLTHIF sp. ISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQK (strain TSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLIGIKLEMEPSL BV3L6) SFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTE UniProtKB KTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQT U2UMQ6 AYAKKTGDQKGYREALCKWIDFTRDFLSKYTKITSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKE IMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLG EKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPIT LNYQAANSPSKENQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREK ERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRIGIAEKAVYQQFEKMLIDK LNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLIGFVDPFVWKTIKNHESRK HFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHR FTGRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPV RDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQIDNO:74) AsCas12a MTQFEGFTNLYQVSKTLRFELIPQGKILKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDW nickase ENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRIDNLTDAINKRHAEIYKGLFKAELFNGKVLKQL (e.g., GTVTTTEHENALLRSFDKFTTYFSGFYENRKNVESAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLR R1226A) EHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETA HIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKILLRNENVLETAEALFNELNSIDLTHIF ISHKKLETISSALCDHWDTLRNALYERRISELIGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQK TSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSL SFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTE KTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQT AYAKKTGDQKGYREALCKWIDFTRDELSKYTKITSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKE IMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLG EKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPIT LNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREK ERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRIGIAEKAVYQQFEKMLIDK LNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLIGFVDPFVWKTIKNHESRK HFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGEMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHR FTGRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTMVALIRSVLQMANSNAATGEDYINSPV RDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN(SEQ IDNO:75) LbCas12a MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQQELKEIMDDYYRTFIEEKLGQIQ (previously GIQWNSLFQKMEETMEDISVRKDLDKIQNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEK knownasCpf1) AEKEQTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMRAFQRIEQQYPEEVCGMEEE Lachnospiraceae YKDMLQEWQMKHIYSVDFYDRELTQPGIEYYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYY bacterium EIPFRFESDQEVYDALNEFIKTMKKKEIIRRCVHLGQECDDYDLGKIYISSNKYEQISNALYGSWDTIRKCIK GAM79 EEYMDALPGKGEKKEEKAEAAAKKEEYRSIADIDKIISLYGSEMDRTISAKKCITEICDMAGQISIDPLVCNS RefSeq. DIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPY WP_119623382.1 STVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLP GPSKMLPKVFITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDFHFSD TKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEEN LKDIVLKLNGEAELFFRKASIKTPIVHKKGSVLVNRSYTQTVGNKEIRVSIPEEYYTEIYNYLNHIGKGKLSS EAQRYLDEGKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKAKSDVAVNERTLAYIAKKEDIHIIGIDRGER NLLYISVVDVHGNIREQRSFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLVVK YNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLTYIPESLKKVGKQC GFIFYVPAGYTSKIDPTTGFVNLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTILAST KWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQM RNSRSESEDREYDRLISPVLNDKGEFFDTATADKILPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNK LVQDNKTWEDEMQKKRYL(SEQIDNO:76) PcCas12a- MAKNFEDFKRLYSLSKTLRFEAKPIGATLDNIVKSGLLDEDEHRAASYVKVKKLIDEYHKVFIDRVLDDGCLP previously LENKGNNNSLAEYYESYVSRAQDEDAKKKFKEIQQNLRSVIAKKLTEDKAYANLFGNKLIESYKDKEDKKKII knownat DSDLIQFINTAESTQLDSMSQDEAKELVKEFWGFVTYFYGFFDNRKNMYTAEEKSTGIAYRLVNENLPKFIDN Cpf1 IEAFNRAITRPEIQENMGVLYSDESEYLNVESIQEMFQLDYYNMLLTQKQIDVYNAIIGGKTDDEHDVKIKGI Prevotella NEYINLYNQQHKDDKLPKLKALFKQILSDRNAISWLPEEFNSDQEVLNAIKDCYERLAENVLGDKVLKSLLGS copri LADYSLDGIFIRNDLQLTDISQKMFGNWGVIQNAIMQNIKRVAPARKHKESEEDYEKRIAGIFKKADSFSISY RefSeq. INDCLNEADPNNAYFVENYFATFGAVNTPTMQRENLFALVQNAYTEVAALLHSDYPTVKHLAQDKANVSKIKA WP_119227726.1 LLDAIKSLQHFVKPLLGKGDESDKDERFYGELASLWAELDTVTPLYNMIRNYMTRKPYSQKKIKLNFENPQLL GGWDANKEKDYATIILRRNGLYYLAIMDKDSRKLLGKAMPSDGECYEKMVYKFFKDVTTMIPKCSTQLKDVQA YFKVNTDDYVLNSKAFNKPLTITKEVEDLNNVLYGKYKKFQKGYLTATGDNVGYTHAVNVWIKFCMDELNSYD STCIYDFSSLKPESYLSLDAFYQDANLLLYKLSFARASVSYINQLVEEGKMYLFQIYNKDFSEYSKGTPNMHT LYWKALFDERNLADVVYKLNGQAEMFYRKKSIENTHPTHPANHPILNKNKDNKKKESLFDYDLIKDRRYTVDK FMFHVPITMNFKSVGSENINQDVKAYLRHADDMHIIGIDRGERHLLYLVVIDLQGNIKEQYSLNEIVNEYNGN TYHTNYHDLLDVREEERLKARQSWQTIENIKELKEGYLSQVIHKITQLMVRYHAIVVLEDLSKGEMRSRQKVE KQVYQKFEKMLIDKLNYLVDKKTDVSTPGGLLNAYQLTCKSDSSQKLGKQSGELFYIPAWNTSKIDPVTGFVN LLDTHSLNSKEKIKAFFSKFDAIRYNKDKKWFEFNLDYDKFGKKAEDTRIKWTLCTRGMRIDTERNKEKNSQW DNQEVDLTTEMKSLLEHYYIDIHGNLKDAISAQTDKAFFTGLLHILKLTLQMRNSITGTETDYLVSPVADENG IFYDSRSCGNQLPENADANGAYNIARKGLMLIEQIKNAEDLNNVKFDISNKAWINFAQQKPYKNG(SEQID NO:77) ErCas12a- MFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLESRFATSFKDYFKNRANCESANDISSSSCHRIVNDNAE previously IFFSNALVYRRIVKNLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNLEMNLYC knownat QKNKENKNLYKLRKLHKQILCIADTSYEVPYKFESDEEVYQSVNGELDNISSKHIVERLRKIGENYNGYNLDK Cpf1 IYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCPDD Eubacterium NIKAETYIHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAE rectale LEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNNAIILMRDNLYYLGIFNAKN RefSeq. KPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTGVETYKPSAYILEGYKQNKHLKSSKDEDITE WP_119223642.1 CHDLIDYFKNCIAIHPEWKNFGFDFSDTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIY NKDFSKKSSGNDNLHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQF GNIQIVRKTIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYFLHMPITINFKA NKTSFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQIAR KEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVEK DISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDS IRYDSDKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTDINW RDGHDLRQDIIDYEIVQHIFEIFKLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANG AYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWEDFIQNKRYL(SEQIDNO:78) CsCas12a- MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQQELKEIMDDYYRAFIEEKLGQIQ previously GIQWNSLFQKMEETMEDISVRKDLDKIQNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEK knownat AEKEQTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMRAFQRIEQQYPEEVCGMEEE Cpf1 YKDMLQEWQMKHIYLVDFYDRVLTQPGIEYYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYY Clostridium EIPFRFESDQEVYDALNEFIKIMKEKEIICRCVHLGQKCDDYDLGKIYISSNKYEQISNALYGSWDTIRKCIK sp. EEYMDALPGKGEKKEEKAEAAAKKEEYRSIADIDKIISLYGSEMDRTISAKKCITEICDMAGQISTDPLVCNS AF34-10BH DIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPY RefSeq: STVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLP WP_118538418.1 GPSKMLPKVFITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDFHFSD TKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEEN LKDIVLKLNGEAELFFRKASIKTPVVHKKGSVLVNRSYTQTVGDKEIRVSIPEEYYTEIYNYLNHIGRGKLST EAQRYLEERKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKAKSDIAVNERTLAYIAKKEDIHIIGIDRGER NLLYISVVDVHGNIREQRSFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLVVK YNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLTYIPESLKKVGKQC GFIFYVPAGYTSKIDPTTGFVNLFSFKNLINRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTMLAST KWKVYINGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQM RNSRSESEDREYDRLISPVLNDKGEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNK LVQDNKTWFDFMQKKRYL(SEQIDNO:79) BhCas12b MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFV Bacillus LKMQKCNSFTHEVDKDEVENILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYN hisashii LKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDM RefSeq. FIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRG WP_095142515.1 LRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEID KKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKG KVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFN MTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAAS IFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQ QFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLS DGRKGLYGISLKNIDEIDRTRKELLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHA LGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQ FSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLILDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTT HADINAAQNLQKRFWIRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLK IKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLINQYS ISTIEDDSSKQSM(SEQIDNO:80) ThCas12b MSEKTTQRAYTLRLNRASGECAVCQNNSCDCWHDALWATHKAVNRGAKAFGDWLLTLRGGLCHTLVEMEVPAK Thermomonas GNNPPQRPTDQERRDRRVLLALSWLSVEDEHGAPKEFIVATGRDSADDRAKKVEEKLREILEKRDFQEHEIDA hydrothermalis WLQDCGPSLKAHIREDAVWVNRRALFDAAVERIKTLTWEEAWDFLEPFFGTQYFAGIGDGKDKDDAEGPARQG RefSeq. EKAKDLVQKAGQWLSARFGIGTGADFMSMAEAYEKIAKWASQAQNGDNGKATIEKLACALRPSEPPTLDTVLK WP_072754838 CISGPGHKSATREYLKTLDKKSTVTQEDLNQLRKLADEDARNCRKKVGKKGKKPWADEVLKDVENSCELTYLQ DNSPARHREFSVMLDHAARRVSMAHSWIKKAEQRRRQFESDAQKLKNLQERAPSAVEWLDRFCESRSMTTGAN TGSGYRIRKRAIEGWSYVVQAWAEASCDTEDKRIAAARKVQADPEIEKFGDIQLFEALAADEAICVWRDQEGT QNPSILIDYVTGKTAEHNQKRFKVPAYRHPDELRHPVFCDFGNSRWSIQFAIHKEIRDRDKGAKQDTRQLQNR HGLKMRLWNGRSMTDVNLHWSSKRLTADLALDQNPNPNPTEVTRADRLGRAASSAFDHVKIKNVFNEKEWNGR LQAPRAELDRIAKLEEQGKTEQAEKLRKRLRWYVSFSPCLSPSGPFIVYAGQHNIQPKRSGQYAPHAQANKGR ARLAQLILSRLPDLRILSVDLGHRFAAACAVWETLSSDAFRREIQGLNVLAGGSGEGDLFLHVEMTGDDGKRR TVVYRRIGPDQLLDNTPHPAPWARLDRQFLIKLQGEDEGVREASNEELWTVHKLEVEVGRTVPLIDRMVRSGF GKTEKQKERLKKLRELGWISAMPNEPSAETDEKEGEIRSISRSVDELMSSALGTLRLALKRHGNRARIAFAMT ADYKPMPGGQKYYFHEAKEASKNDDETKRRDNQIEFLQDALSLWHDLFSSPDWEDNEAKKLWQNHIATLPNYQ TPEEISAELKRVERNKKRKENRDKLRTAAKALAENDQLRQHLHDTWKERWESDDQQWKERLRSLKDWIFPRGK AEDNPSIRHVGGLSITRINTISGLYQILKAFKMRPEPDDLRKNIPQKGDDELENFNRRLLEARDRLREQRVKQ LASRIIEAALGVGRIKIPKNGKLPKRPRTTVDTPCHAVVIESLKTYRPDDLRTRRENRQLMQWSSAKVRKYLK EGCELYGLHFLEVPANYTSRQCSRTGLPGIRCDDVPTGDFLKAPWWRRAINTAREKNGGDAKDRFLVDLYDHL NNLQSKGEALPATVRVPRQGGNLFIAGAQLDDINKERRAIQADLNAAANIGLRALLDPDWRGRWWYVPCKDGT SEPALDRIEGSTAFNDVRSLPTGDNSSRRAPREIENLWRDPSGDSLESGTWSPTRAYWDTVQSRVIELLRRHA GLPTS(SEQIDNO:81) LsCas12b MSIRSFKLKLKTKSGVNAEQLRRGLWRTHQLINDGIAYYMNWLVLLRQEDLFIRNKETNEIEKRSKEEIQAVL Laceyella LERVHKQQQRNQWSGEVDEQTLLQALRQLYEEIVPSVIGKSGNASLKARFFLGPLVDPNNKTTKDVSKSGPTP sacchari KWKKMKDAGDPNWVQEYEKYMAERQTLVRLEEMGLIPLFPMYTDEVGDIHWLPQASGYTRTWDRDMFQQAIER WP_132221894.1 LLSWESWNRRVRERRAQFEKKTHDFASRFSESDVQWMNKLREYEAQQEKSLEENAFAPNEPYALTKKALRGWE RVYHSWMRLDSAASEEAYWQEVATCQTAMRGEFGDPAIYQFLAQKENHDIWRGYPERVIDFAELNHLQRELRR AKEDATFTLPDSVDHPLWVRYEAPGGTNIHGYDLVQDTKRNLTLILDKFILPDENGSWHEVKKVPFSLAKSKQ FHRQVWLQEEQKQKKREVVFYDYSTNLPHLGTLAGAKLQWDRNFLNKRTQQQIEETGEIGKVFFNISVDVRPA VEVKNGRLQNGLGKALTVLTHPDGTKIVTGWKAEQLEKWVGESGRVSSLGLDSLSEGLRVMSIDLGQRTSATV SVFEITKEAPDNPYKFFYQLEGTEMFAVHQRSFLLALPGENPPQKIKQMREIRWKERNRIKQQVDQLSAILRL HKKVNEDERIQAIDKLLQKVASWQLNEEIATAWNQALSQLYSKAKENDLQWNQAIKNAHHQLEPVVGKQISLW RKDLSTGRQGIAGLSLWSIEELEATKKLLTRWSKRSREPGVVKRIERFETFAKQIQHHINQVKENRLKQLANL IVMTALGYKYDQEQKKWIEVYPACQVVLFENLRSYRFSFERSRRENKKLMEWSHRSIPKLVQMQGELFGLQVA DVYAAYSSRYHGRTGAPGIRCHALTEADLRNETNIIHELIEAGFIKEEHRPYLQQGDLVPWSGGELFATLQKP YDNPRILTLHADINAAQNIQKRFWHPSMWFRVNCESVMEGEIVTYVPKNKTVHKKQGKTFRFVKVEGSDVYEW AKWSKNRNKNTFSSITERKPPSSMILFRDPSGTFFKEQEWVEQKTFWGKVQSMIQAYMKKTIVQRMEE(SEQ IDNO:82) DtCas12b MVLGRKDDTAELRRALWTTHEHVNLAVAEVERVLLRCRGRSYWILDRRGDPVHVPESQVAEDALAMAREAQRR Dsulfonatronum NGWPVVGEDEEILLALRYLYEQIVPSCLLDDLGKPLKGDAQKIGTNYAGPLFDSDTCRRDEGKDVACCGPFHE thiodismutans VAGKYLGALPEWATPISKQEFDGKDASHLRFKATGGDDAFFRVSIEKANAWYEDPANQDALKNKAYNKDDWKK WP_031386437 EKDKGISSWAVKYIQKQLQLGQDPRTEVRRKLWLELGLLPLFIPVEDKIMVGNLWNRLAVRLALAHLLSWESW NHRAVQDQALARAKRDELAALFLGMEDGFAGLREYELRRNESIKQHAFEPVDRPYVVSGRALRSWTRVREEWL RHGDTQESRKNICNRLQDRLRGKFGDPDVFHWLAEDGQEALWKERDCVTSFSLLNDADGLLEKRKGYALMTFA DARLHPRWAMYEAPGGSNLRTYQIRKTENGLWADVVLLSPRNESAAVEEKTENVRLAPSGQLSNVSFDQIQKG SKMVGRCRYQSANQQFEGLLGGAEILFDRKRIANEQHGATDLASKPGHVWFKLTLDVRPQAPQGWLDGKGRPA LPPEAKHFKTALSNKSKFADQVRPGLRVLSVDLGVRSFAACSVFELVRGGPDQGTYFPAADGRTVDDPEKLWA KHERSFKITLPGENPSRKEEIARRAAMEELRSLNGDIRRLKAILRLSVLQEDDPRTEHLRLFMEAIVDDPAKS ALNAELFKGFGDDRFRSTPDLWKQHCHFFHDKAEKVVAERFSRWRTETRPKSSSWQDWRERRGYAGGKSYWAV TYLEAVRGLILRWNMRGRTYGEVNRQDKKQFGTVASALLHHINQLKEDRIKTGADMIIQAARGFVPRKNGAGW VQVHEPCRLILFEDLARYRFRIDRSRRENSRLMRWSHREIVNEVGMQGELYGLHVDTTEAGESSRYLASSGAP GVRCRHLVEEDFHDGLPGMHLVGELDWLLPKDKDRTANEARRLLGGMVRPGMLVPWDGGELFATLNAASQLHV IHADINAAQNLQRRFWGRCGEAIRIVCNQLSVDGSTRYEMAKAPKARLLGALQQLKNGDAPFHLTSIPNSQKP ENSYVMTPTNAGKKYRAGPGEKSSGEEDELALDIVEQAEELAQGRKTFFRDPSGVFFAPDRWLPSEIYWSRIR RRIWQVTLERNSSGRQERAEMDEMPY(SEQIDNO:83)
[0449] The base editors described herein may also comprise Cas12a/Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cas12a/Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity.
(8) Cas9 Equivalents with Expanded PAM Sequence
[0450] In some embodiments, the napDNAbp is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5 phosphorylated ssDNA of 24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 2016 Jul;34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference.
[0451] In some embodiments, the napDNAbp is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., et al., Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements, Biol Direct. 2009 Aug. 25; 4:29. doi: 10.1186/1745-6150-4-29, the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein cleaves single-stranded target sequences using 5-phosphorylated guides. The 5 guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5 phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5-hydroxylated guide. See, e.g., Kaya et al., A bacterial Argonaute with noncanonical guide RNA specificity, Proc Natl Acad Sci USA. 2016 Apr. 12; 113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other argonaute proteins may be used, and are within the scope of this disclosure.
[0452] In some embodiments, the napDNAbp is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, C2c1, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (C2c1, C2c2, and C2c3) have been described by Shmakov et al., Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2c1 and C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system, C2c2 contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single-stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpf1. See, e.g., East-Seletsky, et al., Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection, Nature, 2016 Oct. 13;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science, 2016 Aug. 5; 353(6299), the entire contents of which are hereby incorporated by reference.
[0453] The crystal structure of Alicyclobaccillus acidoterrastris C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism, Mol. Cell, 2017 Jan. 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2c1 bound to target DNAs as ternary complexes. See e.g., Yang et al., PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease, Cell, 2016 Dec. 15;167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.
[0454] In some embodiments, the napDNAbp may be a C2c1, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2c1 protein. In some embodiments, the napDNAbp is a C2c2 protein. In some embodiments, the napDNAbp is a C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2c1, C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring C2c1, C2c2, or C2c3 protein.
[0455] Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region (e.g., a editing window), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., Engineered CRISPR-Cas9 nucleases with altered PAM specificities Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
[0456] For example, a napDNAbp domain with altered PAM specificity, such as a domain with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Francisella novicida Cpf1 (SEQ ID NO: 84) (D917, E1006, and D1255), which has the following amino acid sequence:
TABLE-US-00037 (SEQIDNO:84) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT SEVNQRVFSLDEVFEIANFNNYLNQSGITKENTIIGGKFVNGENTKRKGI NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT TMQSFYEQIAAFKTVEEKSIKETLSLLEDDLKAQKLDLSKIYFKNDKSLT DLSQQVEDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQINNLLHKLKIFHISQSED KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKENDEI NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKIGG VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR LINERNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLISVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN
[0457] An additional napDNAbp domain with altered PAM specificity, such as a domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Geobacillus thermodenitrificans Cas9 (SEQ ID NO: 85), which has the following amino acid sequence:
TABLE-US-00038 (SEQIDNO:85) MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPR RLARSARRRLRRRKHRLERIRRLFVREGILTKEELNKLFEKKHEIDVWQL RVEALDRKLNNDELARILLHLAKRRGERSNRKSERINKENSTMLKHIEEN QSILSSYRTVAEMVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAK QREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTFEPKEKRAP KATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFH DVRILLNLPDDTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDSVY GKGAAKSFRPIDFDTFGYALTMEKDDTDIRSYLRNEYEQNGKRMENLADK VYDEELIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYSTACERAGYTF TGPKKKQKTVLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIE LARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLILNPTGLDIVKF KLWSEQNGKCAYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLV LIKENREKGNRTPAEYLGLGSERWQQFETFVLINKQFSKKKRDRLLRLHY DENEENEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVNGRIT AHLRSRWNENKNREESNLHHAVDAAIVACTTPSDIARVTAFYQRREQNKE LSKKTDPQFPQPWPHFADELQARLSKNPKESIKALNLGNYDNEKLESLQP VFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQLDKTG HFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGELGPIIR TIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTIDMMK GILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAV GEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSRTLKRFEKYQV DVLGNIYKVRGEKRVGVASSSHSKAGETIRPL
[0458] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5 phosphorylated ssDNA of 24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 34(7): 768-73 (2016), PubMed PMID: 27136078; Swarts et al., Nature, 507(7491): 258-61 (2014); and Swarts et al., Nucleic Acids Res. 43(10) (2015): 5120-9, each of which is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 63.
[0459] The disclosed fusion proteins may comprise a napDNAbp domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 86), which has the following amino acid sequence:
TABLE-US-00039 (SEQIDNO:86) MTVIDLDSTTTADELTSGHTYDISVTLIGVYDNTDEQHPRMS LAFEQDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNID YEVKDGYENLTATYQTTVENATAQEVGTTDEDETFAGGEPLD HHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLHTY TLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLM LLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLARE LVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDL SVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVK TTYRPRRGHIVWGLRDECATDSLNTLGNQSVVAYHRNNQTPI NTDLLDAIEAADRRVVETRROGHGDDAVSFPQELLAVEPNT HQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRL NGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAH PDETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLN QAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVDAAFV VLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFR DAKIFYTRNVALGLLAAAGGVAFTTEHAMPGDADMFIGIDV SRSYPEDGASGQINIAATATAVYKDGTILGHSSTRPQLGEKL QSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDP ATEFLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAA INQNEPRATVATFGAPEYLATRDGGGLPRPIQIERVAGETD IETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKG YLVQTGAFESNVGFL
(9) Cas9 Circular Permutants
[0460] In various embodiments, the base editors disclosed herein may comprise a circular permutant of Cas9.
[0461] The term circularly permuted Cas9 or circular permutant of Cas9 or CP-Cas9) refers to any Cas9 protein, or variant thereof, that occurs or has been modify to engineered as a circular permutant variant, which means the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein) have been topically rearranged. Such circularly permuted Cas9 proteins, or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al., Protein Engineering of Cas9 for enhanced function, Methods Enzymol, 2014, 546: 491-511 and Oakes et al., CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification, Cell, Jan. 10, 2019, 176: 254-267, each of are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA).
[0462] Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.
[0463] In various embodiments, the circular permutants of Cas9 may have the following structure: [0464] N-terminus-[original C-terminus]-[optional linker]-[original N-terminus]-C-terminus.
[0465] As an example, the present disclosure contemplates the following circular permutants of canonical S. pyogenes Cas9 (1368 amino acids of UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 28)): [0466] N-terminus-[1268-1368]-[optional linker]-[1-1267]-C-terminus; [0467] N-terminus-[1168-1368]-[optional linker]-[1-1167]-C-terminus; [0468] N-terminus-[1068-1368]-[optional linker]-[1-1067]-C-terminus; [0469] N-terminus-[968-1368]-[optional linker]-[1-967]-C-terminus; [0470] N-terminus-[868-1368]-[optional linker]-[1-867]-C-terminus; [0471] N-terminus-[768-1368]-[optional linker]-[1-767]-C-terminus; [0472] N-terminus-[668-1368]-[optional linker]-[1-667]-C-terminus; [0473] N-terminus-[568-1368]-[optional linker]-[1-567]-C-terminus; [0474] N-terminus-[468-1368]-[optional linker]-[1-467]-C-terminus; [0475] N-terminus-[368-1368]-[optional linker]-[1-367]-C-terminus; [0476] N-terminus-[268-1368]-[optional linker]-[1-267]-C-terminus; [0477] N-terminus-[168-1368]-[optional linker]-[1-167]-C-terminus; [0478] N-terminus-[68-1368]-[optional linker]-[1-67]-C-terminus; or [0479] N-terminus-[10-1368]-[optional linker]-[1-9]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
[0480] In particular embodiments, the circular permuant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 28): [0481] N-terminus-[102-1368]-[optional linker]-[1-101]-C-terminus; [0482] N-terminus-[1028-1368]-[optional linker]-[1-1027]-C-terminus; [0483] N-terminus-[1041-1368]-[optional linker]-[1-1043]-C-terminus; [0484] N-terminus-[1249-1368]-[optional linker]-[1-1248]-C-terminus; or [0485] N-terminus-[1300-1368]-[optional linker]-[1-1299]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
[0486] In still other embodiments, the circular permuant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 28): [0487] N-terminus-[103-1368]-[optional linker]-[1-102]-C-terminus; [0488] N-terminus-[1029-1368]-[optional linker]-[1-1028]-C-terminus; [0489] N-terminus-[1042-1368]-[optional linker]-[1-1041]-C-terminus; [0490] N-terminus-[1250-1368]-[optional linker]-[1-1249]-C-terminus; or [0491] N-terminus-[1301-1368]-[optional linker]-[1-1300]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
[0492] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, The C-terminal fragment may correspond to the C-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1300-1368), or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., any one of SEQ ID NO: 28, 8, 10, 12-26). The N-terminal portion may correspond to the N-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1-1300), or the N-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., of SEQ ID NO: 28, 8, 10, 12-26).
[0493] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 28). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas9 (e.g., the Cas9 of SEQ ID NO: 28). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., the Cas9 of SEQ ID NO: 28). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 5). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 28).
[0494] In other embodiments, circular permutant Cas9 variants may be defined as a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 28: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to preceed the original N-terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (relative the S. pyogenes Cas9 of SEQ ID NO: 28) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 would become the new N-terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP.sup.181, Cas9-CP.sup.199, Cas9-CP.sup.230, Cas9-CP.sup.270, Cas9-CP.sup.310, Cas9-CP.sup.1010, Cas9-CP.sup.1016, Cas9-CP.sup.1023, Cas9-CP.sup.1029, Cas9-CP.sup.1041, Cas9-CP.sup.1247, Cas9-CP.sup.1249, and Cas9-CP.sup.1282, respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 28, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entireley. This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant.
[0495] Exemplary CP-Cas9 amino acid sequences, based on the Cas9 of SEQ ID NO: 28, are provided below in which linker sequences are indicated by underlining and optional methionine (M) residues are indicated in bold. It should be appreciated that the disclosure provides CP-Cas9 sequences that do not include a linker sequence or that include different linker sequences. It should be appreciated that CP-Cas9 sequences may be based on Cas9 sequences other than that of SEQ ID NO: 28 and any examples provided herein are not meant to be limiting. Exemplary CP-Cas9 sequences are as follows:
TABLE-US-00040 CPname Sequence SEQIDNO: CP1012 DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN SEQIDNO:87 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE VLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGDKKYSIGL AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINEDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV TVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLILTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLING IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA KLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYG CP1028 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT SEQIDNO:88 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDE YKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC FDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK VYDVRKMIAKSEQ CP1041 NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV SEQIDNO:89 KKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGG SGGSGGSGGSGGSGGSGGDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLIPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAIL RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDREN ASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLE DDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDELKSDGFANRNEMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN EKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE IGKATAKYFFYS CP1249 PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR SEQIDNO:90 EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYET RIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEY KVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECE DSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDELKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNE LYLASHYEKLKGS CP1300 KPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG SEQIDNO:91 LYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGDKKYSIGLAIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRENASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFE DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF LKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL DKVLSAYNKHRD
[0496] The Cas9 circular permutants that may be useful in the base editing constructs described herein. Exemplary C-terminal fragments of Cas9, based on the Cas9 of SEQ ID NO: 28, which may be rearranged to an N-terminus of Cas9, are provided below. It should be appreciated that such C-terminal fragments of Cas9 are exemplary and are not meant to be limiting. These exemplary CP-Cas9 fragments have the following sequences:
TABLE-US-00041 CPname Sequence SEQIDNO: CP1012C- DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN 92 terminal GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA fragment RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKE VLDATLIHQSITGLYETRIDLSQLGGD CP1028C- EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFAT 93 terminal VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGEDSP fragment TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGD CP1041C- NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV 94 terminal KKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE fragment KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD CP1249C- PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR 95 terminal EQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYET fragment RIDLSQLGGD CP1300C- KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG 96 terminal LYETRIDLSQLGGD fragment
Cas9 Variants with Modified PAM Specificities
[0497] The base editors of the present disclosure may also comprise Cas9 variants with modified PAM specificities. For example, the base editors described herein may utilize any naturally occurring or engineered variant of SpCas9 having expanded and/or relaxed PAM specificities which are described in the literate, including in Nishimasu et al., Engineered CRISPR-Cas9 nuclease with expanded targeting space, Science, 2018, 361: 1259-1262; Chatterjee et al., Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants, BioRxiv, Apr. 26, 2019 Some aspects of this disclosure provide Cas9 proteins that exhibit activity on a target sequence that does not comprise the canonical PAM (5-NGG-3, where N is A, C, G, or T) at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NGG-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NNG-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NNA-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NNC-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NNT-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NGT-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NGA-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NGC-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NAA-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NAC-3 PAM sequence at its 3-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NAT-3 PAM sequence at its 3-end. In still other embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5-NAG-3 PAM sequence at its 3-end.
[0498] It should be appreciated that any of the amino acid mutations described herein, (e.g., A262T) from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) may also include mutations from the first amino acid residue to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue. For example, mutation of an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) may be a mutation to a second amino acid with a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation of an alanine to a threonine (e.g., a A262T mutation) may also be a mutation from an alanine to an amino acid that is similar in size and chemical properties to a threonine, for example, serine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a different polar side chain (e.g., serine, threonine, asparagine, or glutamine). Additional similar amino acid pairs include, but are not limited to, the following: phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine. The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a threonine may be an amino acid mutation to a serine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an arginine may be an amino acid mutation to a lysine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a lysine may be an amino acid mutation to an arginine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.
[0499] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5-NAA-3 PAM sequence at its 3-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table A.
1.
TABLE-US-00042 TABLE A NAA PAM Clones Mutations from wild-type SpCas9 (e.g., SEQ ID NO: 28) D177N, K218R, D614N, D1135N, P1137S, E1219V, A1320V, A1323D, R1333K D177N, K218R, D614N, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, $409I, E427G, G715C, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K A367T, K710E, R1114G, D1135N, P1137S, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R753G, D861N, D1135N, K1188R, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, V743I, R753G, E762G, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R753G, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, S1274R, A1320V, R1333K A10T, I322V, S409I, E427G, A589S, R753G, D1135N, E1219V, Q1221H, H1264H, A1320V, R1333K A10T, I322V, S409I, E427G, R753G, E757K, G865G, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, R753G, E757K, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, K599R, M631A, R654L, K673E, V743I, R753G, N758H, E762G, D1135N, D1180G, E1219V, Q1221H, Q1256R, H1264Y, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N869S, N1054D, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, L727I, V743I, R753G, E762G, R859S, N946D, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, N1317T, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, V1290G, L1318S, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, K1151E, D1180G, E1219V, Q1221H, H1264Y, V1290G, L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, V1290G, L1318S, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, N803S, N869S, L921P, Y1016D, G1077D, F1080S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, E630K, R654L, K673E, V743I, R753G, E762G, Q768H, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, Q768H, N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, G1223S, H1264Y, L1318S, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, N803S, N869S, L921P, Y1016D, G1077D, F1801S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y, L1318S, A1320V, A1323D, R1333K A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R, E1219V, Q1221H, H1264Y, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, M673I, N803S, N869S, G1077D, R1114G, D1135N, V1139A, D1180G, E1219V, Q1221H, A1320V, R1333K A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S, R1114G, D1135N, E1219V, Q1221H, A1320V, R1333K
[0500] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table A.
[0501] In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5-NGG-3) at its 3 end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 28. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3 end that is not directly adjacent to the canonical PAM sequence (5-NGG-3) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 28 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5-NGG-3) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 28 on the same target sequence. In some embodiments, the 3 end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5-NAC-3 PAM sequence at its 3-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table B.
TABLE-US-00043 TABLE B NAC PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO: 28) T472I, R753G, K890E, D1332N, R1335Q, T1337N I1057S, D1135N, P1301S, R1335Q, T1337N T472I, R753G, D1332N, R1335Q, T1337N D1135N, E1219V, D1332N, R1335Q, T1337N T472I, R753G, K890E, D1332N, R1335Q, T1337N I1057S, D1135N, P1301S, R1335Q, T1337N T472I, R753G, D1332N, R1335Q, T1337N T472I, R753G, Q771H, D1332N, R1335Q, T1337N E627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, K1156E, E1219V, D1332N, R1335Q, T1337N E627K, T638P, V647I, R753G, N803S, K959N, G1030R, I1055E, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K, E630G, T638P, V647A, G687R, N767D, N803S, K959N, R1114G, D1135N, E1219V, D1332G, R1335Q, T1337N E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N E627K, T638P, R753G, N803S, K959N, I1057T, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N E627K, M631I, T638P, R753G, N803S, K959N, Y1036H, R1114G, D1135N, E1219V, D1251G, D1332G, R1335Q, T1337N E627K, T638P, R753G, N803S, V875I, K959N, Y1016C, R1114G, D1135N, E1219V, D1251G, D1332G, R1335Q, T1337N, I1348V K608R, E627K, T638P, V647I, R654L, R753G, N803S, T804A, K848N, V922A, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N K608R, E627K, T638P, V647I, R753G, N803S, V922A, K959N, K1014N, V1015A, R1114G, D1135N, K1156N, E1219V, N1252D, D1332N, R1335Q, T1337N K608R, E627K, R629G, T638P, V647I, A711T, R753G, K775R, K789E, N803S, K959N, V1015A, Y1036H, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N K608R, E627K, T638P, V647I, T740A, R753G, N803S, K948E, K959N, Y1016S, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N K608R, E627K, T638P, V647I, T740A, N803S, K948E, K959N, Y1016S, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N I670S, K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, K797N, N803S, K866R, K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N K608R, E627K, T638P, V647I, T740A, G752R, R753G, K797N, N803S, K948E, K959N, V1015A, Y1016S, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N I570T, A589V, K608R, E627K, T638P, V647I, R654L, Q716R, R753G, N803S, K948E, K959N, Y1016S, R1114G, D1135N, E1207G, E1219V, N1234D, D1332N, R1335Q, T1337N K608R, E627K, R629G, T638P, V647I, R654L, Q740R, R753G, N803S, K959N, N990S, T995S, V1015A, Y1036D, R1114G, D1135N, E1207G, E1219V, N1234D, N1266H, D1332N, R1335Q, T1337N I562F, V565D, I570T, K608R, L625S, E627K, T638P, V647I, R654I, G752R, R753G, N803S, N808D, K959N, M1021L, R1114G, D1135N, N1177S, N1234D, D1332N, R1335Q, T1337N I562F, I570T, K608R, E627K, T638P, V647I, R753G, E790A, N803S, K959N, V1015A, Y1036H, R1114G, D1135N, D1180E, A1184T, E1219V, D1332N, R1335Q, T1337N I570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N, V1015A, R1114G, D1127A, D1135N, E1219V, D1332N, R1335Q, T1337N I570T, K608R, L625S, E627K, T638P, V647I, R654I, T703P, R753G, N803S, N808D, K959N, M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N I570S, K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, N803S, K866R, K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N I570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N, V1016A, R1114G, D1135N, E1219V, K1246E, D1332N, R1335Q, T1337N K608R, E627K, T638P, V647I, R654L, K673E, R753G, E790A, N803S, K948E, K959N, R1114G, D1127G, D1135N, D1180E, E1219V, N1286H, D1332N, R1335Q, T1337N K608R, L625S, E627K, T638P, V647I, R654I, I670T, R753G, N803S, N808D, K959N, M1021L, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N E627K, M631V, T638P, V647I, K710E, R753G, N803S, N808D, K948E, M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, H1349R
[0502] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table B.
[0503] In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5-NGG-3) at its 3 end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 28. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3 end that is not directly adjacent to the canonical PAM sequence (5-NGG-3) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 28 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5-NGG-3) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 28 on the same target sequence. In some embodiments, the 3 end of the target sequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.
[0504] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5-NAT-3 PAM sequence at its 3-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table C.
TABLE-US-00044 TABLE C NAT PAM Clones MUTATIONS FROM WILD-TYPE SPCAS9 (E. G., SEQ ID NO: 28) K961E, H985Y, D1135N, K1191N, E1219V, Q1221H, A1320A, P1321S, R1335L D1135N, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L V743I, R753G, E790A, D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I F575S, M631L, R654L, V748I, V743I, R753G, D853E, V922A, R1114G D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, N1286K, P1321S, D1322G, R1335L M631L, R654L, R753G, K797E, D853E, V922A, D1012A, R1114G D1135N, G1218S, E1219V, Q1221H, P1249S, N1317K, P1321S, D1322G, R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, D596Y, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, Q1256R, P1321S, D1322G, R1335L F575S, M631L, R654L, R664K, K710E, V750A, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, K649R, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, K1156E, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L F575S, M631L, R654L, R664K, R753G, D853E, V922A, I1057G, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, N1308D, P1321S, D1322G, R1335L M631L, R654L, R753G, D853E, V922A, R1114G, Y1131C, D1135N, E1150V, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L M631L, R654L, R664K, R753G, D853E, I1057V, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L M631L, R654L, R664K, R753G, I1057V, R1114G, Y1131C, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L
[0505] The above description of various napDNAbps which can be use in connection with the presently disclose base editors is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 proteinincluding any naturally occurring variant, mutant, or otherwise engineered version of Cas9that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are dead Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). The base editors described herein may also comprise Cas9 equivalents, including Cas12a/Cpf1 and Cas12b proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequences or a reference Cas9 equivalent (e.g., Cas12a/Cpf1).
[0506] In a particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRQR, having the following amino acid sequence (with the V, R, Q, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 97 show in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRQR) (SpCas9-VRQR). This SpCas9 variant possesses an altered PAM-specificity which recognizes a PAM of 5-NGA-3 instead of the canonical PAM of 5-NGG-3:
TABLE-US-00045 SpCas9-VRQR (SEQIDNO:97) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE IFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRK LINGIRDKQSGKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLNAVVGTAL IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLK GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYEDT TIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD
[0507] In another particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VQR, having the following amino acid sequence (with the V, Q, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 98 show in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRQR) (SpCas9-VQR). This SpCas9 variant possesses an altered PAM-specificity which recognizes a PAM of 5-NGA-3 instead of the canonical PAM of 5-NGG-3:
TABLE-US-00046 SpCas9-VOR (SEQIDNO:98) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE IFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMIRKSEETITPWNFEEVVDKGASAQSFIERMINFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRK LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAL IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLK GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDT TIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD
[0508] In another particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRER, having the following amino acid sequence (with the V, R, E, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 99 are shown in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRER) (SpCas9-VRER). This SpCas9 variant possesses an altered PAM-specificity which recognizes a PAM of 5-NGCG-3 instead of the canonical PAM of 5-NGG-3:
TABLE-US-00047 SpCas9-VRER (SEQIDNO:99) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE IFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRK LINGIRDKQSGKTILDFLKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLNAVVGTAL IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLK GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLETLTNLGAPAAFKYFDT TIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD
[0509] In yet particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9-NG, as reported in Nishimasu et al., Engineered CRISPR-Cas9 nuclease with expanded targeting space, Science, 2018, 361: 1259-1262, which is incorporated herein by reference. SpCas9-NG (VRVRFRR), having the following amino acid sequence substitutions: R1335V, L1111R, D1135V, G1218R, E1219F, A1322R, and T1337R relative to the canonical SpCas9 sequence (SEQ ID NO: 28. This SpCas9 has a relaxed PAM specificity, i.e., with activity on a PAM of NGH (wherein H=A, T, or C). See Nishimasu et al., Engineered CRISPR-Cas9 nuclease with expanded targeting space, Science, 2018, 361: 1259-1262, which is incorporated herein by reference.
TABLE-US-00048 SpCas9-NG (SEQIDNO:100) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQ EIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLILFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLNAVVGTA LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIEINGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPRAFKYFD TTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD
[0510] In addition, any available methods may be utilized to obtain or construct a variant or mutant Cas9 protein. The term mutation, as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include loss-of-function mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. Mutations also embrace gain-of-function mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.
[0511] Mutations can be introduced into a reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3 end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation. More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.
[0512] Mutations may also be introduced by directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted noncontinuous evolution (PANCE). The term phage-assisted continuous evolution (PACE), as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Application, U.S. Pat. No. 9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference. Variant Cas9s may also be obtain by phage-assisted non-continuous evolution (PANCE), which as used herein, refers to non-continuous evolution that employs phage as viral vectors. PANCE is a simplified technique for rapid in vivo directed evolution using serial flask transfers of evolving selection phage (SP), which contain a gene of interest to be evolved, across fresh E. coli host cells, thereby allowing genes inside the host E. coli to be held constant while genes contained in the SP continuously evolve. Serial flask transfers have long served as a widely-accessible approach for laboratory evolution of microbes, and, more recently, analogous approaches have been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.
[0513] Any of the references noted above which relate to Cas9 or Cas9 equivalents are hereby incorporated by reference in their entireties, if not already stated so.
Linkers
[0514] In certain embodiments, linkers may be used to link any of the peptides or peptide domains or moieties of the invention (e.g., a mitoTALE fused to a DddA).
[0515] As defined above, the term linker, as used herein, refers to a chemical group or a molecule linking two molecules or moieties (e.g., a binding domain (e.g., mitoTALE) and a editing domain (e.g., DddA, or portion thereof)). In some embodiments, a linker joins a binding domain (e.g., mitoTALE) and a catalytic domain (e.g., DddA, or portion thereof). In some embodiments, a linker joins a mitoTALE and DddA. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 1-100 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer linkers are also contemplated.
[0516] The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
[0517] In some other embodiments, the linker comprises the amino acid sequence is greater than one amino acid residues in length. In some embodiments, the linker comprises less than six amino acid in length. In some embodiments, the linker is two amino acid residues in length. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs.: 101-117.
[0518] In certain embodiments, linkers may be used to link any of the protein or protein domains described herein (e.g., a deaminase domain and a Cas9 domain). The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
[0519] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 101), which may also be referred to as the XTEN linker. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS).sub.2-SGSETPGTSESATPES-(SGGS).sub.2 (SEQ ID NO: 102), which may also be referred to as (SGGS).sub.2XTEN-(SGGS).sub.2 (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 104). In some embodiments, a linker comprises (SGGS).sub.n(SEQ ID NO: 104), (GGGS).sub.n (SEQ ID NO: 105), (GGGGS).sub.n(SEQ ID NO: 106), (G).sub.n (SEQ ID NO: 107), (EAAAK).sub.n (SEQ ID NO: 108), (SGGS).sub.n-SGSETPGTSESATPES-(SGGS).sub.n(SEQ ID NO: 109), (GGS).sub.n (SEQ ID NO: 110), SGSETPGTSESATPES (SEQ ID NO: 101), or (XP). (SEQ ID NO: 111) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, a linker comprises SGSETPGTSESATPES (SEQ ID NO: 101), and SGGS (SEQ ID NO: 104). In some embodiments, a linker comprises SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 109). In some embodiments, a linker comprises SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 112). In some embodiments, a linker comprises GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 113). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 114). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 115). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSG GS (SEQ ID NO: 116). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 117). It should be appreciated that any of the linkers provided herein may be used to link a first adenosine deaminase and a second adenosine deaminase; an adenosine deaminase (e.g., a first or a second adenosine deaminase) and a napDNAbp; a napDNAbp and an NLS; or an adenosine deaminase (e.g., a first or a second adenosine deaminase) and an NLS.
[0520] In some embodiments, any of the fusion proteins provided herein, comprise an adenosine or a cytidine deaminase and a napDNAbp that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise a first adenosine deaminase and a second adenosine deaminase that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise an NLS, which may be fused to an adenosine deaminase (e.g., a first and/or a second adenosine deaminase), a nucleic acid programmable DNA binding protein (napDNAbp). Various linker lengths and flexibilities between an adenosine deaminase (e.g., an engineered ecTadA) and a napDNAbp (e.g., a Cas9 domain), and/or between a first adenosine deaminase and a second adenosine deaminase can be employed (e.g., ranging from very flexible linkers of the form (GGGGS).sub.n (SEQ ID NO: 106), (GGGGS).sub.n (SEQ ID NO: 106), and (G).sub.n (SEQ ID NO: 107) to more rigid linkers of the form (EAAAK).sub.n (SEQ ID NO: 108), (SGGS).sub.n(SEQ ID NO: 104), SGSETPGTSESATPES (SEQ ID NO: 101) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP). (SEQ ID NO: 111)) in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS).sub.n(SEQ ID NO: 110) motif, wherein n is 1, 3, or 7. In some embodiments, the adenosine deaminase and the napDNAbp, and/or the first adenosine deaminase and the second adenosine deaminase of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 101), SGGS (SEQ ID NO: 104), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 109), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 102), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 113). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 114). In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence (SGGS).sub.2SGSETPGTSESATPES-(SGGS).sub.2 (SEQ ID NO: 102), which may also be referred to as (SGGS).sub.2XTEN-(SGGS).sub.2 (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 115). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSG GS (SEQ ID NO: 116). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 117).
Uracil Glycosylase Inhibitor (UGI)
[0521] In some embodiments, the fusion proteins of the disclosure comprises a UGI. When the DddA enzyme is employed and deaminates the target nucleotide, it may trigger uracil repair activity in the cell, thereby causing excision of the deaminated nucleotide. This may cause degradation of the nucleic acid or otherwise inhibit the effect of the correction or nucleotide alteration induced by the fusion protein. To inhibit this activity, a UGI may be desired. In some embodiments, the first and/or second fusion protein comprises more than one UGI. In some embodiments, the first and/or second fusion protein comprises two UGIs. In some embodiments, the first and/or second fusion protein contains two UGIs. The UGI or multiple UGIs may be appended or attached to any portion of the fusion protein. In some embodiments, the UGI is attached to the first or second portion of a DddA in the first or second fusion protein. In some embodiments, a second UGI is attached to the first UGI which is attached to the first or second portion of a DddA in the first or second fusion protein.
[0522] In other embodiments, the base editors described herein may comprise one or more uracil glycosylase inhibitors. The term uracil glycosylase inhibitor or UGI, as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 118. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 118. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 118, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as UGI variants. A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 118. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 118. In some embodiments, the UGI comprises the following amino acid sequence:
Uracil-DNA Glycosylase Inhibitor:
TABLE-US-00049 >sp|P14739|UNGI_BPPB2 (SEQIDNO:118) MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDE STDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.
[0523] The base editors described herein may comprise more than one UGI domain, which may be separated by one or more linkers as described herein. It will also be understood that in the context of the herein disclosed base editors, the UGI domain may be linked to a deaminase domain.
[0524] In some embodiments, a UGI is absent from a base editor. In some embodiments, where a base editor comprises a ZFP or mitoZFP, UGIs are removed or are absent from the base editor. In some embodiments, the removal and/or absence of UGIs increases the activity of a DddA.
NLS Domains
[0525] In various embodiments, the fusion proteins may comprise one or more nuclear localization sequences (NLS), which help promote translocation of a protein into the cell nucleus. Such sequences are well-known in the art and can include the following examples:
TABLE-US-00050 SEQUENCE DESCRIPTION SEQUENCE IDENTIFIER NLSOFSV40 PKKKRKV 119 LARGET-AG NLSOF VSRKRPRP 120 POLYOMA LARGET-AG NLSOFC- PAAKRVKLD 121 MYC NLSOFTUS- KLKIKRPVK 122 PROTEIN NLSOF EGAPPAKRAR 123 HEPATITISD VIRUS ANTIGEN NLSOF PPQPKKKPLDGE 124 MURINEP53 NLS MKRTADGSEFESPKKKRKV 125 NLSOF AVKRPAATKKAGQAKKKKLD 126 NUCLEOPLAS MIN NLSOFPE1 SGGSKRTADGSEFEPKKKRKV 127 ANDPE2 NLSOFEGL- MSRRRKANPTKLSENAKKLAKEV 128 13 EN NLS MDSLLMNRRKFLYQFKNVRWAKG 129 RRETYLC
[0526] The NLS examples above are non-limiting. The PE fusion proteins may comprise any known NLS sequence, including any of those described in Cokol et al., Finding nuclear localization signals, EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., Mechanisms and Signals for the Nuclear Import of Proteins, Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference.
Split-Intein Domains
[0527] It will be understood that in some embodiments (e.g., delivery of a base editor in vivo using AAV particles), it may be advantageous to split a polypeptide (e.g., a deaminase or a napDNAbp) or a fusion protein (e.g., a base editor) into an N-terminal half and a C-terminal half, delivery them separately, and then allow their colocalization to reform the complete protein (or fusion protein as the case may be) within the cell. Separate halves of a protein or a fusion protein may each comprise a split-intein tag to facilitate the reformation of the complete protein or fusion protein by the mechanism of protein trans splicing.
[0528] Protein trans-splicing, catalyzed by split inteins, provides an entirely enzymatic method for protein ligation. A split-intein is essentially a contiguous intein (e.g. a mini-intein) split into two pieces named N-intein and C-intein, respectively. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction essentially in same way as a contiguous intein does. Split inteins have been found in nature and also engineered in laboratories. As used herein, the term split intein refers to any intein in which one or more peptide bond breaks exists between the N-terminal and C-terminal amino acid sequences such that the N-terminal and C-terminal sequences become separate molecules that can non-covalently reassociate, or reconstitute, into an intein that is functional for trans-splicing reactions. Any catalytically active intein, or fragment thereof, may be used to derive a split intein for use in the methods of the invention. For example, in one aspect the split intein may be derived from a eukaryotic intein. In another aspect, the split intein may be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, the split intein so-derived will possess only the amino acid sequences essential for catalyzing trans-splicing reactions.
[0529] As used herein, the N-terminal split intein (In) refers to any intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions. An In thus also comprises a sequence that is spliced out when trans-splicing occurs. An In can comprise a sequence that is a modification of the N-terminal portion of a naturally occurring intein sequence. For example, an In can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the In.
[0530] As used herein, the C-terminal split intein (Ic) refers to any intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions. In one aspect, the Ic comprises 4 to 7 contiguous amino acid residues, at least 4 amino acids of which are from the last -strand of the intein from which it was derived. An Ic thus also comprises a sequence that is spliced out when trans-splicing occurs. An Ic can comprise a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, an Ic can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the Ic.
[0531] In some embodiments of the invention, a peptide linked to an Ic or an In can comprise an additional chemical moiety including, among others, fluorescence groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, a peptide linked to an Ic can comprise one or more chemically reactive groups including, among others, ketone, aldehyde, Cys residues and Lys residues. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction when an intein-splicing polypeptide (ISP) is present. As used herein, intein-splicing polypeptide (ISP) refers to the portion of the amino acid sequence of a split intein that remains when the Ic, In, or both, are removed from the split intein. In certain embodiments, the In comprises the ISP. In another embodiment, the Ic comprises the ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to In nor to Ic.
[0532] Split inteins may be created from contiguous inteins by engineering one or more split sites in the unstructured loop or intervening amino acid sequence between the12 conserved beta-strands found in the structure of mini-inteins. Some flexibility in the position of the split site within regions between the beta-strands may exist, provided that creation of the split will not disrupt the structure of the intein, the structured beta-strands in particular, to a sufficient degree that protein splicing activity is lost.
[0533] In protein trans-splicing, one precursor protein consists of an N-extein part followed by the N-intein, another precursor protein consists of the C-intein followed by a C-extein part, and a trans-splicing reaction (catalyzed by the N- and C-inteins together) excises the two intein sequences and links the two extein sequences with a peptide bond. Protein trans-splicing, being an enzymatic reaction, can work with very low (e.g. micromolar) concentrations of proteins and can be carried out under physiological conditions.
RNA-Protein Recruitment System
[0534] In various embodiments, two separate protein domains (e.g., a Cas9 domain and a double-stranded deaminase domain) may be colocalized to one another to form a functional complex (akin to the function of a fusion protein comprising the two separate protein domains) by using an RNA-protein recruitment system, such as the MS2 tagging technique. Such systems generally tag one protein domain with an RNA-protein interaction domain (aka RNA-protein recruitment domain) and the other with an RNA-binding protein that specifically recognizes and binds to the RNA-protein interaction domain, e.g., a specific hairpin structure. These types of systems can be leveraged to colocalize the domains of a base editor, as well as to recruitment additional functionalities to a base editor, such as a UGI domain. In one example, the MS2 tagging technique is based on the natural interaction of the MS2 bacteriophage coat protein (MCP or MS2cp) with a stem-loop or hairpin structure present in the genome of the phage, i.e., the MS2 hairpin. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). Thus, in one exemplarly scenario a deaminase-MS2 fusion can recruit a Cas9-MCP fusion.
[0535] A review of other modular RNA-protein interaction domains are described in the art, for example, in Johansson et al., RNA recognition by the MS2 phage coat protein, Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., Organization of intracellular reactions with rationally designed RNA assemblies, Science, 2011, Vol. 333: 470-474; Mali et al., Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering, Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the com hairpin, which specifically recruits the Com protein. See Zalatan et al.
[0536] The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the MS2 aptamer) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 25).
[0537] The amino acid sequence of the MCP or MS2cp is:
TABLE-US-00051 (SEQIDNO:26) GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSV RQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATN SDCELIVKAMQGLLKDGNPIPSAIAANSGIY.
Delivery
[0538] In another aspect, the present disclosure provides for the delivery of fusion proteins in vitro and in vivo using split DddA protein formulations. The presently disclosed methods for delivering fusion proteins via various methods. For example, DddA proteins have exhibited toxic effects in vivo, and so require special solutions. One such solution is formulating the DddA, and fusion protein thereof, split into pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional DddA protein. Several other special considerations to account for the unique features of fusion protein are described, including the optimization of split sites. MitoTALE-DddA and/or mitoZF-DddA and/or Cas9-DddA fusion proteins, mRNA expressing the fusion proteins, or DNA can be packaged into lipid nanoparticles, rAAV, or lentivirus and injected, ingested, or inhaled to alter genomic DNA in vivo and ex vivo, including for the purposes of establishing animal models of human disease, testing therapeutic and scientific hypotheses in animal models of human disease, and treating disease in humans.
[0539] In another aspect, the present disclosure provides for the delivery of base editors in vitro and in vivo using various strategies, including on separate vectors using split inteins and as well as direct delivery strategies of the ribonucleoprotein complex (i.e., the base editor complexed to the gRNA and/or the second-site gRNA) using techniques such as electroporation, use of cationic lipid-mediated formulations, and induced endocytosis methods using receptor ligands fused to the ribonucleoprotein complexes. Any such methods are contemplated herein.
[0540] In some aspects, the invention provides methods comprising delivering one or more base editor-encoding polynucleotides, such as or one or more vectors as described herein encoding one or more components of the base editing system described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0541] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0542] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0543] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[0544] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (AAV) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0545] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
[0546] In various embodiments, the base editor constructs (including, the split-constructs) may be engineered for delivery in one or more rAAV vectors. An rAAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load (i.e., a recombinant nucleic acid vector that expresses a gene of interest, such as a whole or split base editor fusion protein that is carried by the rAAV into a cell) that is to be delivered to a cell. An rAAV may be chimeric.
[0547] As used herein, the serotype of an rAAV refers to the serotype of the capsid proteins of the recombinant virus. Non-limiting examples of derivatives and pseudotypes include rAAV2/1, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y.fwdarw.F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. A non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins is rAAV2/5-1VP1u, which has the genome of AAV2, capsid backbone of AAV5 and VP1u of AAV1. Other non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.
[0548] AAV derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer D V, Samulski R J.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).
[0549] Methods of making or packaging rAAV particles are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV particle can be packaged and subsequently purified.
[0550] Recombinant AAV may comprise a nucleic acid vector, which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest or an RNA of interest (e.g., a siRNA or microRNA), and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g., heterologous nucleic acid regions). Herein, heterologous nucleic acid regions comprising a sequence encoding a protein of interest or RNA of interest are referred to as genes of interest.
[0551] Any one of the rAAV particles provided herein may have capsid proteins that have amino acids of different serotypes outside of the VP1u region. In some embodiments, the serotype of the backbone of the VP1 protein is different from the serotype of the ITRs and/or the Rep gene. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the ITRs. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the Rep gene. In some embodiments, capsid proteins of rAAV particles comprise amino acid mutations that result in improved transduction efficiency.
[0552] In some embodiments, the nucleic acid vector comprises one or more regions comprising a sequence that facilitates expression of the nucleic acid (e.g., the heterologous nucleic acid), e.g., expression control sequences operatively linked to the nucleic acid. Numerous such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer).
[0553] Final AAV constructs may incorporate a sequence encoding the gRNA. In other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA. In still other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA and a sequence encoding the gRNA.
[0554] In various embodiments, the gRNAs and the second-site nicking guide RNAs can be expressed from an appropriate promoter, such as a human U6 (hU6) promoter, a mouse U6 (mU6) promoter, or other appropriate promoter. The gRNAs and the second-site nicking guide RNAs can be driven by the same promoters or different promoters.
[0555] In some embodiments, a rAAV constructs or the herein compositions are administered to a subject enterally. In some embodiments, a rAAV constructs or the herein compositions are administered to the subject parenterally. In some embodiments, a rAAV particle or the herein compositions are administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, a rAAV particle or the herein compositions are administered to the subject by injection into the hepatic artery or portal vein.
[0556] In other aspects, the base editors can be divided at a split site and provided as two halves of a whole/complete base editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete base editor through the self-splicing action of the inteins on each base editor half. Split intein sequences can be engineered into each of the halves of the encoded base editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning base editor.
[0557] These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding base editors is larger than the rAAV packaging limit, and so requires special solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein. Several other special considerations to account for the unique features of prime editing are described, including the optimization of second-site nicking targets and properly packaging base editors into virus vectors, including lentiviruses and rAAV.
[0558] In this aspect, the base editors can be divided at a split site and provided as two halves of a whole/complete base editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete base editor through the self-splicing action of the inteins on each base editor half. Split intein sequences can be engineered into each of the halves of the encoded base editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning base editor.
[0559] In various embodiments, the base editors may be engineered as two half proteins (i.e., a BE N-terminal half and a BE C-terminal half) by splitting the whole base editor as a split site. The split site refers to the location of insertion of split intein sequences (i.e., the N intein and the C intein) between two adjacent amino acid residues in the base editor. More specifically, the split site refers to the location of dividing the whole base editor into two separate halves, wherein in each halve is fused at the split site to either the N intein or the C intein motifs. The split site can be at any suitable location in the base editor fusion protein, but preferably the split site is located at a position that allows for the formation of two half proteins which are appropriately sized for delivery (e.g., by expression vector) and wherein the inteins, which are fused to each half protein at the split site termini, are available to sufficiently interact with one another when one half protein contacts the other half protein inside the cell.
[0560] In some embodiments, the split site is located in the napDNAbp domain. In other embodiments, the split site is located in the RT domain. In other embodiments, the split site is located in a linker that joins the napDNAbp domain and the RT domain.
[0561] In various embodiments, split site design requires finding sites to split and insert an N- and C-terminal intein that are both structurally permissive for purposes of packaging the two half base editor domains into two different AAV genomes. Additionally, intein residues necessary for trans splicing can be incorporated by mutating residues at the N terminus of the C terminal extein or inserting residues that will leave an intein scar.
[0562] In various embodiments, using SpCas9 nickase (SEQ ID NO: 29, 1368 amino acids) as an example, the split can between any two amino acids between 1 and 1368. Preferred splits, however, will be located between the central region of the protein, e.g., from amino acids 50-1250, or from 100-1200, or from 150-1150, or from 200-1100, or from 250-1050, or from 300-1000, or from 350-950, or from 400-900, or from 450-850, or from 500-800, or from 550-750, or from 600-700 of SEQ ID NO: 29. In specific exemplary embodiments, the split site may be between 740/741, or 801/802, or 1010/1011, or 1041/1042. In other embodiments the split site may be between 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8/9, 9/10, 10/11, 12/13, 14/15, 15/16, 17/18, 19/20 . . . 50/51 . . . 100/101 . . . 200/201 . . . 300/301 . . . 400/401 . . . 500/501 . . . 600/601 . . .
[0563] 700/701 . . . 800/801 . . . 900/901 . . . 1000/1001 . . . 1100/1101 . . . 1200/1201 . . . 1300/1301 . . . and 1367/1368, including all adjacent pairs of amino acid residues.
[0564] In various embodiments, the split intein sequences can be engineered by from the following intein sequences.
TABLE-US-00052 2-4INTEIN: (SEQIDNO:17) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSAL LDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAH LLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRF RMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGL TLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAG GSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEG VVVHNC 3-2INTEIN (SEQIDNO:18) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGL RIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSAL LDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAH LLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRF RMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGL TLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHAG GSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEG VVVHNC 30R3-1INTEIN (SEQIDNO:19) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSA LLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQA HLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAG LTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAE GVVVHNC 30R3-2INTEIN (SEQIDNO:20) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSA LLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQA HLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAG LTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAE GVVVHNC 30R3-3INTEIN (SEQIDNO:21) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSA LLDAEPPIPYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQA HLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAG LTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAE GVVVHNC 37R3-1INTEIN ((SEQIDNO:22) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSA LLDAEPPILYSEYNPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQA HLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAG LTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAE GVVVHNC 37R3-2INTEIN (SEQIDNO:23) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVVSWFDQGTRDVIGL RIAGGAIVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSAL LDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAH LLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRF RMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAGL TLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAG GSGASRVQAFADALDDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEG VVVHNC 37R3-3INTEIN (SEQIDNO:24) CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVSWFDQGTRDVIGL RIAGGATVWATPDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLALSLTADQMVSA LLDAEPPILYSEYDPTSPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQA HLLERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSR FRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITDTLIHLMAKAG LTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHA GGSGASRVQAFADALDDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAE GVVVHNC
[0565] In various embodiments, the split inteins can be used to separately deliver separate portions of a complete Base editor fusion protein to a cell, which upon expression in a cell, become reconstituted as a complete Base editor fusion protein through the trans splicing.
[0566] In some embodiments, the disclosure provides a method of delivering a Base editor fusion protein to a cell, comprising: constructing a first expression vector encoding an N-terminal fragment of the Base editor fusion protein fused to a first split intein sequence; constructing a second expression vector encoding a C-terminal fragment of the Base editor fusion protein fused to a second split intein sequence; delivering the first and second expression vectors to a cell, wherein the N-terminal and C-terminal fragment are reconstituted as the Base editor fusion protein in the cell as a result of trans splicing activity causing self-excision of the first and second split intein sequences.
[0567] In other embodiments, the split site is in the napDNAbp domain.
[0568] In still other embodiments, the split site is in the adenosine deaminase domain.
[0569] In yet other embodiments, the split site is in the linker.
[0570] In other embodiments, the base editors may be delivered by ribonucleoprotein complexes.
[0571] In this aspect, the base editors may be delivered by non-viral delivery strategies involving delivery of a base editor complexed with a gRNA (i.e., a BE ribonucleoprotein complex) by various methods, including electroporation and lipid nanoparticles. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0572] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0573] In some aspects, the invention provides methods comprising delivering one or more fusion proteins or polynucleotides encoding such fusion proteins, such as or one or more vectors as described herein encoding one or more components of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor (e.g., deaminating enzyme) as described herein in combination with (and optionally complexed with) a guide domain (e.g., mitoTALE) is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0574] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner: WO 91/17424 and WO 91/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
[0575] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0576] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[0577] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (AAV) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0578] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003-0087817, incorporated herein by reference.
gRNAs
[0579] Some aspects of the invention relate to guide sequences (guide RNA or gRNA) that are capable of guiding a napDNAbp or a base editor comprising a napDNAbp to a target site in a DNA molecule. In various embodiments base editors (e.g., base editors provided herein) can be complexed, bound, or otherwise associated with (e.g., via any type of covalent or non-covalent bond) one or more guide sequences, i.e., the sequence which becomes associated or bound to the base editor and directs its localization to a specific target sequence having complementarity to the guide sequence or a portion thereof. The particular design aspects of a guide sequence will depend upon the nucleotide sequence of a genomic target site of interest and the type of napDNA/RNAbp (e.g., type of Cas protein) present in the base editor, among other factors, such as PAM sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, secondary structures, etc.
[0580] In embodiments relating mtDNA base editors comprising Cas9/gRNA complexes, the Cas9 and gRNA components will need to be localized to the mitochondria. Cas9 can be modified with one or more MTS as discussed herein. In addition, the guide RNA may be localized to the mitochondria using known localization techniques for mRNA localization to mitochondria.
[0581] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence, such as a sequence within an SMN2 gene that comprises a C840T point mutation. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 75, or more nucleotides in length.
[0582] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence (e.g., a HGADFN 167 or HGADFN 188 cell line), such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are are well known in the art and can be used with the base editors described herein.
[0583] Additional exemplary guide sequences are disclosed in, for example, Jinek M., et al., Science 337:816-821(2012); Mali P, Esvelt K M & Church G M (2013) Cas9 as a versatile tool for engineering biology, Nature Methods, 10, 957-963; Li J F et al., (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9, Nature Biotechnology, 31, 688-691; Hwang, W. Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system, Nature Biotechnology 31, 227-229 (2013); Cong L et al., (2013) Multiplex genome engineering using CRIPSR/Cas systems, Science, 339, 819-823; Cho S W et al., (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease, Nature Biotechnology, 31, 230-232; Jinek, M. et al., RNA-programmed genome editing in human cells, eLife 2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acid Res. (2013); Briner A E et al., (2014) Guide RNA functional modules direct Cas9 activity and orthogonality, Mol Cell, 56, 333-339, the entire contents of each of which are herein incorporated by reference.
Methods of Treatment
[0584] The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins). For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease (e.g., MELAS/Leigh syndrome and Leber's hereditary optic neuropathy, other disorders associated with a point mutation as described above), an effective amount of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein that corrects the point mutation or introduces a point mutation comprising desired genetic change. In some embodiments, a method is provided that comprises administering to a subject having such a disease, (e.g., MELAS/Leigh syndrome and Leber's hereditary optic neuropathy, other disorders associated with a point mutation as described above), an effective amount of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a mitochondrial disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.
[0585] The instant disclosure provides methods for the treatment of additional diseases or disorders (e.g., diseases or disorders that are associated with or caused by a point mutation that can be corrected by the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) provided herein). Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins, or nucleic acids thereof, provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different (e.g., in precursors of a mature protein and the mature protein itself), and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art (e.g., by sequence alignment and determination of homologous residues). Exemplary suitable diseases and disorders include, without limitation: MELAS/Leigh syndrome and Leber's hereditary optic neuropathy.
[0586] The mtDNA base editors described herein may be used to treat any mitochrondrial disease or disorder. As used herein, mitochondrial disorders related to disorders which are due to abnormal mitochondria such as for example, a mitochondrial genetic mutation, enzyme pathways etc. Examples of disorders include and are not limited to: loss of motor control, muscle weakness and pain, gastro-intestinal disorders and swallowing difficulties, poor growth, cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual/hearing problems, lactic acidosis, developmental delays and susceptibility to infection.
[0587] The mitochondrial abnormalities give rise to mitochondrial diseases which include, but not limited to: AD: Alzheimer's Disease; ADPD: Alzheimer's Disease and Parkinsons's Disease; AMDF: Ataxia, Myoclonus and Deafness CIPO: Chronic Intestinal Pseudoobstruction with myopathy and Opthalmoplegia; CPEO: Chronic Progressive External Opthalmoplegia; DEAF: Maternally inherited DEAFness or aminoglycoside-induced DEAFness; DEMCHO: Dementia and Chorea; DMDF: Diabetes Mellitus & DeaFness; Exercise Intolerance; ESOC: Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN: Familial Bilateral Striatal Necrosis; FICP: Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; GER: Gastrointestinal Reflux; KSS Kearns Sayre Syndrome LDYT: Leber's hereditary optic neuropathy and DYsTonia; LHON: Leber Hereditary Optic Neuropathy; LFMM: Lethal Infantile Mitochondrial Myopathy; MDM: Myopathy and Diabetes Mellitus; MELAS:
[0588] Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes; MEPR: Myoclonic Epilepsy and Psychomotor Regression; MERME: MERRF/MELAS overlap disease; MERRF: Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM: Maternally Inherited Hypertrophic CardioMyopathy; MICM: Maternally Inherited Cardiomyopathy; MILS: Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Mitochondrial Encephalomyopathy; MM: Mitochondrial Myopathy; MMC: Maternal Myopathy and Cardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NARP: Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; alternate phenotype at this locus is reported as Leigh Disease; NIDDM: Non-Insulin Dependent Diabetes Mellitus; PEM: Progressive Encephalopathy; PME: Progressive Myoclonus Epilepsy; RTT: Rett Syndrome; SIDS: Sudden Infant Death Syndrome.
[0589] In embodiments, a mitochondrial disorder that may be treatable using the mtDNA base editors described herein include Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, and Stroke (MELAS); Maternally Inherited Diabetes and Deafness (MIDD); Leber's Hereditary Optic Neuropathy (LHON); chronic progressive external ophthalmoplegia (CPEO); Leigh Disease; Kearns-Sayre Syndrome (KSS); Friedreich's Ataxia (FRDA); Co-Enzyme QIO (CoQIO) Deficiency; Complex I Deficiency; Complex II Deficiency; Complex III Deficiency; Complex IV Deficiency; Complex V Deficiency; other myopathies; cardiomyopathy; encephalomyopathy; renal tubular acidosis; neurodegenerative diseases; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis (ALS); motor neuron diseases; hearing and balance impairments; or other neurological disorders; epilepsy; genetic diseases; Huntington's Disease; mood disorders; nucleoside reverse transcriptase inhibitors (NRTI) treatment; HIV-associated neuropathy; schizophrenia; bipolar disorder; age-associated diseases; cerebral vascular diseases; macular degeneration; diabetes; and cancer.
Pharmaceutical Compositions
[0590] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the various components of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein (e.g., including, but not limited to, the mitoTALE, DddA, or portions thereof, and fusion proteins (e.g., comprising mitoTALE and portion of DddA)).
[0591] The term pharmaceutical composition, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).
[0592] As used here, the term pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as excipient, carrier, pharmaceutically acceptable carrier or the like are used interchangeably herein.
[0593] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject (e.g., for nucleic acid editing). Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
[0594] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject (e.g., a human). In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
[0595] A pharmaceutical composition for systemic administration may be a liquid (e.g., sterile saline, lactated Ringer's or Hank's solution). In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.
[0596] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in stabilized plasmid-lipid particles (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or DOTAP, are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.
[0597] The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term unit dose when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
[0598] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising: (a) a container containing a compound of the invention in lyophilized form; and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0599] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Delivery Methods
[0600] In another aspect, the present disclosure provides for the delivery of mtDNA base editors in vitro and in vivo using various strategies, including on separate vectors using split inteins and as well as direct delivery strategies of the ribonucleoprotein complex (i.e., the base editor complexed to the gRNA and/or the second-site gRNA) using techniques such as electroporation, use of cationic lipid-mediated formulations, and induced endocytosis methods using receptor ligands fused to the ribonucleoprotein complexes. In addition, mRNA delivery methods may also be employed. Any such methods are contemplated herein. The mtDNA BE fusion proteins, or components thereof, preferably be modified with an MTS or other signal sequence that facilitates entry of the polypeptides and the guide RNAs (in the case where a pDNAbp is Cas9) into the mitochondria.
[0601] In some aspects, the invention provides methods comprising delivering one or more base editor-encoding and/or gRNA-encoding polynucleotides, such as or one or more vectors as described herein encoding one or more components described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0602] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0603] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[0604] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (AAV) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0605] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
[0606] In various embodiments, the base editor constructs (including, the split-constructs) may be engineered for delivery in one or more rAAV vectors. An rAAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load (i.e., a recombinant nucleic acid vector that expresses a gene of interest, such as a whole or split base editor fusion protein that is carried by the rAAV into a cell) that is to be delivered to a cell. An rAAV may be chimeric.
[0607] As used herein, the serotype of an rAAV refers to the serotype of the capsid proteins of the recombinant virus. Non-limiting examples of derivatives and pseudotypes include rAAV2/1, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y.fwdarw.F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. A non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins is rAAV2/5-1VP1u, which has the genome of AAV2, capsid backbone of AAV5 and VP1u of AAV1. Other non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.
[0608] AAV derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer D V, Samulski R J.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).
[0609] Methods of making or packaging rAAV particles are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV particle can be packaged and subsequently purified.
[0610] Recombinant AAV may comprise a nucleic acid vector, which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest or an RNA of interest (e.g., a siRNA or microRNA), and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g., heterologous nucleic acid regions). Herein, heterologous nucleic acid regions comprising a sequence encoding a protein of interest or RNA of interest are referred to as genes of interest.
[0611] Any one of the rAAV particles provided herein may have capsid proteins that have amino acids of different serotypes outside of the VP1u region. In some embodiments, the serotype of the backbone of the VP1 protein is different from the serotype of the ITRs and/or the Rep gene. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the ITRs. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the Rep gene. In some embodiments, capsid proteins of rAAV particles comprise amino acid mutations that result in improved transduction efficiency.
[0612] In some embodiments, the nucleic acid vector comprises one or more regions comprising a sequence that facilitates expression of the nucleic acid (e.g., the heterologous nucleic acid), e.g., expression control sequences operatively linked to the nucleic acid. Numerous such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer).
[0613] Final AAV constructs may incorporate a sequence encoding the gRNA. In other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA. In still other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA and a sequence encoding the gRNA.
[0614] In various embodiments, the gRNAs can be expressed from an appropriate promoter, such as a human U6 (hU6) promoter, a mouse U6 (mU6) promoter, or other appropriate promoter. The gRNAs (if multiple) can be driven by the same promoters or different promoters.
[0615] In some embodiments, a rAAV constructs or the herein compositions are administered to a subject enterally. In some embodiments, a rAAV constructs or the herein compositions are administered to the subject parenterally. In some embodiments, a rAAV particle or the herein compositions are administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, a rAAV particle or the herein compositions are administered to the subject by injection into the hepatic artery or portal vein.
[0616] In other aspects, the base editors can be divided at a split site and provided as two halves of a whole/complete base editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete base editor through the self-splicing action of the inteins on each base editor half. Split intein sequences can be engineered into each of the halves of the encoded base editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning base editor.
[0617] These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding base editors is larger than the rAAV packaging limit, and so requires special solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein.
[0618] In this aspect, the base editors can be divided at a split site and provided as two halves of a whole/complete base editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete base editor through the self-splicing action of the inteins on each base editor half. Split intein sequences can be engineered into each of the halves of the encoded base editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning base editor.
[0619] In various embodiments, the base editors may be engineered as two half proteins (i.e., a ABE N-terminal half and a CBE C-terminal half) by splitting the whole base editor as a split site. The split site refers to the location of insertion of split intein sequences (i.e., the N intein and the C intein) between two adjacent amino acid residues in the base editor. More specifically, the split site refers to the location of dividing the whole base editor into two separate halves, wherein in each halve is fused at the split site to either the N intein or the C intein motifs. The split site can be at any suitable location in the base editor fusion protein, but preferably the split site is located at a position that allows for the formation of two half proteins which are appropriately sized for delivery (e.g., by expression vector) and wherein the inteins, which are fused to each half protein at the split site termini, are available to sufficiently interact with one another when one half protein contacts the other half protein inside the cell.
[0620] In some embodiments, the split site is located in the pDNAbp domain. In other embodiments, the split site is located in the double stranded deaminase domain (DddA). In other embodiments, the split site is located in a linker that joins the napDNAbp domain and the deaminase domain. Preferably, the DddA is split so as to inactive the deaminase activity until the split fragments are co-localized in the mitochondria at the target site.
[0621] In various embodiments, split site design requires finding sites to split and insert an N- and C-terminal intein that are both structurally permissive for purposes of packaging the two half base editor domains into two different AAV genomes. Additionally, intein residues necessary for trans splicing can be incorporated by mutating residues at the N terminus of the C terminal extein or inserting residues that will leave an intein scar.
[0622] In various embodiments, using SpCas9 nickase as an example, the split can be between any two amino acids between 1 and 1368 of SEQ ID NO: 28. Preferred splits, however, will be located between the central region of the protein, e.g., from amino acids 50-1250, or from 100-1200, or from 150-1150, or from 200-1100, or from 250-1050, or from 300-1000, or from 350-950, or from 400-900, or from 450-850, or from 500-800, or from 550-750, or from 600-700 of SEQ ID NO: 28. In specific exemplary embodiments, the split site may be between 740/741, or 801/802, or 1010/1011, or 1041/1042. In other embodiments the split site may be between 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8/9, 9/10, 10/11, 12/13, 14/15, 15/16, 17/18, 19/20 . . . 50/51 . . . 100/101 . . . 200/201 . . . 300/301 . . . 400/401 . . . 500/501 . . . 600/601 . . .
[0623] 700/701 . . . 800/801 . . . 900/901 . . . 1000/1001 . . . 1100/1101 . . . 1200/1201 . . . 1300/1301 . . . and 1367/1368, including all adjacent pairs of amino acid residues.
[0624] In various embodiments, the split inteins can be used to separately deliver separate portions of a complete Base editor fusion protein to a cell, which upon expression in a cell, become reconstituted as a complete Base editor fusion protein through the trans splicing.
[0625] In some embodiments, the disclosure provides a method of delivering a Base editor fusion protein to a cell, comprising: constructing a first expression vector encoding an N-terminal fragment of the Base editor fusion protein fused to a first split intein sequence; constructing a second expression vector encoding a C-terminal fragment of the Base editor fusion protein fused to a second split intein sequence; delivering the first and second expression vectors to a cell, wherein the N-terminal and C-terminal fragment are reconstituted as the Base editor fusion protein in the cell as a result of trans splicing activity causing self-excision of the first and second split intein sequences.
[0626] In other embodiments, the split site is in the napDNAbp domain.
[0627] In still other embodiments, the split site is in the deaminase domain.
[0628] In yet other embodiments, the split site is in the linker.
[0629] In other embodiments, the base editors may be delivered by ribonucleoprotein complexes.
[0630] In this aspect, the base editors may be delivered by non-viral delivery strategies involving delivery of a base editor complexed with a gRNA (i.e., a ABE ribonucleoprotein complex) by various methods, including electroporation and lipid nanoparticles. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0631] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
Kits, Vectors, Cells
[0632] Some aspects of this disclosure provide kits comprising a fusion protein or a nucleic acid construct comprising a nucleotide sequence encoding the various components (e.g., fusion protein) of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein (e.g., including, but not limited to, the mitoTALE-DddA fusion proteins, vectors or cells comprising the same). In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the fusion protein editing system components described herein.
[0633] Some aspects of this disclosure provide kits comprising one or more fusion proteins or nucleic acid constructs encoding the various components of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) described herein, e.g., the comprising a nucleotide sequence encoding the components of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the mtDNA editing system provided herein (e.g., deamination of mitochondrial DNA by a fusion protein or multiple fusion proteins) components.
[0634] In some embodiments, a kit further comprises a set of instructions for using the fusion proteins and/or carrying out the methods herein.
[0635] Some aspects of this disclosure provides kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a fusion protein (e.g., a mitoTALE and portion of a DddA) and (b) a heterologous promoter that drives expression of the sequence of (a).
[0636] Some aspects of this disclosure provide cells comprising any of the constructs disclosed herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr/, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a fusion protein system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a fusion protein complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
OTHER EMBODIMENTS
[0637] In yet further aspects, the present application provides the following embodiments as reflected in the following numbered paragraphs: [0638] 1. A non-naturally occurring polypeptide variant comprising a double-stranded DNA deaminase activity. [0639] 2. The non-naturally occurring polypeptide variant of paragraph 1, wherein the polypeptide comprises a minimum domain conferring double-stranded DNA deaminase activity. [0640] 3. The non-naturally occurring polypeptide variant of paragraph 2, wherein the minimum domain corresponds to amino acid residues 1261-1427 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0641] 4. The non-naturally occurring polypeptide variant of paragraph 2, wherein the minimum domain corresponds to amino acid residues 1290-1425 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0642] 5. The non-naturally occurring polypeptide variant of paragraph 1, wherein the minimum domain corresponds to SEQ ID NO: 19, or to an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19, wherein SEQ ID NO: 19 corresponds to amino acids 1251-1427 of SEQ ID NO: 1. [0643] 6. The non-naturally occurring polypeptide variant of paragraph 1, wherein the minimum domain corresponds to SEQ ID NO: 20, or to an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, wherein SEQ ID NO: 20 corresponds to amino acids 1290-1425 of SEQ ID NO: 1. [0644] 7. A non-naturally occurring polypeptide fragment of a double-stranded DNA deaminase. [0645] 8. The non-naturally occurring polypeptide fragment of paragraph 7, wherein the polypeptide fragment comprises a minimum domain conferring double-stranded DNA deaminase activity. [0646] 9. The non-naturally occurring polypeptide fragment of paragraph 8, wherein the minimum domain corresponds to amino acid residues 1261-1427 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0647] 10. The non-naturally occurring polypeptide fragment of paragraph 8, wherein the minimum domain corresponds to amino acid residues 1290-1425 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0648] 11. The non-naturally occurring polypeptide fragment of paragraph 8 having the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. [0649] 12. The non-naturally occurring polypeptide fragment of paragraph 8 having the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. [0650] 13. A non-naturally occurring polypeptide fragment of a double-stranded DNA deaminase obtained by splitting the deaminase in the deaminase domain at a split site. [0651] 14. The non-naturally occurring polypeptide fragment of paragraph 13, wherein the fragment corresponds to an N-terminal half fragment, wherein said fragment comprises an N-terminal portion of a split deaminase domain. [0652] 15. The non-naturally occurring polypeptide fragment of paragraph 13, wherein the fragment corresponds to a C-terminal half fragment, wherein said fragment comprises a C-terminal portion of a split deaminase domain. [0653] 16. The non-naturally occurring polypeptide fragment of paragraph 14, wherein the deaminase activity is restored upon co-localizing the N-terminal half fragment with a C-terminal half fragment. [0654] 17. The non-naturally occurring polypeptide fragment of paragraph 15, wherein the deaminase activity is restored upon co-localizing the C-terminal half fragment with an N-terminal half fragment. [0655] 18. The non-naturally occurring polypeptide fragment of any one of paragraphs 13-17, wherein the amino acid sequence of the double-stranded DNA deaminase that is split is any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. [0656] 19. The non-naturally occurring polypeptide fragment of any one of paragraphs 13-18, wherein the split site is at the peptide bond immediately following residue G1322, G1333, A1343, N1357, G1371, N1387, or G1397 of the amino acid sequence of SEQ ID NO: 1, or at a corresponding split site in an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0657] 20. The non-naturally occurring polypeptide fragment of paragraphs 14, wherein the N-terminal half fragment comprises an amino acid sequence of SEQ ID NOs: 349 or 351, or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOs: 349 or 351. [0658] 21. The non-naturally occurring polypeptide fragment of paragraphs 15, wherein the C-terminal half fragment comprises an amino acid sequence of SEQ ID NOs: 350 or 352, or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOs: 350 or 352. [0659] 22. A base editor comprising a heterodimer having first and second monomers, said first monomer comprising a first programmable DNA binding protein and an N-terminal or C-terminal fragment of a split double-stranded DNA deaminase, and said second monomer comprising a second programmable DNA binding protein and an N-terminal or C-terminal fragment of a split double-stranded DNA deaminase, wherein dimerization of the first and second monomers reconstitutes the double-stranded DNA deaminase activity. [0660] 23. The base editor of paragraph 22, wherein the first and/or second programmable DNA binding protein are the same. [0661] 24. The base editor of paragraph 22, wherein the first and/or second programmable DNA binding protein are different. [0662] 25. The base editor of paragraph 22, wherein the first and/or second programmable DNA binding protein is a nucleic acid programmable DNA binding protein (napDNAbp). [0663] 26. The base editor of paragraph 25, wherein the napDNAbp is a Cas9 domain. [0664] 27. The base editor of of paragraph 25, wherein the napDNAbp is a nickase. [0665] 28. The base editor of paragraph 25, wherein the napDNAbp comprises an inactivated nuclease activity. [0666] 29. The base editor of paragraph 25, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity. [0667] 30. The base editor of paragraph 25, wherein the napDNAbp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 31, 33, 35, 36-91, 353, and 354, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 31, 33, 35, 36-91, 353, and 354. [0668] 31. The base editor of paragraph 22, wherein the programmable DNA binding protein is a TALE protein. [0669] 32. The base editor of paragraph 31, wherein TALE protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. [0670] 33. The base editor of paragraph 22, wherein the programmable DNA binding protein is a zinc finger protein. [0671] 34. The base editor of paragraph 33, wherein zinc finger protein is a commercially available zinc finger protein. [0672] 35. The base editor of paragraph 22, wherein the programmable DNA binding protein is a mitoTALE protein. [0673] 36. The base editor of paragraph 35, wherein mitoTALE protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-12, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12. [0674] 37. The base editor of paragraph 22, wherein the N-terminal fragment of the split double-stranded DNA deaminase double-stranded DNA deaminase domain comprises an amino acid sequence of SEQ ID NO: 349 or 351, or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 349 or 351. [0675] 38. The base editor of paragraph 22, wherein the C-terminal fragment of the split double-stranded DNA deaminase double-stranded DNA deaminase domain comprises an amino acid sequence of SEQ ID NO: 350 or 352, or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 350 or 352. [0676] 39. The base editor of paragraph 22, wherein the C-terminal fragment and the N-terminal fragment of the split double-stranded DNA deaminase are obtained by splitting a double-stranded DNA deaminase in the deaminase domain at a split site. [0677] 40. The base editor of paragraph 39, wherein the double-stranded DNA deaminase comprises an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. [0678] 41. The base editor of paragraph 39, wherein the double-stranded DNA deaminase comprises a minimum domain conferring double-stranded DNA deaminase activity. [0679] 42. The base editor of paragraph 41, wherein the minimum domain corresponds to amino acid residues 1261-1427 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0680] 43. The base editor of paragraph 41, wherein the minimum domain corresponds to amino acid residues 1290-1425 of (i) full-length DddA of SEQ ID NO: 1 of Burkholderia cenocepacias, (ii) a corresponding region of any one of the DddA homologs of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, or (iii) a corresponding region of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0681] 44. The base editor of paragraph 41, wherein the minimum domain comprises SEQ ID NO: 19, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. [0682] 45. The base editor of paragraph 41, wherein the minimum domain comprises SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. [0683] 46. The base editor of paragraph 39, wherein the split site is at the peptide bond immediately following residue G1322, G1333, A1343, N1357, G1371, N1387, or G1397 of the amino acid sequence of SEQ ID NO: 1, or at a corresponding split site in an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. [0684] 47. The base editor of paragraph 22, wherein the first monomer comprises a linker that joins the first programmable DNA binding protein with the N-terminal or C-terminal fragment of the split double-stranded DNA deaminase. [0685] 48. The base editor of paragraph 22, wherein the second monomer comprises a linker that joins the first programmable DNA binding protein with the N-terminal or C-terminal fragment of the split double-stranded DNA deaminase. [0686] 49. The base editor of paragraph 47 or 48, wherein the linker comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 101-117, 357, 358, and 359, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 101-117, 357, 358, or 359. [0687] 50. The base editor of paragraph 47 or 48, wherein the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. [0688] 51. The base editor of paragraphs 47 or 48, wherein the linker comprises 2 amino acids. [0689] 52. The base editor of any one of paragraphs 22-51, further comprising one or more uracil glycosylase inhibitor (UGI) domains. [0690] 53. The base editor of paragraph 52, wherein the one or more UGI domains comprise an amino acid sequence selected from the group consisting of: SEQ ID NOs: 118 and 355, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 118 or 355. [0691] 54. The base editor of any one of paragraphs 22-53, further comprising one or more targeting sequences. [0692] 55. The base editor of paragraph 54, wherein the one or more targeting sequences is a nuclear localization sequence (NLS). [0693] 56. The base editor of paragraph 55, wherein the NLS comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 119-129 and 356, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 119-129 or 356. [0694] 57. The base editor of paragraph 54, wherein the one or more targeting sequences is a mitochondrial targeting sequence (MTS). [0695] 58. The base editor of paragraph 57, wherein the MTS comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 13, 14, and 299, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 13, 14, or 299. [0696] 59. The base editor of paragraph 57, wherein the MTS is an SOD2 having an amino acid sequence comprising SEQ ID NO: 13, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. [0697] 60. The base editor of paragraph 57, wherein the MTS is a COX8a having an amino acid sequence comprising SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14. [0698] 61. The base editor of paragraph 22, wherein the first and/or second monomers have one of the following structures: [0699] (a) [A]-[programmable DNA binding protein]-[N-terminal or C-terminal fragment of a split double-stranded DNA deaminase]-[B]; or [0700] (b) [A]-[N-terminal or C-terminal fragment of a split double-stranded DNA deaminase]-[programmable DNA binding protein]-[B], [0701] wherein [A] and/or [B] represent optional one or more additional functional domains and wherein ]-[ is an optional linker. [0702] 62. The base editor of paragraph 61, wherein the optional one or more additional functional domains are selected from the group consisting of a uracil glycosylase inhibitor (UGI) domain and a targeting domain. [0703] 63. The base editor of paragraph 62, wherein the UGI domain comprises an amino acid sequence comprising SEQ ID NO: 335, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 335. [0704] 64. The base editor of paragraph 62, wherein the targeting domain comprises an NLS having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 119-129 and 356, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 119-129 or 356. [0705] 65. The base editor of paragraph 62, wherein the targeting domain comprises an MTS having an amino acid sequence comprising SEQ ID NO: 299, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 299. [0706] 66. The base editor of paragraph 22, wherein the first monomer comprises the following structure: [SOD2]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 349 or DddA.sub.tox-C of SEQ ID NO: 350]-[UGI].sub.1-2. [0707] 67. The base editor of paragraph 66, wherein the first monomer comprises an amino acid sequence comprising SEQ ID NO: 360, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 360. [0708] 68. The base editor of paragraph 22, wherein the first monomer comprises the following structure: [COX8A]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 351 or DddA.sub.tox-C of SEQ ID NO: 352]-[UGI].sub.1-2. [0709] 69. The base editor of paragraph 68, wherein the first monomer comprises an amino acid sequence of SEQ ID NO: 360, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 360. [0710] 70. The base editor of paragraph 22, wherein the second monomer comprises the following structure: [SOD2]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 349 or DddA.sub.tox-C of SEQ ID NO: 350]-[UGI].sub.1-2. [0711] 71. The base editor of paragraph 70, wherein the second monomer comprises an amino acid sequence of SEQ ID NO: 361, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 361. [0712] 72. The base editor of paragraph 22, wherein the second monomer comprises the following structure: [COX8A]-[UGI].sub.1-2-[mitoTALE]-[DddA.sub.tox-N of SEQ ID NO: 351 or DddA.sub.tox-C of SEQ ID NO: 352]-[UGI].sub.1-2. [0713] 73. The base editor of paragraph 72, wherein the second monomer comprises an amino acid sequence of SEQ ID NO: 361, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 361. [0714] 74. The base editor of any one of paragraphs 22-74, wherein the first and second monomers bind to first and second nucleotide sequences, respectively, on either side of a target site. [0715] 75. The base editor of paragraph 74, wherein the target site comprises a target base which becomes deaminated by the base editor. [0716] 76. The base editor of paragraph 75, wherein the target base is a C. [0717] 77. The base editor of paragraph 76, wherein the C is within a 5-TC-3 sequence context. [0718] 78. The base editor of paragraph 76, wherein the C is within a 5-TCC-3 sequence context, a 5-CCC-3 context, a 5-TCA-3 sequence context, or a 5-TCT-3 sequence context. [0719] 79. The base editor of paragraph 74, wherein the nucleotide sequences are each on the same strand as the target base which becomes deaminated by the base editor. [0720] 80. The base editor of paragraph 75, wherein the first and second nucleotide sequences are each on the same strand as the strand comprising the target base which becomes deaminated by the base editor. [0721] 81. The base editor of paragraph 75, wherein the first and second nucleotide sequences are each on the opposite strand as the strand comprising the target base which becomes deaminated by the base editor. [0722] 82. The base editor of paragraph 75, wherein the first and second nucleotide sequences are each on the opposite strand as the strand comprising the target base which becomes deaminated by the base editor. [0723] 83. The base editor of paragraph 75, wherein the first and second nucleotide sequences are on opposing strands. [0724] 84. The base editor of any of paragraph 74, further comprising one or more guide RNAs if the first and/or second programmable DNA binding protein is a nucleic acid programmable DNA binding protein (napDNAbp), and wherein the one or more guide RNAs directs the base edito to bind to the first or second nucleotide sequence at the target site. [0725] 85. An isolated nucleic acid encoding the first monomer of the base editor of paragraph 22. [0726] 86. An isolated nucleic acid encoding the second monomer of the base editor of paragraph 22. [0727] 87. An isolated nucleic acid encoding the non-naturally occurring polypeptide variant of any of paragraphs 1-6. [0728] 88. An isolated nucleic acid encoding the non-naturally occurring polypeptide fragment of any of paragraphs 7-12. [0729] 89. A vector comprising the isolated nucleic acid of any one of paragraphs 85-88. [0730] 90. A cell comprising a vector of paragraph 89. [0731] 91. A method of editing a target nucleotide sequence at a target site, comprising contacting a target nucleotide sequence with a base editor of paragraphs 22-84, wherein the first monomer targets a first nucleotide sequence flanking a target site, and the second monomer targets a second nucleotide sequence flanking the target site, thereby inducing deamination of a target base at the target site. [0732] 92. The method of claim 91, wherein the target base is a C. [0733] 93. The method of claim 92, wherein the C is within a 5-TC-3 sequence context. [0734] 94. The method of paragraph 92, wherein the C is within a 5-TCC-3 sequence context, a 5-CCC-3 context, a 5-TCA-3 sequence context, or a 5-TCT-3 sequence context. [0735] 95. The method of paragraph 91, wherein the programmable DNA binding protein of the base editor is a TALE, mitoTALE, zinc finger protein, or napDNAbp. [0736] 96. The method of paragraph 91, wherein the first and/or second programmable DNA binding protein of the base editor is a napDNAbp. [0737] 97. The method of paragraph 96, wherein the napDNAbp is a Cas9 domain. [0738] 98. The method of paragraph 96, wherein the napDNAbp is a nickase. [0739] 99. The method of paragraph 96, wherein the target nucleotide sequence is a mitochondrial sequence within a mitochondria. [0740] 100. The method of paragraph 96, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity. [0741] 101. The method of paragraph 96, wherein the napDNAbp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 33, 35, 36-91, 353, and 354, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 28, 31, 33, 35, 36-91, 353, and 354. [0742] 102. The method of paragraph 91, wherein the first and/or second programmable DNA binding protein of the base editor is a mitoTALE. [0743] 103. The method of paragraph 102, wherein mitoTALE protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-12 or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12. [0744] 104. The method of paragraph 91, wherein the method further comprises contacting the target nucleotide sequence with guide RNAs if the programmable DNA binding protein of the base editor is a napDNAbp, wherein the guide RNAs direct the first and second monomers of the base editor to the target nucleotide sequence. [0745] 105. The method of paragraph 91, wherein the editing occurs in vivo. [0746] 106. The method of paragraph 91, wherein the editing occurs ex vivo. [0747] 107. The method of paragraph 91, wherein the target nucleotide sequence is a disease gene. [0748] 108. The method of paragraph 91, wherein the target nucleotide sequence is a disease gene in a mitochondria. [0749] 109. The method of paragraph 91, wherein the method of editing a nucleotide sequence results in the treatment of a mitochondrial disease. [0750] 110. The method of paragraph 91, wherein the mitochondrial disease is MELAS/Leigh syndrome and Leber's hereditary optic neuropathy. [0751] 111. A method of delivering a base editor of any of claims 22-84 to a cell comprising transforming a cell with one or more vectors encoding the first and second monomers of the base editor, wherein once in the cell the first and second monomers are expressed and dimerize, thereby forming a base editor in the cell. [0752] 112. The method of paragraph 111, wherein the one or more vectors are viral expression vectors. [0753] 113. The method of paragraph 111, wherein one or more vectors are adeno-associated viral vectors (AAV) of any serotype. [0754] 114. The method of paragraph 111, further comprising the step of expressing a guide RNA in the cell if the first and/or second monomer comprise a napDNAbp, and wherein the guide RNA is expressed from the same vector or a different vector. [0755] 115. A method of delivering a base editor of any of paragraphs 22-84 to a mitochondria comprising transforming a cell with one or more vectors encoding the first and second monomers of the base editor, wherein once in the cell the first and second monomers are expressed, transported to the mitochondria, and dimerize therein, thereby forming a base editor in the mitochondria, wherein the first and second programmable DNA binding proteins of the base editor are mitoTALE domains. [0756] 116. The method of paragraph 115, wherein the one or more vectors are viral expression vectors. [0757] 117. The method of paragraph 115, wherein one or more vectors are adeno-associated viral vectors (AAV) of any serotype. [0758] 118. A therapeutic kit comprising one or more nucleic acid constructs, comprising: [0759] (i) one or more nucleic acid sequences encoding the first and second monomers of the base editor of any of paragraphs 22-84, [0760] (ii) a promoter that drives expression of (i). [0761] 119. The therapeutic kit of paragraph 118, further comprising an expression construct encoding one or more guide RNAs where either the first or second monomer comprises a napDNAbp.
Examples
Example 1: A Bacterial Double-Stranded DNA Cytidine Deaminase Toxin Enables RNA-Free Base Editing in the Mitochondrial Genome
Background
[0762] Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Many toxins function in interbacterial antagonism, yet their modes of action remain largely unknown. Here, the discovery, structure, biochemical characterization, and application of DddA, an interbacterial toxin that catalyzes the unprecedented deamination of cytidines within double-stranded DNA, is reported. All previously described cytidine deaminases, including those used in base editing, operate on single-stranded DNA and thus when used for genome editing require unwinding of double-stranded DNA by macromolecules such as CRISPR-Cas9 complexed with a guide RNA. The difficulty of delivering guide RNAs into the mitochondria has thus far precluded base editing in mitochondrial DNA (mtDNA). The ability of DddA to deaminate double-stranded DNA raises the possibility of RNA-free precision base editing, rather than simple elimination of targeted mtDNA copies following double-strand DNA breaks. Split-DddA halves were engineered that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, TALE array proteins, and uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyze C.Math.G-to-T.Math.A conversions efficiently and with high DNA sequence specificity and product purity at targeted sites within mtDNA in human cells. DddA-mediated base editing was used to model a disease-associated mtDNA mutation in human cell lines, resulting in changes in rates of respiration and oxidative phosphorylation. CRISPR-free, DddA-mediated base editing enables precision editing of mtDNA, with important basic science and biomedical implications.
[0763] Enzymes that catalyze the deamination of cytidine and adenosine play pivotal roles in precision genome editing..sup.1,2 The biochemical and functional diversity of deaminases, however, remain largely unexplored. In particular, bacterial genomes contain a wide variety of uncharacterized cryptic deaminases,.sup.3 raising the possibility that some may possess unique activities that could be exploited to enable new genome editing capabilities.
[0764] Inherited or acquired mutations in mitochondrial DNA (mtDNA) can profoundly impact cell physiology and are associated with a spectrum of human diseases, ranging from rare inborn errors of metabolism,.sup.4 certain cancers,.sup.5 age-associated neurodegeneration,.sup.6 and even the aging process itself..sup.7,8 Tools for introducing specific modifications to mtDNA are urgently needed both for modeling diseases and for their therapeutic potential. The development of such tools, however, has been constrained in part by the challenge of transporting RNAs into mitochondria, including guide RNAs required to program CRISPR-associated proteins..sup.9
[0765] Each mammalian cell contains hundreds to thousands of copies of a circular mtDNA.sup.10. Homoplasmy refers to a state in which all mtDNA molecules are identical, while heteroplasmy refers to a state in which a cell contains a mixture of wild-type and mutant mtDNA. Current approaches to engineer mtDNA rely on DNA-binding proteins such as transcription activator-like effectors nucleases (mitoTALENs).sup.11-17 and zinc finger nucleases (mitoZFNs).sup.18-20 fused to mitochondrial targeting sequences to induce double-strand breaks (DSBs). Such proteins do not rely on nucleic acid programmability (e.g., such as with Cas9 domains). Linearized mtDNA is rapidly degraded,.sup.21-23 resulting in heteroplasmic shifts to favor uncut mtDNA genomes. As a candidate therapy however, this approach cannot be applied to homoplasmic mtDNA mutations.sup.24 since destroying all mtDNA copies is presumed to be harmful..sup.22-25 In addition, using DSBs to eliminate heteroplasmic mtDNA mutations, which tend to be functionally recessive,.sup.26 implicitly requires the edited cell to restore its wild-type mtDNA copy number. During this transient period of mtDNA repopulation, the loss of mtDNA copies could result in cellular toxicity.
[0766] A favorable alternative to targeted destruction of DNA through DSBs is precision genome editing, a capability that has not been reported for mtDNA. The ability to precisely install or correct pathogenic mutations, rather than destroy targeted mtDNA, could accelerate our ability to model mtDNA diseases in cells and animal models, and in principle could also enable therapeutic approaches that correct pathogenic mtDNA mutations.
A Predicted Deaminase Functions as an Interbacterial Toxin
[0767] Some predicted bacterial deaminases contain sequence hallmarks that suggest they are substrates for intercellular protein delivery systems..sup.3 These hallmarks include domains that direct transport through the type VI secretion system (T6SS)..sup.3 The T6SS mediates antagonism between Gram-negative bacteria by catalyzing the direct transfer of antibacterial toxins into contacting cells..sup.27,28 Given their sequence divergence and potential functional differences from characterized deaminases, the biochemical activity of T6SS-associated deaminases were sought. Investigations were focused on a predicted deaminase (belonging to the SCP1.201-like family),.sup.3 henceforth referred to as DddA, encoded by Burkholderia cenocepacias (B. cen) (
[0768] Members of the deaminase superfamily are known to catalyze deamination of single-stranded DNA (ssDNA), RNA (including mRNA and tRNA), free nucleosides, nucleotides, nucleobases, and other nucleotide derivatives.sup.3. To begin to define the substrate of DddA, which belongs to a clade of predicted deaminases lacking a characterized member.sup.3, first whether or not deaminases representing the substrate range of the superfamily are toxic if ectopically expressed in bacteria was determined. The growth of E. coli was unaffected by production of deaminases that act on ssDNA, tRNA, or free cytidine (
DddA is a Double-Stranded DNA-Specific Cytidine Deaminase
[0769] To further illuminate the substrate and mechanism of DddA.sub.tox, we determined a 2.5-A resolution co-crystal structure of DddA.sub.tox bound to DddI.sub.A (Table 2). DddA.sub.tox adopts a typical deaminase fold consisting of a five-stranded -sheet with buttressing helices that contribute critical catalytic residues to the active site (
[0770] Given the similarity of DddA.sub.tox and APOBEC proteins, the ability of DddA.sub.tox to catalyze the deamination of cytidine in vitro was tested. To date, all known DNA cytidine deaminases operate on ssDNA, often with a preference for the base immediately 5 of the substrate cytidine..sup.29 Therefore, the activity of DddA.sub.tox on a ssDNA substrate containing cytidine was measured in all four possible 5-NC contexts. While the activity of APOBEC3A was readily detected, DddA.sub.tox did not catalyze uracil formation within ssDNA sequences (
[0771] If DddA.sub.tox converts cytidine to uracil specifically within dsDNA, it was reasoned that the enzyme should be mutagenic in a manner that is dependent on uracil DNA glycosylase (UDG), a protein that initiates base excision repair (BER) through uracil removal..sup.32,33 Indeed, expression of sub-lethal levels of DddA.sub.tox in E. coli substantially increased mutation frequency, and these mutagenic effects of DddA.sub.tox were enhanced >100-fold in an E. coli strain lacking UDG (
Identifying Split DddA.sub.tox Halves that are Non-Toxic and Catalytically Competent
[0772] Current base editors deaminate nucleotides in single-stranded DNA loops created by RNA-guided CRISPR proteins..sup.2,34,35 The ability of DddA.sub.tox to deaminate cytidines in dsDNA raises the possibility of using RNA-free programmable dsDNA-binding proteins such as zinc-finger arrays.sup.36 or TALE arrays.sup.37 to direct DddA.sub.tox to target cytidines in dsDNA. The resulting dsDNA cytosine base editor would enable base editing without requiring guide RNAs, raising the possibility of base editing in systems for which RNA delivery is prohibitive, such as the mitochondria..sup.9
[0773] Consistent with experiments in E. coli (
[0774] Based on the crystal structure of apo-DddA.sub.tox, seven split sites within the loop regions of DddA.sub.tox were tested (
[0775] HEK293T cells were transfected with four plasmids: two plasmids encode DddA.sub.tox-N or DddA.sub.tox-C fused to either dSpCas9 or SaKKH-Cas9(D10A), and the remaining two plasmids encode two guide RNAs that direct the fusion proteins to flank a target TC (
[0776] Among active split-DddA.sub.tox fusions, predominantly C.Math.G-to-T.Math.A conversions were observed in the intervening DNA sequence between the two protospacers. Editing efficiencies varied from 1.1% to 49% depending on the split site, split orientation and dsDNA spacing length (
[0777] Out of the seven split sites tested, G1333 and G1397 yielded the highest editing efficiencies (
TALE-Split-DddA.SUB.tox .Fusions Enable RNA-Free Base Editing
[0778] Despite the potential of mitochondrial DNA editing to illuminate mitochondrial biology and treat diseases that arise from mtDNA mutations, editing mtDNA has been hampered by the lack of an effective way to import RNA into the organelle.sup.9. This constraint has precluded the use of RNA-guided precision editing strategies such as base editing.sup.34,35, as well as other CRISPR methods.sup.43. We speculated that we could use RNA-independent programmable DNA-binding proteins fused to split-DddA.sub.tox halves to enable RNA-free base editing of mtDNA. TALE proteins contain an array of highly conserved 33- or 34-amino acid repeats that each recognize a dsDNA nucleotide. Each repeat can be programmed to target A, C, G/A, or T nucleotides.sup.45,46. Arrays of TALE repeats recognize consecutive nucleotides of sufficient total length to specify a single dsDNA target sequence within a mammalian genome.sup.47-49.
[0779] While deaminases have been previously fused to zinc-finger arrays and TALE arrays, the very low activity of previously described deaminases on dsDNA results in very low editing efficiencies (<2.5% in human cells) that are spread over >150 bp around the DNA-binding site.sup.50. Since our Cas9 fusion results indicate that DddA.sub.tox split at sites G1333 and G1397 deaminate target TC bases in dsDNA efficiently and in a manner that limits their activity to a modest spacing region (
[0780] Each TALE array was designed to target neighboring 17-bp sites within CCR5 in U20S cells and contained a bipartite NLS (bpNLS).sup.51,52 (
[0781] We hypothesized that fusing uracil DNA glycosylase inhibitor (UGI) from B. subtilis bacteriophage PBS1 to the N- or C-terminus of each TALE-DddA.sub.tox fusion could suppress UDG-mediated nuclear base excision repair, a strategy we previously used to develop cytosine base editors.sup.34,53,54. Indeed, appending two copies of UGI (2-UGI) to the N-terminus increased editing efficiency at C.sub.9 by 8-fold to 22-27%, and reduced indels to <2.30.31% (
Optimizing TALE-Split DddA.SUB.tox .Fusions for Mitochondrial Base Editing
[0782] Given that TALE-split DddA.sub.tox fusions mediated efficient RNA-free base editing of nuclear DNA in human cells, we next investigated the possibility of applying this system to achieve programmable C.Math.G-to-T.Math.A conversion in mtDNA. We introduced previously reported mutations into the N-terminal domain (NTD).sup.55 of the 14459A-mitoTALE pair.sup.11 to recognize wild-type ND6, a mitochondrial gene that encodes the NADH dehydrogenase 6 subunit of complex I. Each DddA.sub.tox half from a G1333 or G1397 split was fused to the C-terminus of the mitoTALE array protein through a 2-amino acid linker to form a mitoTALE-DddA.sub.tox pair that flanks a 15-bp mtDNA spacing region in HEK293T cells (
[0783] Among simple mitoTALE-DddA.sub.tox fusions (
[0784] Fluorescence microscopy revealed that while C-terminal MTS-mitoTALE-split-DddA.sub.tox-UGI fusions clearly localized to the mitochondria in HeLa cells, N-terminal MTS-UGI-mitoTALE-split-DddA.sub.tox fusions remained diffused throughout the cytoplasm (
[0785] In contrast with cytosine base editors for nuclear DNA.sup.53, appending a second copy of UGI to the C-terminus of MTS-mitoTALE-split-DddA.sub.tox-UGI did not increase mtDNA editing efficiencies for any tested target, despite exhibiting similar levels of mitochondrial localization (
[0786] These results collectively suggested an optimized architecture for a mitoTALE-split-DddA.sub.tox pair in which each protein consists of (in N- to C-terminus order): an MTS, a TALE array, a 2-amino acid linker, a DddA.sub.tox half from the G1333 or G1397 split, and one UGI protein (
[0787] Given that DddA.sub.tox can edit cytidines on either DNA strand, intermediates containing uracils on opposing DNA strands could produce DSBs following base excision repair, thereby resulting in unwanted indels. While BE4max targeting EMX1 in the nucleus resulted in 1.80.67% indels, typical of nuclear cytosine base editors, indels were not detected (<0.1%) at ND6 in HEK293T cells despite editing by ND6-DdCBE on both DNA strands (
[0788] Taken together, the very high product purity and lack of indels associated with DdCBE suggest that uracil repair processes that lead to indels and other byproducts in nuclear DNA.sup.32 are inefficient in the mitochondria. This model is consistent with previous observations that a ssDNA deaminase targeted to the mitochondria introduced C.Math.G-to-T.Math.A conversions with no indels.sup.62, supporting a model in mitochondria in which lesion-containing genomes are degraded rather than repaired.sup.63,64, resulting in selective maintenance of mtDNA copies that have been cleanly edited.
[0789] We analyzed the ND6 allele distributions produced by ND6-DdCBE and found that alleles containing the G.sub.6G7A-to-A.sub.6A.sub.7A conversion (which corresponds to a TC.sub.7C.sub.6-to-TT.sub.7T.sub.6 conversion in the complementary mtDNA strand) accounted for 380.90% of all edited alleles (
[0790] Taken together, these findings establish a precise mtDNA editing capability that uses an unprecedented dsDNA-specific cytidine deaminase that we split to mitigate its toxicity, programmable dsDNA-binding TALE arrays, and a uracil glycosylase inhibitor to achieve efficient RNA-free base editing in the mitochondria.
Mitochondrial Base Editing of Five mtDNA Genes in Human Cells
[0791] To explore the generality of DdCBE for mtDNA editing, we constructed seven additional pairs of TALE array proteins either engineered de novo or adapted from validated mitoTALE arrays (Table 3) to target five mitochondrial genes: ND1, ND2, ND4, ND5, and ATP8. For some of the targeted sites, we expected DdCBE editing to install disease-relevant mutations (see the Supplementary Discussion section of Example 1). For example, C.Math.G-to-T.Math.A conversion by ND2- and ND4-DdCBE would install the m.5032G>A and m.11922G>A mutations, respectively, which are believed to be disease-causing in cancer-related tumors of the kidney and thyroid.sup.5, 65.
[0792] Mitochondrial base editing efficiencies of DdCBEs in HEK293T cells 3-6 days after treatment varied between 4.6-49% depending on the split type, split orientation, and target cytidine position within the spacing region (
[0793] In contrast, the effect of split orientation on editing efficiencies was more site-dependent for the G1397 split. The Right-G1397-DddA.sub.tox-N+Left-G1397-DddA.sub.tox-C orientation mediated 3.1- to 7.6-fold higher editing than the Right-G1397-DddA.sub.tox-C+Left-G1397-DddA.sub.tox-N orientation at TC.sub.12C within an 18-bp spacing region (
[0794] For a given TC target that was edited by both G1333 and G1397 splits, G1397 generally afforded higher editing efficiencies than G1333 (
[0795] We observed a narrower editing window, which we define as the number of nucleotide positions upstream or downstream of the target TC base that are amenable to deamination, for G1397-split DdCBE compared to G1333-split DdCBE. The G1397 split efficiently converted TCs within a window of 1-2 nucleotides positioned (i) approximately 4-7 nucleotides from the 3 end of a 15- to 18-bp spacing region in the H-strand of mtDNA (
[0796] These results collectively suggest that each split is associated with a distinct preference for editing TCs within a specific editing window in the spacing region. We recommend testing G1397 and G1333 splits in both orientations to determine the fusion that gives the highest base editing efficiency for a given target sequence and spacing length.
[0797] To evaluate the durability of C.Math.G-to-T.Math.A mitochondrial DNA conversions in HEK293T cells, we tracked editing induced by ND6-, ND5.1-, ND5.2- and ATP8-DdCBE over 18 days at 3- or 6-day intervals, spanning approximately 21 cell divisions. Throughout this period, the viability of cells expressing DdCBEs remained indistinguishable from that of untreated cells (
[0798] Across all DdCBEs, editing efficiencies increased by 1.5- to 3.7-fold from day 3 to day 6 as levels of DdCBE protein persisted (
[0799] We further characterized the ND4-DdCBE-edited HEK293T cells to evaluate the functional consequences of mtDNA editing of ND4. Following 8 days of passaging of three independently edited cell lines, we observed persistence of m.11922G>A heteroplasmic mutation in ND4 (
[0800] Collectively, these results show that DdCBEs are capable of inducing durable, C.Math.G-to-T.Math.A conversions in mtDNA that are stably maintained (in the absence of edit-induced fitness changes) over many cell divisions without concomitant loss of cell viability and mtDNA copy number.
Off-Target Editing by DdCBEs
[0801] Virtually all genome editing agents reported to date exhibit some degree of editing at off-target loci, a consequence of the inherently imperfect nature of binding specificity, as well as the high sensitivity of modern genome analyses.sup.1,2,66. To profile potential off-target activity of DdCBE in the human mitochondrial genome, we transfected HEK293T cells with plasmids that constitutively expressed optimized DdCBE halves targeting ND6, ND5.1, ND5.2, ND4 or ATP8, or the corresponding inactive mutant DdCBE (dead-DdCBE) containing the E1347A mutation in DddA.sub.tox. The dead-DdCBE controls enable identification of single-nucleotide variants (SNVs) that arise from background heteroplasmy to be distinguished from those that arise from off-target editing. To test for spontaneous assembly of split DddA.sub.tox in the absence of TALE-directed DNA binding, cells were also transfected with plasmids expressing DddA.sub.tox halves containing MTS-G1397 split DddA.sub.tox-UGI, with no TALE array. Bulk cell populations were collected 3 days after transfection and sequenced by ATAC-seq.sup.67-69 to capture the entire 16.6-kb mitochondrial genome with an average of 5,100 to 9,900-fold coverage per mtDNA base (
[0802] The average frequencies of genome-wide off-target C.Math.G-to-T.Math.A editing by ND5.2-DdCBE, ND4-DdCBE and ATP8-DdCBE were comparable to that of the untreated and TALE-free G1397 DddA.sub.tox controls (0.030-0.034% for the DdCBEs versus 0.029-0.030% for the controls), while ND5.1-DdCBE had 1.6-fold higher average off-target editing frequency (0.049%) compared to the untreated control (
[0803] ND6-targeting DdCBE showed 4.2-fold higher average off-target editing frequency (0.13%) compared to that of the untreated control (
[0804] Among the DdCBEs with standard NTDs, off-target editing levels did not strongly correlate with on-target editing efficiencies (compare
[0805] We noted a strong 5-TC-3 preference across all tested DdCBEs that matched the sequence preferences for overexpression of free DddA.sub.tox in E. coli (compare
[0806] To further probe the nature of off-target edits by DdCBEs, we searched for sequence homology between 20-bp regions flanking each off-target edit and on-target TALE-binding sites. We did not observe any consensus off-target sequences that closely resemble on-target TALE binding sites (
DISCUSSION
[0807] This study describes the discovery of a dsDNA-specific cytidine deaminase toxin, and its development into an RNA-free base editor that can install the first targeted point mutations in the human mitochondrial genome. Additional research will be needed to fully elucidate the principles governing DdCBE efficiency and specificity. Given that DddA.sub.tox activity can be attenuated by the DddI.sub.A immunity protein, this built-in kill switch could be used to control DdCBE activity if necessary. In addition, developing in vitro and in vivo strategies to deliver DdCBEs will be essential for exploring their therapeutic potential in other cell types and in animal models of mitochondrial diseases. Evolving or engineering DddA.sub.tox variants with altered sequence context preferences beyond 5-TC-3.sup.74, or that deaminate nucleosides other than cytidine.sup.35, would further expand the scope of mtDNA editing. The largely untapped natural diversity of bacterial DNA deaminases could provide additional starting points towards these and other novel base editing systems.
[0808] Editing of mitochondrial DNA has previously been limited to heteroplasmy shifts.sup.75 or copy number modulation.sup.76 due to the lack of DSB-initated DNA repair pathways found in the nucleus, and the unavailability of reliable methods to import guide RNAs required for CRISPR methods. Following our discovery of an interbacterial toxin DddA.sub.tox that unprecedentedly deaminates cytidines only in dsDNA, we designed splits of DddA.sub.tox to overcome its inherent toxicity and engineered fusions of the non-toxic halves to DNA-binding TALE proteins. The resulting DdCBEs enable programmable C.Math.G-to-T.Math.A conversions in mtDNA without requiring DSBs, a new capability that has the potential to install or correct pathogenic mtDNA SNPs associated with mitochondrial disorders (
[0809] More broadly, the principles behind DdCBE demonstrate how enzymes that modify double-stranded DNA can be made dependent on DNA-binding proteins to enable efficient CRISPR-free gene editing systems. Finally, while this study has focused on the use of DdCBE for mitochondrial base editing, some features of DdCBE (or zinc-finger array variants), such as its all-protein nature, lack of a PAM requirement, and independence from CRISPR components, may also offer advantages for base editing outside the mitochondria.
Methods
Bacterial Strains and Culture Conditions
[0810] Except as noted, all bacterial strains used in this study were grown in Lysogeny Broth (LB) at 37 C. When required, media was supplemented with the following: carbenicillin 150 g mL.sup.1 gentamycin g mL.sup.1, 80 M IPTG, 0.05% (w/v) rhamnose, chloramphenicol g mL.sup.1 or tetracycline g mL.sup.1 for E. coli, chloramphenicol 15 g mL.sup.1 or gentamycin 30 g mL.sup.1 for P. aeruginosa, and carbenicillin 150 g mL.sup.1, or tetracycline 120 g mL.sup.1 for B. cenocepacia. E. coli strains DH5, XK1502, and BL21 were used for plasmid maintenance, toxicity and mutagenesis assays, and protein expression, respectively. P. aeruginosa strains were derived from the model strain PAO1, and B. cenocepacia strains were derived from the cystic fibrosis clinical isolate H111. A detailed description of the bacterial strains and plasmids used in this study is provided in Tables 8A-8B.
Genetic Techniques and Plasmid Construction for Bacterial Expression
[0811] All procedures for DNA manipulation and transformation were performed with standard methods. Molecular biology reagents, Phusion high fidelity DNA polymerase, restriction enzymes, UDG, and Gibson Assembly Reagent were obtained from New England Biolabs (NEB). GoTaq Green Master Mix was obtained from Promega. Primers and gBlocks used in this study were obtained by Integrated DNA Technologies (IDT). A list of all primers is provided in the Supplementary Sequence section in Example 1.
[0812] Protein expression constructs were generated by Gibson assembly. For functional protein expression assays of DddA.sub.tox, TadA and CDD, the relevant genes or gene fragments were amplified from B. cenocepacia (DddA.sub.tox) or E. coli genomic DNA and cloned into the vector pSCRhaB2. DddA.sub.1 was amplified from B. cenocepacia and cloned into pPSV39, and the expression construct for DddA.sub.tox(E1347A) was generated by splicing by overlap extension PCR followed by Gibson assembly with pSCRhaB2. For the APOBEC3G expression construct, the gene sequence was codon optimized for expression in E. coli, generated by synthesis as a gBLOCK (IDT) and cloned into pSCRhaB2. For protein purification, DddA.sub.tox and DddA.sub.1 were amplified from B. cenocepacia and cloned into pETDuet.
[0813] Deletion of udg in P. aeruginosa was performed using allelic exchange mediated by the vector pEXG2, using counter selection with sucrose as described in detail previously.sup.79. Gene deletions and nucleotide substitutions in B. cenocepacia were performed by homologous recombination using the plasmid pDONRPEX18Tp-SceI-pheS, followed by counter selection using the plasmid pDAI-SceI and plasmid curing using 0.1% (w/v) p-chlorophenylalanine, as described previously.sup.80. Gentamycin resistant B. cenocepacia was generated by insertion of a resistance cassette at the Tn7 site attachment site as described previously.sup.81.
Plasmid Construction for Mammalian Expression
[0814] PCR was performed using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific), Phusion U Green Hot Start DNA Polymerase (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). All plasmids were constructed using USER cloning (New England Biolabs). DddA.sub.tox and mitoTALE genes were synthesized as gene blocks and codon optimized for human expression (Genscript). BE2 and BE4max plasmids were obtained from previous reports.sup.34,54. DddA.sub.tox-Cas9 fusions and DdCBE variants were cloned into pCMV (mammalian codon-optimized) backbones. sgRNA plasmids were constructed by blunt-end ligation of a linear polymerase chain reaction (PCR) product generated by encoding the 20- to 23-nt variable protospacer sequence onto the 5 end of an amplification primer and treating the resulting piece with KLD Enzyme Mix (New England Biolabs) according to the manufacturer's instruction. Mach1 chemically competent E. coli cells (ThermoFisher Scientific) were used for plasmid construction. Plasmids for mammalian transfection were purified using ZymoPURE II Plasmid Midiprep Kits (Zymo Research), as previously described.sup.82. A list of all primers is provided in the Supplementary Sequence section of Example 1.
Bacterial Competition Experiments
[0815] Donor and recipient strains were grown overnight and mixed in a 10:1 (v/v) ratio for donor and recipient, respectively. The cell suspensions were then concentrated to a total OD.sub.600 of 10, and 10 L was spotted on a 0.2 m nitrocellulose membrane placed on LB with 3% (w/v) agar followed by 6 hours incubation at 37 C. After the incubation, cells were scraped from the membranes surface and resuspended in 1 mL LB. The initial donor:recipient ratio and the post-incubation ratio were determined by plating on LB agar (LBA) to determine the total number of colony forming units (CFU) and on LBA to with gentamycin to determine CFUs for the marked recipient strain. The competitive index is defined as the final donor:recipient ratio divided by the initial donor:recipient ratio. Competition experiments used to determine the bactericidal effect of DddA were performed similarly, but instead of cells being plated after 6 hours, they were harvested and plated hourly and CFU were enumerated on LBA with gentamycin to quantify the recipient strain population.
Toxicity Assays
[0816] To evaluate the toxicity of deaminases expressed heterologously, overnight cultures of E. coli XK1502 containing the appropriate plasmids were diluted 1:1000 into fresh medium and grown until reaching exponential phase (OD.sub.600 0.6), at which point deaminase expression was induced with 0.2% (w/v) rhamnose. Aliquots of cultures were then collected periodically until 480 minutes of growth, and were diluted and plated onto LBA for CFU determination.
Crystallization and Structure Determination
[0817] Crystals of the selenomethionine derivative hexahistidine-tagged DddA.sub.tox(a.a. 1264-1427).Math.DddA.sub.1 complex were obtained at 5-10 mg/mL in crystallization buffer (15 mM Tris pH 7.5, 150 mM NaCl, 1.0 mM tris(2-carboxyethyl)phosphine (TCEP)), mixed 1:1 with crystallization solution containing 25% (w/v) PEG 3350, 0.1 M Bis-Tris:HCl pH 6.5, 200 mM MgCl.sub.2. Rectangular crystals grew to 400200100 m over 5 days. Selenomethionine DddA.sub.tox.Math.DddA.sub.1 crystals displayed the symmetry of space group P2.sub.12.sub.12 (a=126.8 , b=145.0 , c=64.2 , ===90), with four dimers in the asymmetric unit. Prior to data collection, crystals were cryoprotected in crystallization solution 15% glycerol, 25% PEG3350, 100 mM MgCl.sub.2, 100 mM NaCl, 7.5 mM Tris pH 7.5, 50 mM Bis-Tris pH 6.5, 0.5 mM TCEP.
[0818] Highly redundant anomalous (SAD) data were obtained at 0.9790 A (peak) wavelength from a single selenomethionine crystal at 100 K temperature at the BL502 beamline (ALS, Lawrence Berkeley National Laboratory). Data were processed using HKL2000.sup.83. Heavy atom searching using phenix.autosol identified 18 possible sites, and refinement yielded an estimated Bayes correlation coefficient of 55.9 to 2.5 resolution. After density modification, the estimated Bayes correlation coefficient increased to 61.2. Approximately 70% of the selenomethionine model was constructed automatically, and the remaining portion was built manually. The current model (Table 2) contains four DddA-DddIA dimers.
[0819] Refinement was carried out against peak anomalous data with Bijvoet pairs kept separate using phenix.refine.sup.84 interspersed with manual model revisions using the program Coot.sup.85 and consisted of conjugate-gradient minimization and calculation of individual atomic displacement and translation/libration/screw parameters.sup.86. Residues that could not be identified in the electron density were: 1250-1289 and 1423-1427 for DddA, and 71-73 for DddIA. Both models exhibit excellent geometry, as determined by MolProbity.sup.87. Ramachandran analysis identified 99.1% favored, 0.9% allowed, and 0% disallowed residues for the model. Coordinates and structure factors are deposited in the RCSB Protein Data Bank (ID 6U08).
Mutation Frequency Determination and SNP Generation Assay
[0820] To determine the frequency of mutations induced by expression of DddA.sub.tox and DddA.sub.tox (E1347 ), overnight cultures of E. coli containing the expression plasmids for these proteins together with the plasmid for expression of DddA.sub.1 were diluted 1:1000 into fresh medium and grown until reaching exponential phase (OD.sub.600 0.6). The cultures were then induced with 0.08 mM IPTG for DddIA and 0.04% rhamnose for DddA.sub.tox or DddA.sub.tox(E1347 ) expression respectively. The combined expression of both toxin and immunity proteins at this low level allows the cells expressing DddA.sub.tox to suffer growth arrest but does not result in a decrease in culture viability. After 1 hour under these inducing conditions, cultures were supplemented with 1 mM of IPTG to increase DddI.sub.A expression and thus block DddA toxicity and were then grown an additional 16 hours. After this recovery period, the cultures were plated onto LBA containing rifampicin or no antibiotics. Mutation frequency was determined by the ratio of the number of rifampicin resistant colonies by the total CFU obtained on non-selective medium.
[0821] For the genome-wide identification of SNPs that accumulate following low level expression of DddA.sub.tox or DddA.sub.tox(E1347 ), E. coli udg carrying plasmids for expression of one of these proteins plus the plasmid for expressing DddI.sub.A was submitted to seven rounds of expression and recovery as described above, with cultures being plated after recovery and single colonies being selected and used to inoculate the subsequent round of expression. Randomly chosen single colonies were used to avoid introducing selection for increased fitness under the culture conditions.sup.87. Five isolated colonies from each starting population subjected to this regimen were selected for whole genome sequencing.
Western Blot for E. coli-Expressed Deaminase
[0822] Western blotting to detect deaminases expressed in E. coli was performed using rabbit -VSV-G (diluted 1:5000, Sigma) and detected with -rabbit horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000, Sigma). Loading control was performed with mouse -RNAP (diluted 1:500, Biolegend) and detected with sheep -mouse (diluted 1:500, Millipore). Western blots were developed using chemiluminescent substrate (SuperSignal West Pico Substrate, Thermo Scientific) and imaged with a C600 imager (Azure biosystems).
Western Blot for Mammalian Cell-Expressed DdCBE
[0823] HEK293T cells were transfected as described below. For preparation of cell lysate for western blot analysis of DdCBE, cells were lysed in 150 L of ice-cold 1 RIPA buffer (Sigma) with added protease inhibitor (Roche Complete Mini) by incubating for 30 min at 4 C. with agitation. Lysates were cleared by pelleting at 12,000 rcf for 10 min at 4 C.
[0824] 60 L of cleared lysate supernatant was added to 20 L of 4 LDS sample loading buffer (ThermoFisher Scientific) with a final DTT (Sigma Aldrich) concentration of 10 mM. Lysates were boiled for 10 min at 95 C. 15-20 L of protein lysate was loaded into the wells of a Bolt 4-12% Bis-Tris Plus (Thermo Fisher Scientific) pre-cast gel. 6 L of Precision Plus Protein Dual Color Standard (Bio-Rad) was used as a reference. Samples were separated by electrophoresis at 180 V for 35 min in Bolt MES SDS running buffer (Thermo Fisher Scientific). Transfer to a PVDF membrane was performed using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific) according to manufacturer's protocols. The membrane was blocked in Odyssey Blocking Buffer (LI-COR) for 1 h at room temperature, then incubated with rat anti-FLAG (ThermoFisher Scientific MA1-142; 1:2000 dilution), mouse anti-HA (ThermoFisher Scientific 26183; 1:2000 dilution) and rabbit anti-actin (CST 4970; 1:2000 dilution) in blocking buffer (0.5% Tween-20 in 1PBS, 0.2 m filtered) overnight at 4 C. The membrane was washed 3 with TBST (1TBS in 0.5% Tween-20) for 10 min each at room temperature, then incubated with IRDye-labeled secondary antibodies goat anti-rat 680RD (LI-COR 926-68076), goat anti-mouse 800CW (LI-COR 926-32210) and donkey anti-rabbit 800CW (LI-COR 926-32213) diluted 1:5000 in blocking buffer for 1 h at room temperature. The membrane was washed as before, then imaged using an Odyssey Imaging System (LI-COR).
Purification of Proteins for Biochemical Assays
[0825] Overnight cultures of E. coli BL21 pETDuet-1::dddA.sub.tox-dddA.sub.1, or E. coli BL21 pETDuet-1::dddA.sub.tox (E1347 ) were used to inoculate 2 L of LB broth in a 1:100 dilution and cultures were grown to approximately OD.sub.600 0.6. At this point, plasmid expression was induced with 0.5 mM IPTG and the cultures were incubated for 16 hours at 18 C. in a shaking incubator. Cell pellets were harvested by centrifugation at 4000 g for 20 min, followed by resuspension in 50 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 30 mM imidazole, 1 mM DTT, and 1 mg/mL lysozyme). Cell pellets were then lysed by sonication (5 pulses, 10 s each) and supernatant was separated by centrifugation at 25,000 g for 30 min.
[0826] The DddA.sub.tox-DddA.sub.1 complex or DddA.sub.tox(E1347 ) was purified from cell lysates by nickel affinity chromatography using 4 mL of Ni-NTA agarose beads loaded onto a gravity-flow column. Supernatant was loaded onto the column and resin was washed with 50 mL of wash buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 30 mM imidazole, 1 mM DTT). Proteins of interest were eluted with 5 mL elution buffer (50 mM Tris-HCl pH 7.5, 300 mM Imidazole, 500 mM NaCl, 30 mM imidazole, 1 mM DTT). When DddA.sub.tox(E1347 ) was purified, the eluted samples were applied directly to size exclusion chromatography. For DddA-DddA.sub.1, the eluted samples underwent a denaturation and renaturation step to isolate only the toxin. In this case, the eluted proteins were added to 50 mL 8 M urea denaturing buffer (50 mM Tris-HCl pH 7.5, 300 mM Imidazole, 500 mM NaCl, and 1 mM DTT) and incubated for 16 hours at 4 C. The 8 M urea denaturing buffer with the eluted proteins was loaded on a gravity-flow column with 4 mL Ni-NTA agarose beads. The column was washed with 50 mL 8 M urea denaturing buffer to remove any remaining DddA.sub.1. While still bound to Ni-NTA agarose beads, DddA.sub.tox was renatured by sequential washes with 25 mL denaturing buffer with decreasing concentrations of urea (6 M, 4 M, 2 M, 1 M), and a last wash with wash buffer to remove remaining traces of urea. Proteins bound to the column were then eluted with 5 mL elution buffer. The eluted samples were purified again by sizing exclusion chromatography using protein liquid chromatography (FPLC) with gel filtration on a Superdex200 column (GE Healthcare) in sizing buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM DTT, 5% (w/v) glycerol). The fraction purity was evaluated by SDS-PAGE gel stained with Coomassie Brilliant Blue and the highest quality factions were stored at 80 C.
DNA Deamination Assays
[0827] All the DNA substrates were designed with the addition of a 5 6-FAM fluorophore for visualization, and they were purchased from Integrated DNA Technologies (IDT). All substrate sequences are present in the Supplementary Sequence section of Example 1. Reactions were performed in 10 L of deamination buffer (20 mM MES pH 6.4, 200 mM NaCl, 1 mM DTT, 8% Ficoll 70, and 1 M substrate) with APOBEC3A, DddA.sub.tox or DddA.sub.tox(E1347 ) at the concentrations indicated in
Poisoned Primer Extension Assay for RNA Deamination
[0828] All substrate sequences are listed in the Supplementary Sequence section of Example 1. The RNA substrates and the oligonucleotide containing a 5 6-FAM fluorophore for visualization were purchased from Integrated DNA Technologies (IDT). Deamination reactions were performed in 10 L of RNA deamination buffer (Tris-HCl pH 7.5, 200 mM NaCl, 1 mM DTT) with the addition of 1 M of DddA.sub.tox or DddA.sub.tox (E1347 ). Substrate combinations and concentrations were added as indicated in
Genome Sequencing and SNP Identification in Bacteria
[0829] Overnight cultures from isolated colonies were used for total gDNA extraction with the DNeasy Blood & Tissue kit (Qiagen), and extraction yield was quantified using a Qubit. Sequencing libraries were constructed using the Nextera DNA Flex Library Prep Kit (Illumina). Library quality and concentration was evaluated with a Qubit and TapeStation System (Agilent). Sequencing was performed with an Illumina MiSeq instrument (300 cycles paired end program). Genome mapping was performed with BWA.sup.88 using the E. coli MG1655 (NC_000913.3) genome as a reference. Pileup data from alignments were generated with SAMtools and variant calling was performed with VarScan2.sup.89. SNPs were considered valid if they were present at a frequency higher than 90%.
Mammalian Cell Culture
[0830] All cells were cultured and maintained at 37 C. with 5% CO.sub.2. Antibiotics were not used for cell culture. HEK293T cells [CRL-3216, American Type Culture Collection (ATCC)] were cultured in Dulbecco's modified Eagle's medium plus GlutaMax (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS). U20S cells (HTB-96, ATCC) were cultured in MyCoy's 5 medium plus GlutaMax (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS. HeLa cells (ATCC CCL-2) were cultured in high glucose DMEM (Gibco) with 10% fetal bovine serum (Atlanta Biological), and 100 U/mL penicillin (Sigma-Aldrich). Cell lines were authenticated by their respective suppliers and tested negative for mycoplasma.
Mammalian Cell Lipofection
[0831] HEK293T cells were seeded on 48-well collagen-coated plates (Corning) at a density of 210.sup.5 cells/mL 18-24 hours before lipofection. Lipofection was performed at a cell density of approximately 70%. For split DddA.sub.tox-Cas9 screening, cells were transfected with 375 ng of split DddA.sub.tox-dSpCas9 monomer expression plasmid, 375 ng of split DddA.sub.tox-SaKKH-Cas9(D10 ) monomer expression plasmid, 125 ng of SpCas9 gRNA expression plasmid and 125 ng of SaKKH gRNA plasmid. pUC19 was used as a filler DNA for monomer and no-gRNA control experiments to make up to 1000 ng of total plasmid DNA. For DdCBE experiments, cells were transfected with 500 ng of each mitoTALE monomer to make up 1000 ng of total plasmid DNA. Lipofectamine 2000 (1.5 L; ThermoFisher Scientific) was used per well. Cells were harvested at the indicated timepoint.
[0832] For western blot analysis of DdCBEs expressed in mammalian cells, HEK293T cells were seeded on 6-well tissue culture-treated plates (Corning) at a density of 210.sup.5 cells/mL 18-24 hours before lipofection. Cells were transfected with 4000 ng of each mitoTALE monomer to make up 8000 ng of total plasmid DNA. Lipofectamine 2000 (12 L; ThermoFisher Scientific) was used per well. Cells were harvested at the indicated timepoint.
[0833] For inducible expression of full-length DddA.sub.tox fused to Cas9 (
Mammalian Cell Nucleofection
[0834] We combined 500 ng of Left DdCBE monomer and 500 ng of Right DdCBE monomer in a volume that did not exceed 2 L. This combined plasmid mixture was nucleofected in a final volume of 22 L per sample in a 16-well Nucleocuvette strip (Lonza). U20S cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 30,000-50,000 cells per sample (program DN-100), according to the manufacturer's protocol.
Cell Viability Assays
[0835] Cell viability was measured every either every 24 h post-transfection for 3 days (
Genomic DNA Isolation from Mammalian Cell Culture
[0836] Medium was removed, and cells were washed once with 1 Dulbecco's phosphate-buffered saline (ThermoFisher Scientific). Genomic DNA extraction was performed by addition of 45 L freshly prepared lysis buffer (10 mM tris-HCl (pH 7.0), 0.05% SDS, and proteinase K (20 g/mL; ThermoFisher Scientific)) directly into the 48-well culture well. The extraction solution was incubated at 37 C. for 60 min and then 80 C. for 20 min. Resulting genomic DNA was subjected to bead cleanup with AMPure DNAdvance beads according to manufacturer's instructions (Beckman Coulter A48705).
Determination of Relative Total Mitochondrial DNA Levels by Quantitative PCR
[0837] Quantitative PCR (qPCR) reactions were performed on a Bio-Rad CFX96/C1000 qPCR machine performed using SYBR green (Lonza). 5 ng of purified DNA was used as template input in a 25 L reaction volume. For all reactions, the protocol used was an initial heating step of 2 min at 98 C. followed by 40 cycles of amplification (10 s at 98 C., 20 s at 62 C., 15 s at 72 C.). Single threshold values (AC) were determined by manufacturer's software. The level of mtDNA was determined by the calculating the ratio of total mtDNA to genomic DNA (-actin)
where E is the efficiency of the qPCR reaction; E.sub.ND6=0.858, E.sub.ND5=0.844, E.sub.ATP8=0.995, E.sub.-actin=1.05). Refer to the Supplementary Sequence section of Example 1 for list of primers used. NC_012920 was used as the reference for mtDNA; NG_003019 was used as the reference for human ACTBP2.
Long-Range PCR to Detect mtDNA Deletions
[0838] Long-range PCR was performed on purified genomic DNA as previously with listed primers (Supplementary Sequence section of Example 1) to capture the whole mtDNA genome as two overlapping fragment of 8 kb each. Briefly, 50-200 ng of purified DNA was used as input for amplification by PRIMESTAR GXL DNA polymerase (Takara). For all reactions, the protocol used was an initial heating step of 1 min at 94 C. followed by 30 cycles of amplification (30 s at 98 C., 30 s at 60 C., 9 min at 72 C.). Unpurified PCR products were run on 0.8% agarose gel and stained with ethidium bromide.
Immunocytochemical Studies of DdCBE Localization
[0839] HeLa cells were transfected with a total of 1 g of plasmid DNA to express left (HA-tagged) or right (FLAG-tagged) monomers of each DdCBE using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. After 24 hours incubation, cells were labelled with MitoTracker Deep Red (ThermoFisher) at a final concentration of 100 nM for 30 minutes at 37 C., 5% CO.sub.2 incubator. Cells were then seeded on an 8-well chamber glass slide (Ibidi) and fixed in 4% paraformaldehyde/PBS for 15 minutes at room temperature. Next, cells were washed twice with PBS and permeabilized in PBS containing 0.1% saponin and 1% BSA for 30 minutes at room temperature. Cells were then immunostained with -HA (Biolegend) or -Flag (Sigma Aldrich), followed by Alexa-fluor conjugated -mouse (HA tag) or -rabbit (FLAG tag) secondary antibodies (Thermo Fisher). Images were taken using a 60 objective with the high-resolution widefield Nikon system. Acquired images were processed in Fiji (http://fiji.sc/).
High-Throughput DNA Sequencing of Genomic DNA Samples
[0840] Genomic sites of interest were amplified from genomic DNA samples and sequenced on an Illumina MiSeq as previously described with the following modifications.sup.43. Amplification primers containing Illumina forward and reverse adapters (Supplementary Sequence section of Example 1) were used for a first round of PCR (PCR 1) to amplify the genomic region of interest. Briefly, 1 L of purified genomic DNA was input into the first round of PCR (PCR1). For PCR1, DNA was amplified to the top of the linear range using Phusion Hot Start II High-Fidelity DNA Polymerase (ThermoFisher Scientific), according to the manufacturer's instructions but with the addition of 0.5 SYBR Green Nucleic Acid Gel Stain (Lonza) in each 25-L reaction. For all amplicons, the PCR1 protocol used was an initial heating step of 2 min at 98 C. followed by an optimized number of amplification cycles (10 s at 98 C., 20 s at 62 C., 30 s at 72 C.). Quantitative PCR was performed to determine the optimal cycle number for each amplicon. The number of cycles needed to reach the top of the linear range of amplification are 27-28 cycles for nuclear DNA amplicons and 17-19 cycles for mtDNA amplicons. Barcoding PCR2 reactions (25 L) were performed with 1 L of unpurified PCR1 product and amplified with Q5 Hot Start MasterMix (New England Biolabs) using the following protocol 98 C. for 2 min, then 12 cycles of [98 C. for 10 s, 61 C. for 20 s, and 72 C. for 30 s], followed by a final 72 C. extension for 2 min. PCR products were evaluated analytically by electrophoresis in a 1.5% agarose gel. After PCR2, up to 240 samples with different barcode combinations were combined and purified by gel extraction using the QIAquick Gel Extraction Kit (QIAGEN). DNA concentration was quantified using the Qubit ssDNA HS Assay Kit (Thermo Fisher Scientific) to make up a 4 nM library. The library concentration was further verified by qPCR (KAPA Library Quantification Kit-Illumina, KAPA Biosystems) and sequenced using an Illumina MiSeq with 210- to 280-bp single-end reads.
Analysis of HTS Data for DNA Sequencing and Targeted Amplicon Sequencing
[0841] Sequencing reads were demultiplexed using MiSeq Reporter (Illumina). Batch analysis with CRISPResso2.sup.90 was used for targeted amplicon and DNA sequencing analysis. A 10-bp window was used to quantify indels centered around the middle of the dsDNA spacing. To set the cleavage offset, a hypothetical 15- or 16-bp spacing region has a cleavage offset of 8. Otherwise, the default parameters were used for analysis. The output file Reference.NUCLEOTIDE_PERCENTAGE_SUMMARY.txt was imported into Microsoft Excel for quantification of editing frequencies. Reads containing indels within the 10-bp window are excluded for calculation of editing frequencies. The output file CRISPRessoBatch_quantification_of_editing_frequency.txt was imported into Microsoft Excel for quantification of indel frequencies. Indel frequencies were computed by dividing the sum of Insertions and Deletions over the total number of aligned reads.
Bulk ATAC-Seg for Whole Mitochondrial Genome Sequencing
[0842] ATAC-seq was performed as previously described.sup.67. In brief, 5,000-10,000 cells were trypsinzed, washed with PBS, pelleted by centrifugation and lysed in 50 L of lysis buffer (0.1% Igepal CA-360 (v/v %), 10 mM Tris-HCl, 10 mM NaCl and 3 mM MgCl2 in nuclease-free water). Lysates were incubated on ice for 3 minutes, pelleted at 500 rcf for 10 minutes at 4 C. and tagmented with 2.5 L of Tn5 transposase (Illumina #15027865) in a total volume of 10 p L containing 1TD buffer (Illumina #15027866), 0.1% NP-40 (Sigma), and 0.3 PBS. Samples were incubated at 37 C. for 30 minutes on a thermomixer at 300 rpm. DNA was purified using the MinElute PCR Kit (Qiagen) and eluted in 10 L elution buffer. All 10 L of the eluate was amplified using indexed primers (1.25 M each) listed in the the Supplementary Sequence section of Example land NEBNext High-Fidelity 2 PCR Master Mix (New England Biolabs) in a total volume of 50 L using the following protocol 72 C. for 5 min, 98 C. for 30 s, then 5 cycles of [98 C. for 10 s, 63 C. for 30 s, and 72 C. for 60 s], followed by a final 72 C. extension for 1 min. After the initial 5 cycles of pre-amplification, 5 L of partially amplified library was used as input DNA in a total volume of 15 L for quantitative PCR using SYBR Green to determine the number of additional cycles needed to reach of the maximum fluorescence intensity. Typically, 3-8 cycles were conducted on the remaining 45 L of partially amplified library. The final library was purified using a MinElute PCR kit (Qiagen) and quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent). All libraries were sequenced using Nextseq High Output Cartridge kits on an Illumina Nextseq 500 sequencer. Libraries were sequenced using paired-end 275 cycles and demultiplexed using the bc12fastq program.
Genome Sequencing and SNP Identification in Mitochondria
[0843] SNP identification in mitochondria was performed similarly to in bacteria, with the following modifications. Genome mapping was performed with BWA (v0.7.17) using NC_012920 genome as a reference. Duplicates were marked using Picard tools (v2.20.7). Pileup data from alignments were generated with SAMtools (v1.9) and variant calling was performed with VarScan2 (v2.4.3). Variants that were present at a frequency greater than 0.1% and a p-value less than 0.05 (Fisher's Exact Test) were called as high-confidence SNPs independently in each biological replicate. Only reads with Q>30 at a given position were taken into account when calling SNPs at that particular position.
Calculation of Average Off-Target C.Math.G-to-T.Math.A Editing Frequency
[0844] To calculate the mitochondrial genome-wide average off-target editing frequency for each DdCBE in
Effect Prediction of the C.Math.G-to-T.Math.A Off-Target SNVs Identified by ATAC-Seq
[0845] SIFT (https://sift.bii.-star.edu.sg/) was used to predict the outcome of nonsynonymous mutations on protein function. High- and low-confidence calls were made using standard SIFT parameters with GRCh37.74 database as the reference genome.
Data Availability
[0846] High-throughput sequencing and whole-mitochondria sequencing data is deposited in the NCBI Seqeunce Read Archive (PRJNA603010). Amino acids sequences of all base editors in this study are provided in the Supplementary Sequences section of Example 1.
REFERENCES FOR EXAMPLE 1
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Supplementary Discussion
Toxicity of Intact DddA.SUB.tox .Fused to DNA-Binding Proteins
[0938] To characterize DddA.sub.tox activity in human cells, we transfected HEK293T cells with plasmids encoding DddA.sub.tox fused to catalytically inactive S.pyogenes Cas9 (dSpCas9).sup.1,2 or SpCas9(D10 ) nickase.sup.1,3 (
Editing Efficiencies of DddA.SUB.tox .Splits G1322, A1343, N1357, G1371 and N1387
[0939] Splits A1343, G1371, and N1387 resulted in <0.1% conversion of C.Math.G-to-T.Math.A at TC target bases across all tested spacing lengths (
Indel Frequencies Associated with Split-DddA.sub.tox-Cas9 Fusions
[0940] The level of indels associated with splits that afforded the highest editing efficiency (G1333 and G1397) were higher than those of canonical cytosine base editors.sup.6, up to 46% for some split fusion combinations (
Preliminary Observations Governing Editing Efficiency and Selectivity of Split-DddA.SUB.tox .for Subsequent DdCBE Editing
[0941] Cytidines in TCC contexts appeared to be favorable substrates for deamination for split-DddA.sub.tox. TCC target bases are generally deaminated at varying efficiencies depending on their positions within the dsDNA spacing region (
[0942] The selectivity of editing also depended on the fusion orientation. For 17-bp spacing length, we observed preferential deamination of target TCs that were closer to the protospacer of the Cas9 monomer fused to DddA.sub.tox-C. (
[0943] In light of the above results, we designed subsequent mitoTALE binding sites to flank a spacing region approximately 15-18 bp long that contains TCC and/or TCA target bases. To assess the substrate selectivity and editing efficiency of mitoTALE-split-DddA.sub.tox, we also included the two possible orientations for each G1333 and G1397 split.
Efforts to Increase Mitochondrial Localization of MTS-mitoTALE-Split-DddA.SUB.tox.-1UGI
[0944] We fused zmLOC100282174, a Zea mays-derived MTS previously reported to induce high mitochondrial localization in human lymphoblasts.sup.10, in tandem to SOD2 or COX8 MTS sequence. However, the dual MTS sequence lowered or had no effect on base editing efficiencies (
Detecting mtDNA Deletions in DdCBE-Treated Human HEK293T Cells
[0945] Long-range PCR of the whole mitochondrial genome resulted in a shorter DNA band for ND6-DdCBE-treated cells compared to amplicons obtained from cells treated with ND6-dead-DdCBE (
DURABILITY OF C.Math.G-TO-T.Math.A MITOCHONDRIAL DNA CONVERSIONS IN HEK293T CELLS
[0946] From day 12 to day 18, cells treated with ND6-DdCBE had a 6.2% decrease out of 27-34% total edits at C6 and C.sub.7 (
Three-Day Time Course Expression Studies on DdCBEs
[0947] We wondered if DdCBE expression levels could explain the differences in average off-target editing frequencies and SNV numbers among standard DdCBEs that share the same architectures of wild-type NTDs and deaminases, but contain different lengths and sequences of TALE array repeats. We compared expression levels of ND5.1-DdCBE, ND5.2-DdCBE and ATP8-DdCBE over a three-day period and observed no significant differences, suggesting that the steady-state expression of DdCBEs cannot account for the differences in off-target activities (
Predicted Effects of Off-Target SNVs on mtDNA Sequence and Protein Function
[0948] Approximately one-third of the total off-target mutations for each DdCBE resulted in missense mutations, while approximately two-thirds of the off-target mutations were in non-coding regions or led to synonymous mutations in coding regions (
REFERENCES FOR SUPPLEMENTARY DISCUSSION
[0949] 1 Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 (2012). [0950] 2 Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183, doi:10.1016/j.cell.2013.02.022 (2013). [0951] 3 Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819-823, doi:10.1126/science.1231143 (2013). [0952] 4 Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420, doi:10.1038/nature17946 nature.com/articles/nature17946 #supplementary-information (2016). [0953] 5 Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Science Advances 3, eaao4774, doi:10.1126/sciadv.aao4774 (2017). [0954] 6 Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nature Biotechnology 36, 843, doi:10.1038/nbt.4172 nature.com/articles/nbt.4172 #supplementary-information (2018). [0955] 7 Krokan, H. E. & Bjsris, M. Base Excision Repair. Cold Spring Harbor Perspectives in Biology 5, doi:10.1101/cshperspect.a012583 (2013). [0956] 8 Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nature Communications 8, 13905, doi:10.1038/ncomms13905 (2017). [0957] 9 Harrison, L., Brame, K. L., Geltz, L. E. & Landry, A. M. Closely opposed apurinic/apyrimidinic sites are converted to double strand breaks in Escherichia coli even in the absence of exonuclease III, endonuclease IV, nucleotide excision repair and AP lyase cleavage. DNA Repair (Amst) 5, 324-335, doi:10.1016/j.dnarep.2005.10.009 (2006). [0958] 10 Chin, R. M., Panavas, T., Brown, J. M. & Johnson, K. K. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6. Cell Rep 22, 2818-2826, doi:10.1016/j.celrep.2018.02.059 (2018). [0959] 11 Ruiz-Pesini, E. et al. An enhanced MITOMAP with a global mtDNA mutational phylogeny. Nucleic Acids Res 35, D823-828, doi:10.1093/nar/gkl927 (2007). [0960] 12 Lott, M. T. et al. mtDNA Variation and Analysis Using Mitomap and Mitomaster. Curr Protoc Bioinformatics 44, 1 23 21-26, doi:10.1002/0471250953.bi0123s44 (2013). [0961] 13 Lin, Y. et al. SAPTA: a new design tool for improving TALE nuclease activity. Nucleic Acids Res 42, e47, doi:10.1093/nar/gkt1363 (2014).
Tables
TABLE-US-00053 TABLE 2 Diffraction data collection and refinement statistics for DddA/DddIA Ddd.sub.A/DddI.sub.A SAD peak.sup.a PDB accession code 61308 Data Collection Space group P2.sub.12.sub.12 Cell dimension a, b, c () 126.8, 145.0, 64.2 , , () 90, 90, 90 Wavelength () 0.9790 Resolution () 38.6-2.5 (2.53-2.50).sup.b No. unique reflections 41658 (1339) R.sub.merge 0.12 (1.2) I/I 26.7 (2.0) Completeness (%) Total 98.9 (96.7) Anomalous 99.0 Redundancy 13.0 (12.6) Wilson B-factor (.sup.2) 46.2 Refinement Resolution () 38.6-2.5 (2.53-2.50) No. reflections 41655 (2789) Rwork Rfree (%) 17.2/22.8 (26.3/34.6) No. atoms 7889 Protein 7796 Ligand/ion 4 Water 89 B-factors (.sup.2) Protein 59.2 Ligand/ion 72.3 Water 50.7 rmsd Bond lengths () 0.012 Bond angles () 1.091 Missing residues chain A 1250-1289, 1423-1427 chain B 71-3 chain C 1250-1289, 1423-1427 chain D 72-3 chain E 1250-1290, 1423-1427 chain F none chain G 1250-1290, 1423-1427 chain H none
TABLE-US-00054 TABLE3 MitoTALEbindingsitesforeachDdCBE. Sequencesstartfromthe5nucleotiderecognizedbythefirstrepeatintheTALEarray (N.sub.1position)andendwiththenucleotiderecognizedbythecarboxy-terminaltruncated halfrepeat.DenovoTALE-bindingsequencesweredesignedusingthefollowing guidelines.sup.13:(i)15-20bplong,(ii)thymidineatthe3-end,(iii)large%Cwithinthe first5nucleotidesifpossibleand(iv)large%Twithinthelast5nucleotidesifpossible. RemainingtargetsequenceswerefrompreviouslypublishedmitoTALEs(green)oradaptedto recognizeadifferentnucleotideattheN.sub.1position(red).SeetheSupplementarySequences sectionbelowformitoTALEsequences.Toptobottomtheleft-mitoTALEsequencescorrespond toSEQIDNO:146-152.Toptobottomtheright-mitoTALEsequencescorrespondtoSEQID NO:153-159. Left-mitoTALE Right-mitoTALE DdCBE targetsequence targetsequence ND1 5-CTAGCCTAGCCGTTT-3 5-GAGTTTGATGCTCACCCT-3 Denovo Denovo ND2 5-CTTAGCATACTCCTCAAT-3 5-AGAACTGCTATTATT-3 Denovo Denovo ND4 5-GCTAGTAACCACGTTCT-3 5-CCTGTAAGTAGGAGAGT-3 Denovo Denovo ND5.1 5-AGCATTAGCAGGAAT-3 5-CTTTGGAGTAGAA-3 FromHashimoto.Metal,2015 AdaptedfromHashimoto.Metal.,2015 torecognizeCoverTattheN,position ND5.2 5-CAAAACCATACCTCT-3 5-GCTAGGCTGCCAATGT-3 Denovo FromBacman.Setal,2013 ND5.3 5-CAGTTCTTCAAATAT-3 AAGATTAGTATGGT Denovo Dsnovo ATP8 5-ATTAAACACAAACTAT-3 5-ATGGGCTTTGGT-3 FromBacman.Setal,2013 Denovo
TABLE-US-00055 TABLE 4 P-values from comparison of editing efficiencies from time course experiments in HEK293T cells and U2OS cells. [0757] For a given DdCBE, the P-value for editing efficiencies of target cytidine across two cumulative timepoints is shown. P-values were calculated using the Student's two-tailed paired t-test. Entries are highlighted in red if the P-value indicated a significant difference (P < 0.05). HEK293T cells Base editor and Day3 VS Day6 VS Day12 VS cytidine position Day6 Day12 Day18 ND6-DdCBE, C6 0.003248338 0.367579396 0.015940359 ND6-DdCBE, C7 0.002586263 0.565703966 0.020813553 ND6-DdCBE, C11 0.007464037 0.3659328 0.013874452 ND6-DdCBE, C13 0.002564148 0.45334421 0.014317529 ND5.1-DdCBE, C10 0.004716876 0.094419717 0.047243751 ND5.2-DdCBE, C12 0.023166261 0.064203104 0.058523608 ND5.2-DdCBE, C13 0.028990618 0.025844251 0.052962149 ATP8-DdCBE, C12 0.004113271 0.048822963 0.052625302 ATP8-DdCBE, C13 0.011630525 0.099114459 0.042920243 BE2, C5 0.732951948 0.035726897 0.00268113 BE2, C6 0.746509043 0.057803697 0.005118562 BE4max, CS 0.630498196 0.838864469 0.010147598 BE4max, C6 0.575297544 0.865037977 0.008872013 U2OS cells Base editor and Day3 VS Day6 VS Day12 VS cytidine position Day6 Day12 Day18 ND6-DdCBE, C6 0.000590938 0.390452684 0.069490317 ND6-DdCBE, C7 0.000492063 0.464334118 0.146219197 ND6-DdCBE, C11 0.007736858 0.191482179 0.042318087 ND6-DdCBE, C13 0.003832983 0.194483146 0.074581243 ND5.1-DdCBE, C10 0.003808745 0.328396021 0.189480814 ND5.2-DdCBE, C12 0.13305717 0.937487147 0.484054682 ND5.2-DdCBE, C13 0.190940372 0.54498276 0.48011839 ATP8-DdCBE, C12 0.107997405 0.427059997 0.041850528 ATP8-DdCBE, C13 0.172196805 0.381406262 0.030673134 CCR5-DdCBE, C9 0.29867603 0.934543801 0.6557219 CCR5-DdCBE, C10 0.299897207 0.904216914 0.56590558 CCR5-DdCBE, C16 0.273229353 0.769787171 0.967771498
TABLE-US-00056 TABLE 5 Unique off-target SNVs mediated by DdCBEs. SN Reference Altered Average position nucleotide nucleotide % frequency 64 C T 1.34% 94 G A 0.35% 120 C T 0.47% 147 C T 0.39% 150 C T 0.86% 162 C T 0.21% 229 G A 0.16% 320 C T 0.09% 431 C T 0.07% 461 C T 0.40% 486 C T 0.18% 505 C T 1.74% 594 C T 0.51% 597 C T 0.55% 622 G A 0.69% 635 C T 0.97% 660 C T 5.79% 705 C T 1.13% 712 C T 0.29% 734 C T 0.07% 740 G A 1.34% 759 C T 0.36% 822 G A 0.17% 880 C T 0.91% 904 C T 0.55% 909 G A 0.34% 951 G A 0.08% 954 C T 0.27% 962 C T 0.08% 1007 G A 0.07% 1018 G A 0.10% 1074 G A 0.59% 1133 C T 0.08% 1170 G A 0.82% 1190 C T 0.44% 1216 C T 0.24% 1217 G A 0.10% 1227 G A 1.69% 1230 C T 0.16% 1236 C T 0.27% 1250 C T 0.41% 1271 C T 0.43% 1282 G A 0.48% 1290 C T 0.07% 1320 G A 0.37% 1330 C T 0.47% 1345 G A 0.08% 1411 G A 0.09% 1412 G A 0.38% 1414 C T 0.55% 1415 G A 0.28% 1422 G A 0.35% 1469 G A 0.21% 1491 C T 1.61% 1497 C T 0.26% 1500 C T 0.37% 1511 C T 0.07% 1516 G A 0.49% 1554 G A 0.11% 1561 C T 0.08% 1595 G A 4.53% 1660 G A 0.11% 1709 G A 0.49% 1750 G A 0.35% 1831 G A 1.35% 1913 G A 2.41% 1951 C T 0.48% 1970 G A 0.64% 1990 G A 1.59% 2024 C T 2.23% 2070 C T 0.08% 2076 C T 0.11% 2100 C T 1.99% 2121 G A 0.56% 2257 C T 0.35% 2269 G A 2.45% 2328 C T 0.10% 2331 C T 0.87% 2347 C T 1.64% 2349 G A 0.07% 2366 G A 0.42% 2423 C T 0.51% 2511 C T 0.40% 2523 C T 0.50% 2553 G A 0.10% 2611 C T 0.16% 2622 G A 0.79% 2643 G A 0.08% 2657 C T 0.07% 2692 G A 0.47% 2716 G A 1.07% 2719 G A 0.07% 2724 G A 0.09% 2779 C T 0.75% 2819 G A 0.66% 2824 C T 0.47% 2826 G A 0.32% 2842 C T 0.14% 2844 G A 0.09% 2865 C T 0.10% 2872 C T 0.07% 2899 C T 1.09% 2909 G A 0.78% 2917 G A 0.50% 2934 G A 0.36% 2948 C T 0.24% 2961 C T 0.61% 2983 G A 0.50% 2988 C T 0.09% 2989 G A 1.08% 2996 G A 1.73% 2999 C T 0.27% 3002 G A 1.10% 3007 C T 2.38% 3010 G A 1.04% 3047 G A 0.31% 3056 C T 0.63% 3063 G A 0.08% 3066 C T 0.19% 3075 G A 0.17% 3080 G A 0.36% 3087 C T 0.97% 3093 C T 0.85% 3119 C T 0.47% 3132 G A 1.40% 3155 C T 0.09% 3168 C T 0.63% 3187 C T 0.09% 3279 C T 0.45% 3324 C T 0.25% 3333 C T 0.37% 3351 C T 0.51% 3453 C T 0.21% 3457 G A 1.53% 3474 C T 0.61% 3503 C T 0.46% 3510 C T 0.40% 3522 C T 1.13% 3531 G A 0.74% 3549 C T 0.57% 3556 C T 0.09% 3573 C T 0.16% 3594 C T 2.49% 3600 C T 0.13% 3612 C T 0.37% 3654 C T 0.37% 3659 G A 0.60% 3662 C T 0.76% 3668 G A 0.08% 3693 G A 0.49% 3696 C T 2.02% 3738 C T 0.20% 3750 C T 0.09% 3804 C T 0.21% 3824 G A 0.07% 3839 C T 0.26% 3842 G A 0.10% 3869 C T 0.26% 3882 G A 0.68% 3900 C T 0.46% 3901 G A 0.62% 3903 C T 0.08% 3910 G A 0.71% 3920 C T 1.20% 3922 G A 0.17% 3930 C T 0.17% 3945 C T 0.82% 3966 C T 0.63% 3978 C T 0.30% 4032 C T 0.52% 4037 G A 0.22% 4048 G A 1.02% 4086 C T 1.15% 4127 G A 0.08% 4142 G A 0.18% 4153 G A 1.98% 4170 C T 0.24% 4185 C T 0.32% 4273 C T 0.45% 4275 G A 0.18% 4333 G A 0.95% 4345 C T 0.08% 4354 C T 0.85% 4364 C T 0.40% 4374 C T 0.08% 4387 C T 0.24% 4397 C T 1.10% 4410 C T 0.59% 4426 C T 0.37% 4461 C T 0.42% 4476 C T 0.71% 4493 C T 1.40% 4496 C T 0.55% 4526 C T 0.63% 4593 C T 0.34% 4620 C T 0.08% 4624 C T 0.07% 4629 G A 0.09% 4640 C T 0.32% 4652 C T 0.60% 4669 C T 2.96% 4676 C T 6.03% 4714 G A 2.27% 4789 G A 0.41% 4808 C T 0.21% 4814 C T 1.60% 4841 G A 0.49% 4846 C T 4.69% 4860 C T 0.40% 4867 G A 0.43% 4908 C T 0.14% 4912 C T 0.07% 4931 C T 0.10% 4934 C T 0.79% 4951 C T 0.60% 4955 C T 0.17% 4969 G A 0.07% 4975 G A 1.31% 5017 C T 0.42% 5032 G A 0.87% 5099 C T 0.47% 5147 G A 0.18% 5185 G A 0.36% 5202 C T 0.32% 5206 C T 3.91% 5207 C T 0.07% 5213 C T 0.35% 5216 C T 0.30% 5218 C T 0.09% 5224 G A 0.26% 5270 C T 0.26% 5271 G A 0.60% 5297 C T 0.18% 5300 C T 1.09% 5303 C T 2.98% 5312 C T 0.33% 5324 C T 0.41% 5330 C T 0.10% 5413 G A 0.20% 5444 C T 0.08% 5447 C T 2.46% 5448 C T 0.39% 5459 C T 1.99% 5532 G A 0.44% 5554 C T 0.08% 5591 G A 0.67% 5614 C T 0.36% 5660 G A 0.36% 5669 G A 0.34% 5790 C T 0.78% 5820 C T 0.46% 5822 G A 0.07% 5866 C T 1.93% 5875 C T 0.20% 5893 C T 0.36% 5903 G A 0.70% 5909 C T 0.10% 5913 G A 1.46% 5920 G A 0.37% 5950 G A 0.23% 5969 C T 0.09% 5987 C T 1.00% 6008 C T 0.26% 6015 C T 0.11% 6054 G A 2.23% 6062 C T 0.18% 6074 C T 0.28% 6077 C T 2.66% 6107 C T 2.37% 6122 C T 0.21% 6128 C T 0.29% 6130 G A 0.35% 6145 G A 0.72% 6164 C T 0.49% 6174 G A 0.57% 6211 G A 0.20% 6222 C T 0.35% 6242 C T 0.14% 6247 C T 0.31% 6258 G A 0.39% 6265 G A 0.36% 6271 G A 0.36% 6287 C T 0.10% 6294 C T 0.12% 6313 C T 0.08% 6322 G A 0.07% 6328 C T 0.48% 6347 C T 0.07% 6349 C T 0.11% 6368 C T 0.43% 6373 C T 0.26% 6398 C T 0.51% 6455 C T 1.23% 6458 C T 2.26% 6460 G A 0.08% 6463 C T 1.96% 6467 C T 4.90% 6473 C T 0.33% 6482 C T 0.88% 6506 C T 1.07% 6521 C T 0.15% 6537 G A 0.19% 6563 C T 1.00% 6564 G A 0.71% 6566 C T 0.24% 6573 G A 0.40% 6574 G A 2.31% 6577 G A 1.18% 6582 G A 0.27% 6621 C T 0.58% 6627 G A 0.09% 6644 C T 0.65% 6656 C T 0.62% 6658 G A 0.08% 6688 C T 0.09% 6725 C T 0.35% 6734 G A 0.07% 6749 C T 0.57% 6761 C T 0.24% 6790 G A 0.08% 6795 G A 0.61% 6801 G A 0.10% 6808 G A 0.08% 6823 C T 0.52% 6839 C T 0.72% 6845 C T 0.42% 6857 C T 2.06% 6871 G A 0.38% 6889 G A 0.44% 6907 C T 0.09% 6931 G A 0.07% 6962 G A 0.43% 6988 C T 0.38% 6993 G A 0.09% 6998 C T 1.08% 7008 G A 0.38% 7034 C T 0.35% 7043 C T 0.90% 7070 C T 0.13% 7075 G A 0.33% 7119 G A 1.72% 7139 C T 0.10% 7160 C T 1.31% 7181 C T 1.48% 7204 C T 1.13% 7207 G A 0.19% 7216 G A 3.45% 7225 C T 0.41% 7227 G A 1.83% 7236 G A 1.14% 7259 C T 1.19% 7267 C T 0.17% 7331 C T 0.20% 7336 C T 0.51% 7337 G A 0.12% 7349 C T 0.37% 7369 C T 0.19% 7384 G A 0.44% 7393 G A 0.95% 7419 G A 0.65% 7444 G A 0.99% 7454 G A 0.45% 7458 G A 0.07% 7502 C T 0.62% 7506 G A 0.09% 7550 C T 0.56% 7573 C T 0.11% 7616 G A 0.43% 7626 C T 0.46% 7637 G A 0.18% 7658 G A 0.39% 7661 C T 0.44% 7669 C T 0.08% 7693 C T 0.52% 7699 C T 1.76% 7754 G A 0.52% 7777 C T 1.16% 7779 G A 0.08% 7786 C T 0.48% 7798 C T 0.40% 7801 C T 1.92% 7807 C T 1.75% 7810 C T 0.36% 7813 C T 1.41% 7819 C T 0.40% 7824 C T 0.97% 7834 C T 1.01% 7847 G A 1.17% 7850 G A 0.18% 7855 C T 0.49% 7862 C T 0.60% 7866 C T 0.35% 7876 C T 0.08% 7919 G A 1.30% 7929 G A 2.92% 7939 C T 0.10% 7955 C T 0.48% 7979 G A 0.40% 7986 G A 0.45% 7994 G A 1.03% 8000 G A 0.42% 8020 G A 0.23% 8052 C T 0.44% 8057 G A 0.30% 8062 C T 0.46% 8080 C T 0.79% 8102 G A 0.25% 8115 G A 5.88% 8120 C T 0.89% 8148 G A 1.16% 8168 C T 0.30% 8187 G A 0.28% 8212 C T 0.64% 8215 C T 6.92% 8287 C T 0.10% 8428 C T 0.07% 8431 C T 0.66% 8434 C T 1.14% 8474 C T 0.34% 8478 C T 0.38% 8544 C T 0.40% 8549 C T 0.09% 8568 C T 0.54% 8592 G A 0.21% 8595 C T 0.35% 8605 C T 0.09% 8619 C T 4.21% 8620 C T 0.11% 8627 C T 0.62% 8635 C T 0.34% 8640 C T 0.58% 8648 G A 1.05% 8655 C T 0.09% 8669 G A 0.31% 8687 C T 0.31% 8720 G A 11.41% 8729 G A 0.27% 8747 C T 0.08% 8778 C T 0.41% 8781 C T 0.91% 8783 G A 1.48% 8844 C T 1.76% 8910 C T 0.07% 8940 C T 1.00% 8958 C T 0.18% 8967 C T 0.19% 9050 G A 0.72% 9085 C T 0.50% 9099 C T 0.43% 9102 C T 0.08% 9105 C T 0.35% 9123 G A 0.30% 9129 C T 0.29% 9144 C T 1.17% 9153 C T 1.83% 9196 G A 1.19% 9253 G A 0.22% 9281 C T 1.03% 9292 C T 0.65% 9317 C T 0.63% 9335 C T 0.42% 9376 G A 0.17% 9384 G A 1.73% 9394 G A 0.08% 9431 C T 1.83% 9444 C T 0.30% 9445 G A 0.13% 9452 G A 0.34% 9458 C T 0.36% 9472 C T 0.47% 9488 C T 0.94% 9544 G A 0.09% 9569 C T 0.39% 9582 C T 0.54% 9593 C T 1.01% 9610 C T 1.93% 9620 C T 0.10% 9625 C T 0.78% 9657 C T 0.07% 9731 C T 0.17% 9742 C T 0.15% 9752 C T 0.34% 9764 C T 0.08% 9772 C T 0.07% 9774 G A 2.59% 9782 C T 0.09% 9815 C T 0.96% 9820 G A 0.79% 9825 C T 0.24% 9830 C T 0.77% 9851 C T 0.90% 9866 C T 1.77% 9892 C T 2.30% 9900 C T 0.34% 9911 C T 0.47% 9912 G A 0.26% 9942 G A 0.23% 9952 G A 0.27% 9968 C T 0.10% 9974 C T 0.20% 9979 G A 0.07% 10022 C T 0.27% 10038 G A 0.08% 10067 C T 0.26% 10094 C T 0.35% 10126 G A 0.08% 10181 C T 0.46% 10182 G A 0.46% 10184 C T 0.10% 10192 C T 1.17% 10205 C T 6.66% 10213 C T 0.20% 10257 C T 0.37% 10271 C T 0.08% 10327 C T 0.15% 10330 C T 0.88% 10346 C T 0.34% 10349 C T 1.36% 10362 C T 0.28% 10375 G A 0.31% 10387 G A 0.64% 10437 G A 0.36% 10478 C T 0.12% 10518 C T 0.07% 10536 C T 0.33% 10552 C T 0.70% 10555 C T 0.69% 10573 G A 0.07% 10585 C T 0.07% 10616 C T 0.07% 10664 C T 0.08% 10677 G A 0.34% 10706 C T 0.18% 10731 G A 0.30% 10757 C T 0.09% 10774 C T 0.18% 10777 C T 4.87% 10801 G A 0.16% 10834 C T 0.08% 10867 C T 0.10% 10870 C T 1.10% 10917 C T 0.46% 10932 C T 1.37% 10933 C T 0.08% 10934 G A 0.33% 10954 C T 0.44% 10971 G A 0.30% 10984 C T 0.07% 11013 C T 0.76% 11125 C T 0.82% 11140 C T 0.77% 11158 C T 0.46% 11163 G A 1.18% 11166 G A 0.29% 11206 C T 0.43% 11234 C T 0.41% 11242 C T 0.07% 11245 C T 0.80% 11391 G A 2.03% 11422 C T 4.55% 11423 G A 1.53% 11434 C T 0.58% 11497 C T 0.19% 11518 G A 1.20% 11542 C T 0.23% 11592 G A 1.44% 11600 G A 0.11% 11628 C T 0.10% 11632 C T 0.57% 11647 C T 2.09% 11668 C T 1.63% 11679 G A 0.17% 11686 C T 0.15% 11698 C T 0.24% 11710 C T 0.29% 11718 G A 0.67% 11727 C T 1.29% 11730 C T 0.33% 11777 C T 0.36% 11782 C T 0.20% 11788 C T 0.37% 11799 G A 0.34% 11815 C T 0.09% 11840 C T 0.21% 11851 C T 2.34% 11860 C T 1.82% 11922 G A 0.63% 11925 C T 0.36% 11949 G A 0.55% 11965 C T 0.37% 12045 C T 0.07% 12054 G A 0.25% 12073 C T 0.08% 12084 C T 0.45% 12097 C T 1.26% 12102 C T 0.64% 12106 C T 0.18% 12113 G A 0.83% 12118 C T 0.56% 12162 C T 0.56% 12176 G A 0.55% 12192 G A 0.47% 12207 G A 0.07% 12246 C T 0.28% 12276 G A 0.55% 12288 C T 0.42% 12296 C T 0.70% 12377 C T 1.39% 12393 C T 1.69% 12405 C T 0.30% 12449 C T 0.91% 12456 C T 2.06% 12461 C T 2.48% 12474 C T 0.32% 12478 C T 0.08% 12483 C T 1.15% 12508 G A 0.72% 12583 G A 0.65% 12591 C T 0.73% 12593 C T 0.46% 12606 C T 0.90% 12621 C T 0.44% 12632 C T 4.47% 12636 C T 0.32% 12667 G A 0.07% 12682 C T 0.37% 12690 C T 0.29% 12708 C T 0.60% 12759 C T 0.14% 12762 C T 1.29% 12788 C T 0.48% 12806 G A 0.10% 12818 G A 0.42% 12823 G A 0.20% 12852 C T 0.30% 12867 C T 0.46% 12871 G A 0.45% 12876 C T 0.54% 12885 C T 1.96% 12888 C T 0.71% 12906 C T 0.45% 12925 G A 0.30% 12958 C T 1.18% 12966 C T 0.09% 12984 C T 0.40% 12987 C T 0.43% 13007 C T 0.09% 13021 C T 0.17% 13031 G A 0.38% 13040 C T 0.36% 13065 C T 0.20% 13119 C T 2.25% 13125 C T 0.91% 13155 C T 0.71% 13185 C T 0.07% 13197 C T 0.23% 13206 C T 0.10% 13230 C T 0.43% 13250 C T 0.36% 13261 C T 0.08% 13268 G A 0.50% 13287 C T 0.26% 13293 C T 0.50% 13323 C T 0.42% 13341 C T 0.20% 13364 C T 0.19% 13370 C 1 1.68% 13374 C T 0.29% 13377 C T 1.61% 13393 G A 0.18% 13415 G A 0.21% 13418 G A 0.86% 13445 C T 0.17% 13451 C T 0.19% 13455 C T 0.07% 13481 G A 0.07% 13494 C T 0.88% 13508 C T 0.18% 13513 G A 0.24% 13521 C T 0.80% 13524 C T 0.97% 13578 C T 0.90% 13586 C T 0.33% 13590 G A 0.70% 13636 C T 0.21% 13642 C T 0.24% 13647 C T 0.92% 13715 G A 1.01% 13754 C T 0.25% 13763 C T 5.08% 13764 C T 0.07% 13770 C T 1.28% 13782 C T 0.83% 13809 C T 0.09% 13815 C T 0.16% 13826 G A 0.23% 13843 G A 0.07% 13880 C T 0.07% 13947 C T 0.16% 13992 C T 1.62% 13996 G A 0.47% 14006 G A 0.49% 14057 C T 0.39% 14061 C T 0.27% 14064 C T 0.79% 14100 C T 0.38% 14115 C T 1.22% 14124 C T 2.34% 14142 C T 0.09% 14155 C T 0.32% 14160 G A 0.37% 14259 G A 0.11% 14262 C T 0.30% 14265 C T 0.81% 14279 G A 0.54% 14292 C T 0.17% 14309 C T 0.99% 14346 C T 0.35% 14372 C T 0.40% 14379 C T 0.43% 14383 C T 0.34% 14438* G* A* 17.85%* 14439* G* A* 20.74%* 14443* C* T* 3.43%* 14445* C* T* 13.25%* 14446 C T 0.20% 14448 C T 0.09% 14471 C T 0.81% 14485 C T 0.21% 14531 C T 0.53% 14560 G A 2.54% 14601 G A 0.51% 14612 G A 0.10% 14671 C T 0.26% 14686 G A 2.60% 14698 G A 4.24% 14720 C T 0.52% 14749 G A 0.20% 14796 C T 0.18% 14803 C T 0.94% 14804 G A 1.80% 14806 C T 0.32% 14809 C T 3.13% 14810 C T 0.89% 14820 C T 4.50% 14827 C T 0.26% 14829 C T 0.30% 14835 G A 0.48% 14845 C T 0.19% 14854 C T 0.08% 14869 G A 0.18% 14872 C T 2.53% 14875 C T 1.47% 14889 G A 1.35% 14896 C T 0.08% 14918 G A 0.65% 14925 C T 0.11% 14940 C T 0.34% 14944 C T 0.33% 14953 C T 0.66% 14960 G A 0.56% 14983 C T 3.36% 14993 C T 0.32% 15009 C T 0.30% 15031 C T 1.39% 15040 C T 2.78% 15045 G A 0.11% 15060 G A 1.48% 15091 C T 1.34% 15100 C T 0.60% 15103 C T 1.37% 15142 C T 1.18% 15145 C T 0.85% 15150 G A 0.34% 15198 C T 0.85% 15205 C T 1.24% 15221 G A 0.07% 15240 G A 0.45% 15257 G A 0.07% 15263 C T 0.81% 15271 C T 0.07% 15295 C T 0.18% 15298 C T 0.16% 15337 C T 0.49% 15357 G A 1.63% 15384 C T 1.34% 15385 C T 0.34% 15390 C T 0.08% 15392 G A 0.20% 15406 C T 0.81% 15428 G A 0.10% 15436 C T 0.19% 15451 C T 0.96% 15493 C T 0.26% 15500 G A 0.31% 15506 G A 0.51% 15542 C T 0.40% 15550 C T 1.32% 15574 C T 0.07% 15591 G A 0.43% 15594 C T 0.84% 15598 C T 3.60% 15612 G A 0.31% 15619 C T 3.41% 15636 C T 0.45% 15640 C T 0.72% 15643 C T 0.21% 15646 C T 1.05% 15664 C T 0.53% 15667 C T 0.35% 15675 C T 6.13% 15676 C T 0.15% 15737 G A 0.09% 15742 C T 0.34% 15745 C T 0.24% 15760 C T 0.09% 15762 G A 0.65% 15765 G A 1.26% 15793 C T 0.07% 15798 G A 1.94% 15810 C T 0.75% 15832 C T 0.08% 15838 C T 0.33% 15890 C T 0.77% 15928 G A 0.09% 15930 G A 0.10% 15950 G A 1.83% 15957 C T 0.07% 16036 G A 0.27% 16173 C T 0.26% 16179 C T 0.29% 16193 C T 0.19% 16239 C T 0.07% 16363 C T 3.22% 16370 G A 0.09% 16393 C T 15.92% 16394 C T 2.80% 16407 C T 0.82% 16410 C T 0.63% 16425 C T 0.44% 16449 C T 0.70% 16494 C T 0.39% 16496 G A 0.41% 16501 C T 0.62% 16563 C T 0.59% 64 C T 0.10% 505 C T 0.60% 660 C T 1.09% 705 C T 0.12% 740 G A 0.09% 1227 G A 0.28% 1491 C T 0.38% 1595 G A 0.62% 1709 G A 0.30% 1831 G A 0.40% 1913 G A 0.33% 1990 G A 0.19% 2024 C T 0.36% 2100 C T 0.09% 2269 G A 0.69% 2347 C T 0.27% 2523 C T 0.27% 2716 G A 0.37% 2899 C T 0.10% 2989 G A 0.08% 2996 G A 0.31% 3007 C T 0.44% 3132 G A 0.17% 3351 C T 0.16% 3457 G A 0.55% 3522 C T 0.08% 3594 C T 0.28% 3662 C T 0.08% 3696 C T 0.41% 3920 C T 0.21% 4048 G A 0.19% 4153 G A 0.54% 4333 G A 0.17% 4397 C T 0.19% 4493 C T 0.08% 4526 C T 0.16% 4669 C T 0.30% 4714 G A 0.42% 4846 C T 0.96% 4975 G A 0.07% 5032 G A 0.07% 5099 C T 0.26% 5206 C T 0.31% 5300 C T 0.14% 5303 C T 2.28% 5447 C T 0.40% 5459 C T 0.38% 5866 C I 0.27% 5875 C T 0.07% 5913 G A 0.26% 5987 C T 0.23% 6054 G A 0.08% 6077 C T 0.53% 6128 C T 0.16% 6398 C T 0.19% 6455 C T 0.31% 6458 C T 0.27% 6463 C T 0.30% 6467 C T 0.76% 6563 C T 0.14% 6574 G A 0.37% 6839 C T 0.23% 6857 C T 0.18% 6998 C T 0.32% 7119 G A 0.28% 7160 C T 0.34% 7181 C T 0.51% 7204 C T 0.32% 7216 G A 0.73% 7227 G A 0.20% 7236 G A 0.07% 7259 C T 0.07% 7336 C T 0.25% 7393 G A 0.10% 7699 C T 0.21% 7801 C T 0.38% 7807 C T 0.37% 7813 C T 0.35% 7824 C T 0.19% 7834 C T 0.17% 7847 G A 0.27% 7919 G A 0.07% 7929 G A 0.39% 7994 G A 0.20% 8115 G A 1.20% 8120 C T 0.17% 8212 C T 0.10% 8215 C T 1.49% 8619 C T 0.89% 8648 G A 0.33% 8720 G A 1.29% 8781 C T 0.17% 8783 G A 0.15% 8844 C T 0.40% 9144 C T 0.09% 9153 C T 0.84% 9384 G A 0.24% 9431 C T 0.39% 9488 C T 0.31% 9610 C T 0.24% 9774 G A 0.50% 9815 C T 0.10% 9830 C T 0.08% 9851 C T 0.26% 9866 C T 0.08% 9892 C T 0.32% 9942 G A 0.07% 9952 G A 0.28% 10192 C T 0.29% 10205 C T 1.53% 10330 C T 0.11% 10349 C T 0.26% 10387 G A 0.11% 10552 C T 0.08% 10555 C T 0.08% 10777 C T 0.77% 10932 C I 0.26% 11013 C T 0.09% 11140 C T 0.15% 11163 G A 0.29% 11518 G A 0.20% 11592 G A 0.13% 11668 C T 0.35% 11727 C T 0.35% 11799 G A 1.05% 11804 C T 0.53% 12084 C T 0.07% 12296 C T 0.08% 12393 C I 0.16% 12456 C T 0.51% 12461 C T 0.44% 12483 C T 0.07% 12606 C T 0.14% 12632 C T 0.99% 12762 C T 0.17% 12885 C T 0.18% 12888 C T 0.08% 13119 C T 0.36% 13370 C T 0.40% 13377 C T 0.67% 13445 C T 0.08% 13494* C* T* 33.00%* 13496 C T 0.56% 13578 C T 0.31% 13590 G A 0.44% 13647 C T 0.37% 13763 C T 0.95% 13770 C T 0.09% 13782 C T 0.08% 13992 C T 0.33% 14006 G A 0.08% 14124 C T 0.48% 14265 C T 0.08% 14383 C T 0.08% 14560 G A 0.69% 14686 G A 0.51% 14698 G A 0.63% 14803 C T 0.20% 14820 C T 0.40% 14872 C T 0.35% 14875 C I 0.31% 14983 C T 0.64% 15031 C T 0.19% 15040 C T 1.67% 15060 G A 0.09% 15091 C T 0.36% 15103 C T 0.22% 15142 C T 0.21% 15198 C T 0.07% 15205 C T 0.16% 15357 G A 0.28% 15550 C T 0.28% 15594 C T 0.33% 15598 C T 0.80% 15619 C T 0.58% 15646 C T 0.21% 15664 C T 0.20% 15765 G A 0.18% 15798 G A 0.23% 15810 C I 0.16% 15950 G A 0.36% 16363 C T 0.61% 16449 C T 0.16% 933 G A 0.09% 1227 G A 0.07% 1625 A G 0.07% 3457 G A 0.16% 3696 C T 0.19% 4846 C T 0.30% 6876 G A 0.07% 7160 C T 0.08% 7216 G A 0.10% 8115 G A 0.20% 8148 G A 0.07% 8215 C T 0.07% 8720 G A 0.16% 9343 G A 0.07% 9384 G A 0.20% 9565 G A 0.14% 9774 G A 0.07% 10205 C T 0.10% 12145 T C 0.18% 12632 C T 0.09% 12871 G A 0.07% 13451 C T 8.44% 13452 C T 5.89% 13763 C T 0.42% 14560 G A 0.08% 14588 C T 0.24% 15040 C T 0.19% 933 G A 0.09% 1227 G A 0.07% 1625 A G 0.07% 3457 G A 0.16% 3696 C T 0.19% 4846 C T 0.30% 6876 G A 0.07% 7160 C T 0.08% 7216 G A 0.10% 8115 G A 0.20% 8148 G A 0.07% 8215 C T 0.07% 8720 G A 0.16% 9343 G A 0.07% 9384 G A 0.20% 9565 G A 0.14% 9774 G A 0.07% 10205 C T 0.10% 12145 T C 0.18% 12632 C T 0.09% 12871 G A 0.07% 13451 C T 8.44% 13452 C T 5.89% 13763 C T 0.42% 14560 G A 0.08% 14588 C T 0.24% 15040 C T 0.19% 933 G A 0.09% 1227 G A 0.07% 1625 A G 0.07% 3457 G A 0.16% 3696 C T 0.19% 4846 C T 0.30% 6876 G A 0.07% 7160 C T 0.08% 7216 G A 0.10% 8115 G A 0.20% 8148 G A 0.07% 8215 C T 0.07% 8720 G A 0.16% 9343 G A 0.07% 9384 G A 0.20% 9565 G A 0.14% 9774 G A 0.07% 10205 C T 0.10% 12145 T C 0.18% 12632 C T 0.09% 12871 G A 0.07% 13451* C* T* 8.44%* 13452* C* T* 5.89%* 13763 C T 0.42% 14560 G A 0.08% 14588 C T 0.24% 15040 C T 0.19% 546 A C 0.11% 574 A C 0.12% 1623 G A 0.24% 8115 G A 0.09% 11922* G* A* 26.41%* 11925 C I 0.08% 12145 T C 0.09% 13111 T C 0.08% 550 A C 0.21% 660 C T 0.20% 933 G A 0.08% 1169 G A 0.00% 1227 G A 0.09% 1415 G A 0.76% 1422 G A 0.14% 2948 C T 0.61% 3594 C T 0.28% 3696 C T 0.09% 5532 G A 0.34% 6467 C T 0.08% 6621 C T 0.09% 6876 G A 0.09% 7216 G A 0.40% 8115 G A 0.09% 8215 C T 0.42% 8474* C* T* 7.21%* 8475* C* T* 3.64%* 8844 C T 0.97% 8886 G A 0.08% 9384 G A 0.81% 10205 C T 0.16% 12632 C T 0.18% 12682 C T 0.16% 13377 C T 0.27% 14588 C T 0.18% 15031 C T 0.65% 15040 C T 0.08% 15205 C T 0.08% 15950 G A 0.97% SNVs called by VarScan 2 were considered high-confidence if the percentage frequency of a given SNV is >0.1% in one or more replicates. Shown are all the unique SNVs across three independent biological replicates. SNV positions for each DdCBE treatment (ND6-DdCBE, italics; ND5.1-DdCBE, italics, underline; ND5.2 DdCBE, bold; ND4-DdCBE, bold, underlined; ATP8-DdCBE, bold, italics) were from the NC_012920 reference genome. On-target SNVs are marked by asterisks.
TABLE-US-00057 TABLE 6 Overlapping off-target SNVs. Shown are the list of overlapping off-target SNVs between ND6- and ND5.1-DdCBE (italics), ND6-, ND5.1- and ATP8-DdCBE (bold) and ND6-, ND5.1-, ND5.2- and ATP8-DdCBE (underlined). The average frequencies for the respective DdCBEs are shown. Average % frequency SNV position ND6-DdCBE ND5.1-DdCBE ND5.2-DdCBE ATP8-DdCBE 64 1.34% 0.10% 505 1.74% 0.60% 705 1.13% 0.12% 740 1.34% 0.09% 1491 1.61% 0.38% 1595 4.53% 0.62% 1709 0.49% 0.30% 1831 1.35% 0.40% 1913 2.41% 0.33% 1990 1.59% 0.19% 2024 2.23% 0.36% 2100 1.99% 0.09% 2269 2.45% 0.69% 2347 1.64% 0.27% 2523 0.50% 0.27% 2716 1.07% 0.37% 2899 1.09% 0.10% 2989 1.08% 0.08% 2996 1.73% 0.31% 3007 2.38% 0.44% 3132 1.40% 0.17% 3351 0.51% 0.16% 3522 1.13% 0.08% 3662 0.76% 0.08% 3920 1.20% 0.21% 4048 1.02% 0.19% 4153 1.98% 0.54% 4333 0.95% 0.17% 4397 1.10% 0.19% 4493 1.40% 0.08% 4526 0.63% 0.16% 4669 2.96% 0.30% 4714 2.27% 0.42% 4975 1.31% 0.07% 5032 0.87% 0.07% 5099 0.47% 0.26% 5206 3.91% 0.31% 5300 1.09% 0.14% 5303 2.98% 2.28% 5447 2.46% 0.40% 5459 1.99% 0.38% 5866 1.93% 0.27% 5875 0.20% 0.07% 5913 1.46% 0.26% 5987 1.00% 0.23% 6054 2.23% 0.08% 6077 2.66% 0.53% 6128 0.29% 0.16% 6398 0.51% 0.19% 6455 1.23% 0.31% 6458 2.26% 0.27% 6463 1.96% 0.30% 6563 1.00% 0.14% 6574 2.31% 0.37% 6839 0.72% 0.23% 6857 2.06% 0.18% 6998 1.08% 0.32% 7119 1.72% 0.28% 7181 1.48% 0.51% 7204 1.13% 0.32% 7227 1.83% 0.20% 7236 1.14% 0.07% 7259 1.19% 0.07% 7336 0.51% 0.25% 7393 0.95% 0.10% 7699 1.76% 0.21% 7801 1.92% 0.38% 7807 1.75% 0.37% 7813 1.41% 0.35% 7824 0.97% 0.19% 7834 1.01% 0.17% 7847 1.17% 0.27% 7919 1.30% 0.07% 7929 2.92% 0.39% 7994 1.03% 0.20% 8120 0.89% 0.17% 8212 0.64% 0.10% 8619 4.21% 0.89% 8648 1.05% 0.33% 8781 0.91% 0.17% 8783 1.48% 0.15% 9144 1.17% 0.09% 9153 1.83% 0.84% 9431 1.83% 0.39% 9488 0.94% 0.31% 9610 1.93% 0.24% 9815 0.96% 0.10% 9830 0.77% 0.08% 9851 0.90% 0.26% 9866 1.77% 0.08% 9892 2.30% 0.32% 9942 0.23% 0.07% 9952 0.27% 0.28% 10192 1.17% 0.29% 10330 0.88% 0.11% 10349 1.36% 0.26% 10387 0.64% 0.11% 10552 0.70% 0.08% 10555 0.69% 0.08% 10777 4.87% 0.77% 10932 1.37% 0.26% 11013 0.76% 0.09% 11140 0.77% 0.15% 11163 1.18% 0.29% 11518 1.20% 0.20% 11592 1.44% 0.13% 11668 1.63% 0.35% 11727 1.29% 0.35% 11799 0.34% 1.05% 12084 0.45% 0.07% 12296 0.70% 0.08% 12393 1.69% 0.16% 12456 2.06% 0.51% 12461 2.48% 0.44% 12483 1.15% 0.07% 12606 0.90% 0.14% 12762 1.29% 0.17% 12885 1.96% 0.18% 12888 0.71% 0.08% 13119 2.25% 0.36% 13370 1.68% 0.40% 13445 0.17% 0.08% 13494 0.88% 33.00% 13578 0.90% 0.31% 13590 0.70% 0.44% 13647 0.92% 0.37% 13770 1.28% 0.09% 13782 0.83% 0.08% 13992 1.62% 0.33% 14006 0.49% 0.08% 14124 2.34% 0.48% 14265 0.81% 0.08% 14383 0.34% 0.08% 14686 2.60% 0.51% 14698 4.24% 0.63% 14803 0.94% 0.20% 14820 4.50% 0.40% 14872 2.53% 0.35% 14875 1.47% 0.31% 14983 3.36% 0.64% 15060 1.48% 0.09% 15091 1.34% 0.36% 15103 1.37% 0.22% 15142 1.18% 0.21% 15198 0.85% 0.07% 15357 1.63% 0.28% 15550 1.32% 0.28% 15594 0.84% 0.33% 15598 3.60% 0.80% 15619 3.41% 0.58% 15646 1.05% 0.21% 15664 0.53% 0.20% 15765 1.26% 0.18% 15798 1.94% 0.23% 15810 0.75% 0.16% 16363 3.22% 0.61% 16449 0.70% 0.16% 660 5.79% 1.09% 0.20% 3594 2.49% 0.28% 0.28% 6467 4.90% 0.76% 0.08% 8844 1.76% 0.40% 0.97% 13377 1.61% 0.67% 0.27% 15031 1.39% 0.19% 0.65% 15205 1.24% 0.16% 0.08% 15950 1.83% 0.36% 0.97% 1227 1.69% 0.28% 0.07% 0.09% 3696 2.02% 0.41% 0.19% 0.09% 7216 3.45% 0.73% 0.10% 0.40% 8215 6.92% 1.49% 0.07% 0.42% 9384 1.73% 0.24% 0.20% 0.81% 10205 6.66% 1.53% 0.10% 0.16% 12632 4.47% 0.99% 0.09% 0.18% 15040 2.78% 1.67% 0.19% 0.08%
TABLE-US-00058 TABLE 7 Disease-associated mitochondrial DNA point mutations. Pathogenic mtDNA point mutations were obtained from the MITOMAP database12 (accessed Dec. 10, 2019). Disease-associated mutations in rRNA/tRNA and coding/non-coding regions were considered only if they had been assigned Cfrm statuses. Italics: SNPs that are corrected by CG-to-TA conversions; Underlined: SNPs that are corrected by AT-to-GC conversions; Bold, underlined: SNPs that are corrected by AT-to-CG conversions; Bold, italics: SNPs that are corrected by CG-to-GC conversions; Italics, underlined: SNPs that are corrected by AT-to-TA conversions Allele SNP Locus Associated disease T616C MT-TF Maternally inherited epilepsy/kidney disease A1555G MT-RNR1 DEAF; autism spectrum intellectual disability; possibly antiatherosclerotic A1630G MT-TV MNGIE-like disease/MELAS A3243G MT-TL1 MELAS/LS/DMDF/MIDD/SNHL/CPEO/MM/FSGS/ASD/ Cardiac + multi-organ dysfunction T3258C MT-TL1 MELAS/Myopathy A3260G MT-TL1 MMC/MELAS T3271C MT-TL1 MELAS/DM A3280G MT-TL1 Myopathy T3291C MT-TL1 MELAS/Myopathy/Deafness + Cognitive Impairment A3302G MT-TL1 MM A4300G MT-TI MICM A5690G MT-TN CPEO + ptosis + proximal myopathy T5728C MT-TN Multiorgan failure/myopathy A7445G MT-TS1 precursor SNHL A7445G MT-CO1 SNHL T7510C MT-TS1 SNHL T7511C MT-TS1 SNHL/Deafness A8344G MT-TK MERRF; Other-LD/Depressive mood disorder/ leukoencephalopathy/HiCM T8356C MT-TK MERRF T8528C MT-ATP8/6 Infantile cardiomyopathy T8851C MT-ATP6 BSN/Leigh syndrome T8993C MT-ATP6 NARP/Leigh Disease/MILS/other T9035C MT-ATP6 Ataxia syndromes A9155G MT-ATP6 MIDD, renal insufficiency T9176C MT-ATP6 FBSN/Leigh Disease T9185C MT-ATP6 Leigh Disease/Ataxia syndromes/NARP-like disease T10010C MT-TG PEM T10158C MT-ND3 Leigh Disease/MELAS T10191C MT-ND3 Leigh Disease/Leigh-like Disease/ESOC T10663C MT-ND4L LHON T12706C MT-ND5 Leigh Disease T13094C MT-ND5 Ataxia + PEO/MELAS, LD, LHON, myoclonus, fatigue A13514G MT-ND5 Leigh Disease/MELAS/Ca2+ downregulation T14484C MT-ND6 LHON T14487C MT-ND6 Dystonia/Leigh Disease/ataxia/ptosis/epilepsy A14495G MT-ND6 LHON T14674C MT-TE Reversible COX deficiency myopathy T14709C MT-TE MM + DMDF/Encephalomyopathy/ Dementia + diabetes + ophthalmoplegia T14849C MT-CYB EXIT/Septo-Optic Dysplasia T14864C MT-CYB MELAS A15579G MT-CYB Multisystem Disorder, EXIT G583A MT-TF MELAS/MM&EXIT C1494T MT-RNR1 DEAF G1606A MT-TV AMDF G1644A MT-TV LS/HCM/MELAS C3256T MT-TL1 MELAS;possibleatherosclerosisrisk C3303T MT-TL1 MMC G3376A MT-ND1 LHONMELASoverlap G3460A MT-ND1 LHON G3635A MT-ND1 LHON G3697A MT-ND1 MELAS/LS/LDYT/BSN G3700A MT-ND1 LHON G3733A MT-ND1 LHON G3890A MT-ND1 ProgressiveEncephalomyopathy/LS/OpticAtrophy G4298A MT-TI CPEO/MS G4308A MT-TI CPEO G4332A MT-TQ Encephalopathy/MELAS G4450A MT-TM Myopathy/MELAS G5521A MT-TW Mitochondrialmyopathy G5650A MT-TA Myopathy G5703A MT-TN CPEO/MM G7497A MT-TS1 MM/EXIT G8340A MT-TK Myopathy/ExerciseIntolerance/Eyedisease+SNHL G8363A MT-TK MICM+DEAF/MERRF/Autism/LS/Ataxia+Lipomas G8969A MT-ATP6 Mitochondrialmyopathy,lacticacidosisand sideroblasticanemia(MLASA)/IgGnephropathy G10197A MT-ND3 LeighDisease/Dystonia/Stroke/LDYT G11778A MT-ND4 LHON/ProgressiveDystonia G12147A MT-TH MERRF-MELAS/Encephalopathy G12276A MT-TL2 CPEO G12294A MT-TL2 CPEO/EXIT+Ophthalmoplegia G12315A MT-TL2 CPEO/KSS/possiblecarotidatherosclerosisrisk,trend towardmyocardialinfarctionrisk G12316A MT-TL2 CPEO G13042A MT-ND5 Opticneuropathy/retinopathy/LD G13051A MT-ND5 LHON G13513A MT-ND5 LeighDisease/MELAS/LHON-MELASOverlapSyndrome/ negativeassociationwCarotidAtherosclerosis G14459A MT-ND6 LDYT/LeighDisease/dystonia/carotidatherosclerosisrisk G14710A MT-TE Encephalomyopathy+Retinopathy C4171A MT-ND1 LHON/Leigh-likephenotype C11777A MT-ND4 LeighDisease C12258A MT-TS2 DMDF/RP+SNHL C14482A MT-ND6 LHON C14482G MT-ND6 LHON A3243T MT-TL1 MM/MELAS/SNHL/CPEQ
Tables 8A-8B List of Bacterial Strains and Plasmids Used in Example1
[0962] Table 8A is a list of bacterial strains used in this study.
TABLE-US-00059 Bacterial species and strains Genotype Purpose Source Escherichia coli DH5 F- 80lacZM15 (lacZYA- General cloning Thermo Fisher argF)U169 recA1 endA1 Scientific hsdR17(rK, mK+) phoA supE44 - Cat#18258012 thi-1 gyrA96 relA1 Escherichia coli DH5:Dddl F- 80lacZM15 (lacZYA- Cloning for full-length This study argF)U169 recA1 endA1 DddA.sub.tox fusions hsdR17(rK, mK+) phoA supE44 A- thi-1 gyrA96 relA1, with chromosomally integrated dddl Escherichia coli Mach1 str. W General cloning Thermo Fisher recA1398 endA1 fhuA 80(lac)M (mammalian plasmids) Scientific Cat# 15 (lac)X74 hsdR(r.sub.K.sup. m.sub.K.sup.+) C862003 Escherichia coli BL21 F.sup. ompT hsdS.sub.B (r.sub.B.sup., m.sub.B.sup.) gal Protein extraction EMD Millipore dcm (DE3) Cat#69450 Escherichia coli XK1502 F lacU169 nalA Protein expression PMID: 16923902 Burkholderia cenocepacia Wild type Amplification of PMID: H111 dddA-dddA.sub.I, parental 10713433 strain for mutant construction Burkholderia cenocepacia I35_RS01770, attTn7::aacC1 Growth competition This study H111 AicmF1 GentR assays Burkholderia cenocepacia I35_RS17395, attTn7::aacC1 Growth competition This study H111 AicmF2 GentR assays Burkholderia cenocepacia I35_RS34180 I35_RS34175, Growth competition This study H111 AdddAAdddAl GentR attTn7::aacC1 assays Burkholderia cenocepacia I35_RS34180.sup.E1347A, attTn7::aacC1 Growth competition This study H111 dddAE1347A assays Pseudomonas aeruginosa Wild type Growth competition PMID: PAO1 assays 10984043
[0963] Table 8B is a list of plasmids used in this study.
TABLE-US-00060 Plasmids Purpose Source pPSV39-CV For inducible expression of proteins in E. coli PMID: 23954347 pScrhaB2-V For inducible expression of proteins in E. coli PMID: 15925406 pBAD24 Protein expression PMID: 7608087 pETDuet-1 Protein expression EMD Millipore Cat#71146-3 pDONRPEX18Gm-Scel-pheS Allellic replacement in B. cenocepacia PMID: 25795676 pDAI-Scel-pheS Allellic replacement in B. cenocepacia PMID: 25795676 pUC18T-mini-Tn7T-Gm For the generation of gentamycin resistant B. PMID: 15908923 cenocepacia pScrhaB2-V ::dddA To express dddA This study pScrhaB2-V ::cdd To express cdd This study pScrhaB2-V::APOBEC3G To express APOBEC3G This study pScrhaB2-V::tadA To express tadA This study pPSV39-CV::dddA.sub.I To express dddAl This study pDONRPEX18Gm-Scel-pheS::icmF1 To delete icmF1 from B. cenocepacia This study pDONRPEX18Gm-Scel-pheS::icmF2 To delete icmF2 from B. cenocepacia This study pDONRPEX18Gm-Scel- To delete dddA and dddA.sub.I from B. cenocepacia This study pheS::dddAdddA.sub.I pDONRPEX18Gm-Scel- To generate the dddAE.sup.1347A catalytic mutant in B. This study pheS::dddA.sup.E1347A cenocepacia pBAD24::ung To express ung in E. coli This study pETDuet-1 mcs1::dddA-his.sub.6 To co-express dddA-dddA.sub.I This study mcs1::dddA.sub.I pTNS3 Tn7 transposase expression PMID: 18156318 pRK2013 Helper plasmid for plasmid mobilization ATCC 37159 pKD46 Recombinase expression plasmid for A Red PMID: 10829079 deletion system pKD4 Template for ung deletion amplicon PMID: 10829079 pCMV For mammalian expression of BE2, BE4max, PMID: 27096365 split DddAtox and all DddAtox fusions PMID: 24531420 for CCR5 TALEs
TABLE-US-00061 TABLE9A showsgRNAsequencesforCas9screening gRNA Sequence(5-to-3) SEQIDNO: spG7 CCACTGGGGCCTCAACACTC 332 spG6 GAGGCCCCCAGAGCAGCCAC 333 saG1 TCTGTGCCCCTCCCTCCCTGGC 334 saG2 GCCCCTCCCTCCCTGGCCCAGGT 335 saG3 CCCTCCCTGGCCCAGGTGAAGG 336 saG4 GTGTGGTTCCAGAACCGGAGGA 337
TABLE-US-00062 dsDNA spacing length (bp) saG1 saG2 saG3 saG4 spG7 28 33 39 60 spG6 12 17 23 44
TABLE-US-00063 TABLE 10A KKH-Cas9 half with saG4 gRNA Batch Nucleotide G G C C C C T A A C C C G1333 DddAtox-N- A 0.19% 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 99.92% 99.68% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.24% 0.03% 0.01% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.00% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.13% 0.03% 99.28% 99.98% 99.97% 99.97% 0.03% 0.05% 0.00% 99.98% 99.98% 99.79% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.44% 99.91% 0.70% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% 0.00% 0.30% 0.01% 0.01% 0.20% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.19% 0.04% 0.00% 0.00% 0.00% 0.00% 0.00% 99.93% 99.63% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.21% 0.03% 0.01% 0.01% 0.01% 0.01% 99.92% 0.01% 0.00% 0.01% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.13% 0.02% 99.27% 99.98% 99.97% 99.97% 0.03% 0.04% 0.01% 99.99% 99.98% 99.73% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.47% 99.91% 0.72% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.04% 0.00% 0.35% 0.00% 0.01% 0.26% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.21% 0.04% 0.01% 0.01% 0.00% 0.01% 0.00% 99.93% 99.62% 0.01% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.23% 0.03% 0.00% 0.01% 0.01% 0.01% 99.94% 0.02% 0.01% 0.00% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.13% 0.03% 99.26% 99.97% 99.98% 99.98% 0.02% 0.03% 0.00% 99.98% 99.99% 99.75% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 99.43% 99.91% 0.73% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% 0.01% 0.36% 0.01% 0.01% 0.23% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.19% 0.03% 0.01% 0.01% 0.00% 0.01% 0.00% 99.91% 99.63% 0.00% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.25% 0.05% 0.01% 0.01% 0.01% 0.01% 99.92% 0.01% 0.00% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.16% 0.01% 99.19% 99.97% 99.98% 99.97% 0.03% 0.04% 0.01% 99.98% 99.98% 99.73% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.40% 99.91% 0.79% 0.02% 0.00% 0.00% 0.01% 0.02% 0.01% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.04% 0.01% 0.35% 0.01% 0.01% 0.26% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide T A T G T A G C C T C A G1333 DddAtox-N- A 0.00% 99.90% 0.00% 0.05% 0.01% 99.94% 0.03% 0.00% 0.00% 0.01% 0.01% 99.96% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 99.97% 0.01% 99.98% 0.03% 99.97% 0.01% 0.04% 0.01% 0.01% 99.98% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.01% 0.03% 0.01% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.01% 99.97% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.00% 0.02% 0.00% 99.92% 0.00% 0.03% 99.93% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.02% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 99.90% 0.01% 0.05% 0.01% 99.94% 0.01% 0.01% 0.00% 0.01% 0.01% 99.96% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 99.97% 0.02% 99.97% 0.03% 99.98% 0.00% 0.05% 0.01% 0.01% 99.99% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 0.01% 0.01% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.01% 99.97% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.00% 0.03% 0.00% 99.92% 0.00% 0.04% 99.94% 0.00% 0.00% 0.00% 0.00% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.02% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.01% 99.90% 0.01% 0.06% 0.02% 99.94% 0.02% 0.00% 0.01% 0.01% 0.01% 99.97% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 99.95% 0.01% 99.97% 0.04% 99.97% 0.01% 0.03% 0.00% 0.01% 99.97% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 0.02% 0.01% 0.01% 0.00% 0.02% 0.00% 99.99% 99.98% 0.02% 99.98% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.00% 0.01% 0.00% 99.90% 0.00% 0.03% 99.95% 0.00% 0.00% 0.01% 0.00% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.03% 0.06% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 99.90% 0.01% 0.07% 0.01% 99.94% 0.02% 0.01% 0.01% 0.01% 0.01% 99.97% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 99.95% 0.01% 99.96% 0.04% 99.97% 0.01% 0.05% 0.01% 0.01% 99.97% 0.02% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.01% 0.03% 0.01% 0.00% 0.01% 0.03% 0.00% 99.99% 99.99% 0.01% 99.97% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.00% 0.03% 0.00% 99.89% 0.00% 0.03% 99.93% 0.00% 0.00% 0.01% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.03% 0.03% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA Nucle- Batch otide G T C T T C C C A T C A G G1333 A 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 99.96% 0.01% 0.01% 99.96% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 T 0.05% 99.99% 0.00% 99.98% 99.97% 0.01% 0.00% 0.01% 0.01% 99.98% 0.01% 0.01% 0.04% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 0.01% 99.99% 0.01% 0.02% 99.98% 99.98% 99.98% 0.01% 0.00% 99.99% 0.01% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 G 99.95% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.02% 99.95% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 A 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% 99.96% 0.00% 0.01% 99.96% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 T 0.03% 99.98% 0.01% 99.98% 99.98% 0.02% 0.00% 0.01% 0.01% 99.99% 0.00% 0.01% 0.05% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 0.01% 99.99% 0.02% 0.01% 99.98% 99.99% 99.98% 0.01% 0.00% 99.98% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 G 99.95% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.02% 99.94% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 A 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 99.95% 0.00% 0.00% 99.96% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 T 0.04% 99.99% 0.00% 99.98% 99.97% 0.01% 0.01% 0.01% 0.00% 99.99% 0.01% 0.01% 0.04% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 C 0.01% 0.01% 99.99% 0.01% 0.02% 99.98% 99.98% 99.98% 0.01% 0.01% 99.99% 0.01% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 G 99.95% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.04% 0.00% 0.00% 0.02% 99.94% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 A 0.00% 0.01% 0.00% 0.01% 0.01% 0.00% 0.01% 0.01% 99.96% 0.01% 0.00% 99.97% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 T 0.04% 99.98% 0.00% 99.98% 99.97% 0.01% 0.01% 0.01% 0.01% 99.98% 0.01% 0.00% 0.03% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 C 0.00% 0.01% 99.99% 0.02% 0.02% 99.98% 99.98% 99.98% 0.01% 0.01% 99.98% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 G 99.95% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.02% 99.96% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA Nucle- Batch otide G C T C T C A G C T C G1333 A 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 99.97% 0.01% 0.00% 0.01% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 T 0.03% 0.00% 99.99% 0.01% 99.99% 0.01% 0.00% 0.01% 0.01% 99.98% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 100.00% 0.01% 99.98% 0.01% 99.98% 0.01% 0.00% 99.99% 0.01% 99.98% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 G 99.96% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 A 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 99.97% 0.00% 0.00% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 T 0.02% 0.01% 99.99% 0.01% 99.98% 0.02% 0.00% 0.01% 0.01% 99.97% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 99.98% 0.00% 99.99% 0.01% 99.98% 0.01% 0.00% 99.99% 0.02% 99.98% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 G 99.97 0.00% 0.00% 0.00% 0.00% 0.00% 0.022% 99.98% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 99.98% 0.01% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 T 0.01% 0.00% 99.99% 0.01% 99.99% 0.01% 0.00% 0.01% 0.01% 99.98% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 C 0.01% 99.99% 0.01% 99.99% 0.01% 99.98% 0.01% 0.00% 99.98% 0.01% 99.98% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 G 99.97% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 99.98% 0.00% 0.01% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 A 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 99.96% 0.01% 0.00% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 T 0.01% 0.00% 99.98% 0.00% 99.98% 0.01% 0.00% 0.01% 0.01% 99.97% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 C 0.01% 99.99% 0.02% 100.00% 0.01% 99.98% 0.01% 0.00% 99.98% 0.01% 99.98% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 G 99.97% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA Nucle- Batch otide A G C C T G A G T G T T G1333 A 99.97% 0.02% 0.00% 0.00% 0.00% 0.01% 99.96% 0.00% 0.01% 0.01% 0.01% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 T 0.00% 0.01% 0.01% 0.01% 99.98% 0.02% 0.00% 0.01% 99.96% 0.01% 99.97% 99.97% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 C 0.01% 0.00% 99.99% 99.99% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.02% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 G 0.02% 99.97% 0.00% 0.00% 0.00% 99.97% 0.02% 99.98% 0.02% 99.97% 0.01% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 A 99.96% 0.01% 0.00% 0.00% 0.00% 0.01% 99.98% 0.00% 0.01% 0.01% 0.00% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 T 0.00% 0.01% 0.01% 0.00% 99.98% 0.01% 0.00% 0.01% 99.96% 0.01% 99.96% 99.97% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 C 0.01% 0.00% 99.99% 100.00% 0.02% 0.00% 0.00% 0.00% 0.01% 0.00% 0.02% 0.02% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 G 0.03% 99.98% 0.00% 0.00% 0.00% 99.99% 0.02% 99.99% 0.02% 99.98% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 A 99.97% 0.01% 0.00% 0.01% 0.00% 0.01% 99.97% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 T 0.01% 0.01% 0.00% 0.01% 99.98% 0.02% 0.00% 0.01% 99.97% 0.01% 99.96% 99.97% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 C 0.01% 0.00% 100.00% 99.99% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.02% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 G 0.01% 99.98% 0.00% 0.00% 0.00% 99.97% 0.01% 99.99% 0.01% 99.97% 0.00% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 A 99.97% 0.01% 0.00% 0.00% 0.01% 0.01% 99.97% 0.01% 0.01% 0.00% 0.01% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 T 0.01% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% 0.00% 99.94% 0.01% 99.97% 99.97% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 C 0.01% 0.00% 99.99% 99.99% 0.02% 0.01% 0.01% 0.00% 0.02% 0.01% 0.02% 0.02% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 G 0.01% 99.98% 0.01% 0.00% 0.00% 99.97% 0.01% 99.99% 0.03% 99.98% 0.01% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA Nucle- Batch otide G A G G C C C C A G T G G1333 A 0.02% 99.95% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 99.94% 0.00% 0.01% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 T 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 99.96% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 0.03% 0.00% 0.00% 99.97% 99.99% 99.97% 99.96% 0.02% 0.00% 0.02% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 G 99.95% 0.01% 99.96% 99.97% 0.00% 0.00% 0.00% 0.00% 0.03% 99.98% 0.01% 99.98% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1333 A 0.02% 99.96% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 99.96% 0.00% 0.02% 0.02% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 T 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 99.94% 0.02% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 C 0.00% 0.02% 0.00% 0.00% 99.99% 99.98% 99.98% 99.97% 0.02% 0.00% 0.02% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 G 99.96% 0.01% 99.97% 99.97% 0.00% 0.00% 0.00% 0.00% 0.02% 99.99% 0.02% 99.96% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 A 0.02% 99.95% 0.01% 0.02% 0.00% 0.01% 0.01% 0.01% 99.98% 0.01% 0.02% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 T 0.01% 0.00% 0.02% 0.00% 0.01% 0.01% 0.01% 0.02% 0.00% 0.01% 99.93% 0.01% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 C 0.00% 0.02% 0.00% 0.00% 99.97% 99.98% 99.98% 99.95% 0.00% 0.00% 0.02% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 G 99.97% 0.02% 99.97% 99.97% 0.01% 0.00% 0.01% 0.01% 0.01% 99.98% 0.03% 99.98% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DddAtox- N-SaKKH- Cas9 with saG4 gRNA G1397 A 0.01% 99.95% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 99.96% 0.00% 0.01% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 T 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 99.95% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 C 0.00% 0.03% 0.00% 0.00% 99.98% 99.98% 99.98% 99.96% 0.01% 0.00% 0.02% 0.01% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 G 99.98% 0.01% 99.97% 99.99% 0.00% 0.01% 0.00% 0.00% 0.02% 99.98% 0.02% 99.97% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox- C-SaKKH- Cas9 with saG4 gRNA Nucle- Batch otide G C T G C T C T G G G G G1333 DddAtox-N- A 0.00% 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 99.97% 0.02% 0.00% 99.97% 0.00% 99.97% 0.02% 0.02% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 99.98% 0.02% 0.00% 99.99% 0.01% 99.99% 0.02% 0.01% 0.00% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.98% 0.00% 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% 99.96% 99.97% 99.95% 99.97% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.02% 0.01% 99.98% 0.02% 0.01% 99.98% 0.00% 99.97% 0.02% 0.03% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 99.98% 0.01% 0.00% 99.98% 0.02% 99.98% 0.02% 0.00% 0.00% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.96% 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% 99.97% 99.95% 99.77% 99.77% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.20% 0.20% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.00% 0.01% 0.03% 0.02% 0.03% 0.02% 0.01% 0.00% 0.01% 0.03% 0.03% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.00% 99.94% 0.01% 0.01% 99.95% 0.01% 99.98% 0.02% 0.02% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 99.98% 0.01% 0.00% 99.96% 0.01% 99.98% 0.01% 0.01% 0.01% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 99.98% 0.01% 0.01% 99.96% 0.00% 0.01% 0.00% 0.01% 99.96% 99.95% 99.34% 97.81% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.60% 2.15% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 0.00% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.02% 0.01% 99.97% 0.02% 0.01% 99.99% 0.01% 99.96% 0.02% 0.02% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.01% 99.98% 0.02% 0.00% 99.98% 0.01% 99.99% 0.02% 0.00% 0.00% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.97% 0.00% 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% 99.97% 99.97% 99.96% 99.97% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA Nucle- Batch otide G C C T C C T G A G T T G1333 DddAtox-N- A 0.01% 0.00% 0.02% 0.01% 0.00% 0.01% 0.01% 0.02% 99.93% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.02% 0.00% 0.00% 99.97% 0.00% 0.01% 99.96% 0.02% 0.00% 0.00% 99.97% 99.97% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 99.99% 99.97% 0.01% 99.99% 99.98% 0.02% 0.00% 0.04% 0.00% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.95% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.95% 0.02% 99.98% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% 0.01% 99.75% 0.00% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.01% 0.01% 99.77% 0.01% 0.00% 99.77% 0.01% 0.01% 0.01% 99.77% 99.78% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 99.78% 99.77% 0.01% 99.78% 99.79% 0.02% 0.00% 0.03% 0.00% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.74% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.77% 0.01% 99.79% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.24% 0.20% 0.20% 0.20% 0.20% 0.20% 0.21% 0.20% 0.20% 0.20% 0.20% 0.20% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.00% 0.03% 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 97.73% 0.01% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.01% 0.01% 97.80% 0.01% 0.01% 97.82% 0.01% 0.01% 0.01% 97.75% 97.76% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 97.81% 97.80% 0.01% 97.84% 97.83% 0.02% 0.00% 0.03% 0.00% 0.03% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 97.80% 0.01% 0.02% 0.03% 0.00% 0.00% 0.00% 97.83% 0.03% 97.78% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 2.18% 2.15% 2.15% 2.15% 2.15% 2.15% 2.15% 2.15% 2.20% 2.20% 2.20% 2.22% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.93% 0.01% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% 99.96% 99.99% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.01% 99.98% 99.98% 0.01% 99.98% 99.99% 0.01% 0.00% 0.03% 0.00% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.93% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.98% 0.03% 99.98% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.04% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide T C T C A T C T G T G C G1333 DddAtox-N- A 0.00% 0.01% 0.02% 0.01% 99.97% 0.00% 0.01% 0.01% 0.01% 0.02% 0.03% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 99.98% 0.01% 99.96% 0.01% 0.00% 99.99% 0.00% 99.97% 0.02% 99.93% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.01% 99.98% 0.02% 99.97% 0.01% 0.01% 99.98% 0.01% 0.00% 0.02% 0.00% 99.97% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.96% 0.01% 99.95% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.00% 0.00% 0.01% 0.01% 99.77% 0.00% 0.01% 0.01% 0.01% 0.02% 0.03% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 99.78% 0.00% 99.77% 0.01% 0.00% 99.79% 0.01% 99.78% 0.02% 99.73% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.01% 99.79% 0.02% 99.78% 0.01% 0.01% 99.78% 0.01% 0.00% 0.02% 0.00% 99.78% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 99.77% 0.02% 99.74% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.20% 0.20% 0.20% 0.20% 0.20% 0.20% 0.20% 0.20% 0.20% 0.20% 0.21% 0.21% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.00% 0.01% 0.01% 0.01% 97.75% 0.00% 0.01% 0.03% 0.02% 0.03% 0.04% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 97.76% 0.01% 97.75% 0.01% 0.01% 97.42% 0.01% 96.67% 0.01% 96.65% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.00% 97.78% 0.03% 97.75% 0.01% 0.01% 97.43% 0.01% 0.00% 0.02% 0.00% 96.67% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.01% 0.01% 0.01% 0.02% 0.02% 0.03% 0.01% 0.01% 96.68% 0.01% 96.67% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 2.22% 2.21% 2.21% 2.21% 2.21% 2.54% 2.54% 3.28% 3.28% 3.28% 3.28% 3.28% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.00% 0.01% 0.01% 0.00% 99.97% 0.00% 0.01% 0.01% 0.01% 0.03% 0.02% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 99.98% 0.01% 99.97% 0.01% 0.00% 99.99% 0.01% 99.97% 0.01% 99.94% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.01% 99.98% 0.01% 99.98% 0.01% 0.00% 99.98% 0.01% 0.00% 0.02% 0.00% 99.98% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 99.97% 0.01% 99.96% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide C C C T C C C T C C C T G1333 DddAtox-N- A 0.00% 0.02% 0.02% 0.01% 0.01% 0.01% 0.02% 0.01% 0.00% 0.02% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.01% 99.96% 0.02% 0.02% 0.01% 99.97% 0.01% 0.01% 0.00% 99.68% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 99.98% 99.96% 99.95% 0.02% 99.96% 99.96% 99.95% 0.01% 99.96% 99.94% 99.95% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% 0.02% 0.02% 0.03% 0.2% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.01% 0.01% 99.75% 0.02% 0.03% 0.01% 99.77% 0.02% 0.01% 0.01% 99.72% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 99.78% 99.78% 99.77% 0.02% 99.76% 99.76% 99.76% 0.01% 99.74% 99.75% 99.76% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.20% 0.20% 0.21% 0.21% 0.20% 0.20% 0.21% 0.20% 0.22% 0.22% 0.23% 0.23% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.03% 0.02% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.02% 0.01% 96.67% 0.03% 0.02% 0.01% 96.6% 0.02% 0.01% 0.01% 97.37% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 96.68% 96.65% 96.67% 0.02% 96.68% 96.67% 96.68% 0.01% 96.65% 97.40% 97.40% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.00% 0.03% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 3.28% 3.28% 3.29% 3.28% 3.28% 3.30% 3.30% 3.30% 3.31% 2.57% 2.58% 2.57% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 0.01% 0.01% 0.01% 0.01% 0.03% 0.01% 0.00% 0.01% 0.03% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.01% 99.93% 0.06% 0.02% 0.00% 99.86% 0.03% 0.01% 0.00% 99.81% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 99.98% 99.98% 99.97% 0.05% 99.93% 99.94% 99.98% 0.12% 99.94% 99.83% 99.84% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.02% 0.13% 0.14% 0.13% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide G G C C C A G G T G A A G1333 DddAtox-N- A 0.02% 0.02% 0.01% 0.01% 0.03% 99.18% 0.01% 0.01% 0.03% 0.01% 99.16% 99.12% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.04% 0.01% 0.01% 0.01% 0.01% 0.01% 98.88% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 0.01% 99.32% 99.26% 99.24% 0.04% 0.00% 0.01% 0.06% 0.00% 0.04% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.53% 99.52% 0.01% 0.00% 0.01% 0.06% 99.26% 99.26% 0.28% 99.22% 0.04% 0.08% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.44% 0.44% 0.62% 0.71% 0.72% 0.72% 0.72% 0.72% 0.75% 0.76% 0.76% 0.76% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.01% 0.01% 0.01% 0.03% 99.65% 0.01% 0.01% 0.03% 0.01% 99.58% 99.55% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.02% 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 99.30% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 0.00% 99.79% 99.74% 99.70% 0.03% 0.00% 0.00% 0.10% 0.00% 0.04% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.78% 99.77% 0.01% 0.00% 0.00% 0.06% 99.73% 99.70% 0.29% 99.69% 0.05% 0.08% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.20% 0.20% 0.19% 0.24% 0.24% 0.24% 0.25% 0.27% 0.29% 0.29% 0.32% 0.32% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.02% 0.01% 0.03% 0.03% 0.02% 97.25% 0.02% 0.01% 0.05% 0.01% 97.22% 97.19% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.01% 0.05% 0.01% 0.01% 0.02% 0.02% 0.01% 96.78% 0.01% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 0.01% 97.39% 97.44% 97.31% 0.04% 0.00% 0.02% 0.25% 0.02% 0.06% 0.05% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 97.42% 97.43% 0.02% 0.01% 0.02% 0.06% 97.30% 97.29% 0.29% 97.31% 0.04% 0.09% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 2.54% 2.54% 2.50% 2.51% 2.64% 2.64% 2.67% 2.67% 2.63% 2.65% 2.65% 2.66% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 0.02% 0.01% 0.01% 0.02% 99.46% 0.01% 0.01% 0.03% 0.01% 99.43% 99.37% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.02% 0.01% 0.02% 0.01% 0.02% 0.01% 99.18% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.00% 0.00% 99.63% 99.64% 99.51% 0.04% 0.00% 0.00% 0.05% 0.00% 0.06% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.86% 99.86% 0.01% 0.01% 0.00% 0.05% 99.52% 99.53% 0.29% 99.51% 0.04% 0.09% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.11% 0.11% 0.33% 0.34% 0.44% 0.44% 0.45% 0.45% 0.46% 0.46% 0.47% 0.50% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide G G T G T G G T T C C A G1333 DddAtox-N- A 0.00% 0.01% 0.04% 0.01% 0.03% 0.02% 0.00% 0.01% 0.00% 0.01% 0.02% 99.10% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 98.73% 0.02% 99.02% 0.01% 0.01% 99.06% 99.06% 0.06% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 0.00% 0.05% 0.00% 0.03% 0.00% 0.00% 0.02% 0.01% 99.00% 99.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.20% 99.14% 0.27% 99.11% 0.06% 99.11% 99.12% 0.03% 0.00% 0.02% 0.00% 0.04% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.78% 0.84% 0.86% 0.86% 0.86% 0.86% 0.87% 0.89% 0.92% 0.92% 0.94% 0.84% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.00% 0.03% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 0.02% 99.50% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.02% 99.23% 0.02% 99.48% 0.01% 0.00% 99.50% 99.51% 0.03% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 0.00% 0.06% 0.01% 0.02% 0.01% 0.00% 0.03% 0.02% 99.51% 99.51% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.65% 99.63% 0.31% 99.58% 0.08% 99.56% 99.56% 0.02% 0.00% 0.00% 0.00% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.33% 0.35% 0.36% 0.38% 0.40% 0.42% 0.42% 0.45% 0.46% 0.45% 0.46% 0.45% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.01% 0.01% 0.04% 0.01% 0.01% 0.02% 0.03% 0.02% 0.01% 0.00% 0.02% 96.50% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.01% 96.22% 0.02% 96.47% 0.01% 0.01% 96.46% 96.51% 0.04% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 0.02% 0.07% 0.01% 0.03% 0.00% 0.01% 0.05% 0.02% 96.51% 96.50% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 97.32% 96.55% 0.26% 96.56% 0.08% 96.54% 96.52% 0.02% 0.01% 0.00% 0.01% 0.04% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 2.66% 3.41% 3.41% 3.41% 3.42% 3.43% 3.44% 3.45% 3.45% 3.45% 3.45% 3.45% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.00% 0.02% 0.05% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 99.28% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.02% 0.03% 99.08% 0.01% 99.32% 0.02% 0.01% 99.32% 99.34% 0.03% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.00% 0.00% 0.05% 0.01% 0.02% 0.00% 0.00% 0.03% 0.02% 99.33% 99.32% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.47% 99.42% 0.27% 99.42% 0.06% 99.38% 99.40% 0.02% 0.01% 0.01% 0.00% 0.05% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.51% 0.53% 0.56% 0.56% 0.58% 0.59% 0.58% 0.61% 0.63% 0.63% 0.64% 0.65% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide G A A C C G G A G G A C G1333 DddAtox-N- A 0.01% 99.11% 99.11% 0.01% 0.01% 0.02% 0.01% 98.94% 0.01% 0.01% 99.12% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 0.02% 0.02% 99.02% 99.00% 0.00% 0.00% 0.02% 0.00% 0.01% 0.04% 99.28% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.15% 0.01% 0.02% 0.00% 0.00% 98.99% 99.00% 0.06% 98.91% 98.93% 0.02% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.84% 0.85% 0.85% 0.95% 0.97% 0.97% 0.97% 0.97% 1.07% 1.04% 0.81% 0.70% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 99.50% 99.50% 0.00% 0.00% 0.02% 0.01% 99.41% 0.01% 0.01% 99.60% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.00% 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.02% 0.01% 0.00% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 0.02% 0.02% 99.52% 99.50% 0.00% 0.00% 0.02% 0.00% 0.00% 0.05% 99.75% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.53% 0.03% 0.02% 0.00% 0.01% 99.48% 99.56% 0.06% 99.41% 99.42% 0.03% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.45% 0.45% 0.46% 0.46% 0.48% 0.48% 0.48% 0.50% 0.57% 0.56% 0.31% 0.22% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.01% 96.51% 96.49% 0.02% 0.02% 0.02% 0.01% 96.43% 0.01% 0.01% 98.49% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.02% 0.00% 0.00% 0.01% 0.01% 0.01% 0.03% 0.01% 0.02% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.00% 0.01% 0.03% 96.51% 96.51% 0.00% 0.00% 0.03% 0.00% 0.01% 0.09% 98.65% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 96.53% 0.02% 0.04% 0.02% 0.01% 96.51% 96.51% 0.06% 96.48% 96.48% 0.04% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 3.45% 3.44% 3.44% 3.44% 3.45% 3.45% 3.47% 3.45% 3.51% 3.49% 1.37% 1.31% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 99.28% 99.25% 0.01% 0.01% 0.01% 0.02% 99.31% 0.01% 0.01% 99.37% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.02% 0.01% 0.03% 0.02% 0.01% 0.01% 0.02% 0.02% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.01% 0.02% 0.04% 99.30% 99.26% 0.00% 0.00% 0.03% 0.00% 0.01% 0.05% 99.60% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.31% 0.03% 0.02% 0.01% 0.01% 99.28% 99.28% 0.05% 99.25% 99.26% 0.02% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.66% 0.66% 0.68% 0.68% 0.69% 0.69% 0.69% 0.59% 0.72% 0.70% 0.55% 0.37% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide A A A G T A C A A A C G G1333 DddAtox-N- A 99.27% 99.27% 99.26% 0.01% 0.11 99.68% 0.02% 99.71% 99.72% 99.72% 0.01% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.02% 0.00% 0.01% 99.27% 0.00% 0.02% 0.00% 0.00% 0.00% 0.02% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.02% 0.03% 0.03% 0.00% 0.05% 0.01% 99.68% 0.02% 0.02% 0.02% 99.72% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.03% 0.01% 0.03% 99.41% 0.07% 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 99.96% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.68% 0.68% 0.67% 0.57% 0.50% 0.28% 0.27% 0.26% 0.25% 0.25% 0.25% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 99.74% 99.75% 99.75% 0.01% 0.14% 99.88% 0.02% 99.90% 99.89% 99.89% 0.01% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.01% 0.01% 0.00% 99.63% 0.02% 0.00% 0.00% 0.00% 0.00% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.02% 0.03% 0.04% 0.00% 0.05% 0.01% 99.90% 0.02% 0.03% 0.02% 99.90% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.02% 0.01% 0.05% 99.84% 0.07% 0.01% 0.00% 0.01% 0.01% 0.02% 0.01% 99.91% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.21% 0.21% 0.15% 0.14% 0.12% 0.08% 0.07% 0.07% 0.06% 0.07% 0.07% 0.05% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 98.75% 98.71% 98.73% 0.01% 0.12% 98.80% 0.02% 98.84% 99.19% 99.19% 0.01% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.02% 0.03% 0.00% 0.02% 98.44% 0.02% 0.01% 0.00% 0.02% 0.01% 0.05% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.02% 0.05% 0.06% 0.00% 0.23% 0.03% 98.85% 0.02% 0.03% 0.03% 99.20% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.03% 0.03% 0.04% 98.80% 0.06% 0.03% 0.00% 0.04% 0.01% 0.03% 0.00% 99.20% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 1.17% 1.17% 1.16% 1.16% 1.15% 1.12% 1.11% 1.10% 0.75% 0.75% 0.74% 0.74% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 99.69% 99.69% 99.65% 0.01% 0.13% 99.91% 0.02% 99.93% 99.94% 99.93% 0.02% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.01% 0.01% 99.66% 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.02% 0.03% 0.04% 0.00% 0.05% 0.02% 99.92% 0.02% 0.03% 0.03% 99.93% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.02% 0.01% 0.05% 99.73% 0.06% 0.02% 0.00% 0.01% 0.01% 0.02% 0.01% 99.94% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.27% 0.26% 0.26% 0.25% 0.10% 0.05% 0.05% 0.03% 0.02% 0.02% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide G C A G A A G C T G G A G1333 DddAtox-N- A 0.03% 0.01% 99.92% 0.00% 99.95% 99.85% 0.00% 0.00% 0.00% 0.01% 0.01% 99.86% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.95% 0.02% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 99.96% 0.01% 0.00% 0.03% 0.05% 0.00% 99.97% 0.02% 0.01% 0.00% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.96% 0.01% 0.06% 99.99% 0.02% 0.09% 99.98% 0.01% 0.02% 99.97% 99.97% 0.10% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.03% 0.02% 99.87% 0.01% 99.93% 99.83% 0.00% 0.00% 0.00% 0.01% 0.01% 99.81% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 99.92% 0.01% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 99.91% 0.03% 0.00% 0.02% 0.04% 0.00% 99.97% 0.03% 0.00% 0.00% 0.05% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.92% 0.01% 0.05% 99.94% 0.01% 0.11% 99.97% 0.00% 0.03% 99.96% 99.95% 0.11% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.05% 0.05% 0.05% 0.05% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.02% 0.02% 99.12% 0.01% 99.16% 99.07% 0.01% 0.01% 0.00% 0.00% 0.01% 99.76% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.02% 0.03% 0.03% 0.03% 0.02% 0.01% 0.02% 99.95% 0.02% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.01% 99.21% 0.05% 0.00% 0.05% 0.06% 0.00% 99.96% 0.03% 0.01% 0.01% 0.11% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 99.21% 0.01% 0.07% 99.23% 0.02% 0.11% 99.24% 0.01% 0.02% 99.97% 99.95% 0.11% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.74% 0.74% 0.73% 0.73% 0.73% 0.74% 0.73% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.03% 0.01% 99.90% 0.01% 99.93% 99.86% 0.00% 0.00% 0.01% 0.01% 0.00% 99.82% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 99.93% 0.01% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.00% 99.96% 0.03% 0.00% 0.03% 0.05% 0.00% 99.98% 0.04% 0.00% 0.00% 0.04% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.93% 0.01% 0.06% 99.98% 0.03% 0.09% 99.98% 0.01% 0.03% 99.97% 99.97% 0.13% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide G G A G G A A G G G C C G1333 DddAtox-N- A 0.00% 0.01% 99.88% 0.01% 0.01% 99.92% 99.91% 0.02% 0.01% 0.01% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.02% 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.04% 0.08% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.00% 0.00% 0.04% 0.00% 0.01% 0.02% 0.02% 0.01% 0.01% 0.00% 99.93% 99.88% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 99.97% 99.97% 0.07% 99.97% 99.97% 0.06% 0.06% 99.97% 99.97% 99.97% 0.02% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.01% 99.85% 0.00% 0.02% 99.90% 99.87% 0.01% 0.00% 0.00% 0.02% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.05% 0.10% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.00% 0.00% 0.02% 0.00% 0.00% 0.02% 0.02% 0.01% 0.00% 0.01% 99.90% 99.85% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 99.95% 99.96% 0.09% 99.97% 99.95% 0.05% 0.09% 99.95% 99.98% 99.96% 0.03% 0.03% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.03% 0.01% 99.83% 0.03% 0.03% 99.89% 99.88% 0.04% 0.02% 0.01% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.02% 0.01% 0.03% 0.00% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.05% 0.09% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.00% 0.00% 0.05% 0.01% 0.02% 0.04% 0.03% 0.01% 0.02% 0.00% 99.92% 99.87% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 99.95% 99.97% 0.09% 99.96% 99.94% 0.05% 0.07% 99.94% 99.95% 99.98% 0.03% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.00% 0.01% 99.84% 0.01% 0.01% 99.91% 99.88% 0.01% 0.01% 0.01% 0.01% 0.04% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.02% 0.01% 0.02% 0.02% 0.01% 0.01% 0.00% 0.01% 0.01% 0.02% 0.03% 0.09% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.00% 0.00% 0.04% 0.00% 0.00% 0.02% 0.02% 0.01% 0.00% 0.00% 99.92% 99.85% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 99.98% 99.97% 0.10% 99.97% 99.97% 0.06% 0.09% 99.98% 99.98% 99.97% 0.04% 0.02% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide T G A G T C C G G1333 DddAtox-N- A 0.01% 0.01% 99.88% 0.01% 0.02% 0.02% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 99.91% 0.01% 0.00% 0.01% 99.80% 0.03% 0.04% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.04% 0.01% 0.04% 0.00% 0.09% 99.95% 99.87% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.03% 99.97% 0.08% 99.97% 0.06% 0.01% 0.01% 99.92% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.01% 0.03% 0.00% 0.07% 0.04% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 0.01% 0.01% 99.86% 0.01% 0.03% 0.01% 0.01% 0.02% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 99.89% 0.02% 0.01% 0.01% 99.79% 0.04% 0.05% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.04% 0.00% 0.04% 0.00% 0.07% 99.94% 99.84% 0.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.05% 99.97% 0.09% 99.95% 0.07% 0.01% 0.03% 99.91% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.00% 0.00% 0.00% 0.03% 0.04% 0.00% 0.08% 0.06% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 0.02% 0.03% 99.83% 0.02% 0.05% 0.02% 0.01% 0.03% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 99.89% 0.01% 0.01% 0.00% 99.75% 0.05% 0.06% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.05% 0.00% 0.06% 0.01% 0.07% 99.92% 99.83% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.04% 99.96% 0.09% 99.95% 0.08% 0.01% 0.01% 99.89% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.00% 0.00% 0.00% 0.02% 0.05% 0.00% 0.09% 0.07% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 0.01% 0.02% 99.86% 0.00% 0.02% 0.01% 0.01% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 99.90% 0.02% 0.01% 0.01% 99.79% 0.03% 0.05% 0.01% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.05% 0.00% 0.03% 0.00% 0.08% 99.95% 99.83% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.04% 99.97% 0.09% 99.96% 0.07% 0.01% 0.02% 99.90% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.00% 0.00% 0.00% 0.02% 0.04% 0.00% 0.09% 0.07% SaKKH-Cas9 with saG4 gRNA Batch Nucleotide A G C A G A A G G1333 DddAtox-N- A 99.85% 0.01% 0.04% 3.03% 0.08% 2.92% 1.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.03% 0.01% 0.01% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- C 0.02% 0.01% 99.89% 0.05% 0.00% 0.02% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- G 0.09% 99.94% 0.02% 0.11% 3.07% 0.05% 0.01% 1.01% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-N- 0.03% 0.04% 0.02% 96.81% 96.84% 97.01% 98.98% 98.98% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- A 99.85% 0.01% 0.03% 2.89% 0.10% 2.75% 0.74% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- T 0.01% 0.01% 0.04% 0.02% 0.01% 0.01% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- C 0.02% 0.01% 99.87% 0.04% 0.00% 0.03% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- G 0.09% 99.92% 0.02% 0.10% 2.93% 0.05% 0.00% 0.74% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1333 DddAtox-C- 0.03% 0.05% 0.03% 96.95% 96.96% 97.17% 99.25% 99.26% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- A 99.81% 0.01% 0.03% 6.28% 0.10% 6.13% 4.18% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- T 0.01% 0.02% 0.04% 0.01% 0.01% 0.01% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- C 0.03% 0.01% 99.88% 0.06% 0.01% 0.02% 0.01% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- G 0.12% 99.92% 0.01% 0.09% 6.31% 0.08% 0.01% 4.19% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-N- 0.03% 0.04% 0.04% 93.55% 93.57% 93.76% 95.80% 95.81% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- A 99.84% 0.01% 0.03% 3.12% 0.11% 2.98% 0.75% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- T 0.01% 0.00% 0.03% 0.01% 0.01% 0.01% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- C 0.04% 0.01% 99.88% 0.03% 0.00% 0.01% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- G 0.08% 99.91% 0.03% 0.12% 3.14% 0.06% 0.00% 0.76% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% SaKKH-Cas9 with saG4 gRNA G1397 DddAtox-C- 0.03% 0.06% 0.03% 96.72% 96.74% 96.94% 99.24% 99.24% SaKKH-Cas9 with saG4 gRNA
TABLE-US-00064 TABLE 10B dSpCas9 half with spG4 gRNA Nucle- Batch otide G G C C C C T A A C C C G1333 A 0.18% 0.03% 0.00% 0.00% 0.00% 0.00% 0.01% 99.93% 99.67% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.20% 0.03% 0.01% 0.00% 0.01% 0.00% 99.93% 0.00% 0.00% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.10% 0.02% 99.34% 99.99% 99.98% 99.98% 0.03% 0.04% 0.01% 99.98% 99.99% 99.78% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.51% 99.92% 0.65% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% 0.00% 0.30% 0.01% 0.00% 0.20% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.21% 0.02% 0.00% 0.00% 0.01% 0.01% 0.01% 99.92% 99.65% 0.01% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.22% 0.03% 0.00% 0.01% 0.00% 0.00% 99.92% 0.00% 0.00% 0.00% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.11% 0.01% 99.30% 99.97% 99.98% 99.99% 0.02% 0.05% 0.00% 99.98% 99.98% 99.74% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.47% 99.94% 0.69% 0.02% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.05% 0.01% 0.33% 0.01% 0.00% 0.24% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.20% 0.04% 0.01% 0.00% 0.00% 0.01% 0.01% 99.94% 99.65% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.23% 0.03% 0.01% 0.00% 0.01% 0.01% 99.93% 0.01% 0.00% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.15% 0.02% 99.23% 99.99% 99.97% 99.97% 0.03% 0.03% 0.01% 99.97% 99.99% 99.73% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.41% 99.91% 0.75% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 0.03% 0.00% 0.34% 0.01% 0.00% 0.26% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.20% 0.05% 0.01% 0.01% 0.00% 0.01% 0.00% 99.93% 99.66% 0.01% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.24% 0.03% 0.00% 0.00% 0.00% 0.01% 99.93% 0.00% 0.01% 0.01% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.13% 0.03% 99.24% 99.98% 99.98% 99.97% 0.02% 0.04% 0.01% 99.98% 99.98% 99.76% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.44% 99.89% 0.75% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.04% 0.01% 0.31% 0.01% 0.01% 0.23% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide T A T G T A G C C T C A G1333 A 0.00% 99.90% 0.01% 0.05% 0.01% 99.95% 0.02% 0.00% 0.00% 0.01% 0.01% 99.98% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 99.97% 0.02% 99.97% 0.04% 99.97% 0.00% 0.04% 0.00% 0.00% 99.97% 0.01% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.02% 0.01% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.02% 99.95% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.02% 0.00% 99.91% 0.00% 0.03% 99.94% 0.00% 0.00% 0.00% 0.03% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.02% 0.03% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 99.93% 0.01% 0.05% 0.01% 99.95% 0.02% 0.00% 0.01% 0.00% 0.00% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 99.96% 0.00% 99.96% 0.04% 99.97% 0.00% 0.05% 0.00% 0.01% 99.98% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.02% 0.02% 0.00% 0.01% 0.01% 0.00% 99.99% 99.98% 0.01% 99.96% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.01% 0.00% 99.90% 0.00% 0.03% 99.93% 0.00% 0.00% 0.00% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.02% 0.03% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.00% 99.91% 0.01% 0.07% 0.01% 99.94% 0.02% 0.01% 0.00% 0.00% 0.00% 99.96% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 99.95% 0.01% 99.97% 0.03% 99.97% 0.00% 0.05% 0.00% 0.01% 99.98% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.02% 0.02% 0.01% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.01% 99.98% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 0.00% 0.02% 0.00% 99.90% 0.00% 0.04% 99.93% 0.00% 0.00% 0.00% 0.00% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.02% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.00% 99.88% 0.00% 0.04% 0.02% 99.94% 0.02% 0.01% 0.00% 0.01% 0.01% 99.96% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 99.96% 0.02% 99.98% 0.03% 99.96% 0.00% 0.05% 0.00% 0.01% 99.97% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.01% 0.02% 0.01% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.02% 99.98% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 0.00% 0.02% 0.00% 99.92% 0.00% 0.03% 99.93% 0.00% 0.00% 0.00% 0.00% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.03% 0.05% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G T C T T C C C A T C A G1333 A 0.01% 0.01% 0.01% 0.00% 0.00% 0.02% 0.01% 0.01% 99.95% 0.01% 0.01% 99.96% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.04% 99.98% 0.01% 99.96% 99.95% 0.01% 0.01% 0.01% 0.01% 99.96% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.01% 99.98% 0.04% 0.01% 99.97% 99.95% 99.97% 0.01% 0.01% 99.98% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.94% 0.00% 0.00% 0.00% 0.03% 0.00% 0.03% 0.00% 0.03% 0.03% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 99.95% 0.01% 0.00% 99.96% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.04% 99.98% 0.01% 99.97% 99.96% 0.01% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.01% 99.99% 0.03% 0.01% 99.98% 99.97% 99.97% 0.01% 0.01% 99.98% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.95% 0.00% 0.00% 0.00% 0.02% 0.01% 0.01% 0.00% 0.03% 0.02% 0.00% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 99.96% 0.00% 0.00% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.03% 99.97% 0.00% 99.98% 99.97% 0.01% 0.01% 0.01% 0.00% 99.98% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.02% 99.99% 0.02% 0.01% 99.99% 99.98% 99.98% 0.01% 0.01% 99.99% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.96% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.03% 0.01% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 99.96% 0.01% 0.01% 99.96% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.04% 99.99% 0.00% 99.98% 99.97% 0.01% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.01% 99.99% 0.01% 0.02% 99.99% 99.98% 99.99% 0.01% 0.01% 99.98% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.95% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G G C T C T C A G C T C G1333 A 0.01% 0.04% 0.00% 0.02% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.04% 0.01% 0.01% 99.97% 0.01% 99.97% 0.01% 0.00% 0.01% 0.01% 99.97% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 99.96% 0.01% 99.98% 0.01% 99.96% 0.01% 0.00% 99.98% 0.01% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.95% 99.93% 0.03% 0.00% 0.00% 0.00% 0.02% 0.04% 99.98% 0.00% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 0.02% 0.00% 0.00% 0.01% 0.01% 0.01% 99.96% 0.02% 0.00% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.04% 0.00% 0.00% 99.97% 0.00% 99.98% 0.00% 0.00% 0.01% 0.00% 99.96% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.01% 99.98% 0.02% 99.99% 0.01% 99.98% 0.01% 0.00% 99.99% 0.01% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.95% 99.97% 0.01% 0.00% 0.00% 0.01% 0.01% 0.03% 99.97% 0.00% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% 99.96% 0.01% 0.00% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.03% 0.01% 0.01% 99.99% 0.01% 99.98% 0.01% 0.00% 0.01% 0.01% 99.98% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.00% 99.99% 0.01% 99.99% 0.01% 99.98% 0.01% 0.00% 99.99% 0.01% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.96% 99.97% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 99.98% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 99.96% 0.01% 0.00% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.04% 0.01% 0.00% 99.99% 0.01% 99.99% 0.01% 0.00% 0.01% 0.01% 99.98% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.01% 100.00% 0.01% 99.99% 0.01% 99.99% 0.01% 0.00% 99.98% 0.01% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.95% 99.97% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 99.98% 0.00% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide A G C C T G A G T G T T G1333 A 99.95% 0.00% 0.03% 0.01% 0.00% 0.01% 99.94% 0.01% 0.02% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.00% 0.00% 0.01% 0.00% 99.97% 0.01% 0.01% 0.01% 99.92% 0.03% 99.92% 99.95% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.01% 99.96% 99.98% 0.01% 0.01% 0.01% 0.01% 0.04% 0.01% 0.02% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 0.04% 99.98% 0.00% 0.00% 0.00% 99.97% 0.04% 99.98% 0.01% 99.94% 0.04% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 99.96% 0.02% 0.01% 0.00% 0.01% 0.01% 99.93% 0.01% 0.01% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.00% 0.01% 0.01% 0.00% 99.96% 0.01% 0.01% 0.01% 99.94% 0.03% 99.95% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.02% 99.98% 99.99% 0.02% 0.01% 0.01% 0.00% 0.03% 0.00% 0.02% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 0.03% 99.95% 0.00% 0.00% 0.00% 99.96% 0.04% 99.98% 0.02% 99.96% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 99.97% 0.01% 0.00% 0.00% 0.01% 0.01% 99.96% 0.00% 0.01% 0.01% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 0.01% 99.98% 0.01% 0.00% 0.01% 99.96% 0.02% 99.96% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.01% 0.00% 99.99% 99.99% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.02% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 0.02% 99.98% 0.00% 0.00% 0.01% 99.97% 0.02% 99.99% 0.02% 99.97% 0.01% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 99.97% 0.01% 0.00% 0.00% 0.00% 0.01% 99.96% 0.01% 0.02% 0.01% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.00% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% 99.95% 0.02% 99.97% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.01% 0.00% 99.99% 99.98% 0.02% 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 0.02% 99.98% 0.00% 0.00% 0.00% 99.98% 0.01% 99.99% 0.01% 99.97% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G A G G C C C C A G T G G1333 A 0.04% 99.93% 0.02% 0.01% 0.00% 0.02% 0.01% 0.01% 99.94% 0.02% 0.02% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.04% 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 99.92% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.04% 0.01% 0.01% 99.98% 99.96% 99.97% 99.96% 0.02% 0.00% 0.04% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.92% 0.01% 99.92% 99.95% 0.00% 0.00% 0.01% 0.00% 0.02% 99.96% 0.01% 99.94% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.01% 0.02% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.04% 99.93% 0.01% 0.01% 0.01% 0.02% 0.00% 0.02% 99.94% 0.02% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.00% 0.05% 0.01% 0.02% 0.01% 0.01% 0.02% 0.01% 0.02% 99.93% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.05% 0.01% 0.01% 99.97% 99.97% 99.97% 99.94% 0.03% 0.00% 0.04% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.94% 0.01% 99.93% 99.96% 0.01% 0.00% 0.01% 0.00% 0.01% 99.95% 0.01% 99.95% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 0.01% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.02% 99.95% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.96% 0.01% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.00% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 99.95% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.02% 0.00% 0.00% 99.99% 99.98% 99.98% 99.96% 0.02% 0.00% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.96% 0.01% 99.96% 99.98% 0.00% 0.00% 0.00% 0.00% 0.02% 99.98% 0.02% 99.98% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 99.95% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.95% 0.01% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 99.94% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.03% 0.00% 0.00% 99.98% 100.00 99.98% 99.95% 0.01% 0.00% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.96% 0.01% 99.96% 99.97% 0.00% 0.00% 0.00% 0.01% 0.03% 99.99% 0.01% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G C T G C T C T G G G G G1333 A 0.03% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.02% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.00% 99.95% 0.02% 0.00% 99.97% 0.01% 99.97% 0.02% 0.02% 0.05% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.98% 0.03% 0.01% 99.96% 0.01% 99.98% 0.01% 0.01% 0.01% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.95% 0.01% 0.00% 99.96% 0.02% 0.01% 0.00% 0.01% 99.96% 99.95% 99.94% 99.95% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 0.01% 0.02% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 99.67% 0.02% 0.00% 99.66% 0.00% 99.69% 0.01% 0.03% 0.04% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.98% 0.03% 0.00% 99.69% 0.01% 99.70% 0.02% 0.01% 0.00% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.96% 0.00% 0.00% 99.68% 0.02% 0.00% 0.00% 0.00% 99.68% 99.67% 99.65% 99.65% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.28% 0.28% 0.29% 0.29% 0.29% 0.29% 0.29% 0.29% 0.29% 0.30% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.00% 99.98% 0.02% 0.00% 99.76% 0.01% 99.75% 0.01% 0.02% 0.02% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 99.99% 0.01% 0.00% 99.76% 0.01% 99.76% 0.01% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.97% 0.00% 0.00% 99.97% 0.00% 0.01% 0.00% 0.00% 99.75% 99.75% 99.74% 99.74% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.22% 0.22% 0.23% 0.23% 0.22% 0.22% 0.22% 0.23% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 99.97% 0.02% 0.01% 99.98% 0.00% 99.97% 0.02% 0.01% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 99.99% 0.01% 0.00% 99.99% 0.01% 99.99% 0.01% 0.01% 0.01% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.98% 0.00% 0.00% 99.97% 0.00% 0.01% 0.00% 0.00% 99.96% 99.97% 99.96% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G C C T C C T G A G T T G1333 A 0.00% 0.00% 0.04% 0.01% 0.01% 0.01% 0.01% 0.01% 99.91% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.01% 99.95% 0.01% 0.01% 99.96% 0.01% 0.02% 0.01% 99.96% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.98% 99.94% 0.02% 99.97% 99.97% 0.02% 0.00% 0.03% 0.01% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.94% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% 99.96% 0.03% 99.97% 0.01% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.04% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 0.00% 0.08% 0.01% 0.01% 0.01% 0.01% 0.02% 99.62% 0.00% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.00% 0.02% 99.68% 0.01% 0.01% 99.66% 0.02% 0.01% 0.01% 99.65% 99.69% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.69% 99.60% 0.01% 99.68% 99.69% 0.02% 0.01% 0.04% 0.00% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.66% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 99.66% 0.02% 99.68% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.31% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.31% 0.31% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.04% 0.01% 0.00% 0.01% 0.01% 0.01% 99.71% 0.00% 0.01% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 99.75% 0.01% 0.01% 99.74% 0.02% 0.00% 0.01% 99.74% 99.77% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.01% 99.76% 99.73% 0.01% 99.76% 99.75% 0.02% 0.00% 0.03% 0.00% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.72% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.74% 0.03% 99.76% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.26% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 99.91% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 99.97% 0.02% 0.01% 99.97% 0.01% 0.01% 0.01% 99.96% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 99.98% 99.97% 0.01% 99.97% 99.98% 0.02% 0.00% 0.04% 0.00% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.95% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 99.98% 0.03% 99.98% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide T C T C A T C T G T G C G1333 A 0.00% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.02% 0.02% 0.02% 0.04% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 99.97% 0.02% 99.95% 0.01% 0.01% 99.97% 0.01% 99.96% 0.02% 99.92% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.96% 0.01% 99.97% 0.02% 0.01% 99.96% 0.01% 0.00% 0.03% 0.01% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.01% 0.02% 0.00% 0.02% 0.00% 0.01% 0.00% 99.96% 0.02% 99.93% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.00% 0.00% 0.01% 0.00% 99.66% 0.01% 0.01% 0.01% 0.02% 0.03% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 99.68% 0.01% 99.65% 0.01% 0.01% 99.67% 0.01% 99.67% 0.01% 99.60% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 99.68% 0.01% 99.67% 0.02% 0.01% 99.67% 0.01% 0.00% 0.04% 0.00% 99.67% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% 0.00% 99.67% 0.02% 99.65% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.31% 0.31% 0.31% 0.31% 0.31% 0.31% 0.31% 0.31% 0.30% 0.30% 0.30% 0.31% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.00% 0.01% 0.01% 99.75% 0.00% 0.01% 0.01% 0.01% 0.02% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 99.76% 0.00% 99.74% 0.01% 0.00% 99.76% 0.01% 99.76% 0.01% 99.71% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.01% 99.76% 0.01% 99.75% 0.01% 0.01% 99.76% 0.01% 0.00% 0.02% 0.01% 99.76% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.75% 0.02% 99.73% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.01% 0.01% 0.01% 99.96% 0.01% 0.00% 0.01% 0.01% 0.02% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 99.98% 0.01% 99.96% 0.01% 0.01% 99.97% 0.01% 99.97% 0.02% 99.93% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.01% 99.97% 0.01% 99.97% 0.02% 0.01% 99.98% 0.01% 0.00% 0.02% 0.01% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 99.96% 0.01% 99.95% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide C C C T C C C T C C C T G1333 A 0.00% 0.02% 0.01% 0.01% 0.01% 0.01% 0.04% 0.01% 0.01% 0.01% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.02% 99.95% 0.02% 0.02% 0.01% 99.97% 0.02% 0.01% 0.00% 99.92% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 99.97% 99.96% 99.95% 0.02% 99.97% 99.96% 99.93% 0.01% 99.95% 99.95% 99.94% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.02% 0.01% 0.02% 0.02% 0.03% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.03% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.00% 0.01% 99.67% 0.02% 0.02% 0.01% 99.66% 0.02% 0.01% 0.01% 99.62% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 99.67% 99.67% 99.65% 0.01% 99.65% 99.66% 99.67% 0.02% 99.65% 99.65% 99.62% 0.03% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.31% 0.31% 0.32% 0.31% 0.31% 0.31% 0.31% 0.31% 0.33% 0.33% 0.33% 0.33% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.03% 0.00% 0.01% 0.03% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.00% 0.01% 0.00% 99.73% 0.02% 0.02% 0.01% 99.75% 0.02% 0.01% 0.00% 99.71% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 99.76% 99.76% 99.74% 0.02% 99.74% 99.74% 99.73% 0.01% 99.73% 99.72% 99.73% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.23% 0.23% 0.24% 0.23% 0.23% 0.23% 0.23% 0.23% 0.24% 0.24% 0.24% 0.24% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.02% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 99.95% 0.01% 0.05% 0.01% 99.96% 0.01% 0.01% 0.01% 99.89% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 99.97% 99.96% 99.96% 0.03% 99.97% 99.93% 99.96% 0.02% 99.95% 99.94% 99.93% 0.05% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.03% 0.03% 0.04% 0.04% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G G C C C A G G T G A A G1333 A 0.02% 0.02% 0.00% 0.01% 0.05% 99.91% 0.00% 0.01% 0.04% 0.01% 99.90% 99.87% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 99.60% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 99.96% 99.97% 99.91% 0.03% 0.00% 0.00% 0.06% 0.00% 0.03% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.96% 99.96% 0.01% 0.00% 0.01% 0.05% 99.97% 99.97% 0.29% 99.97% 0.05% 0.09% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 0.01% 0.01% 0.00% 0.02% 99.86% 0.00% 0.01% 0.02% 0.01% 99.90% 99.84% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.02% 0.04% 0.02% 0.01% 0.01% 0.02% 0.01% 99.58% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.00% 99.63% 99.94% 99.93% 0.04% 0.00% 0.01% 0.09% 0.00% 0.04% 0.03% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.66% 99.66% 0.01% 0.00% 0.00% 0.06% 99.95% 99.95% 0.28% 99.95% 0.03% 0.09% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.31% 0.31% 0.31% 0.03% 0.03% 0.03% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.01% 0.00% 0.00% 0.01% 99.67% 0.00% 0.01% 0.04% 0.01% 99.69% 99.64% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.02% 0.01% 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 99.38% 0.02% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.00% 99.74% 99.76% 99.74% 0.04% 0.00% 0.00% 0.07% 0.00% 0.04% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.74% 99.75% 0.01% 0.00% 0.00% 0.05% 99.76% 99.76% 0.29% 99.75% 0.04% 0.09% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.02% 0.01% 0.01% 0.03% 0.01% 99.89% 0.01% 0.00% 0.03% 0.01% 99.92% 99.86% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.02% 0.02% 0.02% 0.03% 0.01% 0.02% 0.01% 99.59% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.01% 0.00% 99.96% 99.94% 99.94% 0.03% 0.01% 0.00% 0.08% 0.01% 0.04% 0.04% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.96% 99.96% 0.01% 0.01% 0.00% 0.06% 99.96% 99.97% 0.29% 99.96% 0.03% 0.09% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide C G T G T G G T T C C A G1333 A 0.00% 0.01% 0.04% 0.00% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 99.94% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 99.57% 0.02% 99.89% 0.02% 0.01% 99.94% 99.96% 0.01% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 0.06% 0.00% 0.03% 0.00% 0.00% 0.03% 0.01% 99.97% 99.95% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.98% 99.97% 0.33% 99.97% 0.06% 99.96% 99.97% 0.02% 0.00% 0.00% 0.01% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.00% 0.00% 0.02% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.01% 99.94% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.02% 0.02% 99.49% 0.02% 99.87% 0.02% 0.02% 99.91% 99.94% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 0.11% 0.00% 0.03% 0.01% 0.00% 0.04% 0.03% 99.98% 99.96% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.95% 99.96% 0.36% 99.96% 0.08% 99.95% 99.96% 0.02% 0.00% 0.00% 0.01% 0.04% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.02% 0.02% 0.02% 0.02% 0.02% 0.01% 0.01% 0.01% 0.02% 0.01% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.04% 0.01% 0.02% 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 99.95% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 99.37% 0.02% 99.65% 0.02% 0.01% 99.94% 99.97% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.00% 0.07% 0.00% 0.03% 0.00% 0.00% 0.03% 0.02% 99.98% 99.98% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.75% 99.76% 0.29% 99.75% 0.07% 99.75% 99.99% 0.02% 0.00% 0.00% 0.00% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.23% 0.23% 0.23% 0.23% 0.23% 0.23% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 0.04% 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% 0.00% 0.01% 99.95% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.02% 99.59% 0.03% 99.89% 0.02% 0.01% 99.95% 99.96% 0.02% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.01% 0.00% 0.07% 0.01% 0.03% 0.00% 0.01% 0.02% 0.02% 99.97% 99.97% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.97% 99.98% 0.30% 99.96% 0.07% 99.98% 99.98% 0.02% 0.00% 0.01% 0.00% 0.03% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G A A C C G G A G G A C G1333 A 0.01% 99.97% 99.96% 0.01% 0.00% 0.01% 0.01% 99.92% 0.00% 0.01% 99.90% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.01% 0.02% 99.98% 99.97% 0.00% 0.00% 0.01% 0.00% 0.00% 0.06% 99.99% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.97% 0.01% 0.01% 0.00% 0.01% 99.97% 99.98% 0.06% 99.98% 99.97% 0.03% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.01% 99.97% 99.96% 0.01% 0.00% 0.01% 0.01% 99.88% 0.00% 0.01% 99.88% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.01% 0.01% 0.02% 0.00% 0.01% 0.01% 0.02% 0.01% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.01% 0.01% 0.02% 99.97% 99.96% 0.01% 0.00% 0.03% 0.00% 0.00% 0.07% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.97% 0.01% 0.01% 0.00% 0.01% 99.97% 99.97% 0.07% 99.97% 99.97% 0.03% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.01% 99.97% 99.96% 0.00% 0.00% 0.01% 0.01% 99.92% 0.01% 0.01% 99.90% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.01% 0.02% 99.99% 99.97% 0.00% 0.00% 0.02% 0.00% 0.00% 0.06% 99.99% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.98% 0.01% 0.01% 0.00% 0.00% 99.97% 99.97% 0.04% 99.98% 99.98% 0.03% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 99.97% 99.95% 0.01% 0.00% 0.02% 0.01% 99.91% 0.01% 0.00% 99.92% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.01% 0.03% 99.98% 99.97% 0.00% 0.00% 0.04% 0.00% 0.00% 0.05% 99.98% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.98% 0.01% 0.01% 0.00% 0.01% 99.96% 99.98% 0.05% 99.99% 99.98% 0.02% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide A A A G T A C A A A C G G1333 A 99.95% 99.96% 99.91% 0.00% 0.11% 99.96% 0.02% 99.97% 99.98% 99.96% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.00% 0.01% 99.77% 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.02% 0.02% 0.04% 0.00% 0.05% 0.01% 99.97% 0.02% 0.01% 0.02% 99.98% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 0.02% 0.01% 0.04% 99.99% 0.06% 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 99.97% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 99.95% 99.95% 99.90% 0.01% 0.13% 99.96% 0.03% 99.97% 99.97% 99.96% 0.01% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.00% 0.01% 99.75% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.02% 0.03% 0.04% 0.00% 0.06% 0.01% 99.96% 0.01% 0.01% 0.02% 99.98% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 0.02% 0.01% 0.05% 99.98% 0.06% 0.02% 0.00% 0.01% 0.02% 0.01% 0.01% 99.97% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 99.95% 99.95% 99.91% 0.00% 0.09% 99.97% 0.02% 99.96% 99.97% 99.97% 0.02% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 0.01% 99.79% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.02% 0.02% 0.04% 0.00% 0.05% 0.01% 99.97% 0.02% 0.02% 0.02% 99.96% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 0.02% 0.01% 0.04% 99.99% 0.06% 0.02% 0.00% 0.02% 0.01% 0.01% 0.00% 99.96% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 99.94% 99.95% 99.90% 0.01% 0.11% 99.96% 0.02% 99.97% 99.97% 99.95% 0.01% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 0.01% 99.76% 0.01% 0.01% 0.00% 0.00% 0.01% 0.02% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.03% 0.03% 0.05% 0.00% 0.07% 0.02% 99.96% 0.02% 0.02% 0.03% 99.96% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 0.03% 0.01% 0.04% 99.99% 0.07% 0.01% 0.00% 0.01% 0.01% 0.02% 0.01% 99.96% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G C A G A A G C T G G A G1333 A 0.02% 0.00% 99.91% 0.01% 99.95% 99.86% 0.00% 0.00% 0.00% 0.00% 0.01% 99.86% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% 0.02% 99.95% 0.01% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 99.97% 0.02% 0.00% 0.02% 0.04% 0.01% 99.97% 0.02% 0.00% 0.00% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.97% 0.01% 0.06% 99.98% 0.02% 0.09% 99.99% 0.01% 0.02% 99.98% 99.98% 0.10% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.02% 0.02% 99.91% 0.01% 99.95% 99.86% 0.00% 0.25% 0.00% 0.01% 0.01% 99.86% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.00% 99.97% 0.02% 0.00% 0.02% 0.03% 0.00% 99.74% 0.02% 0.00% 0.00% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.97% 0.01% 0.07% 99.98% 0.02% 0.11% 99.98% 0.01% 0.03% 99.98% 99.98% 0.11% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.03% 0.01% 99.93% 0.00% 99.95% 99.88% 0.00% 0.00% 0.00% 0.00% 0.01% 99.85% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 0.00% 0.00% 99.96% 0.01% 0.02% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 99.96% 0.01% 0.00% 0.02% 0.04% 0.00% 99.98% 0.02% 0.00% 0.00% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.96% 0.01% 0.03% 99.99% 0.01% 0.08% 99.99% 0.01% 0.02% 99.98% 99.97% 0.11% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.02% 0.01% 99.92% 0.00% 99.95% 99.87% 0.00% 0.01% 0.01% 0.00% 0.01% 99.84% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 99.96% 0.02% 0.00% 0.03% 0.03% 0.00% 99.98% 0.02% 0.00% 0.01% 0.05% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.96% 0.02% 0.04% 99.99% 0.02% 0.09% 99.98% 0.01% 0.02% 99.98% 99.97% 0.10% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Nucle- Batch otide G G A G G A A G G G C C G1333 A 0.00% 0.01% 99.87% 0.00% 0.01% 99.91% 99.90% 0.01% 0.01% 0.00% 0.01% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 T 0.02% 0.01% 0.02% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% 0.00% 0.03% 0.08% DddAtox-N- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 0.03% 0.00% 0.00% 0.02% 0.02% 0.00% 0.00% 0.00% 99.92% 99.90% DddAtox-N- dSpCas9 with spG4 gRNA G1333 G 99.98% 99.97% 0.08% 99.98% 99.96% 0.05% 0.07% 99.98% 99.98% 99.99% 0.03% 0.01% DddAtox-N- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1333 A 0.00% 0.00% 99.86% 0.00% 0.01% 99.93% 99.89% 0.01% 0.01% 0.01% 0.02% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1333 T 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.03% 0.08% DddAtox-C- dSpCas9 with spG4 gRNA G1333 C 0.00% 0.00% 0.04% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.01% 99.91% 99.88% DddAtox-C- dSpCas9 with spG4 gRNA G1333 G 99.99% 99.98% 0.10% 99.98% 99.98% 0.05% 0.09% 99.98% 99.98% 99.97% 0.04% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1333 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1333 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DddAtox-C- dSpCas9 with spG4 gRNA G1397 A 0.00% 0.00% 99.88% 0.01% 0.01% 99.91% 99.91% 0.01% 0.01% 0.00% 0.02% 0.03% DddAtox-N- dSpCas9 with spG4 gRNA G1397 T 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.04% 0.06% DddAtox-N- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.00% 0.04% 0.00% 0.00% 0.03% 0.03% 0.00% 0.00% 0.00% 99.90% 99.89% DddAtox-N- dSpCas9 with spG4 gRNA G1397 G 99.97% 99.98% 0.06% 99.97% 99.98% 0.05% 0.05% 99.97% 99.98% 99.98% 0.03% 0.02% DddAtox-N- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-N- dSpCas9 with spG4 gRNA G1397 A 0.01% 0.00% 99.84% 0.01% 0.01% 99.94% 99.91% 0.01% 0.01% 0.01% 0.02% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 T 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.07% DddAtox-C- dSpCas9 with spG4 gRNA G1397 C 0.00% 0.00% 0.05% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 99.92% 99.88% DddAtox-C- dSpCas9 with spG4 gRNA G1397 G 99.97% 99.98% 0.09% 99.97% 99.97% 0.03% 0.07% 99.97% 99.98% 99.98% 0.04% 0.02% DddAtox-C- dSpCas9 with spG4 gRNA G1397 N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA G1397 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DddAtox-C- dSpCas9 with spG4 gRNA Batch Nucleotide T G A G T C C G G1333 DddAtox-N- A 0.00% 0.01% 99.87% 0.01% 0.01% 0.01% 0.01% 0.01% dSpCas9 with spG4 gRNA G1333 DddAtox-N- T 99.91% 0.01% 0.00% 0.00% 99.84% 0.03% 0.04% 0.01% dSpCas9 with spG4 gRNA G1333 DddAtox-N- C 0.05% 0.00% 0.03% 0.00% 0.05% 99.96% 99.86% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- G 0.04% 99.97% 0.08% 99.97% 0.05% 0.00% 0.01% 99.93% dSpCas9 with spG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- 0.00% 0.00% 0.00% 0.01% 0.05% 0.00% 0.08% 0.05% dSpCas9 with spG4 gRNA G1333 DddAtox-C- A 0.01% 0.03% 99.89% 0.01% 0.01% 0.03% 0.01% 0.02% dSpCas9 with spG4 gRNA G1333 DddAtox-C- T 99.90% 0.01% 0.01% 0.02% 99.81% 0.03% 0.05% 0.01% dSpCas9 with spG4 gRNA G1333 DddAtox-C- C 0.04% 0.00% 0.02% 0.00% 0.09% 99.93% 99.82% 0.01% dSpCas9 with spG4 gRNA G1333 DddAtox-C- G 0.04% 99.96% 0.07% 99.96% 0.06% 0.01% 0.02% 99.90% dSpCas9 with spG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-C- 0.00% 0.00% 0.00% 0.02% 0.03% 0.00% 0.10% 0.07% dSpCas9 with spG4 gRNA G1397 DddAtox-N- A 0.01% 0.01% 99.86% 0.00% 0.02% 0.01% 0.01% 0.01% dSpCas9 with spG4 gRNA G1397 DddAtox-N- T 99.91% 0.02% 0.01% 0.01% 99.81% 0.05% 0.05% 0.01% dSpCas9 with spG4 gRNA G1397 DddAtox-N- C 0.04% 0.00% 0.03% 0.00% 0.07% 99.93% 99.86% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- G 0.04% 99.96% 0.09% 99.95% 0.05% 0.01% 0.01% 99.89% dSpCas9 with spG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- 0.00% 0.00% 0.01% 0.03% 0.05% 0.00% 0.08% 0.08% dSpCas9 with spG4 gRNA G1397 DddAtox-C- A 0.01% 0.01% 99.86% 0.01% 0.03% 0.01% 0.00% 0.01% dSpCas9 with spG4 gRNA G1397 DddAtox-C- T 99.89% 0.02% 0.01% 0.01% 99.82% 0.04% 0.05% 0.01% dSpCas9 with spG4 gRNA G1397 DddAtox-C- C 0.05% 0.00% 0.04% 0.00% 0.07% 99.93% 99.85% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- G 0.04% 99.97% 0.09% 99.95% 0.05% 0.01% 0.01% 99.89% dSpCas9 with spG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- 0.01% 0.00% 0.01% 0.03% 0.05% 0.00% 0.09% 0.08% dSpCas9 with spG4 gRNA Batch Nucleotide A G C A G A A G G1333 DddAtox-N- A 99.88% 0.01% 0.03% 1.92% 0.07% 1.81% 0.03% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- T 0.01% 0.01% 0.03% 0.01% 0.01% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- C 0.02% 0.00% 99.90% 0.04% 0.00% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- G 0.07% 99.94% 0.02% 0.09% 1.97% 0.04% 0.00% 0.03% dSpCas9 with spG4 gRNA G1333 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-N- 0.02% 0.04% 0.03% 97.94% 97.96% 98.13% 99.97% 99.97% dSpCas9 with spG4 gRNA G1333 DddAtox-C- A 99.86% 0.01% 0.02% 2.35 0.09% 2.23 0.34 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-C- T 0.01% 0.00% 0.05% 0.01% 0.00% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-C- C 0.02% 0.00% 99.89% 0.06% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-C- G 0.08% 99.96% 0.02% 0.11% 2.39% 0.04% 0.00% 0.34% dSpCas9 with spG4 gRNA G1333 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1333 DddAtox-C- 0.03% 0.03% 0.02% 97.48% 97.52% 97.72% 99.66% 99.66% dSpCas9 with spG4 gRNA G1397 DddAtox-N- A 99.87% 0.01% 0.02% 2.42% 0.10% 2.29% 0.25% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- T 0.01% 0.00% 0.04% 0.01% 0.00% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- C 0.02% 0.01% 99.90% 0.06% 0.00% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- G 0.08% 99.94% 0.02% 0.09% 2.46% 0.06% 0.00% 0.25% dSpCas9 with spG4 gRNA G1397 DddAtox-N- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-N- 0.03% 0.04% 0.02% 97.42% 97.44% 97.64% 99.75% 99.75% dSpCas9 with spG4 gRNA G1397 DddAtox-C- A 99.86% 0.01% 0.03% 2.19% 0.10% 2.05% 0.04% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- T 0.01% 0.01% 0.04% 0.01% 0.01% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- C 0.03% 0.01% 99.88% 0.04% 0.00% 0.01% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- G 0.06% 99.93% 0.03% 0.10% 2.20% 0.04% 0.00% 0.04% dSpCas9 with spG4 gRNA G1397 DddAtox-C- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% dSpCas9 with spG4 gRNA G1397 DddAtox-C- 0.03% 0.04% 0.02% 97.66% 97.69% 97.90% 99.95% 99.96% dSpCas9 with spG4 gRNA
TABLE-US-00065 TABLE 10C No gRNA Nucle- Batch otide G G C C C C T A A C C C nogRNA- A 0.23% 0.03% 0.01% 0.00% 0.00% 0.02% 0.00% 99.89% 99.60% 0.00% 0.00% 0.01% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- T 0.25% 0.04% 0.01% 0.01% 0.01% 0.00% 99.89% 0.02% 0.01% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- C 0.18% 0.01% 99.17% 99.97% 99.98% 99.97% 0.06% 0.03% 0.00% 100.00% 99.98% 99.72% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- G 99.34% 99.92% 0.80% 0.01% 0.00% 0.00% 0.00% 0.05% 0.04% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.05% 0.00% 0.34% 0.00% 0.01% 0.27% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- A 0.25% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 99.92% 99.56% 0.01% 0.00% 0.01% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- T 0.27% 0.03% 0.00% 0.01% 0.01% 0.00% 99.93% 0.00% 0.00% 0.01% 0.02% 0.00% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- C 0.15% 0.03% 99.10% 99.98% 99.98% 99.99% 0.01% 0.05% 0.03% 99.96% 99.97% 99.69% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- G 99.33% 99.90% 0.89% 0.00% 0.00% 0.00% 0.00% 0.04% 0.02% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.05% 0.00% 0.39% 0.01% 0.00% 0.29% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- A 0.21% 0.05% 0.02% 0.00% 0.01% 0.01% 0.00% 99.94% 99.59% 0.00% 0.00% 0.01% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- T 0.25% 0.01% 0.01% 0.01% 0.01% 0.00% 99.90% 0.01% 0.01% 0.01% 0.01% 0.01% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- C 0.14% 0.02% 99.18% 99.98% 99.96% 99.97% 0.04% 0.02% 0.02% 99.98% 99.97% 99.74% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- G 99.40% 99.91% 0.79% 0.01% 0.00% 0.00% 0.00% 0.02% 0.02% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 0.05% 0.01% 0.37% 0.01% 0.01% 0.24% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- A 0.24% 0.03% 0.00% 0.00% 0.00% 0.02% 0.00% 99.90% 99.57% 0.01% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- T 0.27% 0.03% 0.01% 0.00% 0.00% 0.02% 99.93% 0.00% 0.01% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- C 0.14% 0.01% 99.11% 99.97% 99.99% 99.96% 0.02% 0.03% 0.01% 99.98% 99.99% 99.73% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- G 99.35% 99.92% 0.87% 0.03% 0.00% 0.01% 0.00% 0.06% 0.03% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.04% 0.01% 0.38% 0.00% 0.01% 0.27% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- A 0.27% 0.03% 0.01% 0.00% 0.01% 0.00% 0.00% 99.89% 99.56% 0.01% 0.01% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- T 0.28% 0.01% 0.00% 0.01% 0.01% 0.01% 99.93% 0.00% 0.00% 0.00% 0.01% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- C 0.19% 0.02% 99.00% 99.97% 99.97% 99.96% 0.02% 0.03% 0.01% 99.97% 99.96% 99.65% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- G 99.25% 99.93% 0.98% 0.01% 0.00% 0.01% 0.01% 0.07% 0.03% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- 0.01% 0.00% 0.00% 0.00% 0.01% 0.02% 0.05% 0.00% 0.40% 0.01% 0.02% 0.34% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- A 0.20% 0.03% 0.02% 0.00% 0.00% 0.00% 0.01% 99.93% 99.52% 0.00% 0.01% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- T 0.35% 0.02% 0.00% 0.01% 0.00% 0.01% 99.90% 0.01% 0.00% 0.00% 0.02% 0.01% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- C 0.19% 0.03% 99.03% 99.96% 99.95% 99.95% 0.05% 0.03% 0.03% 99.98% 99.95% 99.72% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- G 99.26% 99.92% 0.95% 0.02% 0.01% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.04% 0.04% 0.04% 0.01% 0.44% 0.02% 0.02% 0.26% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- A 0.18% 0.05% 0.02% 0.01% 0.01% 0.01% 0.00% 99.93% 99.62% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- T 0.24% 0.03% 0.01% 0.02% 0.00% 0.00% 99.95% 0.01% 0.00% 0.01% 0.02% 0.02% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- C 0.16% 0.02% 99.25% 99.96% 99.98% 99.97% 0.02% 0.01% 0.01% 99.97% 99.98% 99.66% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- G 99.42% 99.90% 0.71% 0.01% 0.00% 0.00% 0.00% 0.04% 0.01% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% 0.01% 0.36% 0.02% 0.00% 0.33% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- A 0.20% 0.06% 0.00% 0.00% 0.01% 0.00% 0.00% 99.92% 99.61% 0.01% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- T 0.28% 0.03% 0.02% 0.02% 0.01% 0.00% 99.93% 0.00% 0.01% 0.01% 0.00% 0.01% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- C 0.16% 0.02% 99.09% 99.96% 99.97% 99.98% 0.02% 0.02% 0.01% 99.96% 99.98% 99.70% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- G 99.35% 99.89% 0.89% 0.01% 0.00% 0.00% 0.02% 0.05% 0.02% 0.00% 0.01% 0.00% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% 0.00% 0.35% 0.01% 0.01% 0.29% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- A 0.22% 0.06% 0.01% 0.01% 0.00% 0.00% 0.00% 99.88% 99.58% 0.01% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- T 0.26% 0.01% 0.01% 0.00% 0.00% 0.00% 99.92% 0.01% 0.01% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- C 0.19% 0.03% 99.10% 99.97% 99.98% 99.98% 0.03% 0.05% 0.01% 99.98% 99.98% 99.74% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- G 99.31% 99.90% 0.87% 0.02% 0.00% 0.00% 0.00% 0.06% 0.02% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 0.05% 0.01% 0.38% 0.01% 0.01% 0.25% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- A 0.27% 0.04% 0.01% 0.00% 0.01% 0.01% 0.01% 99.92% 99.56% 0.00% 0.00% 0.01% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- T 0.29% 0.02% 0.01% 0.01% 0.01% 0.00% 99.92% 0.01% 0.01% 0.01% 0.02% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- C 0.15% 0.03% 98.97% 99.96% 99.98% 99.98% 0.02% 0.02% 0.01% 99.96% 99.97% 99.76% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- G 99.29% 99.90% 1.01% 0.03% 0.00% 0.00% 0.00% 0.03% 0.03% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.05% 0.02% 0.39% 0.02% 0.01% 0.23% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- A 0.26% 0.04% 0.01% 0.00% 0.00% 0.01% 0.00% 99.93% 99.59% 0.01% 0.00% 0.01% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- T 0.32% 0.02% 0.01% 0.01% 0.02% 0.01% 99.94% 0.00% 0.00% 0.01% 0.01% 0.00% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- C 0.17% 0.01% 99.09% 99.97% 99.97% 99.97% 0.01% 0.03% 0.00% 99.97% 99.99% 99.73% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- G 99.25% 99.92% 0.89% 0.01% 0.00% 0.00% 0.00% 0.02% 0.02% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.05% 0.01% 0.38% 0.01% 0.00% 0.27% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- A 0.22% 0.03% 0.03% 0.01% 0.00% 0.00% 0.01% 99.85% 99.57% 0.00% 0.01% 0.00% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- T 0.23% 0.02% 0.01% 0.01% 0.00% 0.00% 99.94% 0.01% 0.01% 0.00% 0.01% 0.01% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- C 0.19% 0.03% 99.18% 99.96% 99.97% 99.98% 0.02% 0.05% 0.03% 99.99% 99.98% 99.76% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- G 99.37% 99.93% 0.78% 0.02% 0.01% 0.00% 0.00% 0.08 0.03% 0.00% 0.00% 0.01% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.02% 0.02% 0.03% 0.02% 0.36% 0.01% 0.01% 0.23% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- A 0.30% 0.04% 0.02% 0.00% 0.01% 0.01% 0.00% 99.90% 99.52% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- T 0.28% 0.03% 0.01% 0.01% 0.01% 0.00% 99.93% 0.00% 0.00% 0.01% 0.00% 0.02% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- C 0.13% 0.02% 99.06% 99.96% 99.96% 99.96% 0.02% 0.04% 0.01% 99.98% 99.98% 99.64% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- G 99.28% 99.91% 0.91% 0.02% 0.00% 0.00% 0.01% 0.04% 0.03% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- 0.00% 0.00% 0.00% 0.00% 0.01% 0.03% 0.04% 0.01% 0.44% 0.01% 0.01% 0.33% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- A 0.26% 0.04% 0.01% 0.01% 0.01% 0.01% 0.00% 99.90% 99.56% 0.01% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- T 0.28% 0.02% 0.01% 0.02% 0.01% 0.02% 99.94% 0.00% 0.01% 0.00% 0.01% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- C 0.20% 0.03% 99.09% 99.96% 99.96% 99.94% 0.02% 0.04% 0.01% 99.99% 99.99% 99.80% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- G 99.25% 99.90% 0.89% 0.02% 0.00% 0.00% 0.00% 0.05% 0.01% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- 0.00% 0.00% 0.00% 0.00% 0.02% 0.03% 0.03% 0.00% 0.40% 0.00% 0.00% 0.20% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N Nucle- Batch otide T A T G T A G C C T C A nogRNA- A 0.01% 99.89% 0.00% 0.04% 0.00% 99.94% 0.03% 0.01% 0.00% 0.00% 0.00% 99.96% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- T 99.94% 0.00% 99.98% 0.02% 99.98% 0.00% 0.03% 0.02% 0.01% 99.99% 0.02% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- C 0.00% 0.01% 0.01% 0.00% 0.01% 0.03% 0.00% 99.97% 99.99% 0.00% 99.96% 0.03% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- G 0.00% 0.05% 0.00% 99.93% 0.00% 0.02% 99.93% 0.00% 0.00% 0.00% 0.01% 0.01% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- 0.05% 0.05% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-N and dSpCas9-A1343-C nogRNA- A 0.00% 99.86% 0.02% 0.04% 0.01% 99.91% 0.02% 0.01% 0.01% 0.00% 0.02% 99.91% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- T 99.97% 0.02% 99.96% 0.03% 99.97% 0.00% 0.09% 0.03% 0.00% 99.96% 0.02% 0.01% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- C 0.01% 0.03% 0.02% 0.01% 0.01% 0.04% 0.00% 99.96% 99.98% 0.02% 99.96% 0.04% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- G 0.01% 0.05% 0.00% 99.93% 0.00% 0.05% 99.89 0.00% 0.00% 0.02% 0.00% 0.04% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- 0.01% 0.05% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% A1343spilt-KKH- Cas9-A1343-C and dSpCas9-A1343-N nogRNA- A 0.00% 99.89% 0.00% 0.07% 0.01% 99.94% 0.02% 0.00% 0.01% 0.01% 0.00% 99.96% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- T 99.96% 0.01% 99.97% 0.03% 99.98% 0.00% 0.06% 0.00% 0.02% 99.98% 0.01% 0.00% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- C 0.01% 0.01% 0.02% 0.00% 0.01% 0.01% 0.00% 100.00% 99.97% 0.01% 99.99% 0.02% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- G 0.00% 0.03% 0.00% 99.89% 0.00% 0.04% 99.92% 0.00% 0.00% 0.00% 0.00% 0.02% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- 0.03% 0.05% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-N and dSpCas9-G1322-C nogRNA- A 0.01% 99.88% 0.00% 0.08% 0.01% 99.93% 0.02% 0.00% 0.00% 0.00% 0.00% 99.95% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- T 99.94% 0.02% 99.97% 0.03% 99.96% 0.03% 0.07% 0.00% 0.01% 99.97% 0.03% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- C 0.00% 0.03% 0.02% 0.01% 0.02% 0.02% 0.00% 99.99% 99.99% 0.01% 99.96% 0.03% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- G 0.01% 0.05% 0.00% 99.88% 0.00% 0.02% 99.91% 0.00% 0.00% 0.01% 0.00% 0.02% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- 0.04% 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1322spilt-KKH- Cas9-G1322-C and dSpCas9-G1322-N nogRNA- A 0.00% 99.88% 0.01% 0.07% 0.02% 99.94% 0.01 0.01% 0.00% 0.01% 0.00% 99.95% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- T 99.92% 0.02% 99.97% 0.03% 99.97% 0.00% 0.05% 0.00% 0.00% 99.98% 0.01% 0.01% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- C 0.01% 0.01% 0.00% 0.00% 0.01% 0.02% 0.00% 99.99% 99.99% 0.01% 99.99% 0.02% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- G 0.01% 0.05% 0.00% 99.90% 0.00% 0.04% 99.94% 0.00% 0.00% 0.00% 0.00% 0.02% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- 0.06% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-N and dSpCas9-G1333-C nogRNA- A 0.00% 99.87% 0.00% 0.05% 0.01% 99.96% 0.00% 0.00% 0.00% 0.00% 0.00% 99.96% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- T 99.95% 0.01% 99.97% 0.04% 99.97% 0.01% 0.06% 0.01% 0.02% 100.00% 0.01% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- C 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% 99.99% 99.98% 0.00% 99.98% 0.02% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- G 0.00% 0.05% 0.00% 99.90 0.00% 0.02% 99.94% 0.00% 0.00% 0.00% 0.00% 0.02% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- 0.05% 0.06% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1333spilt-KKH- Cas9-G1333-C and dSpCas9-G1333-N nogRNA- A 0.01% 99.89% 0.00% 0.05% 0.01% 99.95% 0.02% 0.01% 0.01% 0.01% 0.01% 99.96% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- T 99.93% 0.02% 99.96% 0.03% 99.95% 0.00% 0.04% 0.01% 0.01% 99.98% 0.02% 0.01% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- C 0.02% 0.00% 0.01% 0.00% 0.03% 0.01% 0.00% 99.98% 99.98% 0.01% 99.97% 0.01% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- G 0.00% 0.06% 0.00% 99.92% 0.00% 0.03% 99.94% 0.00% 0.00% 0.00% 0.00% 0.02% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- 0.05% 0.03% 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-N and dSpCas9-G1371-C nogRNA- A 0.00% 99.87% 0.00% 0.05% 0.01% 99.95% 0.01% 0.00% 0.00% 0.01% 0.00% 99.93% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- T 99.96% 0.03% 99.96% 0.05% 99.96% 0.01% 0.06% 0.00% 0.01% 99.97% 0.03% 0.01% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- C 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.00% 100.00% 99.99% 0.01% 99.96% 0.03% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- G 0.01% 0.06% 0.01% 99.89% 0.00% 0.03% 99.92% 0.00% 0.00% 0.01% 0.00% 0.02% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- 0.02% 0.03% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1371spilt-KKH- Cas9-G1371-C and dSpCas9-G1371-N nogRNA- A 0.01% 99.86% 0.00% 0.07% 0.01% 99.94% 0.01% 0.00% 0.00% 0.01% 0.00% 99.93% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- T 99.96% 0.01% 99.96% 0.01% 99.97% 0.01% 0.05% 0.00% 0.02% 99.99% 0.01% 0.01% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- C 0.01% 0.02% 0.01% 0.01% 0.02% 0.03% 0.00% 99.99% 99.98% 0.00% 99.99% 0.03% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- G 0.00% 0.04% 0.00% 99.91% 0.00% 0.02% 99.95% 0.00% 0.00% 0.00% 0.00% 0.03% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- 0.02% 0.07% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-N and dSpCas9-G1397-C nogRNA- A 0.00% 99.87% 0.00% 0.05% 0.02% 99.95% 0.01% 0.00% 0.01% 0.01% 0.00% 99.96% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- T 99.92% 0.01% 99.96% 0.02% 99.95% 0.01% 0.05% 0.01% 0.01% 99.97% 0.01% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- C 0.02% 0.01% 0.02% 0.01% 0.02% 0.01% 0.00% 99.98% 99.98% 0.01% 99.99% 0.01% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- G 0.01% 0.05% 0.01% 99.92% 0.00% 0.02% 99.94% 0.00% 0.00% 0.01% 0.00% 0.02% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- 0.06% 0.06% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% G1397spilt-KKH- Cas9-G1397-C and dSpCas9-G1397-N nogRNA- A 0.00% 99.89% 0.01% 0.07% 0.03% 99.95% 0.02% 0.00% 0.02% 0.00% 0.00% 99.95% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- T 99.97% 0.01% 99.97% 0.02% 99.97% 0.01% 0.06% 0.02% 0.01% 99.97% 0.02% 0.02% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- C 0.02% 0.01% 0.01% 0.00% 0.00% 0.02% 0.00% 99.98% 99.97% 0.03% 99.98% 0.02% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- G 0.00% 0.05% 0.00% 99.92% 0.00% 0.02% 99.92% 0.00% 0.00% 0.00% 0.00% 0.02% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- 0.01% 0.04% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-N and dSpCas9-N1357-C nogRNA- A 0.01% 99.85% 0.01% 0.05% 0.01% 99.92% 0.01% 0.00% 0.01% 0.00% 0.01% 99.93% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- T 99.94% 0.00% 99.95% 0.03% 99.98% 0.02% 0.04% 0.01% 0.00% 99.98% 0.01% 0.01% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- C 0.01% 0.04% 0.02% 0.00% 0.01% 0.04% 0.01% 99.99% 99.99% 0.02% 99.97% 0.04% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- G 0.00% 0.04% 0.01% 99.92% 0.00% 0.02% 99.94% 0.00% 0.00% 0.00% 0.01% 0.02% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- 0.04% 0.07% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1357spilt-KKH- Cas9-N1357-C and dSpCas9-N1357-N nogRNA- A 0.01% 99.85% 0.00% 0.04% 0.02% 99.90% 0.01% 0.00% 0.01% 0.00% 0.00% 99.95% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- T 99.94% 0.01% 99.96% 0.02% 99.96% 0.01% 0.07% 0.01% 0.00% 99.96% 0.03% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- C 0.01% 0.01% 0.02% 0.00% 0.01% 0.03% 0.00% 99.99% 99.99% 0.02% 99.97% 0.03% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- G 0.01% 0.06% 0.00% 99.94% 0.00% 0.06% 99.92% 0.00% 0.00% 0.01% 0.00% 0.02% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- 0.02% 0.07% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-N and dSpCas9-N1387-C nogRNA- A 0.00% 99.86% 0.01% 0.04% 0.03% 99.92% 0.01% 0.00% 0.00% 0.00% 0.00% 99.94% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- T 99.94% 0.00% 99.98% 0.02% 99.96% 0.00% 0.09% 0.01% 0.01% 99.97% 0.03% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- C 0.01% 0.02% 0.01% 0.00% 0.01% 0.03% 0.00% 99.99% 99.99% 0.01% 99.96% 0.04% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- G 0.01% 0.06% 0.00% 99.94% 0.00% 0.04% 99.89% 0.00% 0.00% 0.01% 0.00% 0.02% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N nogRNA- 0.04% 0.05% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% N1387spilt-KKH- Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G T C T T C C C A nogRNA-A1343spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 0.01% 99.97% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.00% 99.99% 0.01% 99.98% 99.95% 0.01% 0.00% 0.02% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 99.99% 0.00% 0.02% 99.98% 99.98% 99.98% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.00% 0.00% 0.01% 0.03% 0.00% 0.01% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 99.95% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 99.98% 0.01% 99.99% 99.95% 0.01% 0.02% 0.02% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.01% 99.99% 0.00% 0.02% 99.99% 99.97% 99.98% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.99% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 99.95% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 99.98% 0.00% 99.98% 99.96% 0.01% 0.00% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.01% 100.00% 0.02% 0.02% 99.99% 99.99% 99.97% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.99% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.94% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 99.98% 0.00% 99.97% 99.95% 0.01% 0.01% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.01% 99.99% 0.02% 0.02% 99.99% 99.99% 99.98% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.97% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.04% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 99.97% 0.00% 100.00% 99.96% 0.01% 0.00% 0.02% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.02% 100.00% 0.00% 0.02% 99.99% 99.99% 99.98% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.99% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.95% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.00% 99.96% 0.02% 99.97% 99.97% 0.01% 0.00% 0.02% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.03% 99.98% 0.02% 0.01% 99.99% 99.98% 99.98% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.98% 0.00% 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.04% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 99.96% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 99.98% 0.00% 99.97% 99.95% 0.01% 0.01% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.01% 100.00% 0.02% 0.03% 99.99% 99.98% 99.98% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.97% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% 99.95% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 99.98% 0.01% 99.96% 99.97% 0.01% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.02% 99.98% 0.02% 0.01% 99.99% 99.99% 99.99% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.99% 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 99.97% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.02% 99.97% 0.00% 99.96% 99.98% 0.02% 0.00% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.02% 99.99% 0.03% 0.02% 99.98% 99.98% 99.95% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.98% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 99.98% 0.01% 99.98% 99.95% 0.02% 0.01% 0.00% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.02% 99.99% 0.01% 0.03% 99.97% 99.98% 99.98% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.99% 0.00% 0.00% 0.01% 0.02% 0.00% 0.01% 0.00% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.01% 0.01% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.00% 99.97% 0.00% 99.98% 99.97% 0.01% 0.01% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.02% 100.00% 0.01% 0.02% 99.99% 99.98% 99.98% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.98% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 99.93% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 99.98% 0.01% 99.97% 99.97% 0.01% 0.00% 0.02% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.01% 99.99% 0.02% 0.01% 99.98% 99.99% 99.98% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.98% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.04% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.02% 99.96% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 99.98% 0.01% 99.98% 99.96% 0.00% 0.00% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.01% 99.98% 0.01% 0.01% 100.00% 99.98% 99.97% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 100.00% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.96% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 100.00% 0.00% 99.97% 99.95% 0.01% 0.01% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.00% 100.00% 0.01% 0.02% 99.98% 99.99% 99.99% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.99% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide T C A G G C T C nogRNA-A1343spilt-KKH- A 0.00% 0.00% 99.98% 0.00% 0.03% 0.00% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 99.99% 0.01% 0.00% 0.00% 0.01% 0.01% 100.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 99.99% 0.00% 0.00% 0.00% 99.99% 0.00% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.02% 99.99% 99.96% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.01% 0.01% 99.95% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 99.99% 0.01% 0.02% 0.00% 0.00% 0.00% 99.98% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 99.97% 0.03% 0.00% 0.00% 100.00% 0.02% 99.99% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 99.99% 10.000% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.01% 0.01% 99.94% 0.00% 0.01% 0.00% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 99.98% 0.01% 0.03% 0.01% 0.00% 0.00% 99.99% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 99.98% 0.00% 0.00% 0.00% 100.00% 0.00% 99.99% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.01% 0.00% 0.03% 99.99% 99.99% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 99.97% 0.01% 0.01% 0.02% 0.01% 0.01% 99.99% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.01% 99.99% 0.02% 0.00% 0.00% 99.99% 0.00% 99.99% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.02% 0.00% 0.01% 99.98% 99.98% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.00% 99.95% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 99.97% 0.00% 0.01% 0.00% 0.00% 0.01% 100.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.02% 100.00% 0.00% 0.00% 0.00% 99.98% 0.00% 99.99% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.03% 99.99% 99.99% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 99.95% 0.02% 0.01% 0.00% 0.01% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 99.98% 0.00% 0.02% 0.00% 0.01% 0.00% 99.98% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.01% 99.98% 0.00% 0.00% 0.00% 100.00% 0.01% 100.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.01% 0.00% 0.02% 99.98% 99.97% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.01% 99.97% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 99.98% 0.02% 0.01% 0.01% 0.02% 0.00% 99.98% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.01% 99.97% 0.00% 0.00% 0.00% 99.99% 0.01% 100.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.02% 99.98% 99.98% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.00% 99.94% 0.00% 0.01% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 99.98% 0.01% 0.02% 0.01% 0.01% 0.00% 99.99% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 99.98% 0.01% 0.00% 0.00% 99.99% 0.01% 99.99% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.03% 99.99% 99.98% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.01% 0.00% 99.94% 0.00% 0.01% 0.00% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 99.99% 0.03% 0.02% 0.00% 0.00% 0.00% 99.99% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 99.96% 0.00% 0.00% 0.00% 100.00% 0.00% 99.98% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.03% 100.00% 99.99% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 0.01% 99.95% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 99.97% 0.02% 0.02% 0.00% 0.00% 0.00% 99.97% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.01% 99.97% 0.00% 0.00% 0.00% 100.00% 0.01% 99.99% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.02% 0.00% 0.02% 99.99% 99.99% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 0.01% 99.96% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 99.98% 0.01% 0.01% 0.00% 0.01% 0.01% 99.99% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 99.98% 0.01% 0.00% 0.00% 99.99% 0.00% 99.99% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.01% 0.00% 0.02% 99.99% 99.98% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 0.01% 99.93% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 99.98% 0.02% 0.01% 0.01% 0.00% 0.00% 99.98% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 99.97% 0.03% 0.00% 0.00% 99.99% 0.01% 99.99% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.01% 0.00% 0.03% 99.99% 99.99% 0.01% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.00% 99.95% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 99.98% 0.01% 0.02% 0.00% 0.00% 0.00% 99.98% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.01% 99.98% 0.01% 0.00% 0.00% 99.98% 0.02% 99.99% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.01% 0.00% 0.02% 100.00% 99.99% 0.01% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 99.96% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 99.97% 0.02% 0.01% 0.00% 0.00% 0.00% 99.98% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.02% 99.97% 0.01% 0.00% 0.00% 100.00% 0.01% 100.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.01% 0.00% 0.02% 99.98% 99.99% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide T C A G C T C nogRNA-A1343spilt-KKH- A 0.01% 0.00% 99.97% 0.02% 0.01% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 99.97% 0.03% 0.00% 0.00% 0.01% 99.99% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.02% 99.96% 0.00% 0.00% 99.98% 0.01% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.02% 99.98% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 99.96% 0.01% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 99.97% 0.02% 0.00% 0.00% 0.01% 99.98% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.02% 99.97% 0.01% 0.00% 99.99% 0.01% 99.99% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.03% 99.98% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.01% 99.98% 0.01% 0.00% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 99.98% 0.01% 0.01% 0.00% 0.00% 99.98% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.02% 99.98% 0.00% 0.00% 100.00% 0.01% 99.97% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.01% 99.98% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.00% 99.98% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 99.97% 0.01% 0.00% 0.01% 0.00% 99.98% 0.03% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.03% 99.99% 0.00% 0.01% 100.00% 0.01% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.02% 99.98% 0.00% 0.01% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.02% 100.00% 0.02% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 99.99% 0.01% 0.00% 0.00% 0.00% 100.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 99.97% 0.00% 0.00% 100.00% 0.00% 99.98% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.00% 99.98% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.00% 99.97% 0.01% 0.00% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 99.98% 0.01% 0.01% 0.00% 0.00% 100.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.01% 99.98% 0.00% 0.00% 10.000% 0.00% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.02% 99.99% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.02% 0.02% 99.98% 0.01% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 99.98% 0.01% 0.00% 0.01% 0.01% 99.99% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 99.98% 0.00% 0.01% 99.99% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.00% 0.00% 0.02% 99.98% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.00% 99.93% 0.01% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 99.99% 0.01% 0.01% 0.00% 0.01% 99.98% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.99% 0.01% 99.96% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.06% 99.99% 0.00% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 99.98% 0.02% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 99.99% 0.03% 0.00% 0.00% 0.01% 99.99% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.01% 99.96% 0.00% 0.00% 99.99% 0.00% 99.99% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.02% 99.98% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.00% 99.96% 0.01% 0.00% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 99.98% 0.01% 0.00% 0.02% 0.00% 99.96% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.01% 99.98% 0.00% 0.00% 100.00% 0.01% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.00% 0.01% 0.04% 99.97% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 0.01% 99.98% 0.03% 0.00% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 99.98% 0.02% 0.00% 0.00% 0.00% 99.99% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 99.97% 0.00% 0.00% 99.99% 0.00% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.00% 0.00% 0.02% 99.97% 0.00% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 0.00% 99.93% 0.01% 0.01% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 99.98% 0.02% 0.00% 0.00% 0.02% 99.98% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 99.97% 0.02% 0.00% 99.98% 0.01% 99.98% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.00% 0.01% 0.04% 99.99% 0.00% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.01% 99.99% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 99.98% 0.00% 0.00% 0.00% 0.00% 99.99% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.01% 99.98% 0.00% 0.01% 100.00% 0.00% 99.96% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.00% 99.99% 0.00% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.00% 99.96% 0.01% 0.00% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 99.99% 0.02% 0.00% 0.00% 0.02% 99.99% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 99.98% 0.00% 0.00% 99.98% 0.00% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.04% 99.98% 0.00% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide A G C C T G A G T nogRNA-A1343spilt-KKH- A 99.97% 0.02% 0.01% 0.00% 0.01% 0.00% 99.96% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.01% 0.01% 0.01% 99.97% 0.00% 0.00% 0.00% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.01% 99.98% 99.99% 0.01% 0.01% 0.01% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.02% 99.96% 0.00% 0.00% 0.00% 99.98% 0.02% 99.99% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 99.95% 0.01% 0.00% 0.01% 0.00% 0.01% 99.94% 0.00% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 99.99% 0.02% 0.00% 0.00% 99.96% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.03% 0.00% 100.00% 99.98% 0.01% 0.00% 0.04% 0.00% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.01% 99.99% 0.00% 0.00% 0.00% 99.97% 0.02% 100.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 99.98% 0.01% 0.00% 0.00% 0.01% 0.01% 99.95% 0.00% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.01% 0.00% 0.01% 99.97% 0.01% 0.00% 0.00% 99.95% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.00% 99.99% 99.98% 0.02% 0.00% 0.02% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.02% 99.98% 0.00% 0.00% 0.00% 99.98% 0.03% 99.99% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 99.98% 0.00% 0.00% 0.00% 0.01% 0.03% 99.97% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 99.97% 0.00% 0.01% 0.00% 99.98% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.01% 0.00% 99.99% 99.99% 0.02% 0.00% 0.01% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.01% 99.99% 0.00% 0.00% 0.00% 99.97% 0.01% 99.99% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 99.99% 0.01% 0.01% 0.00% 0.00% 0.03% 99.99% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.00% 0.01% 0.02% 100.00% 0.01% 0.00% 0.00% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.01% 99.98% 99.98% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.01% 99.98% 0.00% 0.00% 0.00% 99.96% 0.01% 100.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 99.97% 0.00% 0.00% 0.01% 0.02% 0.01% 99.95% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.01% 0.00% 0.02% 99.95% 0.01% 0.02% 0.00% 99.96% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.00% 100.00% 99.96% 0.03% 0.00% 0.00% 0.00% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.02% 99.99% 0.00% 0.00% 0.00% 99.98% 0.01% 99.99% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 99.96% 0.02% 0.00% 0.00% 0.00% 0.01% 99.98% 0.00% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.00% 0.00% 0.01% 99.98% 0.01% 0.00% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.01% 0.00% 100.00% 99.98% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.02% 99.98% 0.00% 0.00% 0.01% 99.98% 0.02% 100.00% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 99.99% 0.01% 0.00% 0.00% 0.00% 0.00% 99.96% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.00% 0.01% 0.01% 0.02% 99.96% 0.01% 0.01% 0.02% 99.95% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.00% 99.98% 99.98% 0.03% 0.00% 0.00% 0.00% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 99.98% 0.01% 0.00% 0.00% 99.98% 0.02% 99.97% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 99.98% 0.00% 0.01% 0.01% 0.00% 0.00% 99.83% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 99.83% 0.00% 0.00% 0.00% 99.84% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.00% 99.85% 99.85% 0.02% 0.00% 0.01% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.01% 99.99% 0.01% 0.00% 0.00% 99.86% 0.02% 99.86% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.13% 0.13% 0.14% 0.14% 0.14% 0.14% 0.14% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 99.98% 0.01% 0.00% 0.00% 0.01% 0.01% 99.96% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 0.01% 0.01% 0.02% 99.96% 0.01% 0.00% 0.00% 99.97% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.00% 99.99% 99.98% 0.02% 0.00% 0.01% 0.00% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.02% 99.99% 0.00% 0.00% 0.00% 99.98% 0.02% 99.99% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 99.96% 0.02% 0.00% 0.01% 0.00% 0.01% 99.96% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 99.98% 0.01% 0.00% 0.00% 99.96% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.01% 0.00% 100.00% 99.98% 0.01% 0.00% 0.00% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.02% 99.98% 0.00% 0.00% 0.00% 99.97% 0.03% 99.99% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 99.92% 0.01% 0.00% 0.00% 0.00% 0.01% 99.93% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.01% 0.01% 0.00% 99.99% 0.02% 0.01% 0.01% 99.97% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.05% 0.00% 99.99% 10.000% 0.01% 0.00% 0.04% 0.00% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.03% 99.99% 0.00% 0.00% 0.00% 99.97% 0.02% 99.99% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 99.98% 0.01% 0.00% 0.00% 0.00% 0.01% 99.96% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 99.94% 0.01% 0.00% 0.00% 99.96% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.01% 99.98% 99.99% 0.04% 0.00% 0.00% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.02% 99.97% 0.00% 0.00% 0.00% 99.97% 0.02% 99.98% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 99.98% 0.00% 0.01% 0.00% 0.01% 0.02% 99.97% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.01% 0.01% 99.96% 0.01% 0.00% 0.00% 99.96% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.00% 99.98% 99.99% 0.03% 0.00% 0.01% 0.00% 0.04% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.02% 100.00% 0.00% 0.00% 0.00% 99.97% 0.02% 100.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G T T G A G G C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.00% 0.02% 99.98% 0.03% 0.02% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 99.97% 99.98% 0.01% 0.00% 0.01% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.02% 0.01% 99.97% 0.01% 99.95% 99.97% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 99.97% 0.02% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.01% 99.99% 99.98% 0.00% 0.00% 0.00% 0.00% 0.03% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.01% 0.00% 0.02% 0.00% 0.00% 99.97% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.98% 0.01% 0.01% 99.98% 0.00% 99.97% 99.98% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.01% 0.01% 0.02% 99.97% 0.03% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 99.97% 99.99% 0.00% 0.01% 0.01% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 99.98% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.99% 0.01% 0.00% 99.96% 0.02% 99.96% 99.98% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 99.95% 0.01% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.02% 99.95% 99.96% 0.00% 0.00% 0.01% 0.01% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.03% 0.03% 0.00% 0.01% 0.00% 0.00% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.97% 0.02% 0.00% 99.97% 0.01% 99.96% 99.97% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.02% 0.02% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.00% 0.02% 99.98% 0.01% 0.01% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 99.96% 99.99% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 99.99% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.98% 0.02% 0.00% 99.95% 0.01% 99.98% 99.98% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.01% 0.01% 99.95% 0.03% 0.01% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.00% 99.96% 99.97% 0.01% 0.02% 0.02% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.02% 0.02% 0.00% 0.01% 0.00% 0.01% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.99% 0.00% 0.00% 99.98% 0.02% 99.95% 99.97% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.02% 0.00% 0.00% 0.02% 99.95% 0.02% 0.00% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 99.98% 99.99% 0.00% 0.01% 0.00% 0.01% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.97% 0.00% 0.00% 99.97% 0.04% 99.97% 99.99% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.01% 0.00% 0.01% 99.95% 0.01% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.00% 99.95% 99.98% 0.00% 0.01% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 99.98% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.99% 0.01% 0.00% 99.97% 0.02% 99.97% 99.98% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 99.85% 0.01% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 99.83% 99.83% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 99.76% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.83% 0.00% 0.00% 99.85% 0.00% 99.75% 99.75% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.14% 0.14% 0.14% 0.14% 0.14% 0.23% 0.23% 0.23% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 99.97% 0.01% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 99.97% 99.98% 0.00% 0.01% 0.01% 0.00% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.01% 0.02% 0.00% 0.01% 0.00% 0.01% 99.98% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.98% 0.01% 0.00% 99.98% 0.01% 99.97% 99.98% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.00% 0.02% 99.97% 0.01% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 99.97% 99.99% 0.01% 0.01% 0.02% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.98% 0.01% 0.00% 99.97% 0.01% 99.97% 99.99% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.00% 0.02% 99.95% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 99.98% 99.98% 0.00% 0.00% 0.02% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.01% 0.02% 0.00% 0.02% 0.00% 0.00% 99.98% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.99% 0.01% 0.00% 99.97% 0.02% 99.96% 99.99% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.01% 0.02% 99.95% 0.01% 0.02% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 99.96% 99.96% 0.00% 0.01% 0.01% 0.01% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.02% 0.01% 0.00% 0.00% 0.01% 0.00% 99.97% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.97% 0.00% 0.01% 99.96% 0.02% 99.96% 99.94% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.02% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 99.96% 0.00% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 99.96% 99.98% 0.00% 0.01% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.01% 0.02% 0.00% 0.01% 0.00% 0.00% 99.96% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.99% 0.01% 0.00% 99.99% 0.01% 99.99% 99.98% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide C C C A G T G nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.00% 99.97% 0.01% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.03% 0.01% 0.01% 0.00% 0.00% 99.98% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 99.97% 99.98% 99.96% 0.00% 0.00% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 99.98% 0.01% 99.99% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.00% 99.95% 0.00% 0.00% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 99.95% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 99.99% 99.97% 99.99% 0.04% 0.00% 0.02% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 100.00% 0.02% 99.97% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.00% 0.01% 99.94% 0.00% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.02% 0.01% 0.01% 0.00% 99.96% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 99.99% 99.98% 99.93% 0.01% 0.00% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.00% 0.05% 10.000% 0.01% 99.97% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.05% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.01% 0.01% 99.96% 0.00% 0.01% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.01% 0.00% 0.00% 0.00% 99.95% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 10.000% 99.97% 99.95% 0.01% 0.00% 0.03% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 99.99% 0.00% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.01% 0.03% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.01% 99.94% 0.00% 0.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.01% 0.01% 0.01% 0.00% 99.95% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 99.99% 99.96% 99.94% 0.01% 0.00% 0.02% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.01% 0.04% 100.00% 0.02% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.01% 0.03% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.02% 0.00% 99.97% 0.01% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.00% 0.02% 0.02% 0.00% 0.01% 99.97% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 99.99% 99.96% 99.94% 0.01% 0.00% 0.03% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.01% 0.02% 99.97% 0.00% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.00% 99.96% 0.00% 0.01% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.00% 0.00% 0.01% 0.01% 0.00% 99.98% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 100.00% 99.98% 99.96% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.00% 0.01% 0.00% 0.03% 99.99% 0.01% 99.96% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.01% 0.01% 99.96% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.02% 0.01% 0.00% 99.95% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 99.96% 99.97% 99.95% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.00% 0.02% 99.99% 0.02% 99.97% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.01% 99.74% 0.01% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.01% 99.74% 0.03% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 99.75% 99.76% 99.72% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 99.75% 0.01% 99.73% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.23% 0.23% 0.26% 0.23% 0.23% 0.23% 0.23% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 0.00% 0.01% 99.96% 0.00% 0.00% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 0.02% 0.01% 0.00% 0.00% 99.94% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 100.00% 99.97% 99.95% 0.00% 0.00% 0.02% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.00% 0.03% 99.98% 0.02% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.03% 0.01% 0.01% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.00% 0.00% 99.98% 0.03% 0.01% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.01% 0.01% 0.00% 0.00% 99.95% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 99.98% 99.98% 99.95% 0.00% 0.00% 0.02% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 99.97% 0.02% 99.96% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.02% 0.00% 0.00% 99.94% 0.01% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.02% 0.02% 0.01% 0.00% 99.97% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 99.98% 99.97% 99.98% 0.04% 0.00% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.00% 0.01% 0.00% 0.01% 99.99% 0.00% 99.98% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.01% 99.96% 0.00% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.02% 0.01% 0.04% 0.00% 0.00% 99.96% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 99.97% 99.97% 99.89% 0.01% 0.00% 0.02% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.01% 0.02% 99.99% 0.00% 99.96% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.04% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.01% 99.95% 0.00% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.00% 0.02% 0.01% 0.01% 99.96% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 99.98% 99.98% 99.92% 0.01% 0.00% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.05% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G C T G C T C T G nogRNA-A1343spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.00% 0.01% 99.98% 0.00% 0.00% 99.98% 0.00% 99.98% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.01% 99.98% 0.02% 0.00% 99.99% 0.01% 100.00% 0.02% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.00% 0.00% 99.99% 0.00% 0.01% 0.00% 0.00% 99.97% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 99.96% 0.01% 0.00% 99.99% 0.00% 99.98% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 99.99% 0.03% 0.00% 99.99% 0.00% 100.00% 0.02% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.99% 0.00% 0.00% 99.98% 0.01% 0.01% 0.00% 0.00% 99.98% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 0.01% 99.98% 0.02% 0.00% 99.98% 0.01% 99.97% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.99% 0.00% 99.99% 0.02% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 0.00% 0.00% 99.97% 0.00% 0.01% 0.00% 0.01% 99.96% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.00% 0.01% 0.03% 0.00% 0.00% 0.01% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.01% 99.98% 0.01% 0.00% 99.97% 0.03% 99.97% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.99% 0.02% 99.97% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.99% 0.00% 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.00% 99.98% 0.02% 0.01% 99.97% 0.01% 99.98% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.99% 0.01% 99.97% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.98% 0.00% 0.00% 99.97% 0.00% 0.01% 0.00% 0.00% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.00% 99.97% 0.02% 0.00% 99.98% 0.00% 99.99% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 100.00% 0.03% 0.00% 99.99% 0.01% 100.00% 0.01% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.97% 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.01% 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.00% 0.00% 99.97% 0.00% 0.01% 99.97% 0.03% 99.97% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 99.99% 0.02% 0.00% 99.99% 0.02% 99.97% 0.02% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.98% 0.00% 0.00% 99.98% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.00% 99.99% 0.00% 0.00% 99.96% 0.01% 99.96% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.99% 0.02% 99.97% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.97% 0.00% 0.00% 99.98% 0.00% 0.01% 0.00% 0.00% 99.97% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.01% 0.03% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.00% 99.70% 0.00% 0.00% 99.70% 0.01% 99.70% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 99.71% 0.01% 0.00% 99.72% 0.01% 99.71% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.76% 0.00% 0.00% 99.69% 0.00% 0.01% 0.00% 0.00% 99.70% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.23% 0.27% 0.27% 0.28% 0.28% 0.28% 0.28% 0.28% 0.28% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.02% 99.96% 0.01% 0.00% 99.97% 0.01% 99.96% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 99.96% 0.01% 0.00% 99.98% 0.00% 99.96% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.97% 0.00% 0.00% 99.95% 0.00% 0.01% 0.00% 0.01% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.01% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.00% 0.01% 99.97% 0.00% 0.01% 99.98% 0.00% 99.99% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 99.98% 0.03% 0.00% 99.99% 0.01% 100.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.99% 0.01% 0.00% 99.99% 0.00% 0.01% 0.00% 0.00% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.02% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.01% 99.99% 0.01% 0.03% 99.97% 0.00% 99.99% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 99.99% 0.01% 0.00% 99.97% 0.02% 99.99% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.97% 0.00% 0.01% 99.98% 0.00% 0.02% 0.00% 0.00% 99.98% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.01% 0.00% 0.01% 0.01% 0.02% 0.00% 0.01% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 0.01% 99.97% 0.01% 0.00% 99.96% 0.00% 99.95% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 99.97% 0.01% 0.00% 99.98% 0.00% 99.98% 0.02% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.98% 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% 99.95% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.00% 0.03% 0.00% 0.01% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 0.00% 99.94% 0.00% 0.00% 99.97% 0.01% 99.98% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 99.99% 0.04% 0.00% 99.99% 0.01% 99.97% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.98% 0.00% 0.00% 99.96% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G G G G C C T C nogRNA-A1343spilt-KKH- A 0.01% 0.00% 0.02% 0.00% 0.00% 0.02% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.02% 0.01% 0.01% 0.01% 0.02% 99.98% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.01% 0.00% 99.99% 99.95% 0.02% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 99.97% 99.95% 99.94% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.01% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.02% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 99.99% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.99% 99.95% 0.01% 100.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.98% 99.98% 99.98% 99.93% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.06% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.01% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 99.98% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 10.000% 99.99% 0.01% 99.98% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 99.99% 99.98% 99.93% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.02% 0.02% 0.02% 0.02% 0.00% 0.01% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.03% 0.02% 0.01% 0.00% 0.00% 0.02% 99.96% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.99% 99.97% 0.03% 99.98% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.95% 99.97% 99.97% 99.94% 0.01% 0.00% 0.01% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.02% 0.01% 0.02% 0.01% 0.01% 0.02% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% 99.97% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.97% 99.97% 0.03% 99.98% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.97% 99.99% 99.97% 99.94% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.05% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.03% 0.03% 0.02% 0.01% 0.00% 0.01% 99.99% 0.05% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.99% 99.99% 0.00% 99.94% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.96% 99.96% 99.97% 99.95% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.01% 0.00% 0.00% 0.02% 0.00% 0.01% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 0.01% 0.01% 0.02% 0.00% 0.00% 99.98% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 100.00% 99.98% 0.01% 100.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.97% 99.98% 99.98% 99.93% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.02% 0.02% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 99.95% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.97% 99.96% 0.01% 99.95% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.96% 99.94% 99.96% 99.94% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.02% 0.02% 0.02% 0.05% 0.02% 0.02% 0.02% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.03% 0.01% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.02% 0.03% 0.01% 0.01% 0.01% 0.01% 99.64% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.66% 99.66% 0.01% 99.67% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.67% 99.67% 99.66% 99.61% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.28% 0.29% 0.32% 0.37% 0.32% 0.32% 0.33% 0.33% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 99.94% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.97% 99.96% 0.02% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.96% 99.94% 99.96% 99.93% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.02% 0.02% 0.02% 0.04% 0.02% 0.02% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.02% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.02% 0.00% 0.02% 0.00% 0.02% 0.02% 99.99% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 99.98% 99.98% 0.01% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.96% 99.99% 99.97% 99.93% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.06% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.01% 0.01% 0.02% 0.02% 0.00% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.02% 0.00% 0.01% 0.03% 0.03% 99.97% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 0.01% 0.00% 0.00% 99.95% 99.97% 0.02% 100.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.99% 99.97% 99.99% 99.93% 0.01% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.05% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 0.01% 0.02% 0.02% 0.00% 0.01% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 99.98% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.01% 0.00% 0.00% 0.00% 99.98% 99.97% 0.00% 99.97% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.95% 99.96% 99.96% 99.94% 0.00% 0.00% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.03% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.04% 0.02% 0.00% 0.01% 0.01% 0.00% 99.96% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.01% 0.00% 0.00% 99.97% 99.98% 0.02% 99.98% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.94% 99.93% 99.97% 99.93% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.05% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide C T G A G T T nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.02% 99.94% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.02% 99.98% 0.01% 0.01% 0.01% 99.98% 99.99% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 99.98% 0.01% 0.00% 0.03% 0.00% 0.02% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 99.97% 0.02% 99.99% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.01% 0.00% 0.02% 99.91% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.01% 99.96% 0.02% 0.00% 0.00% 100.00% 100.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 99.98% 0.03% 0.00% 0.05% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 99.96% 0.04% 100.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.01% 0.00% 0.02% 99.95% 0.01% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 99.97% 0.01% 0.01% 0.00% 99.98% 99.97% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 99.98% 0.03% 0.00% 0.02% 0.00% 0.01% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 0.00% 99.97% 0.03% 99.99% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 0.00% 0.02% 99.93% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 99.97% 0.02% 0.00% 0.00% 99.96% 99.99% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 99.99% 0.02% 0.00% 0.03% 0.00% 0.01% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.00% 0.00% 99.96% 0.04% 100.00% 0.03% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.01% 99.96% 0.00% 0.01% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 99.94% 0.00% 0.01% 0.00% 99.96% 99.98% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 99.98% 0.04% 0.00% 0.01% 0.00% 0.02% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.00% 0.00% 99.98% 0.01% 100.00% 0.01% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.00% 99.90% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 99.98% 0.01% 0.00% 0.00% 99.99% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 99.99% 0.01% 0.00% 0.05% 0.00% 0.01% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.00% 0.00% 99.98% 0.05% 100.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.01% 0.00% 0.02% 99.89% 0.01% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.00% 99.98% 0.01% 0.01% 0.00% 99.98% 99.99% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 99.98% 0.01% 0.00% 0.06% 0.00% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.00% 0.00% 99.97% 0.04% 99.99% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.01% 99.92% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 99.96% 0.01% 0.01% 0.00% 99.94% 99.95% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 99.97% 0.00% 0.00% 0.03% 0.00% 0.02% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.00% 0.01% 99.96% 0.02% 99.97% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.02% 0.02% 0.02% 0.03% 0.03% 0.03% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.00% 0.01% 99.60% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 99.64% 0.01% 0.00% 0.00% 99.66% 99.65% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 99.66% 0.02% 0.00% 0.03% 0.00% 0.00% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.01% 99.64% 0.03% 99.66% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.33% 0.33% 0.33% 0.34% 0.34% 0.33% 0.33% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.00% 0.00% 99.93% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 99.98% 0.01% 0.00% 0.00% 99.95% 99.99% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 99.97% 0.00% 0.00% 0.03% 0.00% 0.02% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.00% 0.00% 99.98% 0.02% 99.99% 0.02% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 0.01% 0.02% 99.95% 0.01% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 99.97% 0.01% 0.01% 0.00% 99.98% 100.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 99.99% 0.02% 0.00% 0.02% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.00% 0.00% 99.98% 0.03% 99.99% 0.02% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.00% 0.01% 99.88% 0.00% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 99.98% 0.02% 0.01% 0.01% 99.97% 99.99% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 99.99% 0.02% 0.00% 0.08% 0.00% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.00% 0.01% 99.97% 0.03% 99.99% 0.02% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.01% 0.02% 99.89% 0.01% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 99.96% 0.01% 0.01% 0.00% 99.97% 99.97% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 99.97% 0.02% 0.00% 0.04% 0.00% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.00% 0.01% 99.96% 0.05% 99.98% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.01% 99.92% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 99.95% 0.00% 0.00% 0.00% 99.96% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 99.97% 0.03% 0.00% 0.02% 0.00% 0.01% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.00% 0.00% 99.98% 0.04% 99.97% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide T C T C A T C T G nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.01% 0.01% 99.95% 0.00% 0.00% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 99.99% 0.01% 99.97% 0.01% 0.01% 99.98% 0.01% 99.99% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.01% 99.98% 0.01% 99.98% 0.00% 0.01% 99.99% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.04% 0.00% 0.00% 0.00% 99.99% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.01% 0.01% 99.95% 0.00% 0.01% 0.01% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 100.00% 0.01% 99.97% 0.03% 0.00% 100.00% 0.00% 99.97% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 99.99% 0.01% 99.96% 0.04% 0.00% 99.97% 0.02% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 99.97% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.00% 0.01% 0.01% 99.96% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 99.99% 0.00% 99.97% 0.01% 0.00% 99.99% 0.00% 99.96% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.01% 99.99% 0.02% 99.97% 0.01% 0.01% 99.99% 0.02% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 99.98% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.00% 0.01% 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 99.97% 0.00% 99.98% 0.01% 0.00% 99.98% 0.01% 99.97% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.02% 100.00% 0.00% 99.98% 0.00% 0.01% 99.97% 0.01% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.01% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 99.95% 0.00% 0.00% 0.01% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 99.97% 0.01% 99.96% 0.01% 0.00% 99.99% 0.02% 99.97% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.01% 99.98% 0.03% 99.97% 0.00% 0.00% 99.97% 0.02% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.01% 0.00% 0.01% 0.00% 0.04% 0.00% 0.00% 0.00% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.00% 0.02% 0.00% 99.98% 0.00% 0.00% 0.02% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 99.97% 0.01% 99.97% 0.03% 0.00% 99.98% 0.01% 99.96% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.02% 99.99% 0.01% 99.97% 0.00% 0.01% 99.99% 0.02% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.01% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.02% 0.01% 99.94% 0.00% 0.02% 0.01% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 99.98% 0.01% 99.97% 0.03% 0.01% 99.98% 0.01% 99.98% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.02% 99.98% 0.02% 99.96% 0.00% 0.01% 99.97% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.05% 0.00% 0.00% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.00% 0.01% 0.00% 99.94% 0.00% 0.00% 0.00% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 99.96% 0.01% 99.94% 0.02% 0.00% 99.95% 0.00% 99.95% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 99.96% 0.02% 99.94% 0.01% 0.01% 99.96% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.93% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.03% 0.03% 0.03% 0.03% 0.04% 0.04% 0.04% 0.03% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.02% 0.00% 99.64% 0.00% 0.01% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 99.66% 0.01% 99.63% 0.00% 0.01% 99.65% 0.01% 99.65% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.01% 99.65% 0.00% 99.66% 0.00% 0.00% 99.65% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.02% 0.01% 0.00% 0.00% 99.65% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.33% 0.33% 0.34% 0.34% 0.33% 0.33% 0.33% 0.33% 0.33% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 0.00% 0.01% 0.00% 99.94% 0.00% 0.01% 0.00% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 99.99% 0.01% 99.96% 0.01% 0.00% 99.97% 0.01% 99.97% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 99.98% 0.01% 99.98% 0.01% 0.01% 99.97% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 99.95% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 0.00% 0.01% 0.00% 99.97% 0.00% 0.00% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 99.98% 0.02% 99.97% 0.02% 0.01% 99.99% 0.01% 99.98% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.01% 99.98% 0.00% 99.98% 0.01% 0.01% 99.99% 0.02% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.02% 0.00% 0.00% 0.00% 99.97% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.00% 0.02% 0.00% 99.93% 0.01% 0.01% 0.01% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 99.99% 0.01% 99.97% 0.02% 0.00% 99.99% 0.03% 99.98% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 99.99% 0.01% 99.98% 0.05% 0.00% 99.97% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.02% 0.00% 0.00% 0.00% 99.97% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.03% 0.01% 99.96% 0.00% 0.01% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 99.97% 0.00% 99.94% 0.04% 0.00% 99.96% 0.04% 99.96% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 99.97% 0.01% 99.94% 0.00% 0.01% 99.94% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 99.97% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.02% 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 0.00% 0.00% 99.97% 0.00% 0.00% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 99.97% 0.02% 99.98% 0.01% 0.00% 99.96% 0.00% 99.95% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.01% 99.96% 0.00% 99.97% 0.00% 0.02% 99.98% 0.03% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide T G C C C C T C nogRNA-A1343spilt-KKH- A 0.00% 0.03% 0.00% 0.02% 0.01% 0.02% 0.01% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 99.98% 0.01% 0.01% 0.01% 0.02% 0.01% 99.96% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.02% 0.01% 99.99% 99.98% 99.97% 99.95% 0.02% 99.97% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 99.95% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.03% 0.00% 0.00% 0.01% 0.01% 0.00% 0.03% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 99.97% 0.00% 0.01% 0.01% 0.00% 0.02% 99.96% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.02% 0.00% 99.98% 99.98% 99.99% 99.97% 0.03% 99.96% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.01% 99.96% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.02% 0.02% 0.00% 0.01% 0.02% 0.02% 0.02% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 99.96% 0.02% 0.01% 0.01% 0.01% 0.00% 99.94% 0.06% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.02% 0.01% 99.99% 99.96% 99.97% 99.98% 0.02% 99.94% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 99.96% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.02% 0.01% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 99.95% 0.00% 0.04% 0.02% 0.03% 0.03% 99.97% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.01% 0.00% 99.95% 99.96% 99.95% 99.93% 0.01% 99.96% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.02% 99.98% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.03% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 0.04% 0.00% 0.03% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 99.95% 0.00% 0.01% 0.02% 0.02% 0.00% 99.95% 0.03% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.02% 0.00% 99.98% 99.97% 99.94% 99.97% 0.00% 99.96% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.01% 99.99% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 99.98% 0.03% 0.00% 0.01% 0.01% 0.01% 99.97% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.00% 99.99% 99.98% 99.99% 99.99% 0.01% 99.97% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.01% 99.97% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.02% 0.02% 0.00% 0.00% 0.00% 0.00% 0.02% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 99.92% 0.01% 0.02% 0.00% 0.00% 0.02% 99.97% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.04% 0.00% 99% 99.99% 99.99% 99.97% 0.01% 99.98% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.01% 99.96% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 99.92% 0.01% 0.01% 0.02% 0.01% 0.01% 99.92% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.02% 0.00% 99.96% 99.94% 99.95% 99.94% 0.04% 99.95% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 99.94% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.03% 0.03% 0.03% 0.03% 0.03% 0.04% 0.03% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.02% 0.00% 0.01% 0.02% 0.01% 0.00% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 99.62% 0.00% 0.01% 0.01% 0.01% 0.01% 99.63% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.02% 0.01% 99.65% 99.64% 99.64% 99.64% 0.02% 99.64% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.02% 99.63% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.33% 0.33% 0.33% 0.33% 0.33% 0.34% 0.34% 0.34% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.02% 0.00% 0.01% 0.02% 0.01% 0.02% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 99.95% 0.00% 0.01% 0.00% 0.01% 0.01% 99.93% 0.07% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.01% 0.01% 99.96% 99.97% 99.95% 99.93% 0.02% 99.89% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.01% 99.95% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.02% 0.02% 0.02% 0.02% 0.02% 0.04% 0.02% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.03% 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 99.96% 0.01% 0.02% 0.00% 0.00% 0.01% 99.97% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.02% 0.00% 99.97% 99.98% 99.99% 99.97% 0.01% 99.97% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.02% 99.96% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 0.02% 0.00% 0.02% 0.01% 0.00% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 99.97% 0.00% 0.01% 0.01% 0.01% 0.01% 99.93% 0.03% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.02% 0.01% 99.99% 99.97% 99.99% 99.98% 0.04% 99.97% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.01% 99.97% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.02% 0.02% 0.01% 0.00% 0.02% 0.02% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 99.92% 0.00% 0.00% 0.03% 0.01% 0.01% 99.93% 0.03% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.03% 0.01% 99.96% 99.94% 99.95% 99.92% 0.03% 99.93% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.01% 99.94% 0.00% 0.01% 0.00% 0.02% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.02% 0.02% 0.02% 0.02% 0.02% 0.04% 0.02% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.04% 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 99.95% 0.01% 0.01% 0.01% 0.01% 0.00% 99.96% 0.04% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.01% 0.00% 99.96% 99.96% 99.96% 99.96% 0.00% 99.95% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.02% 99.94% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide C C T C C C T nogRNA-A1343spilt-KKH- A 0.00% 0.01% 0.00% 0.01% 0.01% 0.02% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.02% 99.97% 0.02% 0.00% 0.01% 99.94% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 99.98% 99.95% 0.02% 99.96% 99.98% 99.96% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.01% 0.03% 0.02% 0.01% 0.00% 0.01% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.01% 0.01% 99.95% 0.01% 0.02% 0.00% 99.92% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 99.98% 99.95% 0.03% 99.97% 99.96% 99.97% 0.04% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.02% 0.01% 0.01% 0.01% 0.01% 0.02% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.03% 0.01% 99.97% 0.02% 0.02% 0.01% 99.92% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 99.95% 99.97% 0.02% 99.96% 99.96% 99.95% 0.04% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.01% 0.00% 0.01% 0.01% 0.02% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.01% 0.05% 0.02% 0.01% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.01% 99.93% 0.10% 0.00% 0.02% 99.92% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 99.98% 99.96% 0.02% 99.87% 99.97% 99.95% 0.04% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.01% 0.02% 0.01% 0.01% 0.00% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.02% 0.01% 0.01% 0.01% 0.00% 0.03% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.03% 0.01% 99.96% 0.03% 0.02% 0.01% 99.91% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 99.95% 99.97% 0.02% 99.95% 99.97% 99.94% 0.04% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.04% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.01% 0.01% 0.02% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.01% 99.96% 0.03% 0.01% 0.01% 99.90% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 99.99% 99.96% 0.01% 99.91% 99.90% 99.93% 0.04% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.02% 0.00% 0.05% 0.05% 0.05% 0.05% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.02% 0.01% 0.00% 0.01% 0.02% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 0.01% 99.96% 0.02% 0.01% 0.00% 99.92% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 99.97% 99.95% 0.02% 99.95% 99.94% 99.94% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.01% 0.01% 0.03% 0.03% 0.04% 0.04% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.02% 0.02% 0.01% 0.00% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.02% 0.01% 99.92% 0.01% 0.02% 0.01% 99.93% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 99.95% 99.95% 0.03% 99.96% 99.95% 99.95% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.01% 0.01% 0.02% 0.00% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.00% 99.62% 0.02% 0.00% 0.00% 99.58% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 99.65% 99.66% 0.02% 99.61% 99.62% 99.64% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.34% 0.34% 0.34% 0.36% 0.36% 0.36% 0.36% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.02% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.04% 0.03% 99.93% 0.06% 0.05% 0.04% 99.91% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 99.91% 99.93% 0.03% 99.90% 99.93% 99.92% 0.04% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.02% 0.02% 0.02% 0.03% 0.02% 0.03% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.00% 0.00% 0.02% 0.03% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.03% 0.01% 99.99% 0.02% 0.03% 0.00% 99.91% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 99.96% 99.96% 0.00% 99.96% 99.92% 99.95% 0.04% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.02% 0.01% 0.02% 0.02% 0.02% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.02% 0.01% 0.01% 0.01% 0.03% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.02% 0.01% 99.96% 0.02% 0.02% 0.00% 99.95% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 99.97% 99.96% 0.03% 99.97% 99.97% 99.94% 0.03% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 0.02% 0.00% 0.02% 0.02% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.02% 0.01% 99.93% 0.06% 0.05% 0.03% 99.92% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 99.94% 99.94% 0.04% 99.89% 99.89% 99.91% 0.03% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.02% 0.03% 0.02% 0.03% 0.04% 0.04% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.04% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.04% 0.04% 99.95% 0.02% 0.04% 0.02% 99.93% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 99.94% 99.93% 0.03% 99.94% 99.90% 99.95% 0.05% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.02% 0.02% 0.02% 0.02% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G G C C C A G G T nogRNA-A1343spilt-KKH- A 0.01% 0.00% 0.01% 0.01% 0.01% 99.93% 0.00% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.00% 99.77% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 99.98% 99.98% 99.98% 0.00% 0.00% 0.00% 0.05% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 100.00% 0.01% 0.00% 0.00% 0.05% 99.98% 99.98% 0.17% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.02% 0.00% 0.00% 0.00% 0.01% 99.84% 0.00% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.00% 99.71% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.00% 99.99% 99.98% 99.97% 0.08% 0.00% 0.00% 0.04% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.98% 99.98% 0.00% 0.00% 0.00% 0.08% 99.99% 99.98% 0.25% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 0.03% 99.90% 0.00% 0.01% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.00% 0.02% 0.01% 0.02% 0.02% 0.01% 0.00% 99.74% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.01% 99.97% 99.98% 99.95% 0.02% 0.01% 0.00% 0.05% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 99.98% 0.01% 0.01% 0.00% 0.06% 99.98% 99.99% 0.19% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 0.02% 0.00% 0.01% 0.02% 99.92% 0.01% 0.00% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.00% 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% 99.71% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.00% 99.96% 99.97% 99.95% 0.01% 0.00% 0.00% 0.05% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.98% 99.97% 0.02% 0.00% 0.00% 0.05% 99.97% 99.99% 0.22% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.03% 0.03% 0.00% 0.00% 0.00% 99.91% 0.00% 0.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.00% 0.01% 0.02% 0.03% 0.02% 0.02% 0.00% 99.70% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.00% 99.97% 99.96% 99.96% 0.03% 0.00% 0.00% 0.06% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.97% 99.96% 0.02% 0.01% 0.00% 0.04% 99.98% 99.99% 0.21% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.01% 0.00% 0.01% 0.00% 0.00% 99.88% 0.00% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.00% 0.01% 0.02% 0.00% 0.03% 0.01% 0.01% 0.01% 99.69% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.00% 99.95% 99.99% 99.95% 0.02% 0.00% 0.00% 0.07% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.99% 99.98% 0.02% 0.00% 0.01% 0.09% 99.99% 99.99% 0.22% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.02% 0.00% 0.00% 0.00% 0.02% 99.91% 0.00% 0.01% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.00% 0.00% 0.04% 0.02% 0.02% 0.02% 0.01% 0.00% 99.71% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.00% 99.94% 99.97% 99.95% 0.01% 0.00% 0.00% 0.08% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.97% 99.98% 0.01% 0.00% 0.00% 0.06% 99.98% 99.98% 0.19% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.01% 0.00% 0.01% 0.01% 99.89% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.00% 0.01% 0.03% 0.02% 0.02% 0.02% 0.02% 0.00% 99.73% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.01% 99.96% 99.96% 99.95% 0.03% 0.00% 0.00% 0.06% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.98% 99.96% 0.00% 0.01% 0.00% 0.06% 99.98% 99.99% 0.20% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.02% 0.00% 0.01% 0.02% 99.62% 0.02% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.00% 0.05% 0.03% 0.01% 0.02% 0.00% 0.00% 99.53% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.00% 99.61% 99.64% 99.65% 0.01% 0.00% 0.00% 0.09% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.66% 99.65% 0.01% 0.00% 0.00% 0.04% 99.66% 99.81% 0.19% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.33% 0.33% 0.32% 0.32% 0.32% 0.31% 0.31% 0.18% 0.18% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.02% 0.02% 0.00% 0.00% 0.01% 99.90% 0.00% 0.02% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 0.00% 0.04% 0.02% 0.01% 0.00% 0.01% 0.00% 99.71% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.00% 99.94% 99.96% 99.97% 0.01% 0.00% 0.00% 0.06% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.95% 99.96% 0.01% 0.01% 0.00% 0.07% 99.97% 99.97% 0.20% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.02% 0.01% 0.01% 0.01% 0.02% 0.02% 0.01% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.00% 0.00% 0.01% 99.91% 0.01% 0.02% 0.03% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.02% 0.02% 0.02% 0.02% 0.01% 0.00% 0.00% 99.71% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.00% 99.95% 99.97% 99.97% 0.02% 0.00% 0.00% 0.08% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.98% 99.97% 0.03% 0.00% 0.00% 0.05% 99.99% 99.98% 0.18% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.02% 0.02% 0.01% 0.00% 0.01% 99.83% 0.00% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.00% 0.01% 0.01% 0.00% 0.02% 0.00% 0.00% 99.63% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 0.01% 99.97% 99.98% 99.98% 0.07% 0.00% 0.00% 0.03% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.98% 99.98% 0.02% 0.01% 0.01% 0.09% 100.00% 99.99% 0.32% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.02% 0.02% 0.00% 0.01% 0.02% 99.86% 0.01% 0.01% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.02% 0.03% 0.02% 0.03% 0.02% 0.01% 0.00% 99.69% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.01% 0.00% 99.93% 99.94% 99.91% 0.01% 0.00% 0.00% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.92% 99.93% 0.00% 0.00% 0.00% 0.08% 99.96% 99.98% 0.25% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.03% 0.04% 0.03% 0.03% 0.04% 0.02% 0.02% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.02% 0.01% 0.00% 0.01% 0.01% 99.91% 0.00% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.02% 0.01% 0.01% 0.02% 0.00% 0.00% 99.71% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.00% 99.96% 99.97% 99.96% 0.03% 0.00% 0.00% 0.06% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.97% 99.98% 0.01% 0.01% 0.00% 0.03% 100.00% 99.99% 0.21% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G A A G G T G T nogRNA-A1343spilt-KKH- A 0.01% 99.95% 99.93% 0.00% 0.00% 0.02% 0.01% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.01% 99.74% 0.01% 99.93% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.02% 0.00% 0.00% 0.00% 0.05% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.02% 0.05% 99.99% 99.99% 0.19% 99.98% 0.04% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 99.86% 99.83% 0.01% 0.01% 0.02% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 0.02% 0.00% 0.01% 99.72% 0.01% 99.91% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.07% 0.07% 0.00% 0.00% 0.05% 0.00% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.99% 0.05% 0.08% 99.99% 99.98% 0.22% 99.98% 0.08% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.01% 99.93% 99.87% 0.01% 0.00% 0.03% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.02% 0.01% 0.01% 0.01% 0.02% 99.76% 0.02% 99.93% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.03% 0.03% 0.00% 0.00% 0.04% 0.00% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 0.02% 0.09% 99.98% 99.98% 0.17% 99.98% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.00% 99.90% 99.85% 0.00% 0.00% 0.02% 0.00% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.02% 0.02% 0.03% 0.01% 0.01% 99.70% 0.02% 99.90% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.01% 0.03% 0.01% 0.01% 0.00% 0.07% 0.00% 0.03% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.97% 0.06% 0.11% 99.98% 99.99% 0.21% 99.98% 0.05% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 99.95% 99.89% 0.00% 0.01% 0.03% 0.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.02% 0.00% 0.00% 99.76% 0.02% 99.90% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.02% 0.03% 0.00% 0.00% 0.05% 0.00% 0.03% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.98% 0.03% 0.06% 100.00% 99.98% 0.16% 99.97% 0.06% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 99.91% 99.86% 0.00% 0.01% 0.01% 0.01% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.01% 99.75% 0.02% 99.94% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.05% 0.04% 0.00% 0.00% 0.06% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.99% 0.03% 0.10% 99.99% 99.98% 0.18% 99.97% 0.04% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.01% 99.92% 99.90% 0.02% 0.02% 0.03% 0.02% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.01% 0.00% 0.01% 99.70% 0.00% 99.93% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.03% 0.03% 0.00% 0.00% 0.05% 0.00% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.98% 0.03% 0.06% 99.98% 99.97% 0.21% 99.98% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 99.88% 99.85% 0.00% 0.01% 0.04% 0.00% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.02% 0.02% 0.02% 0.01% 0.02% 99.65% 0.05% 99.88% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.05% 0.02% 0.00% 0.00% 0.08% 0.00% 0.04% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.97% 0.05% 0.12% 99.99% 99.97% 0.23% 99.95% 0.07% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 99.72% 99.73% 0.00% 0.00% 0.03% 0.01% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.02% 0.01% 0.01% 0.01% 0.02% 99.71% 0.01% 99.89% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.03% 0.02% 0.00% 0.00% 0.05% 0.00% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.79% 0.06% 0.07% 99.82% 99.80% 0.17% 99.93% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.18% 0.17% 0.17% 0.17% 0.17% 0.05% 0.05% 0.05% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 99.92% 99.90% 0.01% 0.00% 0.03% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 0.02% 0.01% 0.01% 0.00% 99.75% 0.01% 99.93% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.02% 0.02% 0.00% 0.00% 0.06% 0.00% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.99% 0.02% 0.06% 99.98% 100.00% 0.16% 99.98% 0.04% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.00% 99.90% 99.89% 0.00% 0.00% 0.05% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.02% 0.02% 0.02% 0.01% 0.01% 99.71% 0.02% 99.93% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.05% 0.02% 0.00% 0.00% 0.06% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.97% 0.03% 0.08% 99.98% 99.99% 0.18% 99.97% 0.05% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 99.87% 99.79% 0.00% 0.00% 0.02% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.01% 0.01% 0.02% 0.01% 99.74% 0.02% 99.90% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.07% 0.06% 0.00% 0.01% 0.03% 0.00% 0.03% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.99% 0.06% 0.14% 99.98% 99.99% 0.22% 99.98% 0.07% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.02% 99.89% 99.85% 0.01% 0.01% 0.02% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.03% 0.01% 0.02% 0.02% 0.04% 99.69% 0.02% 99.93% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.06% 0.03% 0.00% 0.00% 0.06% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.95% 0.04% 0.09% 99.96% 99.94% 0.22% 99.96% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 99.91% 99.89% 0.00% 0.00% 0.02% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.02% 0.00% 0.02% 0.01% 99.73% 0.02% 99.95% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.04% 0.02% 0.00% 0.00% 0.07% 0.00% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.98% 0.04% 0.09% 99.97% 99.98% 0.18% 99.97% 0.03% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G G T T C C A nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 99.97% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.00% 99.96% 99.98% 0.00% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.02% 0.01% 99.99% 99.97% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.99% 99.99% 0.02% 0.00% 0.00% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 99.89% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.02% 0.00% 99.95% 99.99% 0.02% 0.01% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.03% 0.00% 99.97% 99.97% 0.06% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.97% 100.00% 0.02% 0.00% 0.00% 0.00% 0.04% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.02% 0.01% 0.00% 0.00% 0.00% 0.01% 99.94% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.00% 99.96% 99.98% 0.01% 0.02% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.01% 0.02% 0.01% 99.99% 99.95% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 99.98% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 0.00% 0.00% 0.02% 0.01% 0.00% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.00% 99.95% 99.97% 0.05% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.01% 0.01% 99.93% 99.97% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.97% 99.99% 0.03% 0.00% 0.00% 0.00% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 99.95% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.00% 99.96% 99.94% 0.03% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.00% 0.02% 0.04% 99.97% 99.98% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 10.000% 100.00% 0.02% 0.00% 0.00% 0.00% 0.04% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 99.96% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.00% 99.96% 99.98% 0.02% 0.03% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.00% 0.02% 0.01% 99.98% 99.95% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.99% 99.99% 0.02% 0.00% 0.00% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.01% 0.02% 0.01% 0.00% 0.01% 99.93% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 0.00% 99.95% 99.98% 0.02% 0.01% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.02% 0.01% 99.98% 99.97% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.98% 99.98% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% 99.92% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.00% 0.00% 99.96% 99.96% 0.02% 0.03% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.01% 0.02% 99.97% 99.95% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.99% 99.98% 0.02% 0.00% 0.00% 0.00% 0.06% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.01% 0.00% 0.01% 0.02% 99.94% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.00% 99.95% 99.96% 0.01% 0.02% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.02% 0.03% 99.98% 99.95% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.95% 99.94% 0.01% 0.00% 0.00% 0.00% 0.04% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.05% 0.05% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% 99.95% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.00% 99.95% 99.96% 0.02% 0.01% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.03% 0.02% 99.97% 99.94% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.99% 99.99% 0.02% 0.01% 0.00% 0.01% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 99.97% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.00% 99.97% 99.97% 0.01% 0.01% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.00% 0.00% 0.02% 99.98% 99.98% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.98% 99.98% 0.02% 0.00% 0.00% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% 99.91% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.02% 0.00% 99.97% 99.97% 0.02% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.00% 0.02% 0.02% 99.98% 99.97% 0.06% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.98% 99.98% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 0.00% 0.01% 0.01% 0.00% 0.04% 99.95% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 0.01% 99.96% 99.95% 0.01% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.01% 0.01% 0.01% 99.98% 99.94% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.97% 99.96% 0.01% 0.01% 0.00% 0.00% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 99.97% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.01% 99.97% 99.96% 0.02% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.00% 0.01% 0.03% 99.97% 99.97% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.98% 99.99% 0.02% 0.00% 0.00% 0.00% 0.03% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G A A C C G G A G nogRNA-A1343spilt-KKH- A 0.00% 99.98% 99.96% 0.01% 0.00% 0.00% 0.01% 99.92% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.00% 0.00% 0.01% 0.03% 0.01% 0.00% 0.01% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.01% 0.02% 99.98% 99.96% 0.00% 0.00% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.00% 0.02% 0.00% 0.00% 99.98% 99.98% 0.05% 99.98% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.02% 99.91% 99.92% 0.00% 0.00% 0.01% 0.00% 99.83% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 0.01% 0.02% 0.01% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.07% 0.06% 99.99% 99.98% 0.01% 0.00% 0.08% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.98% 0.02% 0.01% 0.00% 0.01% 99.96% 99.98% 0.09% 99.99% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 99.96% 99.98% 0.01% 0.00% 0.01% 0.01% 99.90% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 0.02% 0.00% 0.01% 0.02% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.02% 0.01% 99.99% 99.98% 0.00% 0.00% 0.02% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.99% 0.01% 0.01% 0.00% 0.01% 99.98% 99.98% 0.06% 99.98% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 99.97% 99.96% 0.00% 0.01% 0.03% 0.01% 99.92% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.00% 0.01% 0.00% 0.03% 0.01% 0.01% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.00% 99.99% 99.95% 0.00% 0.00% 0.02% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.98% 0.02% 0.02% 0.00% 0.01% 99.96% 99.97% 0.05% 99.98% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 99.96% 99.98% 0.00% 0.00% 0.02% 0.02% 99.88% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.02% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.03% 0.00% 99.98% 99.99% 0.00% 0.00% 0.04% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 100.00% 0.01% 0.00% 0.00% 0.01% 99.97% 99.98% 0.06% 99.98% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 99.96% 99.97% 0.00% 0.00% 0.02% 0.01% 99.90% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.00% 0.03% 0.01% 0.02% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.02% 0.01% 99.99% 100.00% 0.00% 0.00% 0.03% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.99% 0.02% 0.02% 0.00% 0.00% 99.95% 99.97% 0.05% 99.96% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.01% 99.94% 99.97% 0.00% 0.00% 0.01% 0.01% 99.87% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.02% 0.01% 0.00% 0.00% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.02% 0.01% 99.99% 99.98% 0.00% 0.00% 0.05% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.98% 0.04% 0.02% 0.00% 0.00% 99.98% 99.98% 0.07% 99.98% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 99.96% 99.96% 0.00% 0.00% 0.00% 0.01% 99.88% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.00% 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.01% 0.01% 99.98% 99.98% 0.00% 0.00% 0.04% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.98% 0.03% 0.01% 0.00% 0.00% 99.99% 99.98% 0.07% 99.98% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSnCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.00% 99.98% 99.98% 0.00% 0.00% 0.02% 0.01% 99.92% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 0.03% 0.02% 0.01% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.01% 0.01% 100.00% 99.95% 0.01% 0.00% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.99% 0.01% 0.00% 0.00% 0.01% 99.95% 99.98% 0.03% 99.99% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 99.98% 99.96% 0.00% 0.01% 0.01% 0.01% 99.91% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.02% 0.01% 0.01% 0.02% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.01% 0.01% 99.99% 99.95% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.97% 0.01% 0.02% 0.00% 0.01% 99.98% 99.98% 0.06% 99.99% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.01% 99.97% 99.96% 0.00% 0.00% 0.02% 0.01% 99.93% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.00% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.01% 0.02% 100.00% 99.98% 0.00% 0.00% 0.03% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.98% 0.01% 0.02% 0.00% 0.00% 99.97% 99.98% 0.03% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 99.90% 99.94% 0.00% 0.00% 0.01% 0.01% 99.85% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.00% 0.00% 0.01% 0.02% 0.03% 0.01% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.05% 0.06% 99.98% 99.98% 0.01% 0.01% 0.07% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.98% 0.05% 0.01% 0.01% 0.00% 99.95% 99.97% 0.08% 99.99% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 99.97% 99.96% 0.01% 0.00% 0.01% 0.02% 99.85% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.03% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.01% 0.01% 99.98% 99.96% 0.00% 0.00% 0.03% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.98% 0.01% 0.03% 0.00% 0.02% 99.97% 99.97% 0.09% 99.98% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 99.99% 99.98% 0.00% 0.00% 0.00% 0.02% 99.92% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.01% 0.01% 100.00% 99.98% 0.00% 0.00% 0.02% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.99% 0.00% 0.01% 0.00% 0.00% 99.99% 99.97% 0.04% 99.98% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G A C A A A G T nogRNA-A1343spilt-KKH- A 0.00% 99.92% 0.00% 99.92% 99.92% 99.89% 0.00% 0.07% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.02% 0.00% 0.01% 0.01% 0.00% 0.02% 99.86% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.01% 0.04% 99.99% 0.04% 0.05% 0.06% 0.00% 0.03% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.02% 0.00% 0.03% 0.02% 0.04% 99.98% 0.04% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 99.83% 0.00% 99.87% 99.86% 99.88% 0.00% 0.08% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.02% 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 99.81% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.01% 0.09% 99.99% 0.08% 0.0% 0.07% 0.00% 0.07% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.97% 0.05% 0.00% 0.03% 0.02% 0.04% 99.98% 0.04% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.02% 99.89% 0.00% 99.94% 99.94% 99.94% 0.00% 0.07% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 99.83% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.05% 99.99% 0.01% 0.05% 0.03% 0.00% 0.05% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.98% 0.05% 0.00% 0.04% 0.01% 0.02% 100.00% 0.04% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 99.85% 0.01% 99.94% 99.93% 99.89% 0.01% 0.08% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 99.82% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.06% 99.97% 0.02% 0.03% 0.04% 0.00% 0.03% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.98% 0.06% 0.00% 0.03% 0.03% 0.05% 99.99% 0.07% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 99.88% 0.00% 99.94% 99.94% 99.91% 0.00% 0.08% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.00% 0.01% 0.03% 0.00% 0.01% 0.00% 0.01% 99.81% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.04% 99.97% 0.05% 0.05% 0.03% 0.00% 0.05% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 100.00% 0.06% 0.00% 0.00% 0.00% 0.06% 99.99% 0.06% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.03% 99.89% 0.00% 99.94% 99.93% 99.90% 0.00% 0.09% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 0.01% 0.02% 0.01% 99.80% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.07% 100.00% 0.02% 0.05% 0.05% 0.00% 0.06% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.96% 0.03% 0.00% 0.03% 0.01% 0.03% 99.99% 0.05% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 99.87% 0.01% 99.95% 99.91% 99.93% 0.01% 0.09% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 0.03% 0.02% 0.02% 0.00% 0.00% 0.01% 99.79% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.06% 99.98% 0.01% 0.07% 0.02% 0.00% 0.05% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.98% 0.03% 0.00% 0.02% 0.02% 0.05% 99.98% 0.07% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 99.86% 0.01% 99.96% 99.93% 99.92% 0.00% 0.06% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.02% 0.02% 0.01% 0.01% 0.01% 0.01% 99.85% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.05% 99.97% 0.02% 0.05% 0.04% 0.00% 0.04% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.98% 0.07% 0.00% 0.02% 0.01% 0.03% 99.99% 0.05% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSnCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.01% 99.88% 0.00% 99.93% 99.90% 99.90% 0.00% 0.04% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.01% 0.02% 0.01% 0.02% 0.00% 0.00% 99.88% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.07% 99.98% 0.03% 0.07% 0.04% 0.00% 0.03% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.99% 0.04% 0.00% 0.02% 0.01% 0.05% 99.99% 0.05% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.00% 99.87% 0.00% 99.93% 99.90% 99.93% 0.00% 0.07% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.00% 0.01% 0.04% 0.01% 0.00% 0.00% 0.00% 99.83% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.06% 99.96% 0.05% 0.05% 0.03% 0.00% 0.04% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.99% 0.06% 0.00% 0.01% 0.04% 0.03% 99.99% 0.06% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.03% 99.86% 0.02% 99.95% 99.92% 99.90% 0.00% 0.06% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.00% 0.03% 0.01% 0.00% 0.01% 0.01% 0.00% 99.83% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.08% 99.97% 0.03% 0.06% 0.04% 0.00% 0.06% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.97% 0.03% 0.00% 0.02% 0.01% 0.03% 100.00% 0.04% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 99.80% 0.01% 99.90% 99.90% 99.90% 0.00% 0.10% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.04% 0.03% 0.01% 0.00% 0.01% 0.01% 0.01% 99.80% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.00% 0.10% 99.98% 0.07% 0.08% 0.07% 0.00% 0.05% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.95% 0.07% 0.00% 0.03% 0.01% 0.02% 99.99% 0.05% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 99.85% 0.01% 99.93% 99.90% 99.91% 0.00% 0.12% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.02% 0.01% 0.00% 0.03% 0.01% 0.00% 99.81% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.09% 99.96 0.04% 0.06% 0.04% 0.00% 0.03% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.97% 0.03% 0.00% 0.02% 0.01% 0.04% 99.99% 0.04% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.01% 99.86% 0.01% 99.91% 99.95% 99.91% 0.00% 0.07% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.03% 0.01% 0.02% 0.01% 0.00% 0.00% 99.86% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.04% 99.97% 0.04% 0.03% 0.04% 0.00% 0.04% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.98% 0.07% 0.00% 0.04% 0.00% 0.04% 99.99% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide A C A A A C G nogRNA-A1343spilt-KKH- A 99.94% 0.03% 99.98% 99.99% 99.96% 0.01% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.01% 99.96% 0.00% 0.00% 0.02% 99.97% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.02% 0.00% 0.02% 0.00% 0.01% 0.02% 99.97% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 99.88% 0.05% 99.91% 99.93% 99.92% 0.02% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.03% 0.00% 0.00% 0.00% 0.02% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.09% 99.92% 0.06% 0.05% 0.08% 99.95% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.03% 0.00% 0.02% 0.01% 0.00% 0.01% 99.96% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 99.95% 0.03% 99.96% 99.97% 99.97% 0.02% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 0.02% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.01% 99.94% 0.01% 0.02% 0.02% 99.96% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.02% 0.01% 0.02% 0.01% 0.01% 0.01% 99.99% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 99.97% 0.01% 99.97% 99.97% 99.97% 0.02% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.01% 99.99% 0.02% 0.02% 0.02% 99.95% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.02% 0.00% 0.01% 0.01% 0.00% 0.01% 99.99% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 99.95% 0.03% 99.97% 99.98% 100.00% 0.03% 0.04% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.04% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.02% 99.96% 0.01% 0.00% 0.00% 99.92% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.02% 0.00% 0.02% 0.02% 0.00% 0.01% 99.94% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 99.98% 0.00% 99.96% 99.96% 99.97% 0.01% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.03% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 99.97% 0.02% 0.02% 0.01% 99.96% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.00% 0.01% 0.01% 0.02% 0.01% 0.00% 99.98% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 99.97% 0.02% 99.97% 99.97% 99.95% 0.02% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.01% 99.97% 0.02% 0.02% 0.03% 99.96% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.01% 0.02% 0.01% 0.00% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 99.97% 0.02% 99.95% 99.98% 99.96% 0.01% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.00% 0.02% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.01% 99.96% 0.02% 0.01% 0.03% 99.95% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.01% 0.00% 0.02% 0.01% 0.00% 0.01% 99.97% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSnCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 99.95% 0.00% 99.99% 99.97% 99.95% 0.02% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.02% 99.99% 0.00% 0.00% 0.02% 99.95% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.01% 0.00% 0.01% 0.02% 0.01% 0.01% 99.97% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 99.96% 0.02% 99.98% 99.93% 99.97% 0.01% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.00% 0.03% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 99.96% 0.01% 0.03% 0.02% 99.95% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.03% 0.01% 0.01% 0.03% 0.01% 0.00% 99.98% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 99.94% 0.00% 99.99% 99.98% 99.98% 0.00% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.02% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.02% 99.98% 0.00% 0.00% 0.02% 99.98% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.03% 0.00% 0.00% 0.01% 0.00% 0.00% 99.99% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 99.93% 0.04% 99.94% 99.92% 99.93% 0.01% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.02% 0.00% 0.00% 0.00% 0.02% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.05% 99.94% 0.04% 0.05% 0.05% 99.96% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.02% 0.00% 0.01% 0.03% 0.02% 0.02% 99.97% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 99.96% 0.03% 99.97% 99.96% 99.98% 0.01% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.02% 99.96% 0.01% 0.01% 0.01% 99.97% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.01% 0.00% 0.01% 0.02% 0.00% 0.01% 99.97% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 99.96% 0.02% 99.98% 99.98% 99.97% 0.01% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.02% 0.00% 0.00% 0.00% 0.04% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 99.96% 0.01% 0.01% 0.02% 99.95% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.02% 0.00% 0.00% 0.00% 0.01% 0.01% 99.98% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G C A G A A G C T nogRNA-A1343spilt-KKH- A 0.01% 0.01% 99.93% 0.00% 99.94% 99.92% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.02% 0.02% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 99.95% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 99.96% 0.00% 0.00% 0.01% 0.02% 0.00% 99.98% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 0.01% 0.07% 99.99% 0.04% 0.06% 99.98% 0.00% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.02% 0.02% 99.82% 0.01% 99.91% 99.83% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 99.94% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 99.94% 0.08% 0.00% 0.07% 0.08% 0.00% 99.98% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.97% 0.03% 0.09% 99.98% 0.02% 0.08% 99.99% 0.01% 0.05% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.03% 0.01% 99.93% 0.01% 99.96% 99.93% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 99.96% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.01% 99.96% 0.01% 0.00% 0.03% 0.02% 0.00% 99.98% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.95% 0.02% 0.05% 99.98% 0.01% 0.05% 100.00% 0.01% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.02% 0.01% 99.87% 0.02% 99.95% 99.89% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.03% 99.95% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 99.96% 0.03% 0.00% 0.01% 0.02% 0.00% 99.96% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.97% 0.02% 0.08% 99.98% 0.03% 0.09% 99.99% 0.02% 0.03% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.02% 0.00% 99.94% 0.01% 99.96% 99.85% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 99.97% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 99.96% 0.01% 0.00% 0.02% 0.05% 0.01% 99.98% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.96% 0.02% 0.04% 99.99% 0.02% 0.10% 99.98% 0.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.03% 0.01% 99.93% 0.00% 99.95% 99.88% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 99.94% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.01% 99.95% 0.01% 0.00% 0.03% 0.03% 0.01% 99.99% 0.04% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.95% 0.01% 0.05% 99.99% 0.02% 0.09% 99.99% 0.00% 0.03% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.04% 0.02% 99.92% 0.01% 99.96% 99.90% 0.01% 0.01% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 99.94% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 99.96% 0.01% 0.00% 0.03% 0.01% 0.00% 99.98% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.95% 0.01% 0.06% 99.98% 0.01% 0.08% 99.99% 0.00% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.02% 0.01% 99.92% 0.01% 99.95% 99.93% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 99.94% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 99.98% 0.01% 0.00% 0.03% 0.01% 0.00% 99.98% 0.03% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.96% 0.01% 0.05% 99.99% 0.02% 0.05% 99.98% 0.01% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.04% 0.02% 99.92% 0.01% 99.95% 99.89% 0.00% 0.00% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.02% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 99.95% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 99.95% 0.02% 0.00% 0.03% 0.02% 0.00% 99.97% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.96% 0.01% 0.06% 99.98% 0.02% 0.09% 99.99% 0.01% 0.03% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.05% 0.00% 99.95% 0.00% 99.95% 99.91% 0.00% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 99.96% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 99.97% 0.00% 0.00% 0.04% 0.02% 0.00% 99.96% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.94% 0.01% 0.04% 100.00% 0.01% 0.07% 100.00% 0.01% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.05% 0.00% 99.93% 0.00% 99.96% 99.93% 0.01% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.02% 99.97% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 99.98% 0.01% 0.00% 0.01% 0.00% 0.00% 99.97% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.95% 0.01% 0.06% 99.99% 0.02% 0.07% 99.98% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.05% 0.02% 99.84% 0.01% 99.90% 99.82% 0.02% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.00% 0.02% 0.02% 0.00% 0.01% 0.00% 0.00% 0.01% 99.97% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 99.96% 0.08% 0.00% 0.06% 0.07% 0.00% 99.98% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.94% 0.01% 0.06% 99.99% 0.04% 0.11% 99.98% 0.02% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.03% 0.02% 99.93% 0.01% 99.94% 99.88% 0.01% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 99.92% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.01% 99.96% 0.01% 0.00% 0.03% 0.04% 0.00% 99.96% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.96% 0.00% 0.05% 99.99% 0.03% 0.08% 99.99% 0.00% 0.05% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.02% 0.01% 99.90% 0.01% 99.94% 99.89% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% 99.95% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 99.96% 0.03% 0.00% 0.04% 0.02% 0.00% 99.98% 0.02% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.97% 0.02% 0.05% 99.99% 0.02% 0.09% 99.97% 0.01% 0.03% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide G G A G G A G G nogRNA-A1343spilt-KKH- A 0.00% 0.00% 99.83% 0.00% 0.01% 99.85% 0.02% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.01% 0.00% 0.01% 0.02% 0.02% 0.01% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.03% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 99.98% 99.98% 0.14% 99.99% 99.97% 0.10% 99.97% 99.99% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.00% 0.01% 99.66% 0.00% 0.00% 99.75% 0.00% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.00% 0.02% 0.02% 0.02% 0.01% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.00% 0.01% 0.09% 0.00% 0.00% 0.09% 0.01% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 99.99% 99.98% 0.23% 99.98% 99.98% 0.15% 99.97% 99.98% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.02% 99.80% 0.00% 0.01% 99.83% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.02% 0.00% 0.00% 0.03% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 99.99% 99.98% 0.17% 99.99% 99.98% 0.13% 100.00% 99.98% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 0.04% 99.78% 0.00% 0.01% 99.82% 0.00% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.00% 0.02% 0.00% 0.00% 0.01% 0.00% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.00% 0.00% 0.01% 0.00% 0.00% 0.06% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 99.98% 99.96% 0.19% 100.00% 99.99% 0.10% 99.99% 99.97% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.00% 0.02% 99.74% 0.01% 0.02% 99.80% 0.01% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.00% 0.02% 0.01% 0.02% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.00% 0.04% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 99.99% 99.98% 0.21% 99.96% 99.97% 0.14% 99.99% 99.99% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.03% 99.78% 0.01% 0.02% 99.85% 0.01% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.00% 0.01% 0.05% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 99.98% 99.95% 0.15% 99.97% 99.97% 0.10% 99.99% 99.99% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.00% 99.79% 0.00% 0.01% 99.82% 0.02% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.02% 0.01% 0.00% 0.01% 0.02% 0.01% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.04% 0.00% 0.01% 0.03% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 99.99% 99.98% 0.16% 100.00% 99.97% 0.13% 99.97% 99.97% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.00% 0.01% 99.79% 0.00% 0.00% 99.80% 0.01% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.02% 0.01% 0.01% 0.01% 0.02% 0.00% 0.02% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.00% 0.00% 0.03% 0.00% 0.00% 0.05% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 99.97% 99.98% 0.17% 99.99% 99.98% 0.15% 99.95% 99.98% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.01% 0.00% 99.79% 0.01% 0.02% 99.86% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.01% 0.00% 0.00% 0.02% 0.01% 0.01% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.04% 0.00% 0.00% 0.02% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 99.98% 99.99% 0.16% 99.98% 99.97% 0.10% 99.97% 99.98% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.02% 99.78% 0.01% 0.01% 99.84% 0.01% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.00% 0.02% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.00% 0.00% 0.05% 0.00% 0.00% 0.06% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 99.98% 99.97% 0.16% 99.98% 99.98% 0.09% 99.99% 100.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.02% 0.01% 99.81% 0.01% 0.01% 99.85% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.00% 0.00% 0.02% 0.02% 0.01% 0.01% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.00% 0.00% 0.02% 0.00% 0.00% 0.03% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 99.97% 99.99% 0.17% 99.98% 99.97% 0.11% 99.98% 99.99% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.00% 0.01% 99.71% 0.00% 0.00% 99.76% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.01% 0.01% 0.03% 0.01% 0.01% 0.01% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.01% 0.00% 0.06% 0.01% 0.00% 0.08% 0.00% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 99.99% 99.98% 0.22% 99.96% 99.98% 0.15% 99.98% 99.96% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.01% 0.01% 99.79% 0.00% 0.00% 99.81% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.00% 0.02% 0.01% 0.00% 0.00% 0.01% 0.02% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.00% 0.00% 0.04% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 99.98% 99.97% 0.15% 99.99% 99.99% 0.14% 99.97% 99.98% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.00% 99.74% 0.01% 0.01% 99.82% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.00% 0.00% 0.04% 0.00% 0.00% 0.04% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 99.99% 99.98% 0.20% 99.97% 99.97% 0.13% 99.98% 99.99% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide A A G G G C C nogRNA-A1343spilt-KKH- A 99.87% 99.97% 0.01% 0.01% 0.01% 0.03% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.02% 0.00% 0.01% 0.01% 0.00% 0.05% 0.05% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.00% 0.00% 0.00% 0.00% 0.01% 99.88% 99.90% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.11% 0.02% 99.98% 99.98% 99.98% 0.03% 0.02% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 99.80% 99.89% 0.03% 0.00% 0.01% 0.03% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 0.01% 0.00% 0.01% 0.04% 0.07% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.05% 0.06% 0.00% 0.00% 0.00% 99.91% 99.90% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.13% 0.04% 99.95% 99.99% 99.98% 0.03% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 99.88% 99.95% 0.03% 0.00% 0.00% 0.01% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.00% 0.02% 0.00% 0.01% 0.00% 0.03% 0.09% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.02% 0.01% 0.00% 0.00% 0.00% 99.93% 99.86% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.09% 0.01% 99.97% 99.99% 99.99% 0.03% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 99.77% 99.92% 0.02% 0.00% 0.01% 0.03% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.02% 0.01% 0.04% 0.01% 0.00% 0.05% 0.10% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.03% 0.01% 0.00% 0.00% 0.00% 99.88% 99.86% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.17% 0.05% 99.94% 99.99% 99.99% 0.05% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 99.80% 99.91% 0.03% 0.01% 0.00% 0.02% 0.03% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.02% 0.01% 0.01% 0.00% 0.02% 0.03% 0.06% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.00% 0.02% 0.00% 0.00% 0.00% 99.90% 99.88% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.18% 0.06% 99.96% 99.98% 99.97% 0.05% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 99.83% 99.96% 0.00% 0.02% 0.00% 0.01% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.03% 0.01% 0.01% 0.00% 0.00% 0.04% 0.09% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.01% 0.01% 0.01% 0.00% 0.00% 99.92% 99.88% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.13% 0.02% 99.98% 99.97% 99.99% 0.03% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 99.82% 99.94% 0.02% 0.02% 0.00% 0.02% 0.01% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.00% 0.05% 0.08% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.02% 0.01% 0.00% 0.00% 0.00% 99.89% 99.87% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.14% 0.04% 99.97% 99.98% 99.98% 0.03% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.02% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 99.83% 99.92% 0.02% 0.02% 0.00% 0.02% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.01% 0.00% 0.01% 0.06% 0.09% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.01% 0.02% 0.00% 0.01% 0.00% 99.88% 99.89% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.14% 0.05% 99.96% 99.97% 99.99% 0.03% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 99.83% 99.93% 0.02% 0.02% 0.01% 0.05% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.01% 0.00% 0.00% 0.00% 0.00% 0.06% 0.09% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.02% 0.01% 0.00% 0.00% 0.01% 99.87% 99.87% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.13% 0.05% 99.98% 99.97% 99.98% 0.02% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 99.78% 99.95% 0.01% 0.01% 0.01% 0.02% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.02% 0.00% 0.02% 0.00% 0.00% 0.05% 0.08% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.02% 0.01% 0.00% 0.00% 0.00% 99.89% 99.86% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.18% 0.03% 99.98% 99.99% 99.99% 0.03% 0.02% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 99.82% 99.94% 0.02% 0.01% 0.01% 0.02% 0.03% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.02% 0.01% 0.01% 0.01% 0.01% 0.05% 0.11% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.02% 0.01% 0.00% 0.00% 0.00% 99.90% 99.84% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.15% 0.05% 99.96% 99.98% 99.97% 0.02% 0.01% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 99.65% 99.92% 0.02% 0.00% 0.01% 0.03% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.01% 0.03% 0.13% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.07% 0.04% 0.00% 0.00% 0.00% 99.92% 99.84% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.26% 0.04% 99.98% 100.00% 99.98% 0.03% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 99.79% 99.93% 0.02% 0.01% 0.00% 0.03% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.00% 0.01% 0.00% 0.00% 0.03% 0.16% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.04% 0.00% 0.00% 0.00% 0.00% 99.92% 99.78% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.17% 0.06% 99.97% 99.99% 100.00% 0.03% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 99.79% 99.93% 0.02% 0.01% 0.00% 0.03% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.02% 0.00% 0.02% 0.00% 0.00% 0.05% 0.05% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.01% 0.01% 0.00% 0.00% 0.00% 99.89% 99.91% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.17% 0.04% 99.96% 99.99% 99.99% 0.04% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide T G A G T C C G nogRNA-A1343spilt-KKH- A 0.02% 0.03% 99.90% 0.01% 0.02% 0.02% 0.02% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 99.88% 0.01% 0.00% 0.00% 99.83% 0.06% 0.05% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.05% 0.00% 0.02% 0.00% 0.05% 99.92% 99.81% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.05% 99.96% 0.07% 99.95% 0.04% 0.00% 0.03% 99.92% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.04% 0.07% 0.00% 0.09% 0.05% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 0.01% 0.01% 99.79% 0.00% 0.00% 0.02% 0.02% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 99.92% 0.02% 0.01% 0.00% 99.82% 0.04% 0.08% 0.01% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.03% 0.00% 0.07% 0.00% 0.05% 99.93% 99.75% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.04% 99.96% 0.12% 99.97% 0.03% 0.01% 0.03% 99.90% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.09% 0.00% 0.13% 0.06% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 0.00% 0.01% 99.87% 0.01% 0.02% 0.01% 0.02% 0.02% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 99.93% 0.01% 0.00% 0.00% 99.83% 0.04% 0.08% 0.01% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.03% 0.00% 0.03% 0.00% 0.07% 99.95% 99.79% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.03% 99.97% 0.10% 99.96% 0.05% 0.00% 0.02% 99.89% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.00% 0.03% 0.03% 0.00% 0.10% 0.08% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 0.01% 0.03% 99.85% 0.00% 0.03% 0.01% 0.01% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 99.87% 0.03% 0.00% 0.00% 99.81% 0.06% 0.05% 0.01% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.07% 0.00% 0.03% 0.00% 0.05% 99.92% 99.83% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.04% 99.94% 0.11% 99.97% 0.06% 0.00% 0.02% 99.89% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.00% 0.00% 0.01% 0.02% 0.05% 0.00% 0.09% 0.09% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 0.01% 0.01% 99.87% 0.01% 0.03% 0.01% 0.00% 0.01% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 99.94% 0.00% 0.00% 0.00% 99.85% 0.04% 0.03% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.02% 0.00% 0.03% 0.00% 0.04% 99.94% 99.87% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.02% 99.99% 0.09% 99.96% 0.04% 0.01% 0.00% 99.91% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.01% 0.03% 0.04% 0.00% 0.09% 0.07% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 0.00% 0.01% 99.87% 0.00% 0.00% 0.00% 0.00% 0.02% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 99.91% 0.00% 0.00% 0.01% 99.85% 0.06% 0.02% 0.01% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.05% 0.00% 0.04% 0.00% 0.07% 99.92% 99.86% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.04% 99.99% 0.08% 99.92% 0.04% 0.01% 0.01% 99.90% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.00% 0.00% 0.00% 0.06% 0.04% 0.00% 0.10% 0.06% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 0.00% 0.03% 99.86% 0.02% 0.02% 0.01% 0.02% 0.03% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 99.92% 0.00% 0.00% 0.01% 99.84% 0.04% 0.05% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.04% 0.00% 0.03% 0.00% 0.06% 99.94% 99.83% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.03% 99.97% 0.11% 99.95% 0.04% 0.00% 0.00% 99.91% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.02% 0.05% 0.01% 0.11% 0.06% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 0.01% 0.02% 99.85% 0.00% 0.01% 0.02% 0.01% 0.02% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 99.91% 0.02% 0.01% 0.01% 99.82% 0.05% 0.04% 0.01% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.04% 0.00% 0.04% 0.00% 0.06% 99.92% 99.85% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.05% 99.95% 0.11% 99.98% 0.05% 0.00% 0.02% 99.91% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.00% 0.00% 0.00% 0.01% 0.06% 0.00% 0.08% 0.07% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 0.01% 0.01% 99.84% 0.01% 0.00% 0.02% 0.00% 0.02% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 99.91% 0.02% 0.01% 0.00% 99.87% 0.05% 0.07% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.05% 0.00% 0.03% 0.00% 0.04% 99.92% 99.83% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.02% 99.96% 0.12% 99.96% 0.03% 0.01% 0.02% 99.90% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.03% 0.05% 0.00% 0.08% 0.09% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 0.01% 0.01% 99.87% 0.00% 0.02% 0.02% 0.02% 0.03% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 99.90% 0.01% 0.00% 0.00% 99.85% 0.03% 0.06% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.05% 0.00% 0.04% 0.00% 0.05% 99.94% 99.79% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.02% 99.98% 0.08% 99.97% 0.04% 0.01% 0.03% 99.88% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.00% 0.00% 0.00% 0.03% 0.04% 0.00% 0.10% 0.09% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 0.02% 0.03% 99.84% 0.01% 0.01% 0.01% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 99.91% 0.01% 0.01% 0.00% 99.87% 0.05% 0.06% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.03% 0.00% 0.04% 0.00% 0.04% 99.94% 99.84% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.03% 99.96% 0.11% 99.96% 0.04% 0.01% 0.02% 99.89% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.01% 0.03% 0.03% 0.00% 0.08% 0.09% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 0.01% 0.01% 99.80% 0.01% 0.02% 0.02% 0.02% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 99.91% 0.01% 0.01% 0.01% 99.87% 0.02% 0.06% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.03% 0.00% 0.06% 0.00% 0.04% 99.96% 99.80% 0.01% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.04% 99.99% 0.13% 99.97% 0.03% 0.01% 0.02% 99.86% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.00% 0.00% 0.00% 0.02% 0.05% 0.01% 0.11% 0.10% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 0.00% 0.01% 99.82% 0.00% 0.02% 0.03% 0.03% 0.02% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 99.88% 0.00% 0.01% 0.00% 99.82% 0.04% 0.07% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.07% 0.00% 0.03% 0.00% 0.08% 99.94% 99.77% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.04% 99.98% 0.12% 99.96% 0.04% 0.00% 0.04% 99.89% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.01% 0.00% 0.01% 0.03% 0.04% 0.00% 0.09% 0.09% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 0.00% 0.03% 99.86% 0.01% 0.01% 0.01% 0.00% 0.01% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 99.92% 0.01% 0.00% 0.01% 99.80% 0.05% 0.08% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.04% 0.00% 0.02% 0.00% 0.06% 99.93% 99.76% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.03% 99.96% 0.12% 99.96% 0.08% 0.00% 0.02% 99.85% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.00% 0.00% 0.00% 0.02% 0.05% 0.00% 0.14% 0.13% Cas9-N1387-C and dSpCas9-N1387-N Batch Nucleotide A G C A G A A G nogRNA-A1343spilt-KKH- A 99.85% 0.00% 0.02% 2.31% 0.10% 2.16% 0.01% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- T 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- C 0.03% 0.00% 99.95% 0.05% 0.01% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- G 0.07% 99.96% 0.00% 0.10% 2.34% 0.06% 0.00% 0.01% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- 0.03% 0.03% 0.00% 97.53% 97.54% 97.77% 99.99% 99.99% Cas9-A1343-N and dSpCas9-A1343-C nogRNA-A1343spilt-KKH- A 99.78% 0.01% 0.02% 2.52% 0.09% 2.38% 0.02% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- T 0.00% 0.01% 0.04% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- C 0.07% 0.00% 99.88% 0.08% 0.01% 0.06% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- G 0.11% 99.93% 0.02% 0.09% 2.57% 0.05% 0.00% 0.02% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-A1343spilt-KKH- 0.04% 0.05% 0.04% 97.29% 97.33% 97.51% 99.98% 99.98% Cas9-A1343-C and dSpCas9-A1343-N nogRNA-G1322spilt-KKH- A 99.86% 0.01% 0.03% 2.26% 0.09% 2.15% 0.03% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- T 0.01% 0.00% 0.04% 0.02% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- C 0.02% 0.00% 99.88% 0.04% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- G 0.09% 99.94% 0.03% 0.12% 2.32% 0.03% 0.00% 0.03% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- 0.02% 0.04% 0.02% 97.56% 97.59% 97.82% 99.97% 99.97% Cas9-G1322-N and dSpCas9-G1322-C nogRNA-G1322spilt-KKH- A 99.84% 0.02% 0.03% 2.49% 0.09% 2.32% 0.03% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- T 0.01% 0.01% 0.03% 0.02% 0.02% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- C 0.04% 0.00% 99.90% 0.06% 0.00% 0.02% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- G 0.08% 99.93% 0.00% 0.10% 2.54% 0.09% 0.00% 0.02% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1322spilt-KKH- 0.03% 0.04% 0.05% 97.33% 97.35% 97.58% 99.97% 99.98% Cas9-G1322-C and dSpCas9-G1322-N nogRNA-G1333spilt-KKH- A 99.84% 0.02% 0.05% 2.70% 0.12% 2.52% 0.02% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- T 0.01% 0.00% 0.03% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- C 0.06% 0.00% 99.88% 0.02% 0.00% 0.01% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- G 0.07% 99.92% 0.02% 0.09% 2.67% 0.06% 0.00% 0.02% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- 0.02% 0.06% 0.02% 97.19% 97.21% 97.40% 99.98% 99.98% Cas9-G1333-N and dSpCas9-G1333-C nogRNA-G1333spilt-KKH- A 99.84% 0.02% 0.01% 2.75% 0.10% 2.61% 0.06% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- T 0.02% 0.00% 0.04% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- C 0.05% 0.00% 99.89% 0.03% 0.00% 0.01% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- G 0.08% 99.95% 0.01% 0.10% 2.77% 0.05% 0.00% 0.05% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1333spilt-KKH- 0.02% 0.03% 0.04% 97.12% 97.13% 97.33% 99.94% 99.95% Cas9-G1333-C and dSpCas9-G1333-N nogRNA-G1371spilt-KKH- A 99.84% 0.00% 0.03% 2.35% 0.10% 2.22% 0.06% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- T 0.02% 0.00% 0.03% 0.01% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- C 0.02% 0.00% 99.87% 0.05% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- G 0.07% 99.95% 0.03% 0.11% 2.38% 0.06% 0.00% 0.06% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- 0.04% 0.05% 0.04% 97.48% 97.51% 97.72% 99.94% 99.94% Cas9-G1371-N and dSpCas9-G1371-C nogRNA-G1371spilt-KKH- A 99.85% 0.01% 0.04% 2.43% 0.08% 2.27% 0.05% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- T 0.01% 0.01% 0.07% 0.01% 0.01% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- C 0.01% 0.00% 99.84% 0.04% 0.00% 0.01% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- G 0.09% 99.93% 0.03% 0.10% 2.45% 0.05% 0.00% 0.06% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1371spilt-KKH- 0.03% 0.05% 0.02% 97.43% 97.46% 97.66% 99.94% 99.94% Cas9-G1371-C and dSpCas9-G1371-N nogRNA-G1397spilt-KKH- A 99.87% 0.01% 0.02% 2.85% 0.08% 2.71% 0.36% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- T 0.00% 0.00% 0.04% 0.02% 0.01% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- C 0.02% 0.00% 99.91% 0.03% 0.00% 0.02% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- G 0.09% 99.94% 0.02% 0.09% 2.87% 0.05% 0.00% 0.36% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- 0.01% 0.05% 0.01% 97.01% 97.04% 97.23% 99.64% 99.64% Cas9-G1397-N and dSpCas9-G1397-C nogRNA-G1397spilt-KKH- A 99.82% 0.01% 0.02% 2.72% 0.10% 2.53% 0.05% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- T 0.01% 0.00% 0.02% 0.01% 0.01% 0.01% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- C 0.05% 0.00% 99.90% 0.04% 0.00% 0.02% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- G 0.07% 99.93% 0.02% 0.09% 2.72% 0.08% 0.00% 0.05% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-G1397spilt-KKH- 0.05% 0.06% 0.04% 97.14% 97.17% 97.35% 99.95% 99.95% Cas9-G1397-C and dSpCas9-G1397-N nogRNA-N1357spilt-KKH- A 99.86% 0.00% 0.03% 2.50% 0.10% 2.35% 0.02% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- T 0.01% 0.01% 0.05% 0.01% 0.00% 0.01% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- C 0.03% 0.00% 99.87% 0.05% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- G 0.09% 99.95% 0.01% 0.13% 2.57% 0.05% 0.00% 0.02% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- 0.02% 0.03% 0.03% 97.31% 97.33% 97.59% 99.98% 99.98% Cas9-N1357-N and dSpCas9-N1357-C nogRNA-N1357spilt-KKH- A 99.78% 0.03% 0.06% 2.41% 0.06% 2.29% 0.02% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- T 0.01% 0.01% 0.03% 0.02% 0.01% 0.01% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- C 0.07% 0.01% 99.86% 0.06% 0.00% 0.05% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- G 0.08% 99.90% 0.03% 0.05% 2.45% 0.04% 0.00% 0.02% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1357spilt-KKH- 0.06% 0.06% 0.02% 97.46% 97.48% 97.62% 99.98% 99.98% Cas9-N1357-C and dSpCas9-N1357-N nogRNA-N1387spilt-KKH- A 99.82% 0.01% 0.04% 2.60% 0.08% 2.46% 0.06% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- T 0.01% 0.03% 0.03% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- C 0.06% 0.00% 99.91% 0.03% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- G 0.08% 99.92% 0.01% 0.12% 2.64% 0.05% 0.00% 0.06% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- 0.03% 0.04% 0.01% 97.24% 97.27% 97.48% 99.94% 99.94% Cas9-N1387-N and dSpCas9-N1387-C nogRNA-N1387spilt-KKH- A 99.82% 0.02% 0.02% 2.58% 0.13% 2.37% 0.05% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- T 0.01% 0.00% 0.04% 0.00% 0.01% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- C 0.04% 0.00% 99.85% 0.03% 0.00% 0.01% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- G 0.11% 99.93% 0.02% 0.13% 2.57% 0.08% 0.00% 0.05% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Cas9-N1387-C and dSpCas9-N1387-N nogRNA-N1387spilt-KKH- 0.02% 0.04% 0.06% 97.27% 97.29% 97.53% 99.94% 99.95% Cas9-N1387-C and dSpCas9-N1387-N
TABLE-US-00066 Sample Reads_aligned Insertions Deletions indel % ND5.1-DdCBE_replicate1 27776 0 4 0.014400922 ND5.1-DdCBE_replicate2 35682 0 13 0.036432935 ND5.1-DdCBE_replicate3 38221 0 7 0.018314539 ND5.2-DdCBE_replicate1 75163 0 33 0.043904581 ND5.2-DdCBE_replicate2 3150 0 1 0.031746032 ND5.2-DdCBE_replicate3 100007 0 37 0.03699741 ND5.3-DdCBE_replicate1 55241 0 10 0.018102496 ND5.3-DdCBE_replicate2 64264 0 19 0.029565542 ND5.3-DdCBE_replicate3 75963 0 18 0.023695747 ND4-DdCBE_replicate1 96693 0 15 0.015513015 ND4-DdCBE_replicate2 34406 0 9 0.026158228 ND4-DdCBE_replicate3 67979 1 6 0.010297298 ND2-DdCBE_replicate1 38026 0 8 0.021038237 ND2-DdCBE_replicate2 42224 0 5 0.011841607 ND2-DdCBE_replicate3 38272 0 7 0.018290134 ND1-DdCBE_replicate1 3412 0 1 0.029308324 ND1-DdCBE_replicate2 71984 0 13 0.018059569 ND1-DdCBE_replicate3 27370 0 4 0.014614541 BMATP8mito55mito56replicate1 46836 0 5 0.010675549 BMATP8mito55mito56replicate2 47180 0 12 0.025434506 BMATP8mito55mito56replicate3 50303 0 27 0.053674731 untreated-day3_replicate1 50677 0 17 0.03354579 untreated-day3_replicate2 81585 0 35 0.042900043 untreated-day3_replicate3 70197 0 20 0.028491246 untreated-day6_replicate1 96647 0 6 0.00620816 untreated-day6_replicate2 35744 0 4 0.011190689 untreated-day6_replicate3 57272 0 5 0.00873027
TABLE-US-00067 TABLE 12A Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide T C A C C A A G A C C T ND6- A 0.01% 0.01% 99.94% 0.01% 0.01% 99.94% 99.93% 0.09% 99.95% 0.02% 0.01% 0.01% DdCBE_ replicate1 ND6- T 99.92% 0.23% 0.01% 0.03% 0.02% 0.01% 0.00% 0.02% 0.00% 0.03% 0.01% 99.96% DdCBE_ replicate1 ND6- C 0.07% 99.76% 0.04% 99.94% 99.96% 0.03% 0.04% 0.00% 0.03% 99.95% 99.99% 0.02% DdCBE_ replicate1 ND6- G 0.00% 0.00% 0.02% 0.01% 0.01% 0.02% 0.03% 99.89% 0.02% 0.00% 0.00% 0.01% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- A 0.01% 0.01% 99.95% 0.01% 0.00% 99.96% 99.94% 0.15% 99.95% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND6- T 99.90% 0.21% 0.01% 0.02% 0.02% 0.01% 0.00% 0.02% 0.02% 0.03% 0.00% 99.99% DdCBE_ replicate2 ND6- C 0.09% 99.78% 0.03% 99.97% 99.97% 0.03% 0.05% 0.00% 0.02% 99.97% 99.99% 0.01% DdCBE_ replicate2 ND6- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 99.83% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 0.01% 0.00% 99.9% 0.01% 0.02% 99.95% 99.95% 0.13% 99.98% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND6- T 99.87% 0.17% 0.01% 0.02% 0.01% 0.01% 0.00% 0.02% 0.00% 0.02% 0.01% 99.98% DdCBE_ replicate3 ND6- C 0.11% 99.82% 0.03% 99.97% 99.96% 0.03% 0.04% 0.00% 0.01% 99.97% 99.99% 0.01% DdCBE_ replicate3 ND6- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 99.85% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide C A A C C C C T G A C C ND6- A 0.01% 99.96% 99.95% 0.01% 0.02% 0.01% 0.01% 0.01% 0.11% 99.98% 0.01% 0.01% DdCBE_ replicate1 ND6- T 0.25% 0.00% 0.00% 0.01% 0.01% 0.00% 0.01% 99.98% 0.02% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND6- C 99.74% 0.03% 0.03% 99.98% 99.97% 99.99% 99.98% 0.02% 0.00% 0.00% 99.98% 99.98% DdCBE_ replicate1 ND6- G 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 99.87% 0.01% 0.00% 0.00% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- A 0.00% 99.96% 99.96% 0.00% 0.01% 0.01% 0.01% 0.01% 0.14% 99.97% 0.01% 0.00% DdCBE_ replicate2 ND6- T 0.19% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND6- C 99.80% 0.02% 0.02% 99.99% 99.98% 99.98% 99.98% 0.02% 0.00% 0.01% 99.98% 99.98% DdCBE_ replicate2 ND6- G 0.00% 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 99.84% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 0.00% 99.97% 99.96% 0.01% 0.01% 0.01% 0.01% 0.00% 0.07% 99.98% 0.01% 0.00% DdCBE_ replicate3 ND6- T 0.18% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND6- C 99.81% 0.02% 0.02% 99.98% 99.98% 99.99% 99.98% 0.02% 0.00% 0.01% 99.98% 99.99% DdCBE_ replicate3 ND6- G 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 99.92% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide C C C A T G C C T C A G ND6- A 0.01% 0.02% 0.00% 99.97% 0.00% 0.02% 0.01% 0.00% 0.01% 0.00% 99.96% 30.47% DdCBE_ replicate1 ND6- T 0.01% 0.02% 0.01% 0.00% 99.99% 0.00% 0.03% 0.00% 99.98% 0.17% 0.00% 0.01% DdCBE_ replicate1 ND6- C 99.98% 99.96% 99.97% 0.01% 0.01% 0.01% 99.96% 99.99% 0.01% 99.82% 0.02% 0.03% DdCBE_ replicate1 ND6- G 0.01% 0.00% 0.00% 0.01% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% 0.02% 69.49% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate1 ND6- A 0.00% 0.03% 0.00% 99.95% 0.01% 0.01% 0.03% 0.00% 0.01% 0.00% 99.97% 29.83% DdCBE_ replicate2 ND6- T 0.01% 0.02% 0.01% 0.00% 99.98% 0.00% 0.01% 0.01% 99.96% 0.20% 0.00% 0.01% DdCBE_ replicate2 ND6- C 99.99% 99.96% 99.97% 0.03% 0.01% 0.01% 99.96% 99.98% 0.02% 99.79% 0.01% 0.04% DdCBE_ replicate2 ND6- G 0.00% 0.00% 0.00% 0.01% 0.00% 99.97% 0.00% 0.00% 0.01% 0.00% 0.01% 70.13% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 0.00% 0.02% 0.01% 99.96% 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 99.97% 26.86% DdCBE_ replicate3 ND6- T 0.01% 0.01% 0.01% 0.01% 99.98% 0.01% 0.02% 0.01% 99.96% 0.15% 0.01% 0.01% DdCBE_ replicate3 ND6- C 99.99% 99.97% 99.96% 0.02% 0.01% 0.01% 99.96% 99.99% 0.03% 99.84% 0.01% 0.03% DdCBE_ replicate3 ND6- G 0.00% 0.00% 0.00% 0.01% 0.00% 99.97% 0.00% 0.00% 0.01% 0.00% 0.02% C DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide G A T A C T C C T C A A ND6- A 36.39% 99.91% 0.00% 99.98% 0.02% 0.02% 0.02% 0.01% 0.01% 0.00% 99.97% 99.97% DdCBE_ replicate1 ND6- T 0.01% 0.01% 99.99% 0.01% 5.37% 99.97% 18.62% 0.45% 99.96% 0.11% 0.00% 0.00% DdCBE_ replicate1 ND6- C 0.00% 0.08% 0.01% 0.01% 94.59% 0.01% 81.35% 99.54% 0.03% 99.88% 0.02% 0.02% DdCBE_ replicate1 ND6- G 63.59% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- A 35.45% 99.90% 0.00% 99.96% 0.03% 0.01% 0.02% 0.01% 0.00% 0.00% 99.97% 99.96% DdCBE_ replicate2 ND6- T 0.01% 0.00% 99.99% 0.00% 5.04% 99.97% 17.88% 0.41% 99.97% 0.10% 0.00% 0.01% DdCBE_ replicate2 ND6- C 0.00% 0.07% 0.01% 0.03% 94.92% 0.01% 82.09% 99.58% 0.02% 99.89% 0.02% 0.02% DdCBE_ replicate2 ND6- G 64.53% 0.02% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 31.97% 99.89% 0.01% 99.96% 0.03% 0.02% 0.02% 0.00% 0.01% 0.00% 99.97% 99.96% DdCBE_ replicate3 ND6- T 0.01% 0.01% 99.97% 0.01% 4.50% 99.96% 15.39% 0.34% 99.98% 0.08% 0.00% 0.01% DdCBE_ replicate3 ND6- C 0.00% 0.09% 0.02% 0.03% 95.46% 0.02% 84.59% 99.65% 0.01% 99.91% 0.01% 0.02% DdCBE_ replicate3 ND6- G 68.02% 0.02% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide T A G C C A T C G C T G ND6- A 0.01% 99.95% 0.02% 0.01% 0.02% 99.96% 0.01% 0.07% 0.02% 0.01% 0.00% 0.01% DdCBE_ replicate1 ND6- T 99.97% 0.01% 0.02% 0.01% 0.02% 0.01% 99.98% 0.25% 0.03% 0.01% 99.97% 0.02% DdCBE_ replicate ND6- C 0.01% 0.03% 0.01% 99.99% 99.96% 0.02% 0.01% 99.69% 0.01% 99.98% 0.03% 0.01% DdCBE_ replicate1 ND6- G 0.02% 0.01% 99.96% 0.00% 0.01% 0.01% 0.00% 0.00% 99.94% 0.00% 0.00% 99.97% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- A 0.01% 99.97% 0.03% 0.00% 0.02% 99.98% 0.01% 0.07% 0.01% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND6- T 99.96% 0.01% 0.02% 0.01% 0.02% 0.00% 99.97% 0.23% 0.02% 0.01% 99.97% 0.02% DdCBE_ replicate2 ND6- C 0.01% 0.02% 0.02% 99.98% 99.96% 0.01% 0.02% 99.71% 0.01% 99.98% 0.02% 0.00% DdCBE_ replicate2 ND6- G 0.01% 0.01% 99.94% 0.00% 0.00% 0.01% 0.00% 0.00% 99.96% 0.00% 0.01% 99.97% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 0.01% 99.97% 0.02% 0.01% 0.03% 99.96% 0.01% 0.07% 0.02% 0.00% 0.01% 0.02% DdCBE_ replicate3 ND6- T 99.98% 0.00% 0.02% 0.01% 0.01% 0.01% 99.97% 0.20% 0.02% 0.01% 99.98% 0.02% DdCBE_ replicate3 ND6- C 0.01% 0.02% 0.01% 99.98% 99.96% 0.01% 0.02% 99.72% 0.01% 99.99% 0.01% 0.00% DdCBE_ replicate3 ND6- G 0.01% 0.01% 99.95% 0.00% 0.00% 0.01% 0.00% 0.00% 99.95% 0.00% 0.00% 99.96% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide T A G T A T A T C C A A ND6- A 0.01% 99.95% 0.01% 0.03% 99.97% 0.01% 99.98% 0.02% 0.01% 0.00% 99.97% 99.98% DdCBE_ replicate1 ND6- T 99.86% 0.01% 0.02% 99.74% 0.01% 99.98% 0.02% 99.97% 1.23% 0.02% 0.00% 0.01% DdCBE_ replicate ND6- C 0.12% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 98.75% 99.98% 0.02% 0.00% DdCBE_ replicate1 ND6- G 0.01% 0.02% 99.96% 0.22% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate1 ND6- A 0.02% 99.96% 0.02% 0.03% 99.98% 0.01% 99.98% 0.02% 0.01% 0.01% 99.95% 99.98% DdCBE_ replicate2 ND6- T 99.86% 0.01% 0.02% 99.75% 0.00% 99.98% 0.01% 99.96% 1.21% 0.03% 0.00% 0.01% DdCBE_ replicate2 ND6- C 0.11% 0.01% 0.00% 0.02% 0.01% 0.01% 0.00% 0.02% 98.78% 99.96% 0.02% 0.01% DdCBE_ replicate2 ND6- G 0.01% 0.02% 99.96% 0.20% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 0.01% 99.96% 0.01% 0.02% 99.97% 0.00% 99.98% 0.01% 0.01% 0.01% 99.97% 99.99% DdCBE_ replicate3 ND6- T 99.91% 0.01% 0.03% 99.76% 0.01% 99.99% 0.01% 99.97% 0.98% 0.02% 0.01% 0.00% DdCBE_ replicate3 ND6- C 0.08% 0.02% 0.00% 0.02% 0.01% 0.00% 0.01% 0.01% 99.01% 99.9% 0.01% 0.00% DdCBE_ replicate3 ND6- G 0.00% 0.02% 99.96% 0.21% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND6-DdCBE Batch Nucleotide A G A C A ND6- A 99.98% 0.16% 99.89% 0.01% 99.96% DdCBE_ replicate1 ND6- T 0.01% 0.01% 0.01% 0.02% 0.00% DdCBE_ replicate1 ND6- C 0.01% 0.01% 0.09% 99.97% 0.02% DdCBE_ replicate1 ND6- G 0.00% 99.82% 0.01% 0.00% 0.01% DdCBE_ replicate1 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND6- A 99.98% 0.17% 99.89% 0.01% 99.97% DdCBE_ replicate2 ND6- T 0.00% 0.01% 0.01% 0.01% 0.00% DdCBE_ replicate2 ND6- C 0.01% 0.00% 0.09% 99.98% 0.01% DdCBE_ replicate2 ND6- G 0.00% 99.82% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND6- A 99.95% 0.14% 99.92% 0.01% 99.97% DdCBE_ replicate3 ND6- T 0.01% 0.02% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND6- C 0.02% 0.00% 0.06% 99.98% 0.01% DdCBE_ replicate3 ND6- G 0.01% 99.84% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND6- N 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND6- 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00068 TABLE 12B Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide C T C C C T C A C C A T ND5.1- A 0.01% 0.00% 0.05% 0.02% 0.03% 0.02% 0.04% 99.95% 0.02% 0.02% 99.98% 0.03% DdCBE_ replicate 1 ND5.1- T 0.01% 99.98% 0.14% 0.03% 0.01% 99.96% 0.12% 0.02% 0.02% 0.03% 0.00% 99.97% DdCBE_ replicate1 ND5.1- C 99.97% 0.01% 99.81% 99.95% 99.93% 0.01% 99.84% 0.00% 99.96% 99.95% 0.00% 0.00% DdCBE_ replicate1 ND5.1- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.03% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 0.01% 0.01% 0.06% 0.01% 0.04% 0.01% 0.01% 99.95% 0.01% 0.01% 99.96% 0.01% DdCBE_ replicate2 ND5.1- T 0.01% 99.95% 0.14% 0.03% 0.02% 99.97% 0.14% 0.02% 0.01% 0.03% 0.02% 99.96% DdCBE_ replicate2 ND5.1- C 99.98% 0.04% 99.79% 99.96% 99.92% 0.02% 99.84% 0.01% 99.97% 99.96% 0.00% 0.03% DdCBE_ replicate2 ND5.1- G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 0.01% 0.01% 0.06% 0.01% 0.03% 0.01% 0.06% 99.96% 0.02% 0.01% 99.97% 0.01% DdCBE_ replicate3 ND5.1- T 0.02% 99.96% 0.09% 0.03% 0.02% 99.97% 0.08% 0.02% 0.04% 0.03% 0.02% 99.98% DdCBE_ replicate3 ND5.1- C 99.97% 0.03% 99.85% 99.95% 99.92% 0.02% 99.86% 0.01% 99.94% 99.96% 0.00% 0.01% DdCBE_ replicate3 ND5.1- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide T G G C A G C C T A G C ND5.1- A 0.03% 0.05% 0.02% 0.00% 99.97% 0.15% 0.01% 0.01% 0.01% 99.98% 0.01% 0.01% DdCBE_ replicate 1 ND5.1- T 99.96% 0.03% 0.03% 0.04% 0.00% 0.01% 0.03% 0.01% 99.99% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND5.1- C 0.01% 0.01% 0.00% 99.95% 0.00% 0.00% 99.95% 99.98% 0.01% 0.00% 0.00% 99.97% DdCBE_ replicate1 ND5.1- G 0.00% 99.91% 99.9% 0.00% 0.02% 99.83% 0.01% 0.00% 0.00% 0.01% 99.97% 0.00% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 0.01% 0.04% 0.02% 0.02% 99.95% 0.13% 0.01% 0.01% 0.01% 99.96% 0.01% 0.02% DdCBE_ replicate2 ND5.1- T 99.99% 0.04% 0.02% 0.05% 0.02% 0.03% 0.01% 0.01% 99.99% 0.02% 0.02% 0.02% DdCBE_ replicate2 ND5.1- C 0.00% 0.01% 0.00% 99.92% 0.00% 0.00% 99.97% 99.97% 0.01% 0.00% 0.00% 99.97% DdCBE_ replicate2 ND5.1- G 0.00% 99.91% 99.96% 0.00% 0.03% 99.84% 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 0.03% 0.05% 0.02% 0.01% 99.93% 0.18% 0.01% 0.02% 0.01% 99.96% 0.01% 0.02% DdCBE_ replicate3 ND5.1- T 99.94% 0.03% 0.04% 0.08% 0.03% 0.02% 0.02% 0.01% 99.96% 0.02% 0.01% 0.02% DdCBE_ replicate3 ND5.1- C 0.03% 0.00% 0.00% 99.90% 0.00% 0.00% 99.97% 99.97% 0.02% 0.00% 0.01% 99.96% DdCBE_ replicate3 ND5.1- G 0.00% 99.92% 99.93% 0.01% 0.04% 99.80% 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide A T T A G C A G G A A T ND5.1- A 99.97% 0.00% 0.00% 99.82% 0.01% 0.02% 99.97% 0.02% 0.05% 99.97% 99.97% 0.01% DdCBE_ replicate 1 ND5.1- T 0.01% 99.98% 99.96% 0.01% 0.03% 0.03% 0.02% 0.04% 0.02% 0.02% 0.01% 99.99% DdCBE_ replicate1 ND5.1- C 0.01% 0.02% 0.02% 0.00% 0.00% 99.95% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.1- G 0.01% 0.00% 0.00% 0.15% 99.95% 0.00% 0.01% 99.94% 99.93% 0.01% 0.02% 0.00% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 99.96% 0.01% 0.00% 99.87% 0.03% 0.01% 99.96% 0.01% 0.03% 99.98% 99.97% 0.01% DdCBE_ replicate2 ND5.1- T 0.03% 99.98% 99.98% 0.01% 0.03% 0.02% 0.02% 0.03% 0.03% 0.00% 0.01% 99.97% DdCBE_ replicate2 ND5.1- C 0.00% 0.01% 0.02% 0.00% 0.00% 99.97% 0.01% 0.01% 0.00% 0.01% 0.00% 0.02% DdCBE_ replicate2 ND5.1- G 0.01% 0.00% 0.00% 0.12% 99.94% 0.01% 0.02% 99.95% 99.94% 0.01% 0.02% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 99.98% 0.01% 0.01% 99.79% 0.02% 0.01% 99.94% 0.01% 0.03% 99.94% 99.95% 0.01% DdCBE_ replicate3 ND5.1- T 0.01% 99.99% 99.97% 0.02% 0.05% 0.03% 0.02% 0.04% 0.04% 0.04% 0.02% 99.97% DdCBE_ replicate3 ND5.1- C 0.00% 0.01% 0.02% 0.00% 0.01% 99.96% 0.01% 0.00% 0.01% 0.01% 0.00% 0.02% DdCBE_ replicate3 ND5.1- G 0.02% 0.00% 0.00% 0.18% 99.92% 0.00% 0.03% 99.95% 99.92% 0.02% 0.03% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide A C C T T T C C T C A C ND5.1- A 99.96% 0.00% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.02% 99.97% 0.01% DdCBE_ replicate 1 ND5.1- T 0.03% 0.03% 0.01% 99.81% 99.97% 99.97% 0.16% 0.05% 99.98% 47.95% 0.01% 1.14% DdCBE_ replicate1 ND5.1- C 0.00% 99.96% 99.97% 0.18% 0.03% 0.03% 99.82% 99.94% 0.01% 52.03% 0.00% 98.85% DdCBE_ replicate1 ND5.1- G 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 99.94% 0.00% 0.01% 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 99.98% 0.02% DdCBE_ replicate2 ND5.1- T 0.03% 0.03% 0.02% 99.84% 99.97% 99.94% 0.24% 0.04% 99.96% 47.91% 0.00% 1.36% DdCBE_ replicate2 ND5.1- C 0.00% 99.97% 99.97% 0.13% 0.01% 0.03% 99.73% 99.94% 0.02% 52.07% 0.00% 98.61% DdCBE_ replicate2 ND5.1- G 0.03% 0.00% 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND5.1- A 99.95% 0.01% 0.02% 0.01% 0.01% 0.02% 0.01% 0.02% 0.01% 0.02% 99.95% 0.02% DdCBE_ replicate3 ND5.1- T 0.02% 0.03% 0.02% 99.80% 99.96% 99.96% 0.13% 0.06% 99.97% 43.61% 0.01% 1.03% DdCBE_ replicate3 ND5.1- C 0.01% 99.96% 99.96% 0.19% 0.03% 0.02% 99.86% 99.92% 0.02% 56.36% 0.01% 98.95% DdCBE_ replicate3 ND5.1- G 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide A G G T T T C T A C T C ND5.1- A 99.97% 0.00% 0.02% 0.02% 0.00% 0.00% 0.01% 0.01% 99.95% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.1- T 0.01% 0.02% 0.01% 99.82% 99.97% 99.99% 0.03% 99.98% 0.02% 0.04% 99.97% 0.03% DdCBE_ replicate1 ND5.1- C 0.00% 0.00% 0.00% 0.09% 0.02% 0.00% 99.96% 0.01% 0.01% 99.95% 0.01% 99.95% DdCBE_ replicate1 ND5.1- G 0.02% 99.98% 99.96% 0.07% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 99.96% 0.00% 0.01% 0.02% 0.00% 0.01% 0.02% 0.01% 99.97% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND5.1- T 0.01% 0.03% 0.02% 99.79% 99.97% 99.97% 0.02% 99.98% 0.02% 0.02% 99.96% 0.02% DdCBE_ replicate2 ND5.1- C 0.01% 0.00% 0.00% 0.12% 0.02% 0.01% 99.96% 0.01% 0.00% 99.97% 0.04% 99.96% DdCBE_ replicate2 ND5.1- G 0.02% 99.97% 99.97% 0.08% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 99.96% 0.01% 0.01% 0.02% 0.01% 0.02% 0.01% 0.01% 99.96% 0.01% 0.02% 0.02% DdCBE_ replicate3 ND5.1- T 0.02% 0.02% 0.00% 99.81% 99.98% 99.96% 0.03% 99.96% 0.02% 0.02% 99.96% 0.02% DdCBE_ replicate3 ND5.1- C 0.00% 0.00% 0.00% 0.12% 0.01% 0.02% 99.96% 0.02% 0.01% 99.96% 0.02% 99.96% DdCBE_ replicate3 ND5.1- G 0.02% 99.97% 99.99% 0.05% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide C A A A G A C C A C A T C ND5.1- A 0.08% 99.95% 99.98% 99.94% 0.03% 99.98% 0.02% 0.03% 99.95% 0.01% 99.98% 0.03% 0.03% DdCBE_ replicate 1 ND5.1- T 0.03% 0.01% 0.01% 0.00% 0.04% 0.01% 0.02% 0.02% 0.01% 0.03% 0.01% 99.95% 0.14% DdCBE_ replicate1 ND5.1- C 99.87% 0.01% 0.00% 0.01% 0.00% 0.00% 99.96% 99.95% 0.01% 99.96% 0.00% 0.02% 99.82% DdCBE_ replicate1 ND5.1- G 0.01% 0.03% 0.01% 0.03% 99.94% 0.01% 0.00% 0.00% 0.03% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 0.09% 99.94% 99.98% 99.93% 0.02% 99.97% 0.01% 0.03% 99.96% 0.01% 99.97% 0.02% 0.02% DdCBE_ replicate2 ND5.1- T 0.03% 0.01% 0.01% 0.00% 0.03% 0.01% 0.01% 0.01% 0.03% 0.02% 0.01% 99.96% 0.17% DdCBE_ replicate2 ND5.1- C 99.87% 0.03% 0.00% 0.00% 0.01% 0.01% 99.98% 99.96% 0.01% 99.96% 0.00% 0.01% 99.81% DdCBE_ replicate2 ND5.1- G 0.01% 0.02% 0.01% 0.03% 99.94% 0.01% 0.00% 0.01% 0.01% 0.00% 0.02% 0.00% 0.00% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.01% 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 0.09% 99.96% 99.99% 99.94% 0.03% 99.96% 0.00% 0.04% 99.96% 0.02% 99.97% 0.02% 0.02% DdCBE_ replicate3 ND5.1- T 0.03% 0.00% 0.00% 0.01% 0.01% 0.03% 0.01% 0.02% 0.03% 0.01% 0.01% 99.96% 0.11% DdCBE_ replicate3 ND5.1- C 99.87% 0.02% 0.00% 0.01% 0.01% 0.01% 99.99% 99.93% 0.00% 99.97% 0.01% 0.02% 99.87% DdCBE_ replicate3 ND5.1- G 0.01% 0.02% 0.01% 0.03% 99.95% 0.01% 0.00% 0.00% 0.02% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.1-DdCBE Nu- cle- Batch otide A T C G A A A C C G C A A ND5.1- A 99.97% 0.01% 0.01% 0.05% 99.95% 99.98% 99.98% 0.01% 0.01% 0.05% 0.01% 99.96% 99.98% DdCBE_ replicate 1 ND5.1- T 0.01% 99.97% 0.12% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.03% 0.03% 0.00% 0.00% DdCBE_ replicate1 ND5.1- C 0.01% 0.02% 99.86% 0.00% 0.00% 0.01% 0.01% 99.97% 99.97% 0.01% 99.96% 0.01% 0.00% DdCBE_ replicate1 ND5.1- G 0.01% 0.00% 0.01% 99.93% 0.03% 0.01% 0.00% 0.00% 0.00% 99.91% 0.00% 0.03% 0.01% DdCBE_ replicate1 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.1- A 99.98% 0.02% 0.01% 0.05% 99.98% 99.97% 99.97% 0.01% 0.02% 0.07% 0.01% 99.97% 99.97% DdCBE_ replicate2 ND5.1- T 0.01% 99.97% 0.12% 0.03% 0.01% 0.00% 0.01% 0.01% 0.02% 0.02% 0.03% 0.01% 0.01% DdCBE_ replicate2 ND5.1- C 0.00% 0.01% 99.86% 0.00% 0.00% 0.01% 0.01% 99.97% 99.96% 0.01% 99.96% 0.01% 0.00% DdCBE_ replicate2 ND5.1- G 0.01% 0.00% 0.01% 99.92% 0.01% 0.02% 0.02% 0.00% 0.00% 99.90% 0.00% 0.01% 0.02% DdCBE_ replicate2 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.1- A 99.96% 0.01% 0.02% 0.07% 99.96% 99.97% 99.95% 0.01% 0.02% 0.04% 0.01% 99.94% 99.97% DdCBE_ replicate3 ND5.1- T 0.02% 99.98% 0.14% 0.02% 0.02% 0.01% 0.01% 0.02% 0.02% 0.02% 0.02% 0.02% 0.01% DdCBE_ replicate3 ND5.1- C 0.00% 0.01% 99.83% 0.00% 0.00% 0.00% 0.03% 99.98% 99.95% 0.01% 99.97% 0.01% 0.01% DdCBE_ replicate3 ND5.1- G 0.02% 0.00% 0.01% 99.90% 0.01% 0.02% 0.01% 0.00% 0.01% 99.93% 0.01% 0.04% 0.01% DdCBE_ replicate3 ND5.1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00069 TABLE 12C Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide T C G A A A A A T A G G ND5.2- A 0.01% 0.08% 0.02% 99.94% 99.97% 99.97% 99.98% 99.96% 0.00% 99.96% 0.02% 0.03% DdCBE_ replicate1 ND5.2- T 99.96% 0.02% 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 99.98% 0.02% 0.03% 0.03% DdCBE_ replicate1 ND5.2- C 0.01% 99.88% 0.00% 0.05% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.00% 0.01% DdCBE_ replicate 1 ND5.2- G 0.01% 0.00% 99.96% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 99.95% 99.92% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 0.00% 0.06% 0.03% 99.94% 99.87% 99.94% 99.90% 99.87% 0.00% 99.94% 0.00% 0.03% DdCBE_ replicate2 ND5.2- T 99.94% 0.00% 0.03% 0.03% 0.00% 0.03% 0.03% 0.00% 99.94% 0.03% 0.03% 0.06% DdCBE_ replicate2 ND5.2- C 0.06% 99.94% 0.00% 0.03% 0.06% 0.03% 0.03% 0.03% 0.06% 0.03% 0.00% 0.03% DdCBE_ replicate2 ND5.2- G 0.00% 0.00% 99.90% 0.00% 0.06% 0.00% 0.03% 0.00% 0.00% 0.00% 99.97% 99.87% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% 0.10% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 0.01% 0.07% 0.01% 99.91% 99.97% 99.97% 99.97% 99.96% 0.00% 99.97% 0.02% 0.02% DdCBE_ replicate3 ND5.2- T 99.97% 0.02% 0.02% 0.00% 0.01% 0.01% 0.01% 0.01% 99.99% 0.01% 0.03% 0.02% DdCBE_ replicate3 ND5.2- C 0.01% 99.90% 0.00% 0.07% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND5.2- G 0.01% 0.00% 99.96% 0.02% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 99.95% 99.95% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide A G G A C T A C T C A A ND5.2- A 99.94% 0.01% 0.03% 99.91% 0.09% 0.01% 99.96% 0.03% 0.01% 0.03% 99.96% 99.96% DdCBE_ replicate1 ND5.2- T 0.01% 0.02% 0.04% 0.01% 0.02% 99.97% 0.02% 0.01% 99.97% 0.02% 0.01% 0.01% DdCBE_ replicate1 ND5.2- C 0.03% 0.00% 0.01% 0.05% 99.89% 0.01% 0.01% 99.96% 0.01% 99.95% 0.02% 0.03% DdCBE_ replicate 1 ND5.2- G 0.02% 99.96% 99.91% 0.03% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 99.84% 0.00% 0.00% 99.87% 0.03% 0.00% 99.90% 0.03% 0.00% 0.00% 99.97% 99.97% DdCBE_ replicate2 ND5.2- T 0.03% 0.03% 0.06% 0.00% 0.00% 99.87% 0.03% 0.06% 99.97% 0.06% 0.00% 0.00% DdCBE_ replicate2 ND5.2- C 0.10% 0.00% 0.00% 0.06% 99.97% 0.03% 0.06% 99.90% 0.03% 99.94% 0.03% 0.03% DdCBE_ replicate2 ND5.2- G 0.03% 99.97% 99.94% 0.06% 0.00% 0.10% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 99.95% 0.01% 0.03% 99.92% 0.09% 0.01% 99.96% 0.04% 0.01% 0.02% 99.96% 99.95% DdCBE_ replicate3 ND5.2- T 0.01% 0.02% 0.03% 0.01% 0.01% 99.96% 0.02% 0.01% 99.98% 0.03% 0.01% 0.01% DdCBE_ replicate3 ND5.2- C 0.03% 0.00% 0.00% 0.05% 99.90% 0.01% 0.02% 99.95% 0.01% 99.94% 0.02% 0.03% DdCBE_ replicate3 ND5.2- G 0.01% 99.97% 99.93% 0.02% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide A A C C A T A C C T C T ND5.2- A 99.94% 99.95% 0.17% 0.45% 99.93% 0.02% 99.91% 0.41% 0.22% 0.03% 0.11% 0.02% DdCBE_ replicate1 ND5.2- T 0.01% 0.02% 0.11% 0.13% 0.01% 99.96% 0.02% 0.61% 0.15% 99.94% 0.03% 99.95% DdCBE_ replicate1 ND5.2- C 0.04% 0.02% 99.71% 99.41% 0.04% 0.02% 0.04% 98.96% 99.61% 0.01% 99.85% 0.02% DdCBE_ replicate 1 ND5.2- G 0.02% 0.01% 0.01% 0.00% 0.02% 0.00% 0.04% 0.02% 0.03% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 99.97% 99.97% 0.16% 0.54% 99.94% 0.00% 99.84% 0.32% 0.10% 0.06% 0.06% 0.06% DdCBE_ replicate2 ND5.2- T 0.00% 0.00% 0.06% 0.10% 0.00% 99.97% 0.03% 0.48% 0.13% 99.90% 0.03% 99.90% DdCBE_ replicate2 ND5.2- C 0.03% 0.03% 99.78% 99.37% 0.03% 0.03% 0.06% 99.21% 99.78% 0.00% 99.90% 0.03% DdCBE_ replicate2 ND5.2- G 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% 0.06% 0.00% 0.00% 0.03% 0.00% 0.00% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 99.95% 99.97% 0.16% 0.40% 99.95% 0.01% 99.94% 0.38% 0.19% 0.04% 0.12% 0.02% DdCBE_ replicate3 ND5.2- T 0.00% 0.01% 0.10% 0.07% 0.01% 99.98% 0.01% 0.46% 0.11% 99.94% 0.03% 99.95% DdCBE_ replicate3 ND5.2- C 0.02% 0.01% 99.74% 99.53% 0.03% 0.01% 0.02% 99.14% 99.67% 0.01% 99.84% 0.02% DdCBE_ replicate3 ND5.2- G 0.02% 0.01% 0.00% 0.00% 0.01% 0.00% 0.02% 0.01% 0.03% 0.00% 0.00% 0.01% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide C A C T T C A A C C T C ND5.2- A 0.04% 99.95% 0.07% 0.01% 0.01% 0.15% 99.95% 99.95% 0.04% 0.04% 0.01% 0.05% DdCBE_ replicate1 ND5.2- T 0.15% 0.01% 0.03% 99.97% 99.97% 0.05% 0.01% 0.01% 0.03% 0.03% 99.97% 7.13% DdCBE_ replicate1 ND5.2- C 99.81% 0.03% 99.89% 0.02% 0.02% 99.78% 0.02% 0.02% 99.85% 99.91% 0.01% 92.81% DdCBE_ replicate 1 ND5.2- G 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% 0.01% 0.08% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 0.00% 99.90% 0.10% 0.00% 0.03% 0.10% 99.94% 99.97% 0.03% 0.03% 0.00% 0.03% DdCBE_ replicate2 ND5.2- T 0.10% 0.00% 0.03% 99.97% 99.90% 0.13% 0.00% 0.00% 0.03% 0.00% 99.97% 6.92% DdCBE_ replicate2 ND5.2- C 99.90% 0.10% 99.87% 0.03% 0.06% 99.78% 0.06% 0.03% 99.84% 99.97% 0.03% 93.05% DdCBE_ replicate2 ND5.2- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.10% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 0.04% 99.95% 0.07% 0.01% 0.01% 0.16% 99.95% 99.95% 0.04% 0.05% 0.02% 0.05% DdCBE_ replicate3 ND5.2- T 0.18% 0.01% 0.02% 99.96% 99.96% 0.04% 0.01% 0.01% 0.03% 0.03% 99.95% 7.69% DdCBE_ replicate3 ND5.2- C 99.77% 0.03% 99.90% 0.02% 0.01% 99.78% 0.01% 0.02% 99.85% 99.91% 0.02% 92.26% DdCBE_ replicate3 ND5.2- G 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.02% 0.01% 0.08% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide C C T C A C C A T T G G ND5.2- A 0.07% 0.04% 0.02% 0.07% 99.97% 0.03% 0.02% 99.97% 0.00% 0.01% 0.03% 0.01% DdCBE_ replicate1 ND5.2- T 5.07% 0.02% 99.95% 0.07% 0.00% 0.01% 0.01% 0.01% 99.98% 99.96% 0.02% 0.01% DdCBE_ replicate1 ND5.2- C 94.85% 99.93% 0.01% 99.85% 0.01% 99.95% 99.96% 0.01% 0.01% 0.01% 0.00% 0.01% DdCBE_ replicate 1 ND5.2- G 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% 99.95% 99.96% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 0.13% 0.00% 0.06% 0.10% 100.00% 0.00% 0.00% 100.00% 0.00% 0.00% 0.03% 0.00% DdCBE_ replicate2 ND5.2- T 4.73% 0.00% 99.90% 0.03% 0.00% 0.00% 0.00% 0.00% 100.00% 99.97% 0.03% 0.03% DdCBE_ replicate2 ND5.2- C 95.14% 100.00% 0.00% 99.84% 0.00% 100.00% 100.00% 0.00% 0.00% 0.03% 0.00% 0.00% DdCBE_ replicate2 ND5.2- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 99.94% 99.97% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.03% 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 0.09% 0.02% 0.02% 0.11% 99.97% 0.05% 0.03% 99.97% 0.01% 0.01% 0.03% 0.01% DdCBE_ replicate3 ND5.2- T 5.57% 0.02% 99.97% 0.05% 0.01% 0.01% 0.02% 0.01% 99.97% 99.96% 0.02% 0.02% DdCBE_ replicate3 ND5.2- C 94.33% 99.93% 0.01% 99.83% 0.01% 99.93% 99.94% 0.01% 0.01% 0.02% 0.00% 0.00% DdCBE_ replicate3 ND5.2- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 99.94% 99.96% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE ND5.2- A C A G C C T A G C A T T DdCBE_ 0.04% 99.97% 0.14% 0.03% 0.03% 0.01% 99.96% 0.01% 0.04% 99.97% 0.00% 0.01% replicate1 ND5.2- T DdCBE_ 0.04% 0.00% 0.01% 0.01% 0.01% 99.97% 0.01% 0.02% 0.01% 0.01% 99.98% 99.97% replicate1 ND5.2- C DdCBE_ 99.92% 0.01% 0.00% 99.95% 99.95% 0.01% 0.01% 0.00% 99.94% 0.01% 0.01% 0.01% replicate 1 ND5.2- G DdCBE_ 0.00% 0.01% 99.84% 0.00% 0.00% 0.00% 0.01% 99.97% 0.00% 0.01% 0.00% 0.01% replicate1 ND5.2- N DdCBE_ 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% replicate1 ND5.2- DdCBE_ 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% replicate1 ND5.2- A DdCBE_ 0.03% 99.97% 0.13% 0.00% 0.06% 0.00% 99.97% 0.03% 0.03% 100.00% 0.03% 0.00% replicate2 ND5.2- T DdCBE_ 0.00% 0.00% 0.03% 0.03% 0.06% 99.97% 0.03% 0.00% 0.03% 0.00% 99.97% 100.00% replicate2 ND5.2- C DdCBE_ 99.97% 0.00% 0.00% 99.97% 99.87% 0.03% 0.00% 0.00% 99.94% 0.00% 0.00% 0.00% replicate2 ND5.2- G DdCBE_ 0.00% 0.03% 99.84% 0.00% 0.00% 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% replicate2 ND5.2- N DdCBE_ 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% replicate2 ND5.2- DdCBE_ 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% replicate2 ND5.2- A DdCBE_ 0.04% 99.97% 0.17% 0.02% 0.03% 0.01% 99.96% 0.01% 0.04% 99.98% 0.01% 0.01% replicate3 ND5.2- T DdCBE_ 0.02% 0.00% 0.01% 0.02% 0.01% 99.96% 0.01% 0.01% 0.02% 0.01% 99.97% 99.97% replicate3 ND5.2- C DdCBE_ 99.93% 0.01% 0.00% 99.96% 99.95% 0.03% 0.01% 0.00% 99.94% 0.01% 0.01% 0.01% replicate3 ND5.2- G DdCBE_ 0.00% 0.01% 99.80% 0.00% 0.00% 0.01% 0.01% 99.97% 0.00% 0.01% 0.01% 0.00% replicate3 ND5.2- N DdCBE_ 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% replicate3 ND5.2- DdCBE_ 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide A G C A G G A A T ND5.2- A 99.86% 0.02% 0.03% 99.97% 0.01% 0.02% 99.95% 99.95% 0.01% DdCBE_ replicate1 ND5.2- T 0.01% 0.02% 0.03% 0.01% 0.01% 0.02% 0.02% 0.02% 99.97% DdCBE_ replicate1 ND5.2- C 0.00% 0.01% 99.94% 0.00% 0.00% 0.00% 0.01% 0.01% 0.02% DdCBE_ replicate 1 ND5.2- G 0.13% 99.96% 0.00% 0.02% 99.97% 99.95% 0.02% 0.01% 0.00% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 99.87% 0.03% 0.00% 100.00% 0.00% 0.03% 100.00% 99.94% 0.00% DdCBE_ replicate2 ND5.2- T 0.00% 0.00% 0.00% 0.00% 0.03% 0.03% 0.00% 0.00% 100.00% DdCBE_ replicate2 ND5.2- C 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% DdCBE_ replicate2 ND5.2- G 0.13% 99.97% 0.03% 0.00% 99.97% 99.94% 0.00% 0.03% 0.00% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 99.80% 0.01% 0.02% 99.96% 0.01% 0.01% 99.93% 99.95% 0.01% DdCBE_ replicate3 ND5.2- T 0.01% 0.02% 0.02% 0.01% 0.03% 0.04% 0.03% 0.02% 99.98% DdCBE_ replicate3 ND5.2- C 0.02% 0.01% 99.96% 0.01% 0.00% 0.01% 0.02% 0.02% 0.01% DdCBE_ replicate3 ND5.2- G 0.17% 99.95% 0.00% 0.02% 99.97% 99.94% 0.02% 0.01% 0.00% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 Base Percentages at TALE Binding Sites-ND5.2-DdCBE Batch Nucleotide A C C T T T C C T ND5.2- A 99.97% 0.02% 0.02% 0.03% 0.00% 0.01% 0.02% 0.02% 0.01% DdCBE_ replicate1 ND5.2- T 0.01% 0.02% 0.01% 99.81% 99.98% 99.97% 0.02% 0.01% 99.97% DdCBE_ replicate1 ND5.2- C 0.01% 99.96% 99.96% 0.15% 0.02% 0.01% 99.96% 99.97% 0.01% DdCBE_ replicate 1 ND5.2- G 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.2- A 99.97% 0.00% 0.06% 0.03% 0.00% 0.00% 0.00% 0.03% 0.03% DdCBE_ replicate2 ND5.2- T 0.03% 0.00% 0.00% 99.84% 99.97% 100.00% 0.00% 0.00% 99.97% DdCBE_ replicate2 ND5.2- C 0.00% 100.00% 99.94% 0.13% 0.03% 0.00% 99.97% 99.97% 0.00% DdCBE_ replicate2 ND5.2- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% DdCBE_ replicate2 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.2- A 99.97% 0.02% 0.02% 0.02% 0.00% 0.01% 0.02% 0.02% 0.01% DdCBE_ replicate3 ND5.2- T 0.01% 0.01% 0.01% 99.81% 99.98% 99.97% 0.02% 0.01% 99.98% DdCBE_ replicate3 ND5.2- C 0.01% 99.97% 99.96% 0.16% 0.01% 0.01% 99.96% 99.97% 0.01% DdCBE_ replicate3 ND5.2- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00070 TABLE 12D Base Percentages at TALE Binding Sites-ND5.3-DdCBE Batch Nucleotide A A C T C A G A ND5.3- A 99.96% 99.98% 0.01% 0.00% 0.01% 99.99% 0.03% 99.99% DdCBE_ replicate1 ND5.3- T 0.01% 0.00% 0.01% 99.98% 0.05% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- C 0.01% 0.00% 99.99% 0.01% 99.95% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- G 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 99.97% 0.01% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 99.98% 99.97% 0.01% 0.00% 0.00% 99.98% 0.02% 99.98% DdCBE_ replicate2 ND5.3- T 0.01% 0.00% 0.01% 99.98% 0.03% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND5.3- C 0.01% 0.00% 99.98% 0.01% 99.96% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- G 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 99.97% 0.01% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 99.98% 99.97% 0.01% 0.00% 0.01% 99.97% 0.01% 99.98% DdCBE_ replicate3 ND5.3- T 0.00% 0.00% 0.01% 99.98% 0.04% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND5.3- C 0.00% 0.01% 99.98% 0.02% 99.94% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- G 0.01% 0.01% 0.00% 0.00% 0.00% 0.03% 99.98% 0.01% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T T C T T C A A ND5.3- A 0.02% 0.01% 0.00% 0.01% 0.01% 0.01% 99.97% 99.97% DdCBE_ replicate 1 ND5.3- T 99.94% 99.97% 0.00% 99.97% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- C 0.02% 0.01% 99.99% 0.02% 0.02% 99.98% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- G 0.02% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.02% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- A 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 99.96% 99.96% DdCBE_ replicate2 ND5.3- T 99.95% 99.99% 0.02% 99.98% 99.97% 0.02% 0.01% 0.01% DdCBE_ replicate2 ND5.3- C 0.02% 0.00% 99.98% 0.01% 0.01% 99.96% 0.00% 0.01% DdCBE_ replicate2 ND5.3- G 0.02% 0.00% 0.00% 0.00% 0.01% 0.00% 0.02% 0.02% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 99.96% 99.96% DdCBE_ replicate3 ND5.3- T 99.94% 99.97% 0.01% 99.99% 99.98% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND5.3- C 0.02% 0.01% 99.99% 0.01% 0.01% 99.98% 0.00% 0.01% DdCBE_ replicate3 ND5.3- G 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% 0.03% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C T A A T T A C ND5.3- A 0.00% 0.00% 99.98% 99.98% 0.00% 0.00% 99.99% 0.00% DdCBE_ replicate 1 ND5.3- T 5.40% 99.99% 0.00% 0.00% 99.99% 99.98% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- C 94.60% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 99.99% DdCBE_ replicate1 ND5.3- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 0.00% 0.00% 99.98% 99.96% 0.00% 0.01% 99.98% 0.01% DdCBE_ replicate2 ND5.3- T 5.00% 99.97% 0.00% 0.01% 99.99% 99.97% 0.01% 0.01% DdCBE_ replicate2 ND5.3- C 94.99% 0.01% 0.00% 0.01% 0.00% 0.02% 0.00% 99.98% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 0.01% 99.98% 99.96% 0.00% 0.01% 99.99% 0.00% DdCBE_ replicate3 ND5.3- T 5.27% 99.98% 0.00% 0.00% 99.99% 99.98% 0.00% 0.01% DdCBE_ replicate3 ND5.3- C 94.72% 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% 99.99% DdCBE_ replicate3 ND5.3- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A C C G C T A A ND5.3- A 99.99% 0.00% 0.02% 0.02% 0.00% 0.00% 99.98% 99.96% DdCBE_ replicate 1 ND5.3- T 0.01% 0.01% 0.02% 0.01% 0.01% 99.99% 0.00% 0.01% DdCBE_ replicate1 ND5.3- C 0.00% 99.99% 99.96% 0.01% 99.98% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- G 0.00% 0.00% 0.01% 99.97% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- A 99.97% 0.01% 0.01% 0.02% 0.01% 0.01% 99.98% 99.93% DdCBE_ replicate2 ND5.3- T 0.01% 0.00% 0.02% 0.01% 0.01% 99.97% 0.00% 0.02% DdCBE_ replicate2 ND5.3- C 0.01% 99.99% 99.96% 0.01% 99.98% 0.02% 0.00% 0.02% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.01% 99.96% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND5.3- A 99.96% 0.01% 0.01% 0.02% 0.01% 0.01% 99.98% 99.96% DdCBE_ replicate3 ND5.3- T 0.01% 0.01% 0.01% 0.01% 0.00% 99.99% 0.00% 0.01% DdCBE_ replicate3 ND5.3- C 0.00% 99.98% 99.98% 0.01% 99.99% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND5.3- G 0.02% 0.00% 0.01% 99.97% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide C C C A A A C A ND5.3- A 0.01% 0.01% 0.01% 99.97% 99.98% 99.98% 0.02% 99.96% DdCBE_ replicate1 ND5.3- T 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- C 99.98% 99.97% 99.97% 0.00% 0.00% 0.00% 99.97% 0.02% DdCBE_ replicate1 ND5.3- G 0.00% 0.00% 0.00% 0.02% 0.02% 0.01% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 0.00% 0.01% 0.00% 99.98% 99.98% 99.98% 0.02% 99.96% DdCBE_ replicate2 ND5.3- T 0.01% 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.3- C 99.99% 99.98% 99.98% 0.00% 0.01% 0.01% 99.97% 0.02% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.00% 0.02% 0.01% 0.01% 0.00% 0.02% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 0.01% 0.01% 99.97% 99.97% 99.96% 0.03% 99.96% DdCBE_ replicate3 ND5.3- T 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.01% 0.00% DdCBE_ replicate3 ND5.3- C 99.98% 99.98% 99.97% 0.01% 0.00% 0.01% 99.96% 0.02% DdCBE_ replicate3 ND5.3- G 0.00% 0.00% 0.00% 0.01% 0.02% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A T A T C T A C ND5.3- A 99.99% 0.01% 99.99% 0.01% 0.01% 0.00% 99.97% 0.00% DdCBE_ replicate 1 ND5.3- T 0.00% 99.98% 0.01% 99.99% 0.01% 99.99% 0.02% 22.67% DdCBE_ replicate 1 ND5.3- C 0.00% 0.01% 0.00% 0.00% 99.98% 0.01% 0.01% 77.32% DdCBE_ replicate 1 ND5.3- G 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- A 99.99% 0.01% 99.98% 0.00% 0.00% 0.01% 99.98% 0.00% DdCBE_ replicate2 ND5.3- T 0.00% 99.98% 0.01% 99.99% 0.02% 99.97% 0.01% 21.07% DdCBE_ replicate2 ND5.3- C 0.00% 0.02% 0.00% 0.01% 99.98% 0.02% 0.00% 78.92% DdCBE_ replicate2 ND5.3- G 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 99.98% 0.00% 99.98% 0.01% 0.01% 0.01% 99.99% 0.01% DdCBE_ replicate3 ND5.3- T 0.01% 99.99% 0.01% 99.99% 0.01% 99.99% 0.01% 16.15% DdCBE_ replicate3 ND5.3- C 0.00% 0.01% 0.00% 0.01% 99.98% 0.01% 0.00% 83.84% DdCBE_ replicate3 ND5.3- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C A T A C T A A ND5.3- A 0.01% 99.98% 0.00% 99.97% 0.00% 0.01% 99.99% 99.96% DdCBE_ replicate 1 ND5.3- T 0.01% 0.01% 99.98% 0.01% 0.01% 99.97% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- C 99.98% 0.00% 0.01% 0.01% 99.99% 0.01% 0.00% 0.02% DdCBE_ replicate1 ND5.3- G 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 0.01% 99.97% 0.00% 99.97% 0.01% 0.00% 99.99% 99.96% DdCBE_ replicate2 ND5.3- T 0.01% 0.02% 99.98% 0.01% 0.00% 99.98% 0.00% 0.02% DdCBE_ replicate2 ND5.3- C 99.98% 0.00% 0.01% 0.01% 99.99% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 99.98% 0.01% 99.98% 0.01% 0.01% 99.99% 99.98% DdCBE_ replicate3 ND5.3- T 0.01% 0.01% 99.98% 0.01% 0.01% 99.97% 0.00% 0.01% DdCBE_ replicate3 ND5.3- C 99.97% 0.00% 0.01% 0.00% 99.99% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND5.3- G 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide C A A C C T A T ND5.3- A 0.01% 99.98% 99.96% 0.01% 0.00% 0.00% 99.97% 0.00% DdCBE_ replicate 1 ND5.3- T 0.01% 0.00% 0.01% 0.01% 0.01% 99.98% 0.02% 99.99% DdCBE_ replicate1 ND5.3- C 99.97% 0.00% 0.01% 99.98% 99.98% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- G 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 0.01% 99.99% 99.98% 0.00% 0.02% 0.01% 99.97% 0.00% DdCBE_ replicate2 ND5.3- T 0.01% 0.00% 0.01% 0.01% 0.01% 99.98% 0.01% 99.98% DdCBE_ replicate2 ND5.3- C 99.98% 0.00% 0.01% 99.99% 99.97% 0.01% 0.00% 0.02% DdCBE_ replicate2 ND5.3- G 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 99.98% 99.97% 0.01% 0.01% 0.01% 99.96% 0.00% DdCBE_ replicate3 ND5.3- T 0.01% 0.00% 0.01% 0.01% 0.01% 99.98% 0.02% 99.98% DdCBE_ replicate3 ND5.3- C 99.99% 0.01% 0.01% 99.99% 99.99% 0.01% 0.01% 0.02% DdCBE_ replicate3 ND5.3- G 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T T A A T C A G ND5.3- A 0.00% 0.00% 99.97% 99.95% 0.01% 0.02% 99.97% 0.09% DdCBE_ replicate1 ND5.3- T 100.00% 99.98% 0.02% 0.02% 99.98% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND5.3- C 0.00% 0.01% 0.01% 0.01% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate1 ND5.3- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.02% 99.90% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- A 0.00% 0.01% 99.96% 99.95% 0.01% 0.02% 99.99% 0.09% DdCBE_ replicate2 ND5.3- T 99.98% 99.98% 0.02% 0.03% 99.98% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND5.3- C 0.02% 0.02% 0.00% 0.01% 0.01% 99.96% 0.00% 0.00% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 99.91% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.00% 0.01% 99.96% 99.94% 0.01% 0.02% 99.97% 0.07% DdCBE_ replicate3 ND5.3- T 99.99% 99.98% 0.03% 0.03% 99.98% 0.01% 0.01% 0.00% DdCBE_ replicate3 ND5.3- C 0.01% 0.01% 0.00% 0.02% 0.01% 99.97% 0.01% 0.00% DdCBE_ replicate3 ND5.3- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% 99.93% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T C A T C T T C ND5.3- A 0.02% 0.01% 99.98% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- T 99.98% 0.02% 0.00% 99.98% 0.10% 99.97% 99.98% 28.54% DdCBE_ replicate 1 ND5.3- C 0.00% 99.97% 0.01% 0.01% 99.90% 0.02% 0.02% 71.45% DdCBE_ replicate 1 ND5.3- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- A 0.02% 0.01% 99.96% 0.01% 0.00% 0.01% 0.01% 0.00% DdCBE_ replicate2 ND5.3- T 99.98% 0.02% 0.01% 99.98% 0.11% 99.98% 99.97% 26.39% DdCBE_ replicate2 ND5.3- C 0.01% 99.97% 0.01% 0.01% 99.89% 0.01% 0.02% 73.61% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- A 0.01% 0.01% 99.98% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND5.3- T 99.98% 0.03% 0.00% 99.99% 0.11% 99.97% 99.98% 25.96% DdCBE_ replicate3 ND5.3- C 0.01% 99.96% 0.01% 0.01% 99.89% 0.01% 0.01% 74.04% DdCBE_ replicate3 ND5.3- G 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T C T T A G T T ND5.3- A 0.01% 0.00% 0.01% 0.01% 99.99% 0.01% 0.02% 0.01% DdCBE_ replicate 1 ND5.3- T 99.98% 0.01% 99.98% 99.97% 0.00% 0.01% 99.97% 99.97% DdCBE_ replicate 1 ND5.3- C 0.01% 99.98% 0.01% 0.02% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate1 ND5.3- G 0.00% 0.00% 0.00% 0.00% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND5.3- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND5.3- A 0.00% 0.00% 0.00% 0.01% 99.98% 0.01% 0.02% 0.01% DdCBE_ replicate2 ND5.3- T 99.98% 0.01% 99.98% 99.97% 0.00% 0.02% 99.97% 99.97% DdCBE_ replicate2 ND5.3- C 0.02% 99.99% 0.01% 0.02% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate2 ND5.3- G 0.00% 0.00% 0.00% 0.00% 0.02% 99.97% 0.00% 0.00% DdCBE_ replicate2 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND5.3- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND5.3- A 0.00% 0.00% 0.01% 0.00% 99.99% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND5.3- T 99.99% 0.00% 99.97% 99.97% 0.00% 0.01% 99.98% 99.98% DdCBE_ replicate3 ND5.3- C 0.01% 99.99% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND5.3- G 0.00% 0.00% 0.00% 0.00% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate3 ND5.3- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND5.3- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide T ND5.3- A 0.01% DdCBE_ replicate 1 ND5.3- T 99.98% DdCBE_ replicate1 ND5.3- C 0.01% DdCBE_ replicate 1 ND5.3- G 0.00% DdCBE_ replicate 1 ND5.3- N 0.00% DdCBE_ replicate 1 ND5.3- 0.00% DdCBE_ replicate 1 ND5.3- A 0.01% DdCBE_ replicate2 ND5.3- T 99.98% DdCBE_ replicate2 ND5.3- C 0.01% DdCBE_ replicate2 ND5.3- G 0.00% DdCBE_ replicate2 ND5.3- N 0.00% DdCBE_ replicate2 ND5.3- 0.00% DdCBE_ replicate2 ND5.3- A 0.01% DdCBE_ replicate3 ND5.3- T 99.97% DdCBE_ replicate3 ND5.3- C 0.01% DdCBE_ replicate3 ND5.3- G 0.00% DdCBE_ replicate3 ND5.3- N 0.00% DdCBE_ replicate3 ND5.3- 0.00% DdCBE_ replicate3
TABLE-US-00071 TABLE 12E Base Percentages at TALE Binding Sites-ND4-DdCBE Batch Nucleotide C C T A C T G G ND4- A 0.00% 0.02% 0.01% 99.98% 0.00% 0.01% 0.25% 0.01% DdCBE_ replicate 1 ND4- T 0.01% 0.01% 99.98% 0.00% 0.01% 99.98% 0.01% 0.01% DdCBE_ replicate1 ND4- C 99.99% 99.97% 0.01% 0.00% 99.99% 0.01% 0.00% 0.04% DdCBE_ replicate 1 ND4- G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 99.74% 99.94% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 0.03% 0.01% 99.99% 0.00% 0.01% 0.25% 0.02% DdCBE_ replicate2 ND4- T 0.00% 0.01% 99.96% 0.00% 0.00% 99.97% 0.03% 0.01% DdCBE_ replicate2 ND4 C 99.99% 99.96% 0.02% 0.00% 100.00% 0.02% 0.00% 0.02% DdCBE_ replicate2 ND4 G 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 99.72% 99.94% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4 A 0.00% 0.02% 0.00% 99.98% 0.00% 0.01% 0.20% 0.01% DdCBE_ replicate3 ND4 T 0.00% 0.01% 99.97% 0.01% 0.01% 99.98% 0.02% 0.02% DdCBE_ replicate3 ND4- C 99.99% 99.96% 0.02% 0.01% 99.99% 0.01% 0.00% 0.03% DdCBE_ replicate3 ND4- G 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 99.77% 99.94% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A G T A A C C A ND4- A 99.98% 0.01% 0.01% 99.98% 99.97% 0.01% 0.01% 99.97% DdCBE_ replicate 1 ND4- T 0.00% 0.01% 99.91% 0.00% 0.02% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND4- C 0.01% 0.00% 0.09% 0.01% 0.00% 99.98% 99.98% 0.01% DdCBE_ replicate1 ND4- G 0.01% 99.98% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 99.98% 0.01% 0.01% 99.98% 99.98% 0.00% 0.01% 99.98% DdCBE_ replicate2 ND4- T 0.01% 0.01% 99.92% 0.01% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND4- C 0.01% 0.00% 0.07% 0.00% 0.01% 99.99% 99.97% 0.00% DdCBE_ replicate2 ND4- G 0.01% 99.99% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 99.98% 0.01% 0.01% 99.98% 99.97% 0.00% 0.01% 99.98% DdCBE_ replicate3 ND4- T 0.01% 0.01% 99.93% 0.00% 0.01% 0.01% 0.02% 0.01% DdCBE_ replicate3 ND4- C 0.01% 0.00% 0.06% 0.01% 0.01% 99.99% 99.96% 0.01% DdCBE_ replicate3 ND4- G 0.01% 99.98% 0.00% 0.01% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate3 Batch Nucleotide T A T C A C T C ND4- A 0.00% 99.97% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND4- T 99.99% 0.02% 99.98% 0.02% 0.01% 0.01% 99.98% 0.01% DdCBE_ replicate1 ND4- C 0.01% 0.00% 0.01% 99.97% 0.01% 99.98% 0.01% 99.98% DdCBE_ replicate 1 ND4- G 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- A 0.01% 99.98% 0.00% 0.00% 99.95% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND4- T 99.98% 0.00% 99.99% 0.02% 0.01% 0.01% 99.98% 0.01% DdCBE_ replicate2 ND4- C 0.01% 0.01% 0.01% 99.98% 0.03% 99.99% 0.01% 99.99% DdCBE_ replicate2 ND4- G 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.01% 99.98% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND4- T 99.99% 0.01% 99.97% 0.02% 0.01% 0.01% 99.98% 0.01% DdCBE_ replicate3 ND4- C 0.01% 0.00% 0.01% 99.97% 0.01% 99.98% 0.01% 99.98% DdCBE_ replicate3 ND4- G 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C A A C A T A C ND4- A 0.01% 99.98% 99.95% 0.00% 99.97% 0.01% 99.96% 0.00% DdCBE_ replicate1 ND4- T 0.02% 0.00% 0.01% 0.01% 0.02% 99.99% 0.01% 0.02% DdCBE_ replicate 1 ND4- C 99.97% 0.01% 0.02% 99.98% 0.01% 0.01% 0.02% 99.98% DdCBE_ replicate 1 ND4- G 0.00% 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 99.96% 99.94% 0.01% 99.98% 0.00% 99.97% 0.01% DdCBE_ replicate2 ND4- T 0.01% 0.00% 0.02% 0.01% 0.01% 99.99% 0.01% 0.01% DdCBE_ replicate2 ND4- C 99.98% 0.03% 0.03% 99.98% 0.00% 0.00% 0.01% 99.99% DdCBE_ replicate2 ND4- G 0.00% 0.02% 0.02% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.01% 99.97% 99.96% 0.00% 99.98% 0.00% 99.97% 0.00% DdCBE_ replicate3 ND4- T 0.01% 0.00% 0.01% 0.01% 0.01% 99.99% 0.01% 0.02% DdCBE_ replicate3 ND4- C 99.98% 0.01% 0.02% 99.99% 0.00% 0.01% 0.01% 99.98% DdCBE_ replicate3 ND4- G 0.00% 0.02% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G A G A A C T C ND4- A 0.02% 99.95% 0.01% 99.96% 99.97% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND4- T 0.03% 0.02% 0.02% 0.01% 0.01% 0.01% 99.98% 0.01% DdCBE_ replicate1 ND4- C 0.00% 0.02% 0.00% 0.02% 0.00% 99.98% 0.01% 99.99% DdCBE_ replicate 1 ND4- G 99.93% 0.01% 99.97% 0.01% 0.02% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 99.95% 0.01% 99.96% 99.98% 0.02% 0.00% 0.01% DdCBE_ replicate2 ND4- T 0.04% 0.02% 0.03% 0.01% 0.01% 0.01% 99.99% 0.00% DdCBE_ replicate2 ND4 C 0.00% 0.02% 0.00% 0.01% 0.00% 99.98% 0.01% 99.99% DdCBE_ replicate2 ND4 G 99.93% 0.02% 99.96% 0.02% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4 A 0.03% 99.97% 0.00% 99.96% 99.98% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND4 T 0.03% 0.01% 0.01% 0.01% 0.01% 0.01% 99.98% 0.01% DdCBE_ replicate3 ND4- C 0.00% 0.01% 0.00% 0.01% 0.00% 99.99% 0.02% 99.98% DdCBE_ replicate3 ND4- G 99.93% 0.01% 99.98% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C G T T C T C C ND4- A 0.00% 0.25% 0.00% 0.00% 0.01% 0.01% 0.01% 0.02% DdCBE_ replicate 1 ND4- T 0.02% 0.00% 99.96% 99.98% 0.01% 99.98% 0.02% 0.01% DdCBE_ replicate1 ND4- C 99.98% 0.00% 0.03% 0.01% 99.99% 0.01% 99.97% 99.97% DdCBE_ replicate1 ND4- G 0.00% 99.75% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.00% 0.24% 0.00% 0.00% 0.00% 0.01% 0.01% 0.03% DdCBE_ replicate2 ND4- T 0.03% 0.01% 99.97% 99.99% 0.00% 99.97% 0.03% 0.01% DdCBE_ replicate2 ND4- C 99.97% 0.00% 0.03% 0.01% 99.99% 0.02% 99.96% 99.96% DdCBE_ replicate2 ND4- G 0.00% 99.75% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.01% 0.23% 0.00% 0.01% 0.00% 0.01% 0.01% 0.02% DdCBE_ replicate3 ND4- T 0.02% 0.00% 99.96% 99.99% 0.00% 99.99% 0.01% 0.01% DdCBE_ replicate3 ND4- C 99.97% 0.00% 0.03% 0.01% 99.99% 0.00% 99.98% 99.97% DdCBE_ replicate3 ND4- G 0.00% 99.76% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T C C T A C T T ND4- A 0.01% 0.01% 0.01% 0.01% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ND4- T 99.98% 0.01% 0.02% 99.98% 0.01% 0.01% 99.98% 99.97% DdCBE_ replicate1 ND4- C 0.01% 99.98% 99.97% 0.01% 0.02% 99.99% 0.01% 0.01% DdCBE_ replicate 1 ND4- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND4- A 0.02% 0.01% 0.01% 0.02% 99.96% 0.00% 0.01% 0.00% DdCBE_ replicate2 ND4- T 99.97% 0.00% 0.02% 99.97% 0.01% 0.00% 99.99% 99.98% DdCBE_ replicate2 ND4- C 0.00% 99.99% 99.97% 0.01% 0.02% 100.00% 0.01% 0.01% DdCBE_ replicate2 ND4- G 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND4- A 0.01% 0.02% 0.00% 0.01% 99.97% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND4- T 99.97% 0.01% 0.02% 99.98% 0.01% 0.01% 99.98% 99.96% DdCBE_ replicate3 ND4- C 0.02% 99.98% 99.97% 0.01% 0.01% 99.99% 0.01% 0.01% DdCBE_ replicate3 ND4- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide T A G T C A C A ND4- A 0.02% 99.98% 0.24% 0.02% 0.02% 99.96% 0.01% 99.99% DdCBE_ replicate1 ND4- T 99.97% 0.00% 0.01% 99.95% 0.03% 0.01% 0.01% 0.00% DdCBE_ replicate 1 ND4- C 0.01% 0.00% 0.00% 0.03% 99.95% 0.02% 99.98% 0.00% DdCBE_ replicate 1 ND4- G 0.00% 0.02% 99.76% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate 1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 99.97% 0.23% 0.02% 0.03% 99.96% 0.00% 99.99% DdCBE_ replicate2 ND4- T 99.97% 0.01% 0.01% 99.92% 0.05% 0.01% 0.01% 0.00% DdCBE_ replicate2 ND4- C 0.01% 0.00% 0.00% 0.06% 99.92% 0.01% 99.98% 0.00% DdCBE_ replicate2 ND4- G 0.00% 0.02% 99.77% 0.00% 0.00% 0.02% 0.00% 0.01% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.01% 99.99% 0.21% 0.02% 0.02% 99.97% 0.01% 99.98% DdCBE_ replicate3 ND4- T 99.97% 0.01% 0.01% 99.93% 0.03% 0.01% 0.01% 0.00% DdCBE_ replicate3 ND4- C 0.02% 0.00% 0.00% 0.05% 99.95% 0.00% 99.98% 0.00% DdCBE_ replicate3 ND4- G 0.00% 0.01% 99.78% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T C T G T G C T ND4- A 0.01% 0.01% 0.01% 0.02% 0.02% 0.01% 0.01% 0.00% DdCBE_ replicate 1 ND4- T 99.98% 0.01% 99.97% 0.00% 99.96% 0.00% 0.01% 99.98% DdCBE_ replicate1 ND4- C 0.01% 99.97% 0.01% 0.00% 0.01% 0.00% 99.98% 0.01% DdCBE_ replicate 1 ND4- G 0.00% 0.01% 0.00% 99.98% 0.01% 99.98% 0.00% 0.00% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 0.01% 0.01% 0.01% 0.02% 0.01% 0.01% 0.00% DdCBE_ replicate2 ND4- T 99.98% 0.01% 99.98% 0.00% 99.96% 0.01% 0.01% 99.99% DdCBE_ replicate2 ND4 C 0.01% 99.97% 0.01% 0.00% 0.01% 0.01% 99.99% 0.01% DdCBE_ replicate2 ND4 G 0.00% 0.00% 0.00% 99.99% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4 A 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND4 T 99.97% 0.01% 99.98% 0.01% 99.95% 0.01% 0.01% 99.98% DdCBE_ replicate3 ND4- C 0.01% 99.97% 0.01% 0.00% 0.02% 0.01% 99.98% 0.01% DdCBE_ replicate3 ND4- G 0.00% 0.01% 0.00% 99.98% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T G A T C A A A ND4- A 0.00% 47.28% 99.97% 0.01% 0.01% 99.96% 99.98% 99.98% DdCBE_ replicate 1 ND4- T 99.98% 0.01% 0.02% 99.98% 0.37% 0.01% 0.01% 0.01% DdCBE_ replicate1 ND4- C 0.01% 0.00% 0.00% 0.01% 99.61% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- G 0.00% 52.70% 0.01% 0.00% 0.00% 0.02% 0.01% 0.01% DdCBE_ replicate1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 47.37% 99.98% 0.01% 0.01% 99.97% 99.99% 99.98% DdCBE_ replicate2 ND4- T 99.98% 0.00% 0.01% 99.99% 0.43% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND4- C 0.01% 0.00% 0.01% 0.01% 99.56% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- G 0.00% 52.62% 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.01% 43.55% 99.97% 0.01% 0.00% 99.97% 99.97% 99.98% DdCBE_ replicate3 ND4- T 99.98% 0.00% 0.02% 99.99% 0.36% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND4- C 0.01% 0.01% 0.00% 0.01% 99.64% 0.01% 0.01% 0.00% DdCBE_ replicate3 ND4- G 0.00% 56.43% 0.01% 0.00% 0.00% 0.01% 0.02% 0.01% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A C A G G A C T ND4- A 99.96% 0.01% 99.96% 0.01% 0.01% 99.93% 0.01% 0.01% DdCBE_ replicate 1 ND4- T 0.00% 0.01% 0.01% 0.02% 0.01% 0.00% 0.01% 99.97% DdCBE_ replicate1 ND4- C 0.03% 99.98% 0.00% 0.00% 0.00% 0.05% 99.98% 0.02% DdCBE_ replicate 1 ND4- G 0.01% 0.00% 0.03% 99.97% 99.98% 0.02% 0.00% 0.00% DdCBE_ replicate 1 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND4- A 99.95% 0.01% 99.97% 0.01% 0.01% 99.94% 0.00% 0.01% DdCBE_ replicate2 ND4- T 0.01% 0.01% 0.01% 0.02% 0.00% 0.01% 0.01% 99.98% DdCBE_ replicate2 ND4- C 0.02% 99.98% 0.01% 0.00% 0.01% 0.05% 99.99% 0.01% DdCBE_ replicate2 ND4- G 0.01% 0.00% 0.01% 99.97% 99.98% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND4- A 99.96% 0.01% 99.97% 0.02% 0.01% 99.92% 0.00% 0.01% DdCBE_ replicate3 ND4- T 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% 0.01% 99.98% DdCBE_ replicate3 ND4- C 0.03% 99.98% 0.00% 0.00% 0.00% 0.06% 99.99% 0.02% DdCBE_ replicate3 ND4- G 0.01% 0.00% 0.02% 99.97% 99.98% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G C ND4- A 0.02% 0.01% DdCBE_ replicate1 ND4- T 0.01% 0.01% DdCBE_ replicate 1 ND4- C 0.01% 99.8% DdCBE_ replicate 1 ND4- G 99.96% 0.00% DdCBE_ replicate 1 ND4- N 0.00% 0.00% DdCBE_ replicate1 ND4- 0.00% 0.00% DdCBE_ replicate1 ND4- A 0.01% 0.00% DdCBE_ replicate2 ND4- T 0.01% 0.01% DdCBE_ replicate2 ND4- C 0.00% 99.99% DdCBE_ replicate2 ND4- G 99.98% 0.00% DdCBE_ replicate2 ND4- N 0.00% 0.00% DdCBE_ replicate2 ND4- 0.00% 0.00% DdCBE_ replicate2 ND4- A 0.02% 0.01% DdCBE_ replicate3 ND4- T 0.01% 0.00% DdCBE_ replicate3 ND4- C 0.00% 99.99% DdCBE_ replicate3 ND4- G 99.97% 0.00% DdCBE_ replicate3 ND4- N 0.00% 0.00% DdCBE_ replicate3 ND4- 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00072 TABLE 12F Base Percentages at TALE Binding Sites-ND2-DdCBE Batch Nucleotide C C A A A C C C ND2- A 0.01% 0.01% 99.98% 99.99% 99.97% 0.02% 0.02% 0.01% DdCBE_ replicate 1 ND2- T 0.02% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.04% DdCBE_ replicate 1 ND2- C 99.97% 99.97% 0.00% 0.00% 0.01% 99.97% 99.97% 99.95% DdCBE_ replicate1 ND2- G 0.00% 0.00% 0.02% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- A 0.00% 0.01% 99.98% 99.97% 99.98% 0.03% 0.00% 0.00% DdCBE_ replicate2 ND2- T 0.01% 0.02% 0.00% 0.00% 0.00% 0.00% 0.02% 0.04% DdCBE_ replicate2 ND2- C 99.98% 99.96% 0.01% 0.02% 0.00% 99.96% 99.98% 99.95% DdCBE_ replicate2 ND2- G 0.00% 0.00% 0.02% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.00% 0.01% 99.99% 99.98% 99.98% 0.02% 0.01% 0.01% DdCBE_ replicate3 ND2- T 0.01% 0.01% 0.00% 0.00% 0.00% 0.02% 0.01% 0.03% DdCBE_ replicate3 ND2- C 99.99% 99.97% 0.00% 0.01% 0.01% 99.96% 99.99% 99.96% DdCBE_ replicate3 ND2- G 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A G C A T A C T ND2- A 99.98% 0.02% 0.01% 99.98% 0.01% 99.97% 0.00% 0.01% DdCBE_ replicate1 ND2- T 0.00% 0.01% 0.02% 0.01% 99.97% 0.01% 0.01% 99.97% DdCBE_ replicate1 ND2- C 0.00% 0.00% 99.97% 0.00% 0.01% 0.01% 99.99% 0.02% DdCBE_ replicate1 ND2- G 0.02% 99.97% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- A 99.98% 0.01% 0.00% 99.97% 0.00% 99.98% 0.01% 0.01% DdCBE_ replicate2 ND2- T 0.00% 0.01% 0.02% 0.01% 99.98% 0.01% 0.00% 99.97% DdCBE_ replicate2 ND2- C 0.00% 0.01% 99.97% 0.00% 0.01% 0.00% 99.98% 0.01% DdCBE_ replicate2 ND2- G 0.01% 99.97% 0.00% 0.02% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 99.98% 0.01% 0.01% 99.96% 0.01% 99.97% 0.02% 0.01% DdCBE_ replicate3 ND2- T 0.00% 0.02% 0.02% 0.02% 99.99% 0.01% 0.00% 99.97% DdCBE_ replicate3 ND2- C 0.00% 0.00% 99.98% 0.00% 0.00% 0.01% 99.98% 0.01% DdCBE_ replicate3 ND2- G 0.01% 99.97% 0.00% 0.02% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A G G A T G A A ND2- A 100.00% 0.29% 4.82% 99.95% 0.00% 0.05% 99.97% 99.94% DdCBE_ replicate 1 ND2- T 0.00% 0.02% 0.01% 0.01% 99.99% 0.02% 0.01% 0.02% DdCBE_ replicate 1 ND2- C 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND2- G 0.00% 99.69% 95.18% 0.02% 0.00% 99.93% 0.02% 0.02% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- A 99.99% 0.35% 4.74% 99.97% 0.00% 0.04% 99.98% 99.96% DdCBE_ replicate2 ND2- T 0.00% 0.02% 0.00% 0.01% 99.98% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND2- C 0.00% 0.00% 0.01% 0.02% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND2- G 0.01% 99.64% 95.25% 0.00% 0.00% 99.95% 0.01% 0.02% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 99.99% 0.33% 4.37% 99.98% 0.01% 0.05% 99.97% 99.98% DdCBE_ replicate3 ND2- T 0.01% 0.02% 0.01% 0.00% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND2- C 0.00% 0.01% 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND2- G 0.01% 99.65% 95.61% 0.01% 0.01% 99.93% 0.02% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G T A C A A C C ND2- A 0.01% 0.01% 99.98% 0.01% 99.99% 99.98% 0.01% 0.02% DdCBE_ replicate 1 ND2- T 0.00% 99.94% 0.01% 0.02% 0.00% 0.01% 0.02% 0.01% DdCBE_ replicate 1 ND2- C 0.00% 0.05% 0.00% 99.97% 0.01% 0.00% 99.97% 99.98% DdCBE_ replicate 1 ND2- G 99.98% 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- A 0.02% 0.01% 99.97% 0.00% 99.99% 99.98% 0.00% 0.02% DdCBE_ replicate2 ND2- T 0.02% 99.95% 0.01% 0.02% 0.00% 0.01% 0.02% 0.02% DdCBE_ replicate2 ND2- C 0.00% 0.05% 0.01% 99.98% 0.00% 0.00% 99.98% 99.96% DdCBE_ replicate2 ND2- G 99.96% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.04% 0.01% 99.97% 0.01% 99.98% 99.97% 0.00% 0.02% DdCBE_ replicate3 ND2- T 0.01% 99.95% 0.02% 0.01% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 ND2- C 0.00% 0.04% 0.00% 99.98% 0.01% 0.01% 99.99% 99.97% DdCBE_ replicate3 ND2- G 99.95% 0.00% 0.01% 0.00% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A G C T A C G C ND2- A 99.97% 0.18% 0.00% 0.00% 99.98% 0.01% 0.03% 0.01% DdCBE_ replicate 1 ND2- T 0.01% 0.02% 0.01% 99.98% 0.01% 0.02% 0.01% 0.01% DdCBE_ replicate 1 ND2- C 0.00% 0.00% 99.99% 0.02% 0.01% 99.98% 0.01% 99.99% DdCBE_ replicate1 ND2- G 0.01% 99.80% 0.00% 0.00% 0.01% 0.00% 99.95% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- A 99.97% 0.20% 0.01% 0.00% 99.99% 0.00% 0.05% 0.01% DdCBE_ replicate2 ND2- T 0.02% 0.02% 0.02% 99.99% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND2- C 0.00% 0.00% 99.98% 0.01% 0.00% 99.99% 0.01% 99.98% DdCBE_ replicate2 ND2- G 0.00% 99.78% 0.00% 0.00% 0.00% 0.00% 99.93% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 99.96% 0.14% 0.00% 0.00% 99.98% 0.00% 0.08% 0.00% DdCBE_ replicate3 ND2- T 0.02% 0.03% 0.00% 99.99% 0.01% 0.01% 0.02% 0.01% DdCBE_ replicate3 ND2- C 0.01% 0.00% 99.99% 0.01% 0.00% 99.99% 0.01% 99.98% DdCBE_ replicate3 ND2- G 0.02% 99.83% 0.00% 0.00% 0.01% 0.00% 99.90% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C C T C A A T T ND2- A 0.03% 0.01% 0.01% 0.00% 99.96% 99.98% 0.01% 0.01% DdCBE_ replicate1 ND2- T 0.02% 0.01% 99.98% 0.31% 0.01% 0.00% 99.99% 99.98% DdCBE_ replicate1 ND2- C 99.96% 99.99% 0.01% 99.68% 0.00% 0.02% 0.00% 0.01% DdCBE_ replicate1 ND2- G 0.00% 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ND2- A 0.01% 0.01% 0.00% 0.00% 99.95% 99.98% 0.01% 0.01% DdCBE_ replicate2 ND2- T 0.03% 0.01% 99.97% 0.33% 0.01% 0.01% 99.98% 99.97% DdCBE_ replicate2 ND2- C 99.95% 99.98% 0.02% 99.66% 0.00% 0.01% 0.01% 0.02% DdCBE_ replicate2 ND2- G 0.00% 0.00% 0.01% 0.00% 0.04% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.02% 0.01% 0.01% 0.00% 99.96% 99.97% 0.01% 0.01% DdCBE_ replicate3 ND2- T 0.03% 0.01% 99.98% 0.33% 0.01% 0.01% 99.98% 99.98% DdCBE_ replicate3 ND2- C 99.96% 99.98% 0.01% 99.67% 0.00% 0.02% 0.01% 0.02% DdCBE_ replicate3 ND2- G 0.00% 0.00% 0.01% 0.00% 0.03% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T A A T A G C A ND2- A 0.00% 99.98% 99.96% 0.01% 99.98% 0.02% 0.01% 99.97% DdCBE_ replicate 1 ND2- T 99.98% 0.01% 0.02% 99.82% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- C 0.01% 0.00% 0.01% 0.18% 0.00% 0.00% 99.98% 0.01% DdCBE_ replicate 1 ND2- G 0.01% 0.01% 0.01% 0.00% 0.02% 99.97% 0.01% 0.02% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- A 0.01% 99.99% 99.96% 0.01% 99.97% 0.01% 0.01% 99.97% DdCBE_ replicate2 ND2- T 99.97% 0.00% 0.02% 99.79% 0.00% 0.01% 0.02% 0.01% DdCBE_ replicate2 ND2- C 0.02% 0.00% 0.00% 0.20% 0.00% 0.01% 99.97% 0.00% DdCBE_ replicate2 ND2- G 0.00% 0.01% 0.01% 0.00% 0.02% 99.97% 0.00% 0.02% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.00% 99.99% 99.96% 0.00% 99.98% 0.01% 0.00% 99.97% DdCBE_ replicate3 ND2- T 99.98% 0.00% 0.02% 99.84% 0.01% 0.01% 0.02% 0.00% DdCBE_ replicate3 ND2- C 0.01% 0.00% 0.01% 0.16% 0.00% 0.01% 99.97% 0.01% DdCBE_ replicate3 ND2- G 0.00% 0.01% 0.01% 0.00% 0.01% 99.97% 0.01% 0.02% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C T A A C A T A ND2- A 0.00% 0.01% 99.97% 99.96% 0.01% 99.96% 0.01% 99.97% DdCBE_ replicate 1 ND2- T 0.02% 99.98% 0.00% 0.01% 0.01% 0.02% 99.98% 0.01% DdCBE_ replicate 1 ND2- C 99.98% 0.01% 0.01% 0.01% 99.98% 0.00% 0.01% 0.01% DdCBE_ replicate 1 ND2- G 0.01% 0.00% 0.02% 0.01% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- A 0.01% 0.00% 99.96% 99.98% 0.01% 99.97% 0.00% 99.98% DdCBE_ replicate2 ND2- T 0.02% 99.98% 0.00% 0.00% 0.01% 0.01% 99.99% 0.00% DdCBE_ replicate2 ND2- C 99.97% 0.02% 0.00% 0.01% 99.98% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND2- G 0.00% 0.00% 0.03% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.01% 0.01% 99.98% 99.96% 0.00% 99.97% 0.00% 99.97% DdCBE_ replicate3 ND2- T 0.02% 99.98% 0.00% 0.01% 0.01% 0.02% 100.00% 0.01% DdCBE_ replicate3 ND2- C 99.97% 0.01% 0.00% 0.02% 99.99% 0.00% 0.00% 0.01% DdCBE_ replicate3 ND2- G 0.00% 0.00% 0.02% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A A A T C T T ND2- A 99.97% 99.96% 99.89% 99.92% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- T 0.02% 0.02% 0.02% 0.00% 99.99% 0.00% 100.00% 99.98% DdCBE_ replicate 1 ND2- C 0.00% 0.01% 0.07% 0.06% 0.01% 99.99% 0.00% 0.02% DdCBE_ replicate1 ND2- G 0.01% 0.02% 0.02% 0.02% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- A 99.96% 99.97% 99.87% 99.95% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- T 0.01% 0.01% 0.02% 0.00% 99.99% 0.01% 99.99% 99.98% DdCBE_ replicate2 ND2- C 0.01% 0.00% 0.10% 0.03% 0.01% 99.98% 0.00% 0.01% DdCBE_ replicate2 ND2- G 0.02% 0.01% 0.01% 0.02% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND2- A 99.96% 99.95% 99.90% 99.96% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND2- T 0.01% 0.02% 0.01% 0.01% 99.99% 0.01% 99.99% 99.97% DdCBE_ replicate3 ND2- C 0.01% 0.00% 0.08% 0.03% 0.01% 99.98% 0.01% 0.02% DdCBE_ replicate3 ND2- G 0.01% 0.03% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide A C C C A C A T ND2- A 99.98% 0.01% 0.00% 0.02% 99.96% 0.01% 99.99% 0.00% DdCBE_ replicate1 ND2- T 0.01% 0.01% 0.02% 0.01% 0.02% 0.74% 0.01% 99.99% DdCBE_ replicate1 ND2- C 0.00% 99.98% 99.97% 99.96% 0.00% 99.25% 0.00% 0.01% DdCBE_ replicate1 ND2- G 0.01% 0.00% 0.01% 0.01% 0.02% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- A 99.98% 0.01% 0.00% 0.02% 99.97% 0.00% 99.97% 0.00% DdCBE_ replicate2 ND2- T 0.01% 0.01% 0.01% 0.02% 0.01% 0.79% 0.01% 99.99% DdCBE_ replicate2 ND2- C 0.00% 99.98% 99.98% 99.95% 0.01% 99.20% 0.01% 0.01% DdCBE_ replicate2 ND2- G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.02% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 99.98% 0.00% 0.01% 0.01% 99.97% 0.01% 99.97% 0.01% DdCBE_ replicate3 ND2- T 0.01% 0.03% 0.01% 0.02% 0.01% 0.74% 0.01% 99.99% DdCBE_ replicate3 ND2- C 0.01% 99.96% 99.98% 99.96% 0.00% 99.25% 0.01% 0.01% DdCBE_ replicate3 ND2- G 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G T T C T A C C ND2- A 0.00% 0.01% 0.01% 0.01% 0.02% 99.98% 0.00% 0.01% DdCBE_ replicate 1 ND2- T 0.01% 99.97% 99.98% 0.01% 99.97% 0.01% 0.02% 0.02% DdCBE_ replicate 1 ND2- C 0.01% 0.01% 0.01% 99.98% 0.01% 0.00% 99.97% 99.96% DdCBE_ replicate 1 ND2- G 99.98% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ND2- A 0.01% 0.01% 0.00% 0.00% 0.01% 99.99% 0.00% 0.01% DdCBE_ replicate2 ND2- T 0.00% 99.97% 100.00% 0.01% 99.98% 0.00% 0.01% 0.02% DdCBE_ replicate2 ND2- C 0.00% 0.01% 0.00% 99.99% 0.01% 0.00% 99.98% 99.96% DdCBE_ replicate2 ND2- G 99.99% 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND2- A 0.01% 0.01% 0.01% 0.00% 0.01% 99.96% 0.00% 0.01% DdCBE_ replicate3 ND2- T 0.01% 99.97% 99.97% 0.01% 99.97% 0.01% 0.02% 0.02% DdCBE_ replicate3 ND2- C 0.00% 0.02% 0.02% 99.98% 0.01% 0.00% 99.98% 99.96% DdCBE_ replicate3 ND2- G 99.98% 0.00% 0.00% 0.01% 0.00% 0.03% 0.00% 0.01% DdCBE_ replicate3 ND2- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A C ND2- A 99.96% 0.00% DdCBE_ replicate 1 ND2- T 0.01% 0.02% DdCBE_ replicate 1 ND2- C 0.01% 99.98% DdCBE_ replicate 1 ND2- G 0.01% 0.00% DdCBE_ replicate 1 ND2- N 0.00% 0.00% DdCBE_ replicate1 ND2- 0.02% 0.00% DdCBE_ replicate1 ND2- A 99.96% 0.01% DdCBE_ replicate2 ND2- T 0.01% 0.02% DdCBE_ replicate2 ND2- C 0.01% 99.97% DdCBE_ replicate2 ND2- G 0.02% 0.00% DdCBE_ replicate2 ND2- N 0.00% 0.00% DdCBE_ replicate2 ND2- 0.01% 0.00% DdCBE_ replicate2 ND2- A 99.97% 0.01% DdCBE_ replicate3 ND2- T 0.00% 0.02% DdCBE_ replicate3 ND2- C 0.02% 99.98% DdCBE_ replicate3 ND2- G 0.01% 0.00% DdCBE_ replicate3 ND2- N 0.00% 0.00% DdCBE_ replicate3 ND2- 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00073 TABLE 12G Base Percentages at TALE Binding Sites-ND1-DdCBE Batch Nucleotide T C C T A T T T ND1- A 0.00% 0.03% 0.00% 0.00% 99.56% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- T 99.97% 0.12% 0.00% 99.91% 0.09% 99.97% 100.00% 100.00% DdCBE_ replicate1 ND1- C 0.03% 99.85% 100.00% 0.09% 0.35% 0.03% 0.00% 0.00% DdCBE_ replicate1 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.01% 0.02% 0.01% 0.00% 99.47% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND1- T 99.97% 0.22% 0.03% 99.96% 0.03% 99.98% 99.99% 99.99% DdCBE_ replicate2 ND1- C 0.02% 99.75% 99.96% 0.04% 0.49% 0.01% 0.01% 0.01% DdCBE_ replicate2 ND1- G 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.00% 0.01% 0.01% 0.00% 99.46% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- T 99.98% 0.15% 0.02% 99.95% 0.01% 99.98% 99.98% 99.98% DdCBE_ replicate3 ND1- C 0.02% 99.83% 99.97% 0.05% 0.53% 0.02% 0.01% 0.01% DdCBE_ replicate3 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G C C T A G C C ND1- A 0.03% 0.03% 0.03% 0.00% 99.97% 0.00% 0.00% 0.03% DdCBE_ replicate1 ND1- T 0.00% 0.00% 0.06% 99.91% 0.00% 0.00% 0.03% 0.00% DdCBE_ replicate1 ND1- C 0.00% 99.97% 99.91% 0.09% 0.03% 0.00% 99.97% 99.97% DdCBE_ replicate1 ND1- G 99.97% 0.00% 0.00% 0.00% 0.00% 100.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.01% 0.03% 0.00% 0.01% 99.92% 0.01% 0.01% 0.00% DdCBE_ replicate2 ND1- T 0.01% 0.01% 0.01% 99.94% 0.02% 0.01% 0.01% 0.02% DdCBE_ replicate2 ND1- C 0.00% 99.95% 99.98% 0.05% 0.06% 0.01% 99.98% 99.97% DdCBE_ replicate2 ND1- G 99.98% 0.00% 0.00% 0.00% 0.01% 99.97% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.01% 0.03% 0.00% 0.00% 99.89% 0.01% 0.01% 0.00% DdCBE_ replicate3 ND1- T 0.00% 0.00% 0.00% 99.95% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND1- C 0.00% 99.96% 100.00% 0.05% 0.08% 0.00% 99.98% 99.99% DdCBE_ replicate3 ND1- G 99.99% 0.00% 0.00% 0.00% 0.01% 99.98% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G A T C A G G G ND1- A 5.28% 99.85% 0.00% 0.00% 99.94% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- T 0.00% 0.12% 100.00% 0.06% 0.00% 0.09% 0.00% 0.00% DdCBE_ replicate1 ND1- C 0.00% 0.00% 0.00% 99.94% 0.03% 0.00% 0.03% 0.00% DdCBE_ replicate1 ND1- G 94.72% 0.03% 0.00% 0.00% 0.03% 99.91% 99.97% 100.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 4.93% 99.88% 0.00% 0.02% 99.97% 0.02% 0.01% 0.01% DdCBE_ replicate2 ND1- T 0.02% 0.10% 99.98% 0.03% 0.01% 0.05% 0.03% 0.00% DdCBE_ replicate2 ND1- C 0.05% 0.01% 0.01% 99.94% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- G 95.00% 0.01% 0.00% 0.00% 0.01% 99.93% 99.96% 99.98% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 4.41% 99.92% 0.01% 0.01% 99.98% 0.01% 0.02% 0.00% DdCBE_ replicate3 ND1- T 0.05% 0.07% 99.98% 0.02% 0.00% 0.07% 0.02% 0.01% DdCBE_ replicate3 ND1- C 0.03% 0.00% 0.01% 99.97% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- G 95.51% 0.01% 0.00% 0.00% 0.01% 99.91% 99.96% 99.99% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A C T A C G C C ND1- A 99.97% 0.03% 0.00% 100.00% 0.23% 0.09% 0.00% 0.00% DdCBE_ replicate1 ND1- T 0.00% 0.00% 100.00% 0.00% 0.00% 0.09% 0.00% 0.00% DdCBE_ replicate1 ND1- C 0.03% 99.97% 0.00% 0.00% 99.77% 0.82% 100.00% 100.00% DdCBE_ replicate1 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 99.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 99.97% 0.01% 0.01% 99.98% 0.14% 0.03% 0.01% 0.01% DdCBE_ replicate2 ND1- T 0.00% 0.01% 99.98% 0.01% 0.01% 0.07% 0.01% 0.01% DdCBE_ replicate2 ND1- C 0.01% 99.98% 0.01% 0.01% 99.86% 1.08% 99.98% 99.98% DdCBE_ replicate2 ND1- G 0.01% 0.00% 0.00% 0.00% 0.00% 98.83% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 99.99% 0.01% 0.01% 99.96% 0.15% 0.02% 0.01% 0.00% DdCBE_ replicate3 ND1- T 0.00% 0.00% 99.95% 0.01% 0.00% 0.10% 0.00% 0.01% DdCBE_ replicate3 ND1- C 0.00% 99.99% 0.03% 0.02% 99.85% 0.99% 99.98% 99.99% DdCBE_ replicate3 ND1- G 0.00% 0.00% 0.00% 0.01% 0.00% 98.89% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A T T C T A G C ND1- A 99.85% 0.00% 0.00% 0.00% 0.00% 99.88% 0.03% 0.06% DdCBE_ replicate1 ND1- T 0.06% 99.97% 100.00% 0.06% 99.97% 0.06% 0.06% 0.03% DdCBE_ replicate1 ND1- C 0.09% 0.03% 0.00% 99.94% 0.03% 0.00% 0.00% 99.91% DdCBE_ replicate1 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.06% 99.91% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 99.84% 0.00% 0.00% 0.01% 0.00% 99.89% 0.01% 0.02% DdCBE_ replicate2 ND1- T 0.06% 99.98% 99.99% 0.03% 99.97% 0.06% 0.01% 0.04% DdCBE_ replicate2 ND1- C 0.08% 0.02% 0.01% 99.95% 0.02% 0.04% 0.00% 99.94% DdCBE_ replicate2 ND1- G 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 99.97% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 99.88% 0.00% 0.00% 0.00% 0.00% 99.91% 0.02% 0.00% DdCBE_ replicate3 ND1- T 0.02% 99.98% 99.99% 0.03% 99.99% 0.04% 0.01% 0.02% DdCBE_ replicate3 ND1- C 0.10% 0.01% 0.01% 99.96% 0.01% 0.05% 0.01% 99.97% DdCBE_ replicate3 ND1- G 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 99.96% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide G T T T A C T C ND1- A 0.00% 0.06% 0.00% 0.00% 99.85% 0.06% 0.00% 0.03% DdCBE_ replicate1 ND1- T 0.00% 99.82% 99.94% 100.00% 0.06% 0.03% 99.97% 0.29% DdCBE_ replicate1 ND1- C 0.00% 0.12% 0.06% 0.00% 0.03% 99.91% 0.03% 99.65% DdCBE_ replicate1 ND1- G 100.00% 0.00% 0.00% 0.00% 0.06% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% DdCBE_ replicate1 ND1- A 0.02% 0.03% 0.00% 0.00% 99.92% 0.03% 0.00% 0.01% DdCBE_ replicate2 ND1- T 0.01% 99.82% 99.96% 99.98% 0.03% 0.01% 99.96% 0.30% DdCBE_ replicate2 ND1- C 0.00% 0.09% 0.03% 0.02% 0.04% 99.95% 0.04% 99.68% DdCBE_ replicate2 ND1- G 99.97% 0.07% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.01% 0.03% 0.00% 0.00% 99.93% 0.02% 0.00% 0.01% DdCBE_ replicate3 ND1- T 0.01% 99.79% 99.97% 99.97% 0.03% 0.02% 99.97% 0.33% DdCBE_ replicate3 ND1- C 0.00% 0.13% 0.03% 0.02% 0.03% 99.96% 0.03% 99.65% DdCBE_ replicate3 ND1- G 99.97% 0.05% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide T G A G C A T C ND1- A 0.15% 0.00% 99.97% 0.00% 0.00% 99.97% 0.00% 0.03% DdCBE_ replicate1 ND1- T 98.74% 0.00% 0.00% 0.00% 0.00% 0.00% 100.00 0.00% DdCBE_ replicate1 ND1- C 1.03% 0.00% 0.00% 0.03% 100.00 0.00% 0.00% 99.97% DdCBE_ replicate1 ND1- G 0.09% 100.00% 0.03% 99.97% 0.00% 0.03% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.11% 0.02% 99.91% 0.01% 0.01% 99.92% 0.02% 0.00% DdCBE_ replicate2 ND1- T 98.81% 0.02% 0.02% 0.01% 0.03% 0.04% 99.96 0.02% DdCBE_ replicate2 ND1- C 0.96% 0.00% 0.01% 0.00% 99.96% 0.03% 0.02% 99.98% DdCBE_ replicate2 ND1- G 0.12% 99.96% 0.06% 99.99% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.10% 0.02% 99.93% 0.01% 0.00% 99.92% 0.01% 0.02% DdCBE_ replicate3 ND1- T 98.81% 0.00% 0.02% 0.00% 0.03% 0.03% 99.97% 0.02% DdCBE_ replicate3 ND1- C 0.95% 0.00% 0.02% 0.00% 99.96% 0.04% 0.02% 99.96% DdCBE_ replicate3 ND1- G 0.14% 99.98% 0.04% 99.99% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C T G A T C G G ND1- A 0.00% 0.00% 0.03% 99.85% 0.00% 0.00% 0.03% 0.03% DdCBE_ replicate1 ND1- T 0.00% 99.97% 0.00% 0.00% 99.79% 0.38% 0.00% 0.03% DdCBE_ replicate1 ND1- C 100.00% 0.03% 0.03% 0.15% 0.21% 99.62% 0.00% 0.23% DdCBE_ replicate1 ND1- G 0.00% 0.00% 99.94% 0.00% 0.00% 0.00% 99.97% 99.71% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.01% 0.01% 0.10% 99.89% 0.01% 0.01% 0.02% 0.02% DdCBE_ replicate2 ND1- T 0.00% 99.88% 0.01% 0.01% 99.83% 0.59% 0.03% 0.02% DdCBE_ replicate2 ND1- C 99.98% 0.10% 0.05% 0.08% 0.16% 99.40% 0.03% 0.20% DdCBE_ replicate2 ND1- G 0.00% 0.00% 99.84% 0.02% 0.00% 0.00% 99.92% 99.76% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.01% 0.00% 0.11% 99.90% 0.03% 0.00% 0.03% 0.02% DdCBE_ replicate3 ND1- T 0.01% 99.84% 0.00% 0.01% 99.84% 0.46% 0.04% 0.03% DdCBE_ replicate3 ND1- C 99.97% 0.15% 0.04% 0.09% 0.13% 99.53% 0.02% 0.19% DdCBE_ replicate3 ND1- G 0.00% 0.00% 99.85% 0.00% 0.00% 0.00% 99.91% 99.76% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C A C C T C T A ND1- A 0.03% 99.88% 0.00% 0.00% 0.00% 0.00% 0.00% 99.88% DdCBE_ replicate1 ND1- T 0.00% 0.00% 0.00% 0.00% 99.97% 0.03% 99.97% 0.09% DdCBE_ replicate1 ND1- C 99.97% 0.09% 100.00% 100.00% 0.03% 99.97% 0.03% 0.00% DdCBE_ replicate1 ND1- G 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% 0.03% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.01% 99.94% 0.01% 0.01% 0.01% 0.01% 0.00% 99.86% DdCBE_ replicate2 ND1- T 0.03% 0.01% 0.01% 0.01% 99.98% 0.05% 99.98% 0.04% DdCBE_ replicate2 ND1- C 99.96% 0.04% 99.99% 99.98% 0.01% 99.94% 0.02% 0.09% DdCBE_ replicate2 ND1- G 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.00% 99.90% 0.00% 0.01% 0.01% 0.00% 0.01% 99.81% DdCBE_ replicate3 ND1- T 0.04% 0.02% 0.01% 0.00% 99.98% 0.03% 99.97% 0.04% DdCBE_ replicate3 ND1- C 99.96% 0.08% 99.98% 99.99% 0.01% 99.96% 0.02% 0.13% DdCBE_ replicate3 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.02% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A T C C T C T ND1- A 99.82% 99.79% 0.00% 0.03% 0.00% 0.00% 0.00% 0.03% DdCBE_ replicate1 ND1- T 0.03% 0.00% 100.00% 47.25% 46.54% 100.00% 31.24% 99.91% DdCBE_ replicate1 ND1- C 0.09% 0.21% 0.00% 52.73% 53.46% 0.00% 68.76% 0.06% DdCBE_ replicate1 ND1- G 0.06% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 99.85% 99.83% 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate2 ND1- T 0.05% 0.02% 99.98% 47.68% 46.95% 99.99% 29.89% 99.97% DdCBE_ replicate2 ND1- C 0.08% 0.15% 0.01% 52.31% 53.04% 0.01% 70.11% 0.02% DdCBE_ replicate2 ND1- G 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 99.81% 99.85% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 ND1- T 0.05% 0.01% 99.98% 43.02% 42.41% 99.97% 27.64% 99.97% DdCBE_ replicate3 ND1- C 0.11% 0.15% 0.01% 56.98% 57.58% 0.03% 72.36% 0.02% DdCBE_ replicate3 ND1- G 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A A C T C A A ND1- A 99.97% 100.00% 99.97% 0.15% 0.00% 0.12% 99.97% 100.00% DdCBE_ replicate1 ND1- T 0.00% 0.00% 0.00% 0.00% 99.97% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- C 0.03% 0.00% 0.03% 99.85% 0.03% 99.88% 0.03% 0.00% DdCBE_ replicate1 ND1- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 99.95% 99.97% 99.97% 0.03% 0.01% 0.06% 99.97% 99.95% DdCBE_ replicate2 ND1- T 0.01% 0.00% 0.01% 0.01% 99.96% 0.02% 0.01% 0.01% DdCBE_ replicate2 ND1- C 0.03% 0.02% 0.02% 99.95% 0.03% 99.92% 0.02% 0.03% DdCBE_ replicate2 ND1- G 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 99.93% 99.96% 99.97% 0.08% 0.00% 0.06% 99.97% 99.96% DdCBE_ replicate3 ND1- T 0.00% 0.00% 0.01% 0.00% 99.97% 0.01% 0.01% 0.01% DdCBE_ replicate3 ND1- C 0.05% 0.03% 0.01% 99.92% 0.02% 99.93% 0.01% 0.03% DdCBE_ replicate3 ND1- G 0.02% 0.01% 0.01% 0.00% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C G C ND1- A 0.00% 0.06% 0.03% DdCBE_ replicate1 ND1- T 0.03% 0.00% 0.00% DdCBE_ replicate1 ND1- C 99.94% 0.03% 99.97% DdCBE_ replicate1 ND1- G 0.03% 99.91% 0.00% DdCBE_ replicate1 ND1- N 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- 0.00% 0.00% 0.00% DdCBE_ replicate1 ND1- A 0.01% 0.01% 0.01% DdCBE_ replicate2 ND1- T 0.03% 0.04% 0.00% DdCBE_ replicate2 ND1- C 99.97% 0.01% 99.98% DdCBE_ replicate2 ND1- G 0.00% 99.94% 0.00% DdCBE_ replicate2 ND1- N 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- 0.00% 0.00% 0.00% DdCBE_ replicate2 ND1- A 0.00% 0.00% 0.02% DdCBE_ replicate3 ND1- T 0.03% 0.03% 0.00% DdCBE_ replicate3 ND1- C 99.97% 0.01% 99.98% DdCBE_ replicate3 ND1- G 0.00% 99.96% 0.00% DdCBE_ replicate3 ND1- N 0.00% 0.00% 0.00% DdCBE_ replicate3 ND1- 0.00% 0.00% 0.00% DdCBE_ replicate3
TABLE-US-00074 TABLE 12H Base Percentages at TALE Binding Sites-ATP8-DdCBE Batch Nucleotide C C T C A T C A ATP8- A 0.07% 0.03% 0.01% 0.01% 99.97% 0.02% 0.02% 99.96% DdCBE_ replicate 1 ATP8- T 0.06% 0.03% 99.96% 0.18% 0.01% 99.97% 0.04% 0.03% DdCBE_ replicate 1 ATP8- C 99.86% 99.93% 0.03% 99.81% 0.01% 0.02% 99.93% 0.01% DdCBE_ replicate1 ATP8 G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 0.07% 0.02% 0.01% 0.04% 99.97% 0.01% 0.03% 99.95% DdCBE_ replicate2 ATP8- T 0.06% 0.02% 99.96% 0.23% 0.01% 99.99% 0.07% 0.02% DdCBE_ replicate2 ATP8- C 99.87% 99.95% 0.03% 99.73% 0.01% 0.00% 99.90% 0.02% DdCBE_ replicate2 ATP8- G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 0.06% 0.03% 0.02% 0.01% 99.96% 0.03% 0.02% 99.93% DdCBE_ replicate3 ATP8- T 0.04% 0.02% 99.96% 0.18% 0.01% 99.97% 0.07% 0.03% DdCBE_ replicate3 ATP8- C 99.90% 99.94% 0.02% 99.80% 0.02% 0.00% 99.92% 0.03% DdCBE_ replicate3 ATP8- G 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate3 Batch Nucleotide A A A C A C A A ATP8- A 99.97% 99.98% 99.95% 0.01% 99.97% 0.01% 99.98% 99.96% DdCBE_ replicate 1 ATP8- T 0.01% 0.01% 0.01% 0.05% 0.01% 0.03% 0.00% 0.02% DdCBE_ replicate1 ATP8- C 0.01% 0.00% 0.01% 99.94% 0.00% 99.96% 0.01% 0.01% DdCBE_ replicate 1 ATP8- G 0.01% 0.01% 0.01% 0.00% 0.02% 0.00% 0.01% 0.01% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 99.95% 99.96% 99.95% 0.02% 99.97% 0.03% 99.97% 99.95% DdCBE_ replicate2 ATP8- T 0.01% 0.03% 0.02% 0.06% 0.01% 0.02% 0.00% 0.02% DdCBE_ replicate2 ATP8- C 0.02% 0.00% 0.01% 99.91% 0.00% 99.95% 0.01% 0.02% DdCBE_ replicate2 ATP8- G 0.02% 0.00% 0.01% 0.00% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.95% 99.96% 99.95% 0.02% 99.96% 0.01% 99.98% 99.93% DdCBE_ replicate3 ATP8- T 0.02% 0.02% 0.02% 0.04% 0.02% 0.02% 0.00% 0.02% DdCBE_ replicate3 ATP8- C 0.01% 0.01% 0.01% 99.94% 0.01% 99.96% 0.01% 0.03% DdCBE_ replicate3 ATP8- G 0.02% 0.02% 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C T C A C C A A ATP8- A 0.04% 0.03% 0.05% 99.98% 0.02% 0.02% 99.97% 99.98% DdCBE_ replicate 1 ATP8- T 0.02% 99.95% 0.07% 0.01% 0.01% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ATP8- C 99.94% 0.02% 99.87% 0.00% 99.97% 99.96% 0.01% 0.00% DdCBE_ replicate 1 ATP8- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- A 0.01% 0.01% 0.05% 99.97% 0.01% 0.03% 99.97% 99.96% DdCBE_ replicate2 ATP8- T 0.01% 99.97% 0.07% 0.01% 0.02% 0.02% 0.01% 0.03% DdCBE_ replicate2 ATP8- C 99.97% 0.02% 99.87% 0.01% 99.97% 99.95% 0.01% 0.00% DdCBE_ replicate2 ATP8- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 0.02% 0.02% 0.04% 99.96% 0.02% 0.02% 99.98% 99.96% DdCBE_ replicate3 ATP8- T 0.02% 99.96% 0.04% 0.01% 0.01% 0.02% 0.01% 0.02% DdCBE_ replicate3 ATP8- C 99.94% 0.01% 99.92% 0.01% 99.97% 99.96% 0.01% 0.01% DdCBE_ replicate3 ATP8- G 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.02% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A A T T A T A ATP8- A 99.99% 99.97% 99.93% 0.01% 0.14% 99.98% 0.04% 99.98% DdCBE_ replicate1 ATP8- T 0.00% 0.02% 0.02% 99.95% 99.84% 0.01% 99.94% 0.01% DdCBE_ replicate 1 ATP8- C 0.00% 0.00% 0.00% 0.03% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ATP8- G 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- 0.00% 0.00% 0.05% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate1 ATP8- A 99.99% 99.97% 99.94% 0.02% 0.16% 99.97% 0.04% 99.96% DdCBE_ replicate2 ATP8- T 0.00% 0.01% 0.02% 99.93% 99.83% 0.01% 99.94% 0.03% DdCBE_ replicate2 ATP8- C 0.00% 0.01% 0.00% 0.05% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate2 ATP8- G 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.98% 99.98% 99.93% 0.01% 0.13% 99.97% 0.02% 99.98% DdCBE_ replicate3 ATP8- T 0.00% 0.01% 0.03% 99.94% 99.85% 0.02% 99.96% 0.01% DdCBE_ replicate3 ATP8- C 0.01% 0.00% 0.00% 0.04% 0.02% 0.00% 0.01% 0.01% DdCBE_ replicate3 ATP8- G 0.01% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.04% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C C C A A C T A ATP8- A 0.01% 0.01% 0.03% 99.98% 99.25% 0.01% 0.02% 99.97% DdCBE_ replicate 1 ATP8- T 0.01% 0.01% 0.02% 0.01% 0.01% 0.01% 99.97% 0.02% DdCBE_ replicate 1 ATP8- C 99.97% 99.97% 99.93% 0.01% 0.72% 99.97% 0.01% 0.00% DdCBE_ replicate1 ATP8 G 0.00% 0.00% 0.00% 0.00% 0.02% 0.00% 0.00% 0.01% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 0.02% 0.01% 0.02% 99.97% 99.30% 0.03% 0.01% 99.96% DdCBE_ replicate2 ATP8- T 0.03% 0.03% 0.02% 0.01% 0.02% 0.00% 99.97% 0.02% DdCBE_ replicate2 ATP8- C 99.95% 99.96% 99.95% 0.01% 0.64% 99.97% 0.01% 0.01% DdCBE_ replicate2 ATP8- G 0.00% 0.00% 0.00% 0.01% 0.03% 0.00% 0.00% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 0.03% 0.02% 0.03% 99.98% 99.25% 0.01% 0.02% 99.97% DdCBE_ replicate3 ATP8- T 0.00% 0.02% 0.03% 0.00% 0.02% 0.02% 99.97% 0.01% DdCBE_ replicate3 ATP8- C 99.96% 99.95% 99.94% 0.01% 0.72% 99.95% 0.01% 0.02% DdCBE_ replicate3 ATP8- G 0.00% 0.00% 0.00% 0.01% 0.02% 0.00% 0.00% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.01% 0.00% DdCBE_ replicate3 Batch Nucleotide A C T A C C A C ATP8- A 99.97% 0.02% 0.03% 99.96% 0.02% 0.15% 99.96% 0.01% DdCBE_ replicate 1 ATP8- T 0.01% 0.04% 99.95% 0.01% 0.03% 0.04% 0.01% 0.02% DdCBE_ replicate1 ATP8- C 0.01% 99.94% 0.02% 0.02% 99.95% 99.80% 0.00% 99.97% DdCBE_ replicate 1 ATP8- G 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 99.97% 0.02% 0.01% 99.96% 0.03% 0.13% 99.96% 0.02% DdCBE_ replicate2 ATP8- T 0.01% 0.04% 99.96% 0.01% 0.01% 0.06% 0.02% 0.02% DdCBE_ replicate2 ATP8- C 0.01% 99.93% 0.02% 0.01% 99.96% 99.81% 0.01% 99.96% DdCBE_ replicate2 ATP8- G 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.97% 0.02% 0.02% 99.96% 0.03% 0.15% 99.96% 0.02% DdCBE_ replicate3 ATP8- T 0.01% 0.04% 99.95% 0.02% 0.04% 0.06% 0.02% 0.02% DdCBE_ replicate3 ATP8- C 0.01% 99.93% 0.03% 0.02% 99.93% 99.78% 0.01% 99.96% DdCBE_ replicate3 ATP8- G 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.00% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.01% 0.01% 0.00% DdCBE_ replicate3 Batch Nucleotide A G C C C A T A ATP8- A 99.97% 0.06% 0.02% 0.02% 0.06% 99.95% 0.02% 99.98% DdCBE_ replicate 1 ATP8- T 0.01% 0.04% 0.01% 0.02% 0.03% 0.03% 99.97% 0.01% DdCBE_ replicate 1 ATP8- C 0.01% 0.06% 99.97% 99.95% 99.91% 0.01% 0.01% 0.00% DdCBE_ replicate 1 ATP8- G 0.01% 99.83% 0.00% 0.00% 0.00% 0.02% 0.00% 0.01% DdCBE_ replicate1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- A 99.98% 0.02% 0.01% 0.04% 0.06% 99.96% 0.01% 99.97% DdCBE_ replicate2 ATP8- T 0.00% 0.03% 0.01% 0.03% 0.04% 0.03% 99.98% 0.01% DdCBE_ replicate2 ATP8- C 0.00% 0.07% 99.97% 99.93% 99.90% 0.01% 0.01% 0.00% DdCBE_ replicate2 ATP8- G 0.01% 99.88% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.01% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.96% 0.06% 0.02% 0.02% 0.05% 99.96% 0.01% 99.99% DdCBE_ replicate3 ATP8- T 0.00% 0.04% 0.02% 0.02% 0.03% 0.03% 99.97% 0.01% DdCBE_ replicate3 ATP8- C 0.01% 0.05% 99.96% 99.96% 99.91% 0.01% 0.02% 0.00% DdCBE_ replicate3 ATP8- G 0.02% 99.85% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.01% 0.00% 0.00% 0.00% 0.01% 0.00% 0.01% 0.00% DdCBE_ replicate3 Batch Nucleotide A C A A ATP8- A 99.97% 0.07% 99.98% 99.99% DdCBE_ replicate1 ATP8- T 0.01% 0.02% 0.00% 0.00% DdCBE_ replicate 1 ATP8- C 0.01% 99.91% 0.00% 0.00% DdCBE_ replicate 1 ATP8- G 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- A 99.94% 0.08% 99.99% 99.99% DdCBE_ replicate2 ATP8- T 0.02% 0.01% 0.00% 0.01% DdCBE_ replicate2 ATP8- C 0.03% 99.91% 0.00% 0.00% DdCBE_ replicate2 ATP8- G 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.94% 0.05% 99.98% 99.97% DdCBE_ replicate3 ATP8- T 0.02% 0.01% 0.00% 0.02% DdCBE_ replicate3 ATP8- C 0.02% 99.94% 0.00% 0.01% DdCBE_ replicate3 ATP8- G 0.01% 0.00% 0.01% 0.01% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A A A T A T T ATP8- A 99.98% 99.97% 99.97% 99.94% 0.01% 99.97% 0.01% 0.03% DdCBE_ replicate 1 ATP8- T 0.01% 0.02% 0.01% 0.01% 99.99% 0.01% 99.98% 99.95% DdCBE_ replicate 1 ATP8- C 0.00% 0.00% 0.01% 0.01% 0.01% 0.02% 0.01% 0.02% DdCBE_ replicate1 ATP8 G 0.01% 0.01% 0.01% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 99.97% 99.96% 99.97% 99.92% 0.00% 99.95% 0.02% 0.01% DdCBE_ replicate2 ATP8- T 0.01% 0.03% 0.02% 0.01% 99.99% 0.02% 99.97% 99.97% DdCBE_ replicate2 ATP8- C 0.01% 0.00% 0.01% 0.02% 0.00% 0.02% 0.01% 0.01% DdCBE_ replicate2 ATP8- G 0.01% 0.01% 0.00% 0.01% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.98% 99.97% 99.96% 99.95% 0.00% 99.97% 0.02% 0.02% DdCBE_ replicate3 ATP8- T 0.01% 0.01% 0.01% 0.00% 99.99% 0.00% 99.96% 99.95% DdCBE_ replicate3 ATP8- C 0.00% 0.01% 0.01% 0.02% 0.00% 0.02% 0.02% 0.02% DdCBE_ replicate3 ATP8- G 0.01% 0.01% 0.01% 0.01% 0.00% 0.01% 0.01% 0.00% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide C T A C C T C C ATP8- A 0.01% 0.03% 99.95% 0.02% 0.02% 0.01% 0.01% 0.01% DdCBE_ replicate 1 ATP8- T 0.03% 99.96% 0.03% 0.04% 0.02% 99.97% 9.61% 4.46% DdCBE_ replicate1 ATP8- C 99.96% 0.01% 0.02% 99.94% 99.96% 0.02% 90.37% 95.53% DdCBE_ replicate 1 ATP8- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate 1 ATP8- A 0.03% 0.02% 99.93% 0.01% 0.03% 0.02% 0.03% 0.01% DdCBE_ replicate2 ATP8- T 0.02% 99.95% 0.03% 0.02% 0.02% 99.97% 12.09% 5.71% DdCBE_ replicate2 ATP8- C 99.95% 0.03% 0.02% 99.97% 99.94% 0.01% 87.88% 94.27% DdCBE_ replicate2 ATP8- G 0.00% 0.00% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 0.04% 0.02% 99.94% 0.03% 0.03% 0.03% 0.02% 0.01% DdCBE_ replicate3 ATP8- T 0.02% 99.96% 0.03% 0.01% 0.03% 99.95% 10.85% 5.12% DdCBE_ replicate3 ATP8- C 99.94% 0.02% 0.02% 99.95% 99.93% 0.02% 89.13% 94.86% DdCBE_ replicate3 ATP8- G 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 Batch Nucleotide A A A A T A A A ATP8- A 99.97% 99.96% 99.98% 99.93% 0.02% 99.96% 99.97% 99.99% DdCBE_ replicate 1 ATP8- T 0.01% 0.01% 0.00% 0.03% 99.97% 0.01% 0.02% 0.00% DdCBE_ replicate 1 ATP8- C 0.00% 0.01% 0.00% 0.02% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate 1 ATP8- G 0.01% 0.02% 0.01% 0.00% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate1 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- 0.00% 0.00% 0.00% 0.01% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate1 ATP8- A 99.97% 99.97% 99.98% 99.90% 0.02% 99.97% 99.97% 99.99% DdCBE_ replicate2 ATP8- T 0.02% 0.01% 0.01% 0.04% 99.96% 0.01% 0.02% 0.01% DdCBE_ replicate2 ATP8- C 0.00% 0.00% 0.00% 0.01% 0.01% 0.01% 0.00% 0.00% DdCBE_ replicate2 ATP8- G 0.00% 0.01% 0.00% 0.02% 0.00% 0.01% 0.01% 0.01% DdCBE_ replicate2 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- 0.00% 0.00% 0.00% 0.03% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate2 ATP8- A 99.97% 99.97% 99.97% 99.91% 0.01% 99.97% 99.98% 99.99% DdCBE_ replicate3 ATP8- T 0.01% 0.02% 0.01% 0.04% 99.96% 0.01% 0.01% 0.01% DdCBE_ replicate3 ATP8- C 0.01% 0.00% 0.01% 0.02% 0.02% 0.01% 0.00% 0.00% DdCBE_ replicate3 ATP8- G 0.01% 0.01% 0.01% 0.00% 0.00% 0.01% 0.00% 0.00% DdCBE_ replicate3 ATP8- N 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% DdCBE_ replicate3 ATP8- 0.00% 0.00% 0.00% 0.03% 0.01% 0.00% 0.00% 0.00% DdCBE_ replicate3
Supplementary Sequences
[0964] Sequences used for DddA.sub.tox characterization in bacteria.
TABLE-US-00075 SEQIDNOS PrimersforcloningdddAdeaminasedomainintopScrhaB2-V NdeI-DddA TCAAGTACTACATATGATAGGACTCAACGGTGGGGC 160 DddA-NS- TACTGATTGATCTAGAACAACCTCCTTTCGTGGGGGA 161 XbaI PrimersforcloningdddAdeaminasedomainintopPSV39-CV DddAI- TACTGATTGATCTAGATTACAACTCGCTCCATGTCAGTTG 162 XbaI SacI-RBS- TCAAGTACTAGAGCTCACGGGAGGAAAGATGTACGCAGACG 163 DddAI ATTTCGACG PrimersforcloningdddAdeaminasedomainintopETDuet-1mcs1 BamHI- TATCAGAAACGGATCCATAGGACTCAACGGTGGGGC 164 DddA_duet DddA_duet- TATGTTACTAGCGGCCGCTCAACAACCTCCTTTCGTGGG 165 NotI PrimersforcloningdddAdeaminasedomainintopETDuet-1mcs1 NdeI_DddAI TCAAGTACTACATATGTACGCAGACGATTTCGACG 166 DddAI-BglII TATGTTACTAAGATCTTTACAACTCGCTCCATGTCAGTTG 167 PrimersforcloningdddA(E1347A)deaminasedomainintopETDuet-1mcs1 DddA_E1347A- CGGACTGACCGGCAACGTGCCCGGCGTTTGC 168 3 DddA_E1347A- GGCACGTTGCCGGTCAGTCCGCCTTATTTATGC 169 4 PrimersforconstructionofdeletioncassetteforicmF1using pDONRPEX18Gm-SceI-pheS HindIII- TGTTAAGCTAAAGCTTTAGGGATAACAGGGTAATCTGCTGG 170 ISecI-IcmF1 ATCCGGATTTCCG IcmF1-2 TTCAGCATGCTTGCGGCTCGAGTTGATGCGTTGCATAGGAC 171 GTTCA Icmf1-3 AACTCGAGCCGCAAGCATGCTGAAAGGGCGCAATGACGCAA 172 ACC Bcen_IcmF1- TCAATCAGTATCTAGAGTAGAACGGATCGACCGGCA 173 XbaI HindIII- TGTTAAGCTAAAGCTTTAGGGATAACAGGGTAATCGCTCAT 174 ISecI-IcmF2 TGTCCGTTTGCAGC IcmF2-2 TTCAGCATGCTTGCGGCTCGAGTTGCGAACGATCATGTGTG 175 ATACAC IcmF2-3 AACTCGAGCCGCAAGCATGCTGAATTTCGAGACCCGCGATG 176 ACG IcmF2-XbaI TCAATCAGTATCTAGACGAGCCGCTCGATACGATTG 177 PrimersforconstructionofdeletioncassettefordddA-dddArusing pDONRPEX18Gm-Scel-pheS HindIII- TGTTAAGCTAAAGCTTTAGGGATAACAGGGTAATGTGGTAC 178 ISecI- TTCAACGAAGCAGATG DddADddAI DddADddAI- TTCAGCATGCTTGCGGCTCGAGTTATCGGATCAGTGACTCG 179 2 TGC DddADddA AACTCGAGCCGCAAGCATGCTGAAAGCGAGTTGTAAGAAAC 180 I-3 GGAGC Bcen_E1- TCAATCAGTATCTAGAAGTGAGCTCTCCGAAATCGAAC 181 I1-XbaI PrimersforconstructionofdddA(E147A)usingpDONRPEX18Gm-SceI-pheS Bcen_E1347A- TAAAACGACGGCCAGTGCCAAGCTTAGGGATAACAGGGTAA 182 1 TAGCAGCTACGTGTACAGTCCGGACGCACCGTATTCGC Bcen_E1347A- AATAAGGCGGACTGACCGGCAACGTGCCCGGCGTTTGCGTA 183 2 GTTGG Bcen_E1347A- CCGGGCACGTTGCCGGTCAGTCCGCCTTATTTATGCGC 184 3 Bcen_E1347A- GCTCGGTACCCGGGGATCCTCTAGACTCGCTCCATGTCAGT 185 4 TGCTCGGGCCG PrimersforcloningofcddintopScrhaB2-V CDD_fwd TGAAATTCAGCAGGATCACATATGCATCCACGTTTTCAAAC 186 CGC CDD_rev TGCATGCCTGCAGGTCGACTCTAGAAGCGAGAAGCACTCGG 187 TC PrimersforcloningoftadAintopScrhaB2-V tadAfwd AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA 188 CGACGCTCTTCCGATCT tadA_rev TGCATGCCTGCAGGTCGACTCTAGACTTAAAAATACGTATC 189 GCTTTAG PrimersforcloningofA3GintopScrhaB2-V A3G_fwd TGAAATTCAGCAGGATCACATATGGACCCACCAACTTTTAC 190 TTTTAATTTTAAC A3G_rev TGCCTGCAGGTCGACTCTAGAGTTTTCCTGGTTTTGTAAGA 191 TTG PrimersforungdeletioninE.coli ung_del-F TAGAAAGAAGCAGTTAAGCTAGGCGGATTGAAGATTCGCAG 192 GAGAGCGAGATGGTGTAGGCTGGAGCTGCTTC ung_del-R TGATAAATCAGCCGGGGGCAACTCTGCCATCCGGCATTTC 193 CCCGCAAATTTACATATGAATATCCTCCTTAG PrimersforcloningungintopBAD24 EcoRI_ung TAGTACAGAGAATTCATGGCTAACGAATTAACCTGGCATGA 194 C ung_XbaI TCAATCAGTATCTAGATTACTCACTCTCTGCCGGTAATACT 195 G Sequencesforinvitrodeaminationassays S36- AAAAAAAAAAAAAAACTCGCCAAAAAAAAAAAAAAA 196 CTCGCC S35- AAAAAAAAAAAAAAAGACGGAAAAAAAAAAAAAAA 197 GACGG S35- AAAAAAAAAAAAAAAGTCGGAAAAAAAAAAAAAAA 198 GTCGG S35- AAAAAAAAAAAAAAAGGCGGAAAAAAAAAAAAAAA 199 GGCGG S35- AAAAAAAAAAAAAAAGCCGGAAAAAAAAAAAAAAA 200 GCCGG
[0965] Sequences used for characterization of DddA.sub.tox and its fusions in mammalian cells.
TABLE-US-00076 SEQIDNOS PrimersusedforgeneratingsgRNAtransfectionplasmids. Rev_sgRNA_plasmidwasusedinallcases rev_sgRNA_ GGTGTTTCGTCCTTTCCACAAG 201 plasmid fwd_saG1 GTCTGTGCCCCTCCCTCCCTGGCGTTTTAGTACTCTGT 202 AATGAAAATTACAGAATCTAC fwd_saG2 GCCCCTCCCTCCCTGGCCCAGGTGTTTTAGTACTCTGT 203 AATGAAAATTACAGAATCTAC fwd_saG3 GCCCTCCCTGGCCCAGGTGAAGGGTTTTAGTACTCTGT 204 AATGAAAATTACAGAATCTAC fwd_saG4 GTGTGGTTCCAGAACCGGAGGAGTTTTAGTACTCTGTA 205 ATGAAAATTACAGAATCTAC fwd_spG6 GAGGCCCCCAGAGCAGCCACGTTTTAGAGCTAGAAATA 206 GCAAGTTAAAATAAGGC fwd_spG7 GCCACTGGGGCCTCAACACTCGTTTTAGAGCTAGAAAT 207 AGCAAGTTAAAATAAGGC fwd_EMX1 GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATA 208 GCAAGTTAAAATAAGGC PrimersforHTSofon-targetsitesfromallmammaliancellculture experiments fwd_CCR5_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 209 AAGTGTGATCACTTGGGTGG rev_CCR5_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGATTCCCG 210 AGTAGCAGATG fwd_EMX1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 211 GCCCCTAACCCTATGTAGC rev_EMX1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTTCTGCTC 212 GGACTCAGGC fwd_ND1_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 213 TCACCATCGCTCTTCTACTATG rev_ND1_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCTAGGGT 214 GACTTCATATGAG fwd_ND2_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 215 GTAAGCCTTCTCCTCACT rev_ND2_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTGAGTAG 216 TAGGAATGCGGTAG fwd_ND4_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 217 ACTTCAAACTCTACTCCCACTAATAG rev_ND4_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTGTGGTA 218 AATATGTAGAGGGAG fwd_ND5.1/5.2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 219 HTS GGGTCCATCATCCACAAC rev_ND5.1/5.2 TGGAGTTCAGACGTGTGCTCTTCCGATCTAGAGTAATA 220 HTS GATAGGGCTCAGGC fwd_ND5.3_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 221 TGTAGCATTGTTCGTTACATGG rev_ND5.3_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGATGAGC 222 AAGAAGGATATAATTCC fwd_ND6_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 223 TCTTTCACCCACAGCACC rev_ND6_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGATTGTTAG 224 CGGTGTGGTCG fwd_ATP8_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 225 TTTACAGTGAAATGCCCCAAC rev_ATP8_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGCAAT 226 GAATGAAGCG PrimersforHTSofoff-targetsitesfromallmammaliancellculture experiments fwd_5303_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 227 CTAACATGACTAACACCCTTAATTC rev_5303_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTAGGAGTA 228 GCGTGGTAAGG fwd_7994/8115_ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 229 HTS CCCCATTATTCCTAGAACCAG rev_7994/8115_ TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTATAGGG 230 HTS TAAATACGGGCC fwd_8619/8648/ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 231 8720_HTS ATCATTCTATTTCCCCCTCTATTG rev_8619/8648/ TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTGCC 232 8720_HTS CGCTCATAAG fwd_10192/10205/ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNG 233 10349_HTS AAAAATCCACCCCTTACGAG rev_10192/10205/ TGGAGTTCAGACGTGTGCTCTTCCGATCTCGTTTTGTT 234 10349_HTS TAAACTATATACCAATTCGG fwd_13763_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 235 CCCACCCTTACTAACATTAACG rev_13763_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTTGTTGG 236 TTAGGTAGTTGAGG fwd_15598/15619/ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 237 15646/15675 CTATTCGCCTACACAATTCTC HTS rev_15598/15619/ TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTGGTATT 238 15646/15675 AGGATTAGGATTGTTGTG HTS fwd_15950_HTS ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 239 TCAAATGGGCCTGTCCTTG rev_15950_HTS TGGAGTTCAGACGTGTGCTCTTCCGATCTGTACTACAG 240 GTGGTCAAGTATTTATG fwd_16363/16393/ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNC 241 16394_HTS CATTTACCGTACATAGCACATTAC rev_16363/16393/ TGGAGTTCAGACGTGTGCTCTTCCGATCTCGTGAGTGG 242 16394_HTS TTAATAGGGTGATAG PrimersformtDNAcopynumberanaylsisbyquantitativePCR fwd_ND5.1/5.2_ GAACAAGATATTCGAAAAATAGGAGGAC 243 qPCR rev_ND5.1/5.2_ GCGGTTTCGATGATGTGG 244 qPCR fwd_ND6_qPCR CACTCACCAAGACCTCAACC 245 rev_ND6_qPCR GAATGATGGTTGTCTTTGGATATACTAC 246 fwd_ATP8_qPCR CTTACACTATTCCTCATCACCCAAC 247 rev_ATP8_qPCR GTTCATTTTGGTTCTCAGGGTTTG 248 fwd_- AGGCACCAGGGCGTGAT 249 actin_qPCR rev_- CAGGGTGAGGATGCCTC 250 actin_qPCR Primersforlong-rangePCRofwholemitochondrialgenomeastwo amplicons fwd_2478-10858 GCAAATCTTACCCCGCCTG 251 rev_2478-10858 AATTAGGCTGTGGGTGGTTG 252 fwd_2688-10653 GCCATACTAGTCTTTGCCGC 253 rev_2688-10653 GGCAGGTCAATTTCACTGG 254 PrimerforamplificationofND4genefragementfromND4-editedcells fwd_ND4_PCR GCCATTCTCATCCAAACC 255 Rev_ND4_PCR GGTTGAGGGATAGGAGGAG 256 PrimersforqPCRandRT-qPCRofND4-editedcells fwd_ND4_RT- CAAGCTCCATCTGCCTACGA 257 qPCR rev_ND4_RT- GCGATTATGAGAATGACTGC 258 qPCR fwd_RNR1_RT- ATTACACATGCAAGCATCCC 259 qPCR rev_RNR1_RT- CACGAAATTGACCAACCCTG 260 qPCR fwd_ND1_RT- TAGCAGAGACCAACCGAACC 261 qPCR rev_ND1_RT- ATGAAGAATAGGGCGAAGGG 262 qPCR fwd_ND2_RT- CTATCTCGCACCTGAAACAAGC 263 qPCR rev_ND2_RT- GGTGGAGTAGATTAGGCGTAGG 264 qPCR fwd_ATP6/8_ TGTTAGCGGTTAGGCGTA 265 RT-qPCR rev_ATP6/8_ TTACACCAACCACCCAAC 266 RT-qPCR fwd_CO3_RT- TTTACCCTCCTACAAGCC 267 qPCR rev_CO3_RT- GCGGATGAAGCAGATAGT 268 qPCR fwd_CYB_RT- GCCTGCCTGATCCTCCAAAT 269 qPCR rev_CYB_RT- AAGGTAGCGGATGATTCAGCC 270 qPCR fwd_B2M_RT- CAGGTACTCCAAAGATTCAGG 271 qPCR_qPCR rev_B2M_RT- GTCAACTTCAATGTCGGATGG 272 qPCR_qPCR NexteraprimersforATAC-seq i5_common AATGATACGGCGACCACCGAGATCTACACTCGTCGGCA 273 GCGTCAGATGTG i7_1 CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCG 274 TGGGCTCGGAGATGT i7_2 CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCG 275 TGGGCTCGGAGATGT i7_3 CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCG 276 TGGGCTCGGAGATGT i7_4 CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCG 277 TGGGCTCGGAGATGT i7_5 CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCG 278 TGGGCTCGGAGATGT i7_6 CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCG 279 TGGGCTCGGAGATGT i7_7 CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCG 280 TGGGCTCGGAGATGT i7_8 CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCG 281 TGGGCTCGGAGATGT i7_9 CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCG 282 TGGGCTCGGAGATGT
Split-DDA.SUB.tox.-Cas9 Fusions Sequences
TABLE-US-00077 G1397DddA.sub.tox-N (SEQIDNO:283) GGCAGCTACGCCCTGGGTCCGTATCAGATTAGCGCCCCGCAGCTGCCAGCTTACAATGGTCAGA CCGTGGGTACCTTCTACTATGTGAACGACGCGGGCGGTCTGGAGAGCAAGGTGTTTAGCAGCGG CGGTCCAACCCCGTACCCAAACTATGCCAATGCCGGTCATGTGGAGGGTCAGAGCGCCCTGTTC ATGCGTGATAACGGCATCAGCGAGGGTCTGGTGTTCCACAACAACCCGGAAGGCACCTGCGGTT TTTGCGTGAACATGACCGAGACCCTGCTGCCGGAAAACGCGAAAATGACCGTGGTGCCGCCGGA AGGT Translatedaminoacidsequence: (SEQIDNO:284) GSYALGPYQISAPOLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALF MRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG G1397DddA.sub.tox-C (SEQIDNO:285) GCCATTCCAGTGAAGCGCGGCGCTACCGGTGAAACCAAAGTGTTTACCGGTAACAGCAACAGCC CGAAGAGCCCGACCAAAGGCGGTTGC (SEQIDNO:286) Translatedaminoacidsequence: AIPVKRGATGETKVFTGNSNSPKSPTKGGC G1333DddA.sub.tox-N-SaKKH-Cas9(D10A)-UGI (SEQIDNO:287) GGCAGCTACGCCCTGGGTCCGTATCAGATTAGCGCCCCGCAGCTGCCAGCTTACAATGGTCAGA CCGTGGGTACCTTCTACTATGTGAACGACGCGGGCGGTCTGGAGAGCAAGGTGTTTAGCAGCGG CGGTTCTGGTGGTTCTTCTGGTGGTTCTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCC ACACCCGAAAGTAGTGGCGGCAGCAGCGGCGGCAGCGGGAAACGGAACTACATCCTGGGGCTTG CCATTGGGATAACCAGCGTTGGCTACGGAATTATTGATTATGAGACACGCGATGTGATTGACGC CGGGGTTAGGCTGTTCAAAGAGGCCAACGTTGAAAACAACGAGGGAAGACGGAGTAAGCGCGGA GCAAGAAGACTCAAGCGCAGACGGAGACATCGGATTCAGAGGGTGAAAAAGCTGCTCTTCGATT ACAATCTCCTGACCGATCATAGTGAGCTGAGCGGAATCAACCCCTACGAGGCGCGAGTGAAAGG GCTTTCCCAGAAGCTGTCCGAAGAGGAGTTCTCCGCCGCGTTGCTGCACCTGGCCAAACGGAGG GGGGTTCACAATGTAAACGAAGTGGAGGAGGACACGGGCAATGAACTTAGTACGAAAGAACAGA TCAGTAGGAACTCTAAGGCTCTCGAAGAGAAATACGTCGCTGAGTTGCAGCTTGAGAGACTGAA AAAAGACGGCGAAGTACGCGGATCTATTAATAGGTTCAAGACTTCAGATTACGTAAAGGAAGCC AAGCAGCTCCTGAAAGTACAGAAAGCGTACCATCAGCTCGATCAGAGCTTCATCGATACCTACA TAGATTTGCTGGAGACACGGAGGACATACTACGAGGGCCCAGGGGAAGGATCTCCTTTTGGGTG GAAGGACATCAAGGAATGGTACGAGATGCTTATGGGACATTGTACATATTTTCCGGAGGAGCTC AGGAGCGTCAAGTACGCCTACAATGCCGACCTGTACAATGCCCTCAATGACCTCAATAACCTCG TGATTACCAGGGACGAGAACGAGAAGCTGGAGTACTATGAAAAGTTCCAGATTATCGAGAATGT GTTTAAGCAGAAGAAGAAGCCGACACTTAAGCAGATTGCAAAGGAAATCCTCGTGAATGAGGAA GATATCAAGGGATACAGAGTGACAAGTACAGGCAAGCCCGAGTTCACAAATCTGAAGGTGTACC ACGATATTAAGGACATAACCGCACGAAAGGAGATAATCGAAAACGCTGAGCTCCTCGATCAGAT CGCAAAAATTCTTACCATCTACCAGTCTAGTGAGGACATTCAGGAGGAACTGACTAATCTGAAC AGTGAGCTCACCCAAGAGGAAATTGAGCAGATTTCAAACCTGAAAGGCTACACCGGGACGCACA ATCTGAGCCTCAAAGCAATCAACCTCATTCTGGATGAACTTTGGCACACAAATGACAACCAAAT TGCCATATTCAACCGCCTGAAACTGGTGCCAAAAAAAGTGGATCTGTCACAGCAAAAGGAAATC CCTACAACCTTGGTTGACGATTTTATTCTGTCCCCCGTTGTCAAGCGGAGCTTCATCCAGTCAA TCAAGGTGATCAATGCCATCATTAAAAAATACGGATTGCCAAACGATATAATTATCGAGCTTGC ACGAGAGAAGAACTCAAAGGACGCCCAGAAGATGATTAACGAAATGCAGAAGCGCAACCGCCAG ACAAACGAACGCATAGAGGAAATTATAAGAACAACCGGCAAAGAGAATGCCAAGTATCTGATCG AGAAAATCAAGCTGCACGACATGCAAGAAGGCAAGTGCCTGTACTCTCTGGAAGCTATCCCACT CGAAGATCTGCTGAATAATCCATTCAATTACGAGGTGGACCACATCATCCCTAGATCCGTAAGC TTTGACAATTCCTTCAATAACAAAGTTCTGGTTAAACAGGAGGAAAATTCTAAAAAAGGGAACC GGACCCCGTTCCAGTACCTGAGCTCCAGTGACAGCAAGATTAGCTACGAGACTTTTAAGAAACA TATTCTGAATCTGGCCAAAGGCAAAGGCAGGATCAGCAAGACCAAGAAGGAGTACCTCCTCGAA GAACGCGACATTAACAGATTTAGTGTGCAGAAAGATTTCATCAACCGAAACCTTGTCGATACTC GGTACGCCACGAGAGGCCTGATGAATCTCCTCAGGAGCTACTTCCGCGTCAATAATCTGGACGT TAAAGTCAAGAGCATAAATGGGGGATTCACCAGCTTTCTGAGGAGAAAGTGGAAGTTTAAGAAG GAACGAAACAAAGGATACAAGCACCATGCTGAGGATGCTTTGATCATCGCTAACGCGGACTTTA TCTTTAAGGAATGGAAAAAGCTGGATAAGGCAAAGAAAGTGATGGAAAACCAGATGTTCGAGGA GAAGCAGGCAGAGTCAATGCCTGAGATCGAGACAGAGCAGGAATACAAGGAAATTTTCATCACC CCTCATCAGATTAAACACATAAAGGACTTCAAAGACTATAAATACTCTCATAGGGTGGACAAAA AACCCAATCGCAAGCTCATTAATGACACCCTGTACTCAACACGGAAGGATGATAAAGGTAATAC CTTGATTGTGAATAATCTTAATGGATTGTATGACAAAGATAACGACAAGCTCAAGAAGCTGATC AACAAGTCTCCAGAGAAGCTCCTTATGTATCACCACGACCCACAGACTTATCAGAAATTGAAAC TGATCATGGAGCAATACGGGGATGAGAAGAACCCACTCTACAAATATTATGAGGAAACAGGTAA TTACCTGACCAAGTACTCCAAGAAGGATAACGGACCAGTGATCAAAAAGATAAAGTACTATGGC AACAAACTTAATGCGCATTTGGACATAACTGACGATTACCCCAATTCTCGAAACAAGGTTGTGA AGCTCTCCCTGAAGCCTTATAGATTTGACGTGTACCTGGATAATGGGGTTTATAAATTCGTCAC CGTGAAAAATCTGGACGTGATCAAAAAGGAGAACTATTATGAAGTAAACTCAAAGTGCTATGAG GAGGCGAAGAAGCTGAAGAAGATCTCCAATCAGGCCGAGTTCATCGCTTCCTTCTATAAGAACG ATCTCATCAAGATCAATGGAGAGCTTTATCGCGTCATTGGTGTGAACAATGACTTGCTGAACAG GATCGAAGTCAATATGATAGACATTACCTACCGGGAGTATCTCGAAAACATGAATGATAAACGG CCGCCTCACATCATCAAGACAATCGCATCTAAAACTCAGTCAATAAAAAAGTACTCTACCGATA TCCTGGGGAATCTCTATGAAGTGAAGTCAAAGAAGCACCCACAAATCATTAAAAAAGGTGGATC CCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCAT
Split-DddA.SUB.tox.-CCR5 TALE Fusions Sequences
TABLE-US-00078
General DdCBE Architecture and mitoTALE Sequences
Legend:
TABLE-US-00079
[0966] All right-side halves of DdCBEs have the general architecture of (from N- to C-terminus): COX8 MTS-3FLAG-mitoTALE-2aa linker-DddA.sub.tox half-4aa linker-1-UGI-ATP5B 3UTR
[0967] All left-side halves of DdCBEs have the general architecture of (from N- to C-terminus):
TABLE-US-00080 SOD2MTS-3xHA-mitoTALE-2aalinker-DddA.sub.toxhalf-4aalinker-1x-UGI-SOD23'UTR (A)SOD2MTS (SEQIDNO:298) MLSRAVCGTSRQLAPVLGYLGSROKHSLPD (B)COX8AMTS (SEQIDNO:299) MSVLTPLLLRGLTGSARRLPVPRAKIHSL (C)SOD23'UTR (SEQIDNO:300) ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCTGTCTTCTAAGA TGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCCTGGATAATTTTTGTTTGATTAT TCATTGAAGAAACATTTATTTTCCAATTGTGTGAAGTTTTTGACTGTTAATAAAAGAATCTGTC AACCATCAAAAAAAAAAAAAAA (D)ATP5B3'UTR (SEQIDNO:301) ACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCAAGATAATGTCCTGTCTTCTAAGA TGTGCATCAAGCCTGGTACATACTGAAAACCCTATAAGGTCCTGGATAATTTTTGTTTGATTAT TCATTGAAGAAACATTTATTTTCCAATTGTGTGAAGTTTTTGACTGTTAATAAAAGAATCTGTC AACCATCAAAAAAAAAAAAAAA (E)ND6-DdCBE:LeftmitoTALE-G1397-DddAtox-N-1x-UGI (SEQIDNo:302) ATGGCCCTGTCCCGTGCGGTTTGTGGCACCTCCCGTCAACTGGCTCCGGTTCTGGGTTATCTGG GTTCCCGTCAAAAACACTCCCTGCCGGACTACCCGTATGATGTTCCGGATTACGCTGGCTACCC ATACGACGTCCCAGACTACGCTGGCTACCCATACGACGTCCCAGACTACGCTATGGACATCGCG GATCTGCGTACCCTGGGTTACAGCCAGCAGCAGCAGGAGAAGATCAAGCCGAAGGTGCGCAGCA CCGTGGCTCAGCACCACGAAGCCCTGGTGGGCCACGGTTTCACCCACGCTCACATTGTGGCCCT GAGCCAGCACCCAGCCGCGCTGGGCACCGTGGCCGTGAAATATCAGGATATGATTGCTGCCCTG CCAGAGGCCACCCATGAAGCTATTGTGGGCGTGGGCAAGCAGTGGAGCGGTGCTCGTGCGCTGG AGGCGCTGCTGACCGTGGCTGGTGAACTGCGTGGTCCGCCGCTGCAGCTGGACACCGGTCAGCT GCTGAAAATCGCGAAACGTGGCGGTGTGACCGCGGTGGAAGCCGTGCATGCTTGGCGTAATGCT CTGACCGGTGCGCCGCTGAACCTGACCCCGCAGCAGGTGGTGGCTATTGCCAGCAACAACGGCG GTAAACAGGCCCTGGAGACCGTGCAGCGCCTGCTGCCGGTGCTGTGCCAGGCCCATGGTCTGAC CCCGGAGCAGGTGGTGGCGATCGCTAGCAACATCGGCGGCAAGCAGGCCCTGGAAACCGTGCAG GCGCTGTTACCGGTGCTGTGCCAGGCTCATGGCCTGACCCCGGAACAAGTTGTGGCTATTGCCA GCCATGATGGCGGTAAACAGGCTCTGGAAACCGTGCAGCGTCTGTTGCCGGTGCTGTGCCAAGC CCATGGCCTGACCCCGGAGCAAGTTGTGGCTATTGCGAGCCATGATGGCGGCAAGCAGGCGCTG GAAACCGTTCAGCGCCTGTTACCGGTGCTGTGCCAAGCTCATGGTCTGACCCCGGAACAGGTGG TGGCCATTGCTTCCCATGATGGCGGTAAACAGGCCCTGGAAACCGTTCAGCGTCTGCTTCCGGT GCTGTGCCAGGCCCATGGGCTGACCCCGGAACAAGTGGTTGCTATTGCCAGCCACGATGGCGGC AAGCAGGCTCTGGAGACCGTTCAGCGCCTGCTTCCGGTGCTGTGCCAGGCCCATGGCTTAACCC CGGAACAAGTTGTTGCTATTGCTAGTCATGATGGCGGTAAACAGGCGCTGGAGACCGTTCAGCG TCTGTTACCGGTGCTGTGCCAGGCGCATGGCTTAACCCCGGAGCAGGTTGTTGCCATTGCCTCC AATATCGGCGGCAAGCAGGCTCTGGAAACCGTTCAGGCCCTGTTGCCGGTGCTGTGCCAGGCCC ATGGACTGACCCCGCAGCAAGTTGTTGCCATTGCCAGCAATGGCGGTGGCAAACAGGCGCTGGA AACTGTTCAGCGCCTGCTCCCGGTGCTGTGCCAAGCGCATGGTCTGACCCCGCAGCAAGTGGTT GCTATTGCTAGCAATGGTGGCGGTCGTCCGGCGCTGGAAAGCATTGTGGCTCAGCTGAGCCGTC CAGACCCGGCCCTGGCGGCTCTGACCAACGATCACCTGGTGGCGCTGGCTTGCCTGGGCGGTCG TCCGGCCCTGGATGCGGTGAAGAAAGGCCTGGGTGGATCCGGCAGCTACGCCCTGGGTCCGTAT CAGATTAGCGCCCCGCAGCTGCCAGCTTACAATGGTCAGACCGTGGGTACCTTCTACTATGTGA ACGACGCGGGCGGTCTGGAGAGCAAGGTGTTTAGCAGCGGCGGTCCAACCCCGTACCCAAACTA TGCCAATGCCGGTCATGTGGAGGGTCAGAGCGCCCTGTTCATGCGTGATAACGGCATCAGCGAG GGTCTGGTGTTCCACAACAACCCGGAAGGCACCTGCGGTTTTTGCGTGAACATGACCGAGACCC
Supplementary Sequences 2|Split-DddA.SUB.tox.-Cas9 Fusions Sequences
[0968] All split-DddA.sub.tox-Cas9 have the general architecture of (from N- to C-terminus): bpNLS-DddA.sub.tox half-32aa linker-dSpCas9 or SaKKH-Cas9(D10 )-10aa linker-UGI-10aa linker-UGI-4aa linker-bpNLS
TABLE-US-00081 G1333DddAtox-N- (SEQIDNO:349) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG G1333DddAtox-C (SEQIDNO:350) PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMT VVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC G1397DddAtox-N- (SEQIDNO:351) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVESSGGPTPYPNYANAGHVE GQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG G1397DddAtox-C (SEQIDNO:352) AIPVKRGATGETKVFTGNSNSPKSPTKGGC dSpCas9 (SEQIDNO:353) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNL SDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGD SaKKH-Cas9(D10A) (SEQIDNO:354) GKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRR RRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGV HNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYV KEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLM GHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKK KPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKI LTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQI AIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIEL AREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYS LEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDS KISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGL MNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFI FKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSH RVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYH HDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLN AHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYL ENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGSPKKKRKVSSD YKDHDGDYKDHDIDYKDDDDK UGI (SEQIDNO:355) TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKML bpNLS (SEQIDNO:356) KRTADGSEFEPKKKRKV 32aalinker (SEQIDNO:357) SGGSSGGSSGSETPGTSESATPESSGGSSGGS 10aalinker (SEQIDNO:358) SGGSGGSGGS 4aalinker (SEQIDNO:359) SGGS
Supplementary Sequences 4 General DdCBE Architecture and mitoTALE Amino Acid Sequences
[0969] All right-side halves of DdCBEs have the general architecture of (from N- to C-terminus): COX8 MTS-3FLAG-mitoTALE-2aa linker-DddA.sub.tox half-4aa linker-1-UGI-ATP5B 3UTR
[0970] All left-side halves of DdCBEs have the general architecture of (from N- to C-terminus): SOD2 MTS-3HA-mitoTALE-2aa linker-DddA.sub.tox half-4aa linker-1-UGI-SOD2 3UTR
[0971] mitoTALE domains are annotated as: bold for N-terminal domain, underlined for RVD and bolded italics for C-terminal domain.
TABLE-US-00082 ND6-DdCBE:LeftmitoTALE-G1397-DddAtox-N-1x-UGI(Note:TerminalNGRVD recognizesamismatchedTinsteadofaGinthereferencegenome) (SEQIDNO:360) MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVP DYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAAL GTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQL LKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETV QALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGP YQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFM RDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETG KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQ DSNGENKIKML** ND6-DdCBE:RightmitoTALE-G1397-DddAtox-N-1x-UGI(Note:TerminalNGRVD recognizesamismatchedTinsteadofaGinthereferencegenome.The NTDwasalsoengineeredtobepermissiveforA,T,CandGnucleotides attheNOposition) (SEQIDNO:361) MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIAD LRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQ DMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGV TAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVAL ACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDI IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKP WALVIQDSNGENKIKML** ND1-DdCBERightmitoTALErepeat (SEQIDNO:362) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV QALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAAL TNDHLVALACLGGRPALDAVKKGLG ND1-DdCBELeftmitoTALErepeat (SEQIDNO:363) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND2-DdCBERightmitoTALErepeat (SEQIDNO:364) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGG KQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND2-DdCBELeftmitoTALErepeat (SEQIDNO:365) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNG GKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPV LCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVA IASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV QALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNI GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALT NDHLVALACLGGRPALDAVKKGLG ND4-DdCBERightmitoTALErepeat (SEQIDNO:366) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG GKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQA HGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND4-DdCBELeftmitoTALErepeat (SEQIDNO:367) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGL TPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV QALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASN GGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.1-DdCBERightmitoTALErepeat (SEQIDNO:368) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLG ND5.1-DdCBELeftmitoTALErepeat (SEQIDNO:369) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG GKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA LETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVA IASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.2-DdCBERightmitoTALErepeat(Note:TerminalNGRVDrecognizesa mismatchedTinsteadofaGinthereferencegenome) (SEQIDNO:370) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAH GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALAC LGGRPALDAVKKGLG ND5.2-DdCBELeftmitoTALErepeat (SEQIDNO:371) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQ ALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALET VQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVA QLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ND5.3-DdCBERightmitoTALErepeat (SEQIDNO:372) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGG KQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV KKGLG ND5.3-DdCBELeftmitoTALErepeat (SEQIDNO:373) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGL TPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG KQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQ LSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ATP8-DdCBERightmitoTALErepeat(Note:TerminalNGRVDrecognizesa mismatchedTinsteadofaCinthereferencegenome) (SEQIDNO:374) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVA IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG ATP8-DdCBELeftmitoTALErepeat (SEQIDNO:375) DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV KYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGG KQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVL CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA LLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLG GRPALDAVKKGLG
Example 2: New DddA Mutants with Improved Editing Activity at TC Context
[0972] As noted in Example 1, DdCBE editing activity at selected sites, including MT-ATP8 and MT-ND5.2, remained low (<10%) across all possible G1333 and G1397 split orientations. Phage-assisted non-continuous and continuous evolution (PANCE and PACE) was applied to evolve split-DddA (Thuronyi, B. W. et al. Nat Biotechnol 37, 1070-1079(2019)). (
TABLE-US-00083 SEQID Mutation(s) Sequence NO: DddA(residues GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTP 338 1290-1427) YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTET LLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC T1380I GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTP 377 YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMIETL LPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC T1314A/T1380I GSYALGPYQISAPQLPAYNGQTVGAFYYVNDAGGLESKVFSSGGPTP 378 YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMIETL LPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC Q1310R/S1330I/ GSYALGPYQISAPQLPAYNGRTVGTFYYVNDAGGLESKVFISGGPTP 379 T1380I YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMIETL LPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC T1380I/T1413I GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTP 380 YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMIETL LPENAKMTVVPPEGAIPVKRGATGETKVFIGNSNSPKSPTKGGC T1314A/T1380I/ GSYALGPYQISAPQLPAYNGQTVGAFYYVNDAGGLESKVFSSGGPTP 381 E1396K YPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMIETL LPENAKMTVVPPKGAIPVKRGATGETKVFTGNSNSPKSPTKGGC
[0973] These DddA mutations were cloned into the DdCBE architecture (MTS-right TALE-G1397 split DddA-(N/C)-UGI) and plasmid transfected into human HEK293T cells (
Example 3. Lentiviral Delivery of DdCBEs into Mouse Cells
[0974] The ability of DdCBEs to install disease-causing mutations in animal models will accelerate the study of disease etiology and facilitate preclinical testing of drug candidates. In particular, mutations in mitochondrial genes encoding for Complex I subunits are increasingly studied for their role in cancer (Gopal, R. K. et al. Cancer Cell 34, 242-255.e245(2018)). DdCBEs were designed that target various Complex I genes in the mouse mtDNA to install a pathogenic missense mutation. The right and left halves of each DdCBE were cloned separately into a lentiviral vector and co-transfected into mouse embryonic fibroblasts (
Example 4. Alternative DdCBE Architectures
[0975] The original DdCBE design, described in Example 1, contains a Left-TALE that targets the top coding DNA strand and a Right-TALE that targets the bottom coding DNA strand (
[0976] In general, the editing efficiencies observed for the alternative DdCBE architectures were generally lower than the original DdCBE design (
TABLE-US-00084 MTS-splitDddAhalfG1397-N-ND1RightTALE repeat-UGI (SEQIDNO:420) SVLTPLLLRGLTGSARRLPVPRAKIHSLMGSYALGPYQISAPQLPAYNG QTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNG ISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGGSDIADLRTL GYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVA VKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLD TGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQ ALETVQALLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN GGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV LCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQALLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAA LTNDHLVALACLGGRPALDAVKKGLGSGGSTNLSDIIEKETGKQLVIQE SILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWA LVIQDSNGENKIKML
Example 5. Mitochondrial Base Editing with ZF-BE
[0977] A process for ZF-BE base editing was designed as an alternative to TALE-DdCBE described in the Examples above. As ZFs (0.5kb) are smaller than TALE (2kb), it was believed that ZF-BEs would allow for better expression and lower immunogenicity, as well as single AAV base editing (21.2 kb). However, to date, no published pairs of mitoZFs exist which target the wild-type genome sequence. As a result, ZFs were designed to be used in combination previously reported mitoZFs R13-1 and R8 (Gammage et al. EMBO Mol Med. 2014 Apr;6(4):458-66.)
ZF-BEs Design
[0978] ZF-BEs were designed to comprise the following components: [0979] Mitochondrial Targeting Sequence (MTS) for import of a cytosolic-located protein into the mitochondria. Localisation of the ZF-BE in the mitochondria is critical for its ability to edit mitochondrial DNA. [0980] FLAG tag which serves two rolesfirstly as an affinity tag allowing both expression levels of the ZF-BE to be determined by Western Blot using an anti-FLAG antibody, and intracellular localization to be imaged by antibody staining of transfected cells by microscopy. Secondly, as a disordered sequence following the MTS which improves mitochondrial import via the MTS, facilitating better loading and protein unfolding through mitochondrial import channel. [0981] Nuclear Export Sequence (NES) for export of a nuclear-located protein into the cytosol, where it can then be transported into the mitochondria via its MTS. Since ZFs inherently encode a Nuclear Localisation Sequence (NLS), this is important to counteract their natural tendency to localize in the nucleus which is problematic for editing mitochondrial DNA. Utilising two separate NES boosts the cytoplasmic localization of ZF-BEs by further reducing their nuclear localization, enabling stronger uptake of ZF-BEs into the mitochondria for BE to occur. [0982] Zinc Finger (ZF) as a DNA-binding domain able to specify the target sequence for the ZF-BE to bind to within mitochondrial DNA and target the split DddA deaminase towards site-specific base editing. [0983] Split DddA deaminase, capable of performing deamination on dsDNA when reconstituted into a full-length DddA at the target site within mitochondrial DNA when both halves of the DddA deaminase are colocalized. [0984] Uracil glycosylase inhibitor (UGI) for binding to mitochondrial uracil DNA glycosylase (UDG) and inhibiting the activity of UDG to excise modified uracil bases from edited DNA. [0985] P2 peptide enabling coexpression of two independent polypeptides from a single gene-encoding sequence due to the inability of the mammalian ribosome to form complete polypeptide bond formation during translation. This is used to enable coexpression of an additional copy of UGI from the same gene-encoding sequence which can independently localize into mitochondria via its own MTS, increasing the level of mitochondrial UDG inhibition.
Optimisation of ZF-BE Architecture
[0986] ZFs were designed to target two sites in the mitochondrial genome: ATP8 (referred to as R8) and ND5.1 (referred to as R13). ZFs were designed using a modular assembly approach, using a modified Zif268 scaffold. At each site ZFs were designed to form one half of a pair alongside R8 or R13-1. The resulting set of ZFs bound to sequences and created an editing window between the ZF pair, ranging from 4-18 bp. Binding motifs are shown in
[0987] The architecture of the lead v1 ZF-BE candidates were varied with regards to MTS, NES and linker length (v2) (
[0988] The ZF-BE architecture was further optimized by retaining the Link13 flexible linker and the N-terminal MTS and NES from v2 experiments. V3 systematically varied the MTS in the presence or absence of an affinity tag sequence (FLAG or HA) immediately following the MTS (
[0989] The next round of optimization tested additional NES sequences (x3), different UGI homologs (x2) and ZF scaffold mutations (
[0990] The D mutations were combined with previous architecture improvements (
[0991] The final round of optimization included an additional NES, improvement of the ZF scaffold sequence, and coexpression of a separate mitochondrially-targeted UGI (
TABLE-US-00085 R8v6 MLGFVGRVAAAPASGALRRLTPSASLPP MTS AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSTSG FLAGtag SLSRHIRTHTGEKPFQCDICMRNFSQSGS DYKDDDDK(SEQIDNO:395) LTRHIRTHTGSEKPFQCDICMRNFSRSDA NES LSQHIRTHTGEKPFQCDICMRNFSRNDN VDEMTKKFGTLTIHDTEK(SEQIDNO:396) RITHIRTHTGEKPFQCDICMRNFSRSDHL Linker TQHTKIHLRGSGGGGSGGSGGSGSYAL GS GPYQISAPQLPAYNGQTVGTFYYVNDA NES2 GGLESKVFSSGGPTPYPNYANAGHVEG LQKKLEELELD(SEQIDNO:397) QSALFMRDNGISEGLVFHNNPEGTCGFC Linker VNMTETLLPENAKMTVVPPEGSGGSTN AA LSDIIEKETGKQLVIQESILMLPEEVEEVI ZF(R8) GNKPESDILVHTAYDESTDENVMLLTSD MAERPFQCDICMRNFSTSGSLSRHIRTHTGEKPF APEYKPWALVIQDSNGENKIKMLGSGA QCDICMRNFSQSGSLTRHIRTHTGSEKPFQCDIC TNFSLLKQAGDVEENPGPMASVLTPLLL MRNFSRSDALSQHIRTHTGEKPFQCDICMRNFS RGLTGSARRLPVPRAKIHSLGSTNLSDII RNDNRITHIRTHTGEKPFQCDICMRNFSRSDHL EKETGKQLVIQESILMLPEEVEEVIGNKP TQHTKIHLR(SEQIDNO:398) ESDILVHTAYDESTDENVMLLTSDAPEY Linker KPWALVIQDSNGENKIKML(SEQIDNO: GSGGGGSGGSGGS(SEQIDNO:399) 382) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDA GGLESKVFSSGGPTPYPNYANAGHVEGQSALF MRDNGISEGLVFHNNPEGTCGFCVNMTETLLP ENAKMTVVPPEG(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-4- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R8v6 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSQAS FLAGtag NLISHIRTHTGEKPFQCDICMRNFSTSHS DYKDDDDK(SEQIDNO:395) LTEHIRTHTGSEKPFQCDICMRNFSERSH NES LREHIRTHTGEKPFQCDICMRNFSQSGN VDEMTKKFGTLTIHDTEK(SEQIDNO:396) LTEHIRTHTGEKPFQCDICMRNFSSKKA Linker LTEHTKIHLRGSGGGGSGGSGGSAIPVK GS RGATGETKVFTGNSNSPKSPTKGGCSGG NES2 STNLSDIIEKETGKQLVIQESILMLPEEVE LQKKLEELELD(SEQIDNO:397) EVIGNKPESDILVHTAYDESTDENVMLL Linker TSDAPEYKPWALVIQDSNGENKIKMLGS AA GATNFSLLKQAGDVEENPGPMASVLTP ZF(5xZnF-4-R8) LLLRGLTGSARRLPVPRAKIHSLGSTNLS MAERPFQCDICMRNFSQASNLISHIRTHTGEKPF DIIEKETGKQLVIQESILMLPEEVEEVIGN QCDICMRNFSTSHSLTEHIRTHTGSEKPFQCDIC KPESDILVHTAYDESTDENVMLLTSDAP MRNFSERSHLREHIRTHTGEKPFQCDICMRNFS EYKPWALVIQDSNGENKIKML(SEQID QSGNLTEHIRTHTGEKPFQCDICMRNESSKKAL NO:383) TEHTKIHLR(SEQIDNO:402) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-10- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R8v6 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSQAS FLAGtag NLISHIRTHTGEKPFQCDICMRNFSQRAN DYKDDDDK(SEQIDNO:395) LRAHIRTHTGSEKPFQCDICMRNFSQAS NES NLISHIRTHTGEKPFQCDICMRNFSTSHS VDEMTKKFGTLTIHDTEK(SEQIDNO:396) LTEHIRTHTGEKPFQCDICMRNFSERSHL Linker REHTKIHLRGSGGGGSGGSGGSAIPVKR GS GATGETKVFTGNSNSPKSPTKGGCSGGS NES2 TNLSDIIEKETGKQLVIQESILMLPEEVEE LQKKLEELELD(SEQIDNO:397) VIGNKPESDILVHTAYDESTDENVMLLT Linker SDAPEYKPWALVIQDSNGENKIKMLGS AA GATNFSLLKQAGDVEENPGPMASVLTP ZF(5xZnF-10-R8) LLLRGLTGSARRLPVPRAKIHSLGSTNLS MAERPFQCDICMRNFSQASNLISHIRTHTGEKPF DIIEKETGKQLVIQESILMLPEEVEEVIGN QCDICMRNFSQRANLRAHIRTHTGSEKPFQCDI KPESDILVHTAYDESTDENVMLLTSDAP CMRNFSQASNLISHIRTHTGEKPFQCDICMRNFS EYKPWALVIQDSNGENKIKML(SEQID TSHSLTEHIRTHTGEKPFQCDICMRNFSERSHLR NO:384) EHTKIHLR(SEQIDNO:403) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) R13-1v6 MLGFVGRVAAAPASGALRRLTPSASLPP MTS AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSRSD FLAGtag NLSTHIRTHTGEKPFQCDICMRNFSDRS DYKDDDDK(SEQIDNO:395) DLSRHIRTHTGEKPFQCDICMRNFSQSG NES DLTRHIRTHTGSEKPFQCDICMRNFSRSD VDEMTKKFGTLTIHDTEK(SEQIDNO:396) SLSAHIRTHTGEKPFQCDICMRNFSQKA Linker TRITHTKIHLRGSGGGGSGGSGGSGSYA GS LGPYQISAPQLPAYNGQTVGTFYYVND NES2 AGGLESKVFSSGGPTPYPNYANAGHVE LqKKLEELELD(SEQIDNO:397) GQSALFMRDNGISEGLVFHNNPEGTCGF Linker CVNMTETLLPENAKMTVVPPEGSGGST AA NLSDIIEKETGKQLVIQESILMLPEEVEEV ZF(R13-1) IGNKPESDILVHTAYDESTDENVMLLTS MAERPFQCDICMRNFSRSDNLSTHIRTHTGEKP DAPEYKPWALVIQDSNGENKIKMLGSG FQCDICMRNFSDRSDLSRHIRTHTGEKPFQCDIC ATNFSLLKQAGDVEENPGPMASVLTPLL MRNFSQSGDLTRHIRTHTGSEKPFQCDICMRNF LRGLTGSARRLPVPRAKIHSLGSTNLSDI SRSDSLSAHIRTHTGEKPFQCDICMRNFSQKAT IEKETGKQLVIQESILMLPEEVEEVIGNK RITHTKIHLR(SEQIDNO:404) PESDILVHTAYDESTDENVMLLTSDAPE Linker YKPWALVIQDSNGENKIKML(SEQID GSGGGGSGGSGGS(SEQIDNO:399) NO:385) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDA GGLESKVFSSGGPTPYPNYANAGHVEGQSALF MRDNGISEGLVFHNNPEGTCGFCVNMTETLLP ENAKMTVVPPEG(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-9- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R13v6 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSQSS FLAGtag SLVRHIRTHTGEKPFQCDICMRNFSRSD DYKDDDDK(SEQIDNO:395) NLVRHIRTHTGSEKPFQCDICMRNFSQA NES GHLASHIRTHTGEKPFQCDICMRNFSRK VDEMTKKFGTLTIHDTEK(SEQIDNO:396) DNLKNHIRTHTGEKPFQCDICMRNFSRK Linker DALRGHTKIHLRGSGGGGSGGSGGSAIP GS VKRGATGETKVFTGNSNSPKSPTKGGCS NES2 GGSTNLSDIIEKETGKQLVIQESILMLPEE LQKKLEELELD(SEQIDNO:397) VEEVIGNKPESDILVHTAYDESTDENVM Linker LLTSDAPEYKPWALVIQDSNGENKIKML AA GSGATNFSLLKQAGDVEENPGPMASVL ZF(5xZnF-9-R13) TPLLLRGLTGSARRLPVPRAKIHSLGSTN MAERPFQCDICMRNFSQSSSLVRHIRTHTGEKP LSDIIEKETGKQLVIQESILMLPEEVEEVI FQCDICMRNFSRSDNLVRHIRTHTGSEKPFQCDI GNKPESDILVHTAYDESTDENVMLLTSD CMRNFSQAGHLASHIRTHTGEKPFQCDICMRNF APEYKPWALVIQDSNGENKIKML(SEQ SRKDNLKNHIRTHTGEKPFQCDICMRNFSRKDA IDNO:386) LRGHTKIHLR(SEQIDNO:405) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-12- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R13v6 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKGSLQKK RAAPTAVHPVRDYAAQ(SEQIDNO:394) LEELELDAAMAERPFQCDICMRNFSRSD FLAGtag HLTTHIRTHTGEKPFQCDICMRNFSQSSS DYKDDDDK(SEQIDNO:395) LVRHIRTHTGSEKPFQCDICMRNFSRSD NES NLVRHIRTHTGEKPFQCDICMRNFSQAG VDEMTKKFGTLTIHDTEK(SEQIDNO:396) HLASHIRTHTGEKPFQCDICMRNFSRKD Linker NLKNHTKIHLRGSGGGGSGGSGGSAIPV GS KRGATGETKVFTGNSNSPKSPTKGGCSG NES2 GSTNLSDIIEKETGKQLVIQESILMLPEEV LqKKLEELELD(SEQIDNO:397) EEVIGNKPESDILVHTAYDESTDENVML Linker LTSDAPEYKPWALVIQDSNGENKIKML AA GSGATNFSLLKQAGDVEENPGPMASVL ZF(5xZnF-12-R13) TPLLLRGLTGSARRLPVPRAKIHSLGSTN MAERPFQCDICMRNFSRSDHLTTHIRTHTGEKP LSDIIEKETGKQLVIQESILMLPEEVEEVI FQCDICMRNFSQSSSLVRHIRTHTGSEKPFQCDI GNKPESDILVHTAYDESTDENVMLLTSD CMRNFSRSDNLVRHIRTHTGEKPFQCDICMRNF APEYKPWALVIQDSNGENKIKML(SEQ SQAGHLASHIRTHTGEKPFQCDICMRNFSRKDN IDNO:387) LKNHTKIHLR(SEQIDNO:406) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) Linker GSG P2APeptide ATNFSLLKQAGDVEENPGP(SEQIDNO:400) MTS MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQIDNO:401) Linker GS UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) R8v3 MLGFVGRVAAAPASGALRRLTPSASLPP MTS AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSTSGSLSRHIRTHTGEKP FLAGtag FACDICGRKFAQSGSLTRHTKIHTGGQR DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSRSDALSQHIRTHTGEKP NES FACDICGRKFARNDNRITHTKIHTGEKPF VDEMTKKFGTLTIHDTEK(SEQIDNO:396) QCRICMRKFARSDHLTQHTKIHLRGSGG Linker GGSGGSGGSGSYALGPYQISAPQLPAYN AA GQTVGTFYYVNDAGGLESKVFSSGGPT ZF(R8) PYPNYANAGHVEGQSALFMRDNGISEG MAERPFQCRICMRNFSTSGSLSRHIRTHTGEKPF LVFHNNPEGTCGFCVNMTETLLPENAK ACDICGRKFAQSGSLTRHTKIHTGGQRPFQCRI MTVVPPEGSGGSTNLSDIIEKETGKQLVI CMRNFSRSDALSQHIRTHTGEKPFACDICGRKF QESILMLPEEVEEVIGNKPESDILVHTAY ARNDNRITHTKIHTGEKPFQCRICMRKFARSDH DESTDENVMLLTSDAPEYKPWALVIQD LTQHTKIHLR(SEQIDNO:407) SNGENKIKML(SEQIDNO:388) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDA GGLESKVFSSGGPTPYPNYANAGHVEGQSALF MRDNGISEGLVFHNNPEGTCGFCVNMTETLLP ENAKMTVVPPEG(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-4- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R8v3 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSQASNLISHIRTHTGEKP FLAGtag FACDICGRKFATSHSLTEHTKIHTGSQKP DYKDDDDK(SEQIDNO:395) FQCRICMRNFSERSHLREHIRTHTGEKPF NES ACDICGRKFAQSGNLTEHTKIHTGEKPF VDEMTKKFGTLTIHDTEK(SEQIDNO:396) QCRICMRKFASKKALTEHTKIHLRGSGG Linker GGSGGSGGSAIPVKRGATGETKVFTGNS AA NSPKSPTKGGCSGGSTNLSDIIEKETGKQ ZF(5xZnF-4-R8) LVIQESILMLPEEVEEVIGNKPESDILVHT MAERPFQCRICMRNFSQASNLISHIRTHTGEKPF AYDESTDENVMLLTSDAPEYKPWALVI ACDICGRKFATSHSLTEHTKIHTGSQKPFQCRIC QDSNGENKIKML(SEQIDNO:389) MRNFSERSHLREHIRTHTGEKPFACDICGRKFA QSGNLTEHTKIHTGEKPFQCRICMRKFASKKAL TEHTKIHLR(SEQIDNO:408) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-10- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R8v3 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSQASNLISHIRTHTGEKP FLAGtag FACDICGRKFAQRANLRAHTKIHTGSQK DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSQASNLISHIRTHTGEKP NES FACDICGRKFATSHSLTEHTKIHTGEKPF VDEMTKKFGTLTIHDTEK(SEQIDNO:396) QCRICMRKFAERSHLREHTKIHLRGSGG Linker GGSGGSGGSAIPVKRGATGETKVFTGNS AA NSPKSPTKGGCSGGSTNLSDIIEKETGKQ ZF(5xZnF-10-R8) LVIQESILMLPEEVEEVIGNKPESDILVHT MAERPFQCRICMRNFSQASNLISHIRTHTGEKPF AYDESTDENVMLLTSDAPEYKPWALVI ACDICGRKFAQRANLRAHTKIHTGSQKPFQCRI QDSNGENKIKML(SEQIDNO:390) CMRNFSQASNLISHIRTHTGEKPFACDICGRKFA TSHSLTEHTKIHTGEKPFQCRICMRKFAERSHLR EHTKIHLR(SEQIDNO:409) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) R13-1v3 MLGFVGRVAAAPASGALRRLTPSASLPP MTS AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSRSDNLSTHIRTHTGEKP FLAGtag FACDICGRKFADRSDLSRHTKIHTGEKPF DYKDDDDK(SEQIDNO:395) QCRICMRKFAQSGDLTRHTKIHTGSQKP NES FQCRICMRNFSRSDSLSAHIRTHTGEKPF VDEMTKKFGTLTIHDTEK(SEQIDNO:396) ACDICGRKFAQKATRITHTKIHLRGSGG Linker GGSGGSGGSGSYALGPYQISAPQLPAYN AA GQTVGTFYYVNDAGGLESKVFSSGGPT ZF(R13-1) PYPNYANAGHVEGQSALFMRDNGISEG MAERPFQCRICMRNFSRSDNLSTHIRTHTGEKP LVFHNNPEGTCGFCVNMTETLLPENAK FACDICGRKFADRSDLSRHTKIHTGEKPFQCRIC MTVVPPEGSGGSTNLSDIIEKETGKQLVI MRKFAQSGDLTRHTKIHTGSQKPFQCRICMRNF QESILMLPEEVEEVIGNKPESDILVHTAY SRSDSLSAHIRTHTGEKPFACDICGRKFAQKAT DESTDENVMLLTSDAPEYKPWALVIQD RITHTKIHLR(SEQIDNO:410) SNGENKIKML(SEQIDNO:391) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397N) GSYALGPYQISAPQLPAYNGQTVGTFYYVNDA GGLESKVFSSGGPTPYPNYANAGHVEGQSALF MRDNGISEGLVFHNNPEGTCGFCVNMTETLLP ENAKMTVVPPEG(SEQIDNO:284) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-9- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R13v3 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSQSSSLVRHIRTHTGEKP FLAGtag FACDICGRKFARSDNLVRHTKIHTGSQK DYKDDDDK(SEQIDNO:395) PFQCRICMRNFSQAGHLASHIRTHTGEK NES PFACDICGRKFARKDNLKNHTKIHTGEK VDEMTKKFGTLTIHDTEK(SEQIDNO:396) PFQCRICMRKFARKDALRGHTKIHLRGS Linker GGGGSGGSGGSAIPVKRGATGETKVFT AA GNSNSPKSPTKGGCSGGSTNLSDIIEKET ZF(5xZnF-9-R13) GKQLVIQESILMLPEEVEEVIGNKPESDI MAERPFQCRICMRNFSQSSSLVRHIRTHTGEKP LVHTAYDESTDENVMLLTSDAPEYKPW FACDICGRKFARSDNLVRHTKIHTGSQKPFQCR ALVIQDSNGENKIKML(SEQIDNO:392) ICMRNFSQAGHLASHIRTHTGEKPFACDICGRK FARKDNLKNHTKIHTGEKPFQCRICMRKFARK DALRGHTKIHLR(SEQIDNO:411) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341) 5xZnF-12- MLGFVGRVAAAPASGALRRLTPSASLPP MTS R13v3 AQLLLRAAPTAVHPVRDYAAQDYKDD MLGFVGRVAAAPASGALRRLTPSASLPPAQLLL DDKVDEMTKKFGTLTIHDTEKAAMAER RAAPTAVHPVRDYAAQ(SEQIDNO:394) PFQCRICMRNFSRSDHLTTHIRTHTGEKP FLAGtag FACDICGRKFAQSSSLVRHTKIHTGSQKP DYKDDDDK(SEQIDNO:395) FQCRICMRNFSRSDNLVRHIRTHTGEKP NES FACDICGRKFAQAGHLASHTKIHTGEKP VDEMTKKFGTLTIHDTEK(SEQIDNO:396) FQCRICMRKFARKDNLKNHTKIHLRGSG Linker GGGSGGSGGSAIPVKRGATGETKVFTG AA NSNSPKSPTKGGCSGGSTNLSDIIEKETG ZF(5xZnF-12-R13) KQLVIQESILMLPEEVEEVIGNKPESDIL MAERPFQCRICMRNFSRSDHLTTHIRTHTGEKP VHTAYDESTDENVMLLTSDAPEYKPWA FACDICGRKFAQSSSLVRHTKIHTGSQKPFQCRI LVIQDSNGENKIKML(SEQIDNO:393) CMRNFSRSDNLVRHIRTHTGEKPFACDICGRKF AQAGHLASHTKIHTGEKPFQCRICMRKFARKD NLKNHTKIHLR(SEQIDNO:412) Linker GSGGGGSGGSGGS(SEQIDNO:399) SplitDddA(DddA-G1397C) AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQIDNO:286) Linker SGGS(SEQIDNO:348) UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPW ALVIQDSNGENKIKML(SEQIDNO:341)
Optimisation of the ZF Scaffold Sequence Encompasses Two Separate Improvements
[0992] First, ZFs designed according to the Zif268 scaffold consisted of an N-terminal sequence (MAERP (SEQ ID NO: 421)), followed by a number of ZF repeats separated by linkers, and ended with a C-terminal sequence (HTKIHLR (SEQ ID NO: 422)). ZFs are typically composed of multiple ZF repeats, and each repeat will have a N-terminal scaffold half (FQCRICMRNFS (SEQ ID NO: 423) or FACDICGRKFA (SEQ ID NO: 424) or FQCRICMRKFA (SEQ ID NO: 425), a seven amino acid DNA-binding region, a C-terminal scaffold half (HIRTH (SEQ ID NO: 426) or HTKIH (SEQ ID NO: 427), and a linker (TGEKP (SEQ ID NO: 428) or TGQKP (SEQ ID NO: 429), or TGSQKP (SEQ ID NO: 430)). An example of this structure is noted below:
TABLE-US-00086 (SEQIDNO:421) MAERP (SEQIDNO:431) FQCRICMRNFS...ZF1...HIRTHTGEKP (SEQIDNO:432) FACDICGRKFA...ZF2...HTKIHTGSQKP (SEQIDNO:431) FQCRICMRNFS...ZF3...HIRTHTGEKP (SEQIDNO:433) FACDICGRKFA...ZF4...HTKIHTGEKP (SEQIDNO:434) FQCRICMRKFA...ZF5...HTKIHLR
[0993] Wherein . . . ZF # . . . was replaced with seven amino acids (XXXXXXX) which specify the 3 bp DNA sequence to which this ZF repeat binds.
[0994] Improvements in ZF-BE editing efficiencies were only found in sequences that were scanned computationally to encode a weaker NLS. This implies designing ZFs such that every repeat will have an N-terminal scaffold half of FQCRICMRNFS (SEQ ID NO: 423) only, the seven amino acid DNA-binding region, a C-terminal scaffold half of HIRTH (SEQ ID NO: 426) only, and a linker of TGEKP (SEQ ID NO: 428) or TGSEKP (SEQ ID NO: 435) only. This strategy reduces the number of positively-charged residues (particularly Lys, K) which contribute to the inherent NLS of ZFs. As a result, the above example sequence would be converted into the sequence below, which produced improved levels of mitochondrial BE:
TABLE-US-00087 (SEQIDNO:421) MAERP (SEQIDNO:431) FQCRICMRNFS...ZF1...HIRTHTGEKP (SEQIDNO:436) FQCRICMRNFS...ZF2...HIRTHTGSEKP (SEQIDNO:431) FQCRICMRNFS...ZF3...HIRTHTGEKP (SEQIDNO:431) FQCRICMRNFS...ZF4...HIRTHTGEKP (SEQIDNO:437) FQCRICMRNFS...ZF5...HTKIHLR
[0995] Second, within each ZF repeat, mutation of the FQCRICMRNFS (SEQ ID NO: 423)N-terminal scaffold half to FQCDICMRNFS (SEQ ID NO: 438) (a single R to D mutation in each repeat), removes a positively-charged amino acid and replaces it with a negatively-charged amino acid. This weakens the NLS inherently encoded within ZFs without disrupting its ability to bind to DNA.
[0996] In combination, these improvements are significant because they specify a way to construct not just ZFs but specifically mitochondrially-optimised ZFs for the first time. Previously, ZFs have been used for nuclear genome editing, or repurposed without any optimization (beyond addition of an MTS) for mitochondrial genome editing. However, these advancements represent optimisations being made to ZFs to specifically tailor their architecture towards mitochondrial genome editingimproving their activity by boosting mitochondrial localization and reducing nuclear localization without impairing the DNA-binding ability of the ZFs themselves.
Co-Expression and Targeting of an Additional Copy of UGI to the Mitochondria in Trans
[0997] Previous BEs rely on inhibition of UDG, either in the nucleus or mitochondria, by fusion of one or multiple copies of UGI to the BE protein itself and act via colocalization. However, co-expression of additional copies of UGI as separate polypeptides (targeted to mitochondria via their own MTS) in order to suppress mitochondrial UDG to even lower levels has not previously been reported. This is an additional way to suppress mitochondrial UDG activity which can be applied to any mitochondrial BE (ZF- or TALE-based).
Example 6. Tale-DdCBE Mitochondrial Base Editing with Alternative UGI Homologs
[0998] New UGI homologs were identified by computational search for homology to known UGI proteins, namely from bacteriophage PBS2, bacteriophage phi29 and S. aureus, in addition to literature review (see UGI2 below). These newly-identified UGI homologs were tested for their ability to inhibit mitochondrial UDG in the context of TALE-DdCBEs (
TABLE-US-00088 UGI Sequence SEQIDNO: CanonicalUGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDE 341 STDENVMLLTSDAPEYKPWALVIQDSNGENKIKML UGI2 MTLELQLKHYITNLFNLPKDEKWHCESIEEIADDILPDQYVRLGAL 445 SNKILQTYTYYSDTLHESNIYPFILYYQKQLIAIGYIDENHDMDFLY LHNTIMPLLDQRYLLTGGQ UGI3 MNKNFDEVKADLRTVTGKKIEFKERLKNILRVQMNQLGFEDSYMI 446 QVQVSSDQEEWVECHENMSLSDFEVMYGNISGEIKRMTVVKYEE ANIEKLVELKFEYEYAKAHQEYIRAYTKLMSNTLYGRKPSL UGI5 MNEEKMHYRDAIKEVELTMMSLDSHFRTHKEFTDSYLLVLILEDV 447 VGETRVEVSEGLTFDEASYIIGGTSDNILNMHMINYCEKNREEIYK WLKVSRVNTFKSNYAKMLLNTAYGKDLLKGVVK UGI7 MNNHFMSIGRNCSKCNNVRLNEDFSKSEEICNECFDKEERFVDSY 375 TLIYITEDETGKRFEAILENQTIEETEIIYGNIIDKIIVWNVILTM UGI12 DGNEHWEVHPGLSLSDFEVVYGNNPHQIVKLRLDKEVGGSGGSM 376 VQNDFIDSYTLCWLLRDDSGGGGSMVQNDFIDSYTLCWLLRDDD GNEHWEVHPGLSLSDFEVVYGNNPHQIVKLRLDKEV UGI12 DGNEHWEVHPGLSLSDFEVVYGNNPHQIVKLRLDKEV 439 Monomer1-C UGI12 GGSGGS 440 Monomer1-C Linker UGI12 MVQNDFIDSYTLCWLLRDD 441 Monomer2-N UGI12 SGGGGS 442 Monomer2-N Linker UGI12 MVQNDFIDSYTLCWLLRDDDGNEHWEVHPGLSLSDFEVVYGNNP 443 Monomer2 HQIVKLRLDKEV
[0999] Three of the top perfuming UGI homologs were tested in the context of BE4 max. The remaining two were unable to be cloned in the BE4max architecture as they were toxic to E. coli in UGI-UGI (BE4max) architecture. However, results show that the UGI homologs did not improve editing efficiency in BE4max (
REFERENCES
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EQUIVALENTS AND SCOPE
[1110] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[1111] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[1112] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[1113] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.