METHOD FOR IDENTIFYING DNA BASE EDITING BY MEANS OF CYTOSINE DEAMINASE

Abstract

Provided are: a composition for DNA double-strand breaks (DSBs), comprising (1) a cytosine deaminase and an inactivated target-specific endonuclease, (2) a guide RNA, and (3) a uracil-specific excision reagent (USER); a method for producing DNA double-strand breaks by means of a cytosine deaminase using the composition; a method for analyzing a DNA nucleic acid sequence to which base editing has been introduced by means of a cytosine deaminase; and a method for identifying (or measuring or detecting) base editing, base editing efficiency at an on-target site, an off-target site, and/or target specificity by means of a cytosine deaminase.

Claims

1-23. (canceled)

24. A method of generating a double strand break in DNA using a cytosine deaminase, comprising: (i) introducing or contacting (a) a cytosine deaminase and an inactivated target-specific endonuclease, or (b) a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, or (c) a plasmid comprising a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, into a cell or with DNA isolated from a cell, together with a guide RNA; and (ii) treating a uracil-specific excision reagent (USER), wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein amino acid residue D10 is substituted with other amino acid, the DNA isolated from a cell in step (i) is a genomic DNA, and the nucleic acid sequence analysis of step (iii) is performed by a whole genome sequencing.

25. A method of analyzing nucleic acid sequence of DNA in which a base editing is introduced by cytosine deaminase, comprising: (i) introducing or contacting (a) a cytosine deaminase and an inactivated target-specific endonuclease, or (b) a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, or (c) a plasmid comprising a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, into a cell or with DNA isolated from a cell, together with a guide RNA; (ii) treating a uracil-specific excision reagent (USER), to generate double strand cleavage in DNA; and (iii) analyzing nucleic acid sequence of the cleaved DNA fragment, wherein the DNA isolated from a cell in step (i) is a genomic DNA.

26. The method of claim 25, wherein the inactivated target-specific endonuclease is a Cas9 protein or Cpf1 protein, which lacks endonuclease activity of cleaving a DNA double strand.

27. The method of claim 25, wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein amino acid residue D10 is substituted with other amino acid.

28. The method of claim 25, wherein the cytosine deaminase and inactivated target-specific endonuclease are in a form of a fusion protein, or the cytosine deaminase coding gene and inactivated target-specific endonuclease coding gene encode a fusion protein comprising the cytosine deaminase and inactivated target-specific endonuclease.

29. The method of claim 25, wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein both of amino acid residues D10 and H840 are substituted with other amino acids, and the method further comprises a step of treating an endonuclease specifically cleaving a single strand region of DNA, after step (ii).

30. The method of claim 25, wherein the guide RNA is a crRNA:tracrRNA duplex in which crRNA and tracrRNA is coupled to each other, or a single-strand guide RNA (sgRNA).

31. The method of claim 25, which is performed in vitro.

32. The method of claim 25, wherein the DNA isolated from a cell in step (i) is a genomic DNA, and the nucleic acid sequence analysis of step (iii) is performed by a whole genome sequencing.

33. A method of identifying a base editing site of cytosine deaminase, comprising: (i) introducing or contacting (a) a cytosine deaminase and an inactivated target-specific endonuclease, or (b) a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, or (c) a plasmid comprising a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, into a cell or with DNA isolated from a cell, together with a guide RNA; (ii) treating a uracil-specific excision reagent (USER), to generate double strand cleavage in DNA; (iii) analyzing nucleic acid sequence of the cleaved DNA fragment; and (iv) identifying the double strand cleavage site in the nucleic acid sequence read obtained by the analysis, wherein the DNA isolated from a cell in step (i) is a genomic DNA.

34. The method of claim 33, wherein the inactivated target-specific endonuclease is a Cas9 protein or Cpf1 protein, which lacks endonuclease activity of cleaving a DNA double strand.

35. The method of claim 33, wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein amino acid residue D10 is substituted with other amino acid.

36. The method of claim 33, wherein the cytosine deaminase and inactivated target-specific endonuclease are in a form of a fusion protein, or the cytosine deaminase coding gene and inactivated target-specific endonuclease coding gene encode a fusion protein comprising the cytosine deaminase and inactivated target-specific endonuclease.

37. The method of claim 33, wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein both of amino acid residues D10 and H840 are substituted with other amino acids, and the method further comprises a step of treating an endonuclease specifically cleaving a single strand region of DNA, after step (ii).

38. The method of claim 33, wherein the guide RNA is a crRNA:tracrRNA duplex in which crRNA and tracrRNA is coupled to each other, or a single-strand guide RNA (sgRNA).

39. The method of claim 33, which is performed in vitro.

40. The method of claim 33, wherein the DNA isolated from a cell in step (i) is a genomic DNA, and the nucleic acid sequence analysis of step (iii) is performed by a whole genome sequencing.

41. A method of identifying an off-target site of cytosine deaminase, comprising: (i) introducing or contacting (a) a cytosine deaminase and an inactivated target-specific endonuclease, or (b) a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, or (c) a plasmid comprising a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, into a cell or with DNA isolated from a cell, together with a guide RNA; (ii) treating a uracil-specific excision reagent (USER), to generate double strand cleavage in DNA; (iii) analyzing nucleic acid sequence of the cleaved DNA fragment; and (iv) identifying the double strand cleavage site in the nucleic acid sequence read obtained by the analysis, wherein the DNA isolated from a cell in step (i) is a genomic DNA.

42. The method of claim 41, which further comprises a step of determining the cleaved site as an off-target site if the site is not an on-target site, after step (iv).

43. The method of claim 41, wherein the cleavage site identified in step (iv) is a position at which the 5 end is straightly aligned, or a position showing a double peak pattern at the 5 end plot, when the obtained nucleotide sequence reads are aligned.

44. The method of claim 43, wherein the alignment is performed using BWA/GATK or ISAAC after mapping the sequence reads to a reference genome.

45. The method of claim 43, which further comprises a step of determining a site where two or more sequence reads corresponding to each of Watson strand and Crick strand are straightly aligned as an off-target site.

46. The method of claim 43, which further comprises a step of determining a site where 20% or more of sequence reads are straightly aligned and the number of sequence reads having the same 5 end in each of the Watson and Crick strands is 10 or more as an off-target site.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0145] FIG. 1a shows the base editing efficiency resulted by BE1 (APOBEC1-dCas9), BE2 (APOBEC1-dCas9-UGI) and BE3 (APOBEC1-nCas9-UGI) (Reference Example 1) on 7 endogenous on-target sites (EMX1, FANCF, HEK2, RNF2, HEK3, HEK4, HBB) in HEK293T cells.

[0146] FIG. 1b shows the frequency of Cas9 nuclease-induced mutation measured by targeted deep sequencing at 7 endogenous on-target sites in HEK293T cells.

[0147] FIG. 1c is a graph representatively showing base editing efficiency or ranking of indel frequency at 7 endogenous target sites.

[0148] FIG. 2a is a graph showing mutation frequency at one of 3 endogenous sites (EMX1) of HEK293T cells which are co-transfected with sgRNA having 0 to 4 mismatches and a plasmid encoding BE3 or Cas9 (wherein the nucleic acid sequences listed are sequentially numbered from SEQ ID NO: 1 to SEQ ID NO: 31 in the downward direction on the graph).

[0149] FIG. 2b is a graph showing mutation frequency at one of 3 endogenous sites (HBB) of HEK293T cells which are co-transfected with sgRNA having 0 to 4 mismatches and a plasmid encoding BE3 or Cas9 (wherein the nucleic acid sequences listed are sequentially numbered from SEQ ID NO: 32 to SEQ ID NO: 62 in the downward direction on the graph).

[0150] FIG. 2c is a graph showing mutation frequency at one of 3 endogenous sites (RNF2) of HEK293T cells which are co-transfected with sgRNA having 0 to 4 mismatches and a plasmid encoding BE3 or Cas9 (wherein the nucleic acid sequences listed are sequentially numbered from SEQ ID NO: 63 to SEQ ID NO: 93 in the downward direction on the graph).

[0151] FIG. 3a is a graph showing Cas9 nuclease associated indel frequency and BE associated base editing frequency at EMX1 site.

[0152] FIG. 3b is a graph showing Cas9 nuclease associated indel frequency and BE associated base editing frequency at HBB site.

[0153] FIG. 3c is a graph showing Cas9 nuclease associated indel frequency and BE associated base editing frequency at RNF2 site.

[0154] FIG. 4a is a schematic view of BE3 Digenome-seq.

[0155] FIG. 4b is an electrophoresis image showing the PCR products cleaved by treating BE3 and/or USER.

[0156] FIG. 4c is a Sanger sequencing result showing C-to-U conversion by BE3 and DNA cleavage by USER.

[0157] FIG. 4d is an IGV image showing straight alignment of the sequence read at on-target site of EMX1.

[0158] FIG. 5 is an IGV image showing straight alignment of sequence reads at 6 different on-target sites.

[0159] FIGS. 6a (EMX1) and 6b (HBB) are genome-wide circus plots representing DNA cleavage scores obtained with intact genomic DNA (gray: first layer from the center) and genomic DNA digested with BE3 and USER (blue: second layer from the center) or with Cas9 (red; third layer from the center, only present in FIG. 6b), where the arrow indicates on-target site.

[0160] FIGS. 6c (EMX1) and 6d (HBB) show sequence logos obtained via WebLogo using DNA sequences at Digenome-capture sites (Tables 2-8) (DNA cleavage score >2.5).

[0161] FIGS. 6e (EMXI) and 6f (HBB) represent scatterplots of BE3-mediated substitution frequencies vs Cas9-mediated indel frequencies determined using targeted deep sequencing, wherein circled dots indicate off-target sites validated by BE3 but invalidated by Cas9.

[0162] FIGS. 6g (EMX1) and 6h (HBB) show BE3 off-target sites validated in HEK293T cells by targeted deep sequencing, wherein PAM sequences are the last 3 nucleotides at 3 end, mismatched bases are shown in small letters, and dashes indicate RNA bulges (Error bars indicate s.e.m. (n=3)).

[0163] FIG. 7 is a Venn diagram showing the number of sites with DNA cleavage scores 2.5 or higher identified by Digenome-seq of Cas9 nuclease- and Base editor-treated genomic DNA.

[0164] FIG. 8 is a graph showing the number of total sites (.square-solid.) and the number of PAM-containing sites with ten or fewer mismatches () for a range of DNA cleavage scores.

[0165] FIG. 9 is a Venn diagram showing the number of PAM-containing homologous sites with DNA cleavage scores over 0.1 or higher identified by Digenome-seq of Cas9 nuclease- and Base editor-treated genomic DNA.

[0166] FIG. 10 shows fractions of homologous sites captured by Digenome-seq, wherein bars represent the number of homologous sites that differ from on-target sites by up to 6 nt, squares (BE3) and triangles (Cas9) represent the fraction of Digenome-seq captured sites for a range of mismatch numbers.

[0167] FIGS. 11a and 11b are graphs showing the significant correlation between the number of BE3- and Cas9-associated sites identified by Digenome 1.0 (11a) and Digenome 2.0 (11b).

[0168] FIGS. 12a and 12b are graphs showing the significant correlation between the number of BE3-associated sites identified by Digenome 1.0 (12a) or Digenome 2.0 (12b) and the number of sites with 6 or fewer mismatches.

[0169] FIG. 13 shows examples of Digenome-captured off-target sites associated only with Cas9, which contain no cytosines at positions 4-9.

[0170] FIGS. 14a-14c show base editing efficiencies at Digenome-captured sites associated only with 3 different Cas9 nucleases.

[0171] FIGS. 15a-15c show base editing efficiencies of 3 different BE3 deaminases at Digenome-negative sites.

[0172] FIG. 16a is a schematic view showing conventional (gX.sub.19 sgRNA), truncated (gX.sub.18 or gX.sub.17 sgRNA), and extended sgRNAs (gX.sub.20 or ggX.sub.20 sgRNA).

[0173] FIG. 16b shows base-editing frequencies at the HBB on- and off-target sites in HEK293T cells measured by targeted deep sequencing.

[0174] FIG. 17 shows the result of reducing BE3 off-target effects using modified sgRNAs, wherein 17a shows a schematic view of conventional sgRNAs (GX.sub.19 sgRNA) and modified sgRNAs (GX.sub.17 sgRNA, gX.sub.18 sgRNA, gX.sub.20 sgRNA, and ggX.sub.20 sgRNA), and 17b shows base editing efficiencies (frequencies) measured at the EMX1 on- and off-target sites by targeted deep sequencing in HEK293T cells.

[0175] FIG. 18a is a cleavage map of plasmid rAPOBEC1-XTEN-dCas9-NLS.

[0176] FIG. 18b is a cleavage map of plasmid rAPOBEC1-XTEN-dCas9-UGI-NLS.

[0177] FIG. 18C is a cleavage map of plasmid rAPOBEC1-XTEN-Cas9n-UGI-NLS.

[0178] FIG. 19 is a cleavage map of Cas9 expression plasmid.

[0179] FIG. 20 is a cleavage map of plasmid pET28b-BE1 encoding His6-rAPOBEC1-XTEN-dCas9.

[0180] FIGS. 21a to 21c are schematic overviews of genome-wide off-target profiling by a method other than Digenome-seq, wherein FIG. 21a illustrates a method using circle-seq, FIG. 21b illustrates a method using Bless, and FIG. 21c illustrates a method using DSBCapture.

[0181] FIG. 22 shows process and results of BE1 (rAPOBEC1-dCas9)-mediated double strand breaks (DSBs), wherein (a) schematically shows precesses to generate DBS using BE1 (rAPOBEC1-dCas9), USER enzyme, and 51 nuclease, and (B) is an agarose gel electrophoresis image showing BE1-mediated DSB results in PCR amplicons obtained after treating BE1/sgRNA, USER enzyme, and 51 nuclease.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0182] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Reference Example

[0183] 1. Cell Culture and Transfection

[0184] HEK293T cells (ATCC CRL-11268) were maintained in DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% (w/v) FBS and 1% (w/v) penicillin/streptomycin (Welgene). HEK293T cells (1.510.sup.5) were seeded on 24-well plates and transfected at 80% confluency with sgRNA plasmid (500 ng) and Base Editor plasmid (Addgene plasmid #73019 (Expresses BE1 with C-terminal NLS in mammalian cells; rAPOBEC1-XTEN-dCas9-NLS; FIG. 18a), #73020 (Expresses BE2 in mammalian cells; rAPOBEC1-XTEN-dCas9-UGI-NLS; FIG. 18b), #73021 (Expresses BE3 in mammalian cells; rAPOBEC1-XTEN-Cas9n-UGI-NLS; FIG. 18c)) (1.5 g) or Cas9 expression plasmid (Addgene plasmid #43945; FIG. 19), using Lipofectamine 2000 (Invitrogen). Genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen) at 72 hours after transfection. The cells were not tested for mycoplasma contamination. The sgRNA used in the following Examples was constructed by converting T to U on the overall sequence at an on-target site (on-target sequence; see Tables 1-8), except the 5-terminal PAM sequence (5-NGG-3; wherein N is A, T, G, or C), and employing the converted sequence as the targeting sequence (N.sub.cas9).sub.I of the following General Formula 3: 5-(N.sub.cas9).sub.1-(GUUUUAGAGCUA)-(GAAA)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC)-3 (General Formula 3; oligonucleotide linker: GAAA).

[0185] 2. Protein Purification

[0186] The His6-rAPOBEC1-XTEN-dCas9 protein-coding plasmid (pET28b-BE1; Expresses BE1 with N-terminal His6 tag in E. coli; FIG. 20) was generously given by David Liu (Addgene plasmid #73018). The His6-rAPOBEC1-XTEN-dCas9 protein-coding plasmid pET28b-BE1 was converted into a His6-rAPOBEC1-nCas9 protein (BE3 delta UGI; BE3 variant lacking a UGI domain)-coding plasmid (pET28b-BE3 delta UGI) by site directed mutagenesis for substituting A840 with H840 in the dCas9.

[0187] Rosetta expression cells (Novagen, catalog number: 70954-3CN) were transformed with the prepared pET28b-BE1 or pET28b-BE3 delta UGI and cultured overnight in Luria-Bertani (LB) broth containing 100 g/ml kanamycin and 50 mg/ml carbenicilin at 37 C. Ten ml of the overnight cultures of Rosetta cells containing pET28b-BE1 or pET28b-BE3 delta UGI was inoculated into 400 ml LB broth containing 100 g/ml kanamycin and 50 mg/ml carbenicilin and cultured at 30 C. until the OD600 reached 0.5-0.6. The cells were cooled to 16 C. for 1 hour, supplemented with 0.5 mM IPTG (Isopropyl -D-1-thiogalactopyranoside), and cultured for 14-18 hours.

[0188] For protein purification, cells were harvested by centrifugation at 5,000g for 10 min at 4 C. and lysed by sonication in 5 ml lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 1 mM DTT, and 10 mM imidazole, pH 8.0) supplemented with lysozyme (Sigma) and a protease inhibitor (Roche complete, EDTA-free). The soluble lysate obtained after centrifugation of the cell lysis mixture at 13,000 rpm. for 30 min at 4 C. was incubated with Ni-NTA agarose resin (Qiagen) for 1 hour at 4 C. The cell lysate/Ni-NTA mixture was applied to a column and washed with a buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, and 20 mM imidazole, pH 8.0). The BE3 protein was eluted with an elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). The eluted protein was buffer exchanged with a storage buffer (20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM DTT, and 20% glycerol) and concentrated with centrifugal filter units (Millipore) to give purified rAPOBEC1-XTEN-dCas9 protein and rAPOBEC1-nCas9 protein.

[0189] 3. Desamination and USER Treatment of PCR Amplification Products

[0190] PCR amplification products (10 g) containing EMX1 site were incubated with purified rAPOBEC1-nCas9 protein (4 g) and EMX1-specific sgRNA (3 g) at 100 l reaction volume for 1 hour at 37 C. The cultures were then incubated for 30 min at 37 C. in a uracil-specific excitation reagent (6 units) (New England Biolabs; https://www.neb.com/products/m5505-user-enzyme; containing a mixture of Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, BSA 175 mg/ml, and 50% (w/v) glycerol) glycerol) and then subjected to agarose gel electrophoresis.

[0191] 4. Deamination and USER Treatment of Genomic DNA

[0192] Genomic DNA was purified (extracted) from HEK293T cells with a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA (10 g) was incubated with the rAPOBEC1-nCas9 protein (300 nM) purified in Reference Example 2 and an sgRNA (900 nM) in a reaction volume of 500 L for 8 hours at 37 C. in a buffer (100 mM NaCl, 40 mM Tris-HCl, 10 mM MgCl.sub.2, and 100 g/ml BSA, pH 7.9). After removal of sgRNA using RNase A (50 g/mL), uracil-containing genomic DNA was purified with a DNeasy Blood & Tissue Kit (Qiagen). The on-target site was amplified by PCR using a SUN-PCR blend and subjected to Sanger sequencing to check BE3-mediated cytosine deamination and USER-mediated DNA cleavage.

[0193] 5. Sequencing of Whole Genome and Digenome

[0194] Genomic DNA (1 g) was fragmented to the 400- to 500-bp range using the Covaris system (Life Technologies) and blunt-ended using End Repair Mix (Thermo Fischer). Fragmented DNA was ligated with adapters to produce libraries, which were then subjected to WGS (whole genome sequencing) using HiSeq X Ten Sequencer (Illumina) at Macrogen.

[0195] 6. Targeted Deep Sequencing

[0196] On-target and potential off-target sites were amplified with a KAPA HiFi HotStart PCR kit (KAPA Biosystems #KK2501) for deep sequencing library generation. Pooled PCR amplicons were sequenced using MiniSeq (Illumina) or Illumina Miseq (LAS Inc. Korea) with TruSeq HT Dual Index system (Illumina).

Example 1. Comparison of BE3-Associated Base Editing Efficiency and Cas9-Associated Indel Frequency in Human Cells

[0197] Base editing efficiencies, defined by single-nucleotide substitution frequencies, of three different forms of BEs, at seven genomic loci (EMX1, FANCF, HEK2, RNF2, HEK3, HEK4 and HBB) in HEK293T cells were determined, and compared with genome editing efficiencies, defined by indel frequencies at target sites, of Cas9 nucleases (FIG. 1a,b). FIG. 1a shows the base editing efficiencies resulted from BE1 (APOBEC1-dCas9), BE2 (APOBEC-dCas9-UGI) and BE3 (APOBEC-nCas9-UGI) (Reference Example 1) in seven endogenous target sites (EMX1, FANCF, HEK2, RNF2, HEK3, HEK4, HBB) of HEK293T cells. The base editing efficiency was measured by targeted deep sequencing (Reference Example 6). The efficiency of BE3 [APOBEC-nCas9-UGI (uracil DNA glycosylase inhibitor), 296%] is superior to that of BE1 (APOBEC1-dCas9, 51%) and BE2 (APOBEC-dCas9-UGI, 82%). FIG. 1b shows the Cas9 nuclease-induced mutation frequencies measured by the target deep-sequnctation at 7 endogenous target sites in HEK293T cells (the results were obtained by using the Cas9 expression plasmid of Reference Example 1 (Addgene plasmid #43945; FIG. 19)). These results show that BE3 activity is independent of Cas9 nuclease activity. FIG. 1c is a graph representatively showing the ranking of indel frequency or base editing efficiency at the 7 endogenous on-target sites (see Table 2-8). As shown in FIG. 1c, several sgRNAs exhibit low activity when working together with Cas9, but highly activity when working together with BE3; while some sgRNAs show opposite correlation.

Example 2. Tolerance of BE3 and Cas9 to Mismatched sgRNAs

[0198] To assess specificities of BE3 deaminases, it was examined in a cell whether BE3 can tolerate mismatches in small guide RNAs (sgRNAs). To this end, plasmids encoding BE3 or Cas9 (Reference Example 1) and sgRNAs with one to four mismatches were co-transfected into HEK293T cells, to measure mutation frequencies at three endogenous sites (EMX1, HBB, RNF2).

[0199] The used target sites (including the PAM sequence (in bold)) of the sgRNA with 1 to 4 mismatches are summarized in Table 1 below:

TABLE-US-00004 TABLE1 SEQ EMX1 SEQ SEQ RNF2 ID mismatched ID HBBmismatched ID mismatched NO: sgRNAs NO: sgRNAs NO: sgRNAs 1 GgactCGAGC 32 GccatCCCAC 63 GctgcCTTAG AGAAGAAGAA AGGGCAGTAA TCATTACCTG GGG CGG AGG 2 GAGTttagGC 33 GTTGttttAC 64 GTCActccAG AGAAGAAGAA AGGGCAGTAA TCATTACCTG GGG CGG AGG 3 GAGTCCGAat 34 GTTGCCCCgtgaG 65 GTCATCTTgactA gaAAGAAGAA GCAGTAACGG TTACCTGAGG GGG 4 GAGTCCGAGC 35 GTTGCCCCACAG 66 GTCATCTTAGTC AGggagAGAA aatgGTAACGG gccgCCTGAGG GGG 5 GAGTCCGAGC 36 GTTGCCCCACAG 67 GTCATCTTAGTC AGAAGAgagg GGCAacggCGG ATTAttcaAGG GGG 6 GAactCGAGC 37 GTcatCCCACAGG 68 GTtgcCTTAGTCA AGAAGAAGAA GCAGTAACGG TTACCTGAGG GGG 7 GAGTCtagGC 38 GTTGCtttACAGGG 69 GTCATtccAGTCA AGAAGAAGAA CAGTAACGG TTACCTGAGG GGG 8 GAGTCCGAat 39 GTTGCCCCgtgGG 70 GTCATCTTgacC gGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 9 GAGTCCGAGC 40 GTTGCCCCACAaa 71 GTCATCTTAGTtg AaggGAAGAA aCAGTAACGG cTACCTGAGG GGG 10 GAGTCCGAGC 41 GTTGCCCCACAG 72 GTCATCTTAGTC AGAAaggGAA GGtgaTAACGG ATcgtCTGAGG GGG 11 GAGTCCGAGC 42 GTTGCCCCACAG 73 GTCATCTTAGTC AGAAGAAagg GGCAGcggCGG ATTACtcaAGG GGG 12 GAacCCGAGC 43 GTcaCCCCACAG 74 GTtgTCTTAGTCA AGAAGAAGAA GGCAGTAACGG TTACCTGAGG GGG 13 GAGTttGAGC 44 GTTGttCCACAGG 75 GTCActTTAGTCA AGAAGAAGAA GCAGTAACGG TTACCTGAGG GGG 14 GAGTCCagGC 45 GTTGCCttACAGG 76 GTCATCccAGTC AGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 15 GAGTCCGAat 46 GTTGCCCCgtAGG 77 GTCATCTTgaTC AGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 16 GAGTCCGAGC 47 GTTGCCCCACga 78 GTCATCTTAGctA gaAAGAAGAA GGCAGTAACGG TTACCTGAGG GGG 17 GAGTCCGAGC 48 GTTGCCCCACAG 79 GTCATCTTAGTC AGggGAAGAA aaCAGTAACGG gcTACCTGAGG GGG 18 GAGTCCGAGC 49 GTTGCCCCACAG 80 GTCATCTTAGTC AGAAagAGAA GGtgGTAACGG ATcgCCTGAGG GGG 19 GAGTCCGAGC 50 GTTGCCCCACAG 81 GTCATCTTAGTC AGAAGAgaAA GGCAacAACGG ATTAttTGAGG GGG 20 GAGTCCGAGC 51 GTTGCCCCACAG 82 GTCATCTTAGTC AGAAGAAGgg GGCAGTggCGG ATTACCcaAGG GGG 21 GgGTCCGAGC 52 GcTGCCCCACAG 83 GcCATCTTAGTC AGAAGAAGAA GGCAGTAACGG ATTACCTGAGG GGG 22 GAGcCCGAGC 53 GTTaCCCCACAG 84 GTCgTCTTAGTC AGAAGAAGAA GGCAGTAACGG ATTACCTGAGG GGG 23 GAGTCtGAGC 54 GTTGCtCCACAGG 85 GTCATtTTAGTC AGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 24 GAGTCCGgGC 55 GTTGCCCtACAGG 86 GTCATCTcAGTC AGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 25 GAGTCCGAGt 56 GTTGCCCCAtAGG 87 GTCATCTTAaTC AGAAGAAGAA GCAGTAACGG ATTACCTGAGG GGG 26 GAGTCCGAGC 57 GTTGCCCCAC 88 GTCATCTTAGTt AaAAGAAGAA AaGGCAGTAACGG ATTACCTGAGG GGG 27 GAGTCCGAGC 58 GTTGCCCCACAG 89 GTCATCTTAGTC AGAgGAAGAA GaCAGTAACGG AcTACCTGAGG GGG 28 GAGTCCGAGC 59 GTTGCCCCACAG 90 GTCATCTTAGTC AGAAGgAGAA GGCgGTAACGG ATTgCCTGAGG GGG 29 GAGTCCGAGC 60 GTTGCCCCACAG 91 GTCATCTTAGTC AGAAGAAaAA GGCAGcAACGG ATTACtTGAGG GGG 30 GAGTCCGAGC 61 GTTGCCCCACAG 92 GTCATCTTAGTC AGAAGAAGAg GGCAGTAgCGG ATTACCTaAGG GGG 31 GAGTCCGAGC 62 GTTGCCCCACAG 93 GTCATCTTAGTC AGAAGAAGAA GGCAGTAACGG ATTACCTGAGG GGGontar- (ontarget (ontarget getse- sequence) sequence) quence)

[0200] (In Table 1, the base position in a lower-case letter refers to the mismatched site)

[0201] The results (Indel frequency and cytosine conversion frequency) obtained in the mismatched sequence and the on-target sequence listed in Table 1 are shown in FIGS. 2a to 2c (2a: EMX1, 2b: HBB and 2c: RNF2; Error bars indicate s.e.m. (n=3)). In FIGS. 2a to 2c, the bars indicated as Cn show a mutation (substitution with other base or deletion) frequency of cytosine (C) at the n-th position from 5 end of mismatched sequence or on-target sequence. The Indel frequency and the cytosine conversion frequency (base editing frequency) were measured using the target deep sequencing (Reference Example 6). The primers used for the target deep sequencing are as follows:

TABLE-US-00005 EMX1 1stPCR Forward(5.fwdarw.3): (SEQIDNO:94) AGTGTTGAGGCCCCAGTG; Reverse(5.fwdarw.3): (SEQIDNO:95) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCAGCAAGCAGCA CTCT; 2ndPCR Forward(5.fwdarw.3): (SEQIDNO:96) ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGGCCTCCTGAGTTTC TCAT; Reverse(5.fwdarw.3) (SEQIDNO:97) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCAGCAAGCAGCA CTCT; HBB 1stPCR Forward(5.fwdarw.3): (SEQIDNO:98) GGCAGAGAGAGTCAGTGCCTA; Reverse(5.fwdarw.3): (SEQIDNO:99) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGGGCTGGGCATAA AAGT; 2ndPCR Forward(5.fwdarw.3): (SEQIDNO:100) ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCTCCACATGCCCAG TTTC; Reverse(5.fwdarw.3) (SEQIDNO:101) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGGGCTGGGCATAA AAGT; RNF2 1stPCR Forward(5.fwdarw.3): (SEQIDNO:102) CCATAGCACTTCCCTTCCAA; Reverse(5.fwdarw.3): (SEQIDNO:103) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCAACATACAGAAG TCAGGAA; 2ndPCR Forward(5.fwdarw.3): (SEQIDNO:104) ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTTCCAGCAATGTCT CAGG; Reverse(5.fwdarw.3) (SEQIDNO:105) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCAACATACAGAAG TCAGGAA.

[0202] In addition, the Cas9 nuclease-associated indel frequency and BE3-associated base editing frequency in EMX1 site (FIG. 3a), HBB site (FIG. 3b), and RNF2 site (FIG. 3c) were measured using mismatched sgRNAs (Table 1), and the obtained results are shown in FIGS. 3a to 3c. As shown in FIGS. 3a-3c, there is a statistically significant correlation (R2=0.70, 0.83, and 0.72 at three sites, respectively) between the Cas9-induced indel frequency and the BE3 induced substitution frequency.

[0203] BE3 deaminases and Cas9 nucleases tolerated one-nucleotide (nt) mismatches at almost every position and 2-nt mismatches in the protospacer-adjacent motif (PAM)-distal region but did not tolerate most of the 3-nt or 4-nt mismatches in either the PAM-proximal or distal regions. We noticed, however, that several sgRNAs (indicated by asterisks in FIG. 2) with two or three mismatches were highly active with BE3 but not with Cas9 or vice versa. For example, BE3 with the fully-matched sgRNA or with a 3-nt mismatched sgRNA induced substitutions at comparable frequencies (33% vs. 14%) at the EMX1 site, whereas Cas9 with the same matched and 3-nt mismatched sgRNAs showed widely different indel frequencies (50% vs. 2%) (FIG. 2a). Conversely, BE3 with two 2-nt mismatched sgRNAs was poorly active (substitution frequencies <1%), whereas Cas9 with the same mismatched sgRNAs was highly active (indel frequencies >10%) (FIG. 2a). These results indicate that the tolerance of Cas9 nucleases and BE3 deaminases for mismatched sgRNAs can differ and imply that BE3 and Cas9 could have separate sets of off-target sites in the genome, calling for a method to profile genome-wide specificities of RNA-programmable deaminases.

Example 3. Digenome-Seq for Identifying BE3 Off-Target Sites in Human Genome

[0204] Several different cell-based methods, which include GUIDE-seq (Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature biotechnology 33, 187-197 (2015)), HTGTS (Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nature biotechnology (2014)), BLESS (Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191 (2015)), and IDLV capture (Wang, X. et al. Unbiased detection of custom-character cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nature biotechnology 33, 175-178 (2015)), have been developed for identifying genome-wide off-target sites at which Cas9 nucleases induce DSBs. None of these methods, at least in their present forms, are suitable for assessing the genome-wide specificities of programmable deaminases, simply because deaminases do not yield DSBs. We reasoned that DSBs could be produced at deaminated, uracil-containing sites in vitro using appropriate enzymes and that these DNA cleavage sites could be identified via Digenome-seq (digested-genome sequencing; Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target-specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome research 26, 406-415 (2016); Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature biotechnology 34, 863-868 (2016); Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nature methods 12, 237-243, 231 p following 243 (2015)), an in vitro method used for assessing genome-wide specificities of Cas9 and Cpf1 nucleases.

[0205] To test this idea, a PCR amplicon containing a target sequence was incubated (1) with the recombinant rAPOBEC1-nCas9 protein (Reference Example 2), a derivative of BE3 with no UGI domain, and its sgRNA in vitro to induce C-to-U conversions and a nick in the Watson and Crick strands, respectively, and then (2) with USER (Uracil-Specific Excision Reagent), a mixture of E. coli Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, to generate a gap at the location of the uracils, giving rise to a composite DSB (FIG. 4a). Next it was investigated whether Digenome-seq could be used to assess genome-wide target-specificities of BE3 deaminases. Human genomic DNA, purified from HEK293T cells, was incubated with each of 7 different BE3 ribonucleoproteins (RNPs) (300 nM rAPOBEC1-nCas9 protein and 900 nM sgRNA each) for 7 hours three times, and then with USER for 3 hours (FIG. 4a).

[0206] FIG. 4a shows an outline of the BE3 Digenome-seq, showing the BE3-mediated cleavage of uracil-containing site by USER, a mixture of E. coli Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII. FIG. 4b is an electrophoresis image showing the PCR products cleaved by treating BE3 and/or USER. As shown in FIG. 4b, the PCR amplicon was cleaved, when incubated with both BE3 and USER.

[0207] C-to-U conversions induced by BE3 and uracil removal by USER were confirmed by Sanger sequencing (FIG. 4c). FIG. 4c is a Sanger sequencing result showing C-to-U conversion by BE3 and DNA cleavage by USER. Each genomic DNA sample was subjected to whole genome sequencing (WGS) after end repair and adaptor ligation (FIG. 4a).

[0208] After sequence alignment to the human reference genome (hg19), we used Integrative Genomics Viewer (IGV) to monitor alignment patterns at each on-target site, and the results are shown in FIGS. 4d and 5, respectively. After sequencing for the human reference genome (hg19), an alignment pattern at the target position was monitored using an Integrative Genomics Viewer (IGV) FIG. 4d is an IGV image showing straight alignment of the sequence read at on-target site of EMX1, and FIG. 5 is an IGV image showing straight alignment of sequence reads at 6 different on-target sites. As shown in FIGS. 4d and 5, uniform alignments of sequence reads, signature patterns associated with DSBs produced in vitro, were observed at all 7 on-target sites.

Example 4. Genome-Wide BE3 Off-Target Sites Revealed by Digenome-Seq

[0209] To identify BE3 off-target sites in the human genome, a DNA cleavage score was assigned, based on the number of sequence reads whose 5 ends aligned at a given position, to each nt position across the genome and listed all the sites with scores over 2.5, a cutoff value that was used for finding off-target sites of Cas9 nucleases with the same set of 7 sgRNAs in the inventor's previous study (Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target-specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome research 26, 406-415 (2016)) (FIG. 6a-d and Tables 2-8).

[0210] The DNA cleavage score at site i of each nucleotide (i.e., the nucleotide position on genomic DNA) was calculated by the following formula:

[00001] $Score at the .Math. .Math. i .Math. .Math. site = {.Math.}_{a = 1}^{5} .Math. .Math. \frac{C (F_{i} - 1)}{D_{i}} \frac{C (R_{i - 4 + a} - 1)}{D_{i - 4 + a}} (F_{i} + R_{i - 4 + a} - 2) + {.Math.}_{a = 1}^{5} .Math. .Math. \frac{C (R_{i - 1} - 1)}{D_{i - 1}} \frac{C (F_{i - 3 + a} - 1)}{D_{i - 3 + a}} (R_{i - a} + F_{i - 3 + a} - 2)$

F.sub.j: Number of forward sequence reads starting at the i site
R.sub.i: Number of reverse sequence reads starling at the i site
D.sub.j: Sequencing depth at the i site
C Arbitrary constant

[0211] In the above formula, the number of nucleotide sequence data means the number of nucleotide leads, the sequencing depth means the number of sequencing leads at a specific site, and the C value is 1.

[0212] Digenome-captured sites (cleavage site+PAM) and DNA cleavage score are shown in Tables 2 to 8 below:

TABLE-US-00006 TABLE2 (Ontarget:EMX1_4) EMX1 DNA DNAseqat SEQ cleavage acleavage ID ID Chr Position Score sites NO Bulge EMX1_1 chr15 44109763 30.53 GAGTCtaAGCAG 106 x AAGAAGAAGAG EMX1_2 chr11 62365273 26.44 GAaTCCaAGCAG 107 x AAGAAGAgAAG EMX1_3 chr5 9227162 23.66 aAGTCtGAGCAc 108 x AAGAAGAATGG EMX1_4 chr2 73160998 14.55 GAGTCCGAGCAG 31 x AAGAAGAAGGG EMX1_5 chr4 131662222 11.14 GAaTCCaAG-AG 109 RNA AAGAAGAATGG bulge EMX1_6 chr8 128801258 9.60 GAGTCCtAGCAG 110 x gAGAAGAAGAG EMX1_7 chr19 24250503 8.35 GAGTCCaAGCAG 111 x tAGAgGAAGGG EMX1_8 chr1 4515013 8.12 GtGTCCtAG-AG 112 RNA AAGAAGAAGGG bulge EMX1_9 chr1 23720618 5.96 aAGTCCGAGgAG 113 x AgGAAGAAAGG EMX1_10 chr2 219845072 5.47 GAGgCCGAGCAG 114 x AAGAAagACGG EMX1_11 chr8 102244551 4.70 agtTCCaAGCAG 115 x AAGAAGcATGG EMX1_12 chr3 45605387 3.11 GAGTCCacaCAG 116 x AAGAAGAAAGA EMX1_13 chr16 12321159 3.01 GAGTCCaAG-AG 117 RNA AAGAAGtgAGG bulge EMX1_14 chr9 111348573 1.56 GAGTCCttG-AG 118 RNA AAGAAGgAAGG bulge EMX1_15 chr3 5031614 1.50 GAaTCCaAGCAG 119 x gAGAAGAAGGA EMX1_16 chr14 31216733 1.34 GtacCaGAG-AG 120 RNA AAGAAGAgAGG bulge EMX1_17 chr14 48932119 1.16 GAGTCCcAGCAa 121 x AAGAAGAAAAG EMX1_18 chr11 107812992 1.04 aAGTCCaAGt-G 122 RNA AAGAAGAAAGG bulge EMX1_19 chr12 106646090 1.03 aAGTCCatGCAG 123 x AAGAgGAAGGG EMX1_20 chr2 71969823 0.80 GAGTCCtAG-AG 124 RNA AAGAAaAAGGG bulge EMX1_21 chr3 145057362 0.48 GAGTCCct-CAG 125 RNA gAGAAGAAAGG bulge EMX1_22 chr6 9118799 0.45 acGTCtGAGCAG 126 x AAGAAGAATGG EMX1_23 chr1 59750259 0.27 GAGTtCcAGaAG 127 x AAGAAGAAGAG EMX1_24 chr11 79484079 0.22 GAGTCCtAa-AG 128 RNA AAGAAGcAGGG bulge EMX1_25 chr9 135663403 0.21 cAGTCCaAaCAG 129 x AAGAgGAATGG

TABLE-US-00007 TABLE3 (Ontargetsequence:FANCF_2) FANCF DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge FANCF_1 chr10 73463135 13.34 tGAATCCCaTCT 130 x cCAGCACCAGG FANCF_2 chr11 22647338 7.04 GGAATCCCTTCT 131 x GCAGCACCTGG FANCF_3 chr10 43410030 6.53 GGAgTCCCTcCT 132 x aCAGCACCAGG FANCF_4 chr10 37953199 5.67 GGAgTCCCTcCT 133 x aCAGCACCAGG FANCF_5 chr11 47554037 5.13 GGAATCCCTTCT 134 x aCAGCAtCCTG FANCF_6 chr16 49671025 3.00 GGAgTCCCTcCT 135 x GCAGCACCTGA FANCF_7 chr18 8707528 1.26 GGAAcCCCgTCT 136 x GCAGCACCAGG FANCF_8 chr7 44076496 0.95 GtctcCCCTTCT 137 x GCAGCACCAGG FANCF_9 chr9 113162294 0.46 aaAATCCCTTCc 138 x GCAGCACCTAG FANCF_10 chr15 49119756 0.42 tGtATttCTTCT 139 x GCctCAggCTG FANCF_11 chr2 54853314 0.39 GGAATatCTTCT 140 x GCAGCcCCAGG FANCF_12 chr8 21374810 0.37 GagtgCCCTgaa 141 x GCctCAgCTGG FANCF_13 chrX 86355179 0.35 accATCCCTcCT 142 x GCAGCACCAGG FANCF_14 chr3 35113165 0.20 tGAATCCtaaCT 143 x GCAGCACCAGG FANCF_15 chr10 3151994 0.13 ctctgtCCTTCT 144 x GCAGCACCTGG

TABLE-US-00008 TABLE4 (Ontargetsequence:RNF2_1) RNF2 DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge RNF2_1 chr1 185056773 27.66 GTCATCTTAGTC 93 x ATTACCTGAGG

TABLE-US-00009 TABLE5 (Ontargetsequence:HBB_1) HBB DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge HBB_1 chr11 5248214 17.68 CTTGCCCCACAG 145 x GGCAGTAACGG HBB_2 chr17 8370252 13.64 tTgctCCCACAG 146 x GGCAGTAAACG HBB_3 chr12 124803834 10.88 gcTGCCCCACAG 147 x GGCAGcAAAGG HBB_4 chrX 75006256 2.34 gTgGCCCCACAG 148 x GGCAGgAATGG HBB_5 chr12 93549201 0.55 aTTGCCCCACgG 149 x GGCAGTgACGG HBB_6 chr10 95791920 0.27 acTctCCCACAa 150 x GGCAGTAAGGG HBB_7 chr9 104595883 0.18 tcaGCCCCACAG 151 x GGCAGTAAGGG

TABLE-US-00010 TABLE6 (Ontargetsequence:HEK2_2) HEK2 DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge HEK2_1 chr4 90522183 18.27 GAACACAAtGCA 152 x TAGAtTGCCGG HEK2_2 chr5 87240613 7.54 GAACACAAAGCA 153 x TAGACTGCGGG HEK2_3 chr2 19844956 0.93 aActcCAAAGCA 154 x TAtACTGCTGG

TABLE-US-00011 TABLE7 (Ontargetsequence:HEK3_2) HEK3 DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge HEK3_1 chr1 47005705 29.27 aGCtCAGACTGA 155 x GCAaGTGAGGG HEK3_2 chr9 110184636 11.38 GGCCCAGACTGA 156 x GCACGTGATGG HEK3_3 chr19 882560 10.90 GGCCCAGA--GA 157 RNA GCACGTGtGGG bulge HEK3_4 chr15 79749930 3.03 caCCCAGACTGA 158 x GCACGTGcTGG HEK3_5 chr17 34954539 2.10 GGCCCa-ACTGA 159 RNA GCAaGTGATGG bulge HEK3_6 chrX 114764149 1.66 aGaCCAGACTGA 160 x GCAaGaGAGGG HEK3_7 chr6 73097166 0.15 GGCCactcaTGg 161 x cCACaTacTGG

TABLE-US-00012 TABLE8 (Ontargetsequence:HEK4_1) HEK4 DNA DNAseqat SEQ Cleavage acleavage ID ID Chr Position Score sites NO Bulge HEK4_1 chr20 31349772 19.26 GGCACTGCGGCT 162 x GGAGGTGGGGG HEK4_2 chr6 160517881 15.45 GGCACTGCtGCT 163 x GGgGGTGGTGG HEK4_3 chr6 168787137 15.37 GGCACTGCa-CT 164 RNA GGAGGTtGTGG bulge HEK4_4 chr19 33382081 13.83 GGCtCTGCGGCT 165 x GGAGGgGGTGG HEK4_5 chr20 60080553 12.71 aGCACTGCaGaT 166 x GGAGGaGGCGG HEK4_6 chr5 141232853 10.87 GGCACTGCGGCa 167 x GGgaGgaGGGG HEK4_7 chr20 60010562 10.51 tGCACTGCGGCc 168 x GGAGGaGGTGG HEK4_8 chr13 70136736 8.76 GGCACT-gGGCT 169 RNA GaAGGTaGAGG bulge HEK4_9 chr20 1151854 8.41 GGCACTGtGGCT 170 x GcAGGTGGAGG HEK4_10 chr15 71686928 7.70 tGCtCTGCGGCa 171 x GGAGGaGGAGG HEK4_11 chr7 1397398 6.71 aGCACTGCaGCT 172 x GGgaGTGGAGG HEK4_12 chr20 45343010 6.57 GGCACTGaGGgT 173 x GGAGGTGGGGG HEK4_13 chr8 20854500 5.57 GGCACTGgGGCT 174 x GGAGacGGGGG HEK4_14 chr7 54561437 5.40 aGgACTGCGGCT 175 x GGgGGTGGTGG HEK4_15 chr15 60790561 5.29 GGCACTGCaaCT 176 x GGAaGTGaTGG HEK4_16 chr13 27629410 4.40 GGCACTGgGGtT 177 x GGAGGTGGGGG HEK4_17 chr7 110143150 3.69 GcCACTGCaGCT 178 x aGAGGTGGAGG HEK4_18 chr7 139244406 3.59 GcCACTGCGaCT 179 x GGAGGaGGGGG HEK4_19 chr19 2474643 3.56 GGCACTG-GGCT 180 RNA GGAGGcGGGGG bulge HEK4_20 chr2 6961255 3.17 aGCtCTGCGGCa 181 x GGAGtTGGAGG HEK4_21 chr17 75429280 2.90 GaCACcaCGGCT 182 x GGAGaTGGTGG HEK4_22 chr7 17979717 2.66 GcactgGCaGCc 183 DNA GGAGGTGGTGG bulge HEK4_23 chr9 5020590 2.64 tGCACTGCaGCT 184 x GcAGGTGGAGG HEK4_24 chrX 122479548 2.52 GGCACTG-GGCT 185 RNA GGAGaTGGAGG bulge HEK4_25 chr12 104739608 2.48 ccttCTGCGGCT 186 x GGAaGTGGTGG HEK4_26 chr17 40693638 2.38 GcactgcaGGCa 187 DNA GGAGGTGaGTG bulge HEK4_27 chr8 144781301 2.38 GaCACTGCaGCT 188 x GGAGGTGGGGT HEK4_28 chr9 74103955 2.36 GGCACTGCaGCa 189 x GGgGaTGGGGG HEK4_29 chr18 37194558 2.31 GGCACTGCGGgT 190 x GGAGGcGGGGG HEK4_30 chr20 60895671 2.12 GGCACaGCaGCT 191 x GGAGGTGcTGG HEK4_31 chr12 113935460 1.63 GGCcCTGCGGCT 192 x GGAGaTatGGG HEK4_32 chrX 70597642 1.57 GaCACTGC-tCT 193 RNA GGAGGTGGTGG bulge HEK4_33 chr15 41044242 1.31 GGCgGGAGCTGC 194 x GGCgGTGGAGG HEK4_34 chr17 176302 1.18 tGCACTGtGGCT 195 x GGAGaTGGGGG HEK4_35 chr10 77103119 1.15 GGCAtcaCGGCT 196 x GGAGGTGGAGG HEK4_36 chr7 134872032 0.93 aGCACTGtGGCT 197 x GGgGGaGGCGG HEK4_37 chr9 133039175 0.86 GtCACTGCaGCT 198 x GGAGGaGGGGG HEK4_38 chr10 73435248 0.79 GtaACTGCGGCT 199 x GGcGGTGGTGG HEK4_39 chr14 21993455 0.78 GGtACaGCGGCT 200 x GGgGGaGGCGG HEK4_40 chr17 29815563 0.59 GGCgCTGCGGCc 201 x GGAGGTGGGGC HEK4_41 chr16 50300346 0.56 aGCACTGtGGCT 202 x GGgGGaGGGGG HEK4_42 chr11 78127584 0.53 tGCACTGCaGCT 203 x GGAGGcaaCGG HEK4_43 chr19 1295086 0.52 GaCACTGaGGCa 204 x GGAGGTGGGGG HEK4_44 chr2 162283033 0.51 GGCAtctgGGTG 205 x GCTGGgaGGGG HEK4_45 chr20 24376056 0.47 GGCACTGaGaCc 206 x aGAGGTGGTGG HEK4_46 chr16 1029977 0.42 GGCACTGCaGac 207 x GGAGGTGtGGG HEK4_47 chr19 47503406 0.39 GGCACTG-GGCT 208 RNA GGAGGgGaGAG bulge HEK4_48 chr2 231467380 0.39 GGCACTGCaGCT 209 x GGgGGTtGGTG HEK4_49 chr10 13692636 0.38 GGCACTGgGGCT 210 x GGgGGaGGGGG HEK4_50 chr1 32471659 0.34 GGCACTtCaGCT 211 x GGAGGcaGAGG HEK4_51 chr17 8634933 0.33 GGCACat-GGaT 212 RNA GGAGGTGGAGG bulge HEK4_52 chr6 83388605 0.30 aGCACTGtGG-T 213 RNA GGAGGTGGAGG bulge HEK4_53 chr10 27700491 0.29 GGCACTG-GGtT 214 RNA GGgGGTGGTGG bulge HEK4_54 chr1 143662284 0.27 GGCACat-GGCT 215 RNA GGgGGTGGTGG bulge HEK4_55 chr16 49777696 0.22 tGCACTGCGaCT 216 x GGAGGgaGAGG HEK4_56 chr19 38616186 0.19 GGCACTGaGaCT 217 x GGgGGTGGGGG HEK4_57 chr10 126752487 0.18 GGCACTGCaGCc 218 x tGgGGgtGGGG HEK4_58 chr16 28266968 0.17 GGCtCTtCGGCT 219 x GGAGGTaGCGG HEK4_59 chr2 149886210 0.15 GaCACTG-GGCT 220 RNA GGAGGTtGCGG bulge HEK4_60 chr20 37471343 0.15 aGCACTGtGcCT 221 x GGgGGTGGGGG HEK4_61 chr12 53453556 0.13 tGgACTGCGGCT 222 x GGAGagGGAGG HEK4_62 chr15 30501337 0.13 GGCACTG-GGCT 223 RNA GGAtGTGGTGG bulge HEK4_63 chr5 139284047 0.12 GGCACTGaGGCT 224 x GcAGGcGGCGG HEK4_64 chr8 119227145 0.12 GGCACaatGGCT 225 x GGAGGTGaAGG HEK4_65 chr14 95761249 0.11 GGCACTctGGCT 226 x GGAGcTGGGGG HEK4_66 chr3 23651529 0.11 GGCACaGCaGgT 227 x GGAGGTGGAGG HEK4_67 chr12 9287415 0.10 GGCtCTGCaGCc 228 x aGgGGTGGAGG

[0213] (In Tables 2 to 8, the bases in lower case letters represent mismatched bases)

[0214] FIGS. 6a and 6b are genome-wide circus plots representing DNA cleavage scores obtained with intact genomic DNA (gray: first layer from the center) and genomic DNA digested with BE3 and USER (blue: second layer from the center) or with Cas9 (red; third layer from the center, only present in FIG. 6b), where the arrow indicates on-target site. FIGS. 6c and 6d show sequence logos obtained via WebLogo using DNA sequences at Digenome-capture sites (Tables 2-8) (DNA cleavage score >2.5). FIGS. 6e and 6f represent scatterplots of BE3-mediated substitution frequencies vs Cas9-mediated indel frequencies determined using targeted deep sequencing, wherein circled dots indicate off-target sites validated by BE3 but invalidated by Cas9. FIGS. 6g and 6h show BE3 off-target sites validated in HEK293T cells by targeted deep sequencing, wherein PAM sequences are the last 3 nucleotides at 3 end, mismatched bases are shown in small letters, and dashes (-) indicate RNA bulges (Error bars indicate s.e.m. (n=3)).

[0215] The primers used in the deep sequencing are summarized in Tables 9 to 15 below:

TABLE-US-00013 TABLE9 EMX1 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) EMX1_1 GCCTTTTTCCG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GACACATAA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAT CTCTTCCGATCT GCCTCATTATCA CTCACCTGGGC GCCTCATTATCA TCAGTGTTGG GAGAAAG TCAGTGTTGG EMX1_2 ACACTCTTTCCC GTCTCTGTGAAT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GGCGTCAC TACACGACGCT TCAGACGTGTG CTTCCGATCTGT CTTCCGATCTGT CTCTTCCGATCT CCCAGACCTTC CCCAGACCTTC CACTGTCTGCA ATCTCCA ATCTCCA GGGCTCTCT EMX1_3 ACACTCTTTCCC TCAAATTGTTTA ACACTCTTTCCC GTGACTGGAGT TACACGACGCT ATAGCTCTGTTG TACACGACGCT TCAGACGTGTG CTTCCGATCTTT TT CTTCCGATCTTT CTCTTCCGATCT GGTCCCACAGG GGTCCCACAGG TTTTTGGTCAAT TGAATAAC TGAATAAC ATCTGAAAGGTT EMX1_4 AGTGTTGAGGC GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT (on CCCAGTG TCAGACGTGTG TACACGACGCT TCAGACGTGTG target) CTCTTCCGATCT CTTCCGATCTG CTCTTCCGATCT CAGCAGCAAGC GGCCTCCTGAG CAGCAGCAAGC AGCACTCT TTTCTCAT AGCACTCT EMX1_5 ACACTCTTTCCC AAAAGATGTGG ACACTCTTTCCC GTGACTGGAGT TACACGACGCT TATATACATACG TACACGACGCT TCAGACGTGTG CTTCCGATCTCT ATGG CTTCCGATCTCT CTCTTCCGATCT GAAAATTTATGA GAAAATTTATGA CAAACAAAGAA CAATTTACTACC CAATTTACTACC GGAAAGTCCTC A A A EMX1_6 ACACTCTTTCCC TGTCTCATTGGC ACACTCTTTCCC GTGACTGGAGT TACACGACGCT TTTTTCTTTTC TACACGACGCT TCAGACGTGTG CTTCCGATCTG CTTCCGATCTG CTCTTCCGATCT CTTGCCTGTGT CTTGCCTGTGT GCCCAGCTGTG GACTTGAC GACTTGAC CATTCTATC EMX1_7 ACACTCTTTCCC CCCAGCTACAC ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GTCACAATG TACACGACGCT TCAGACGTGTG CTTCCGATCTTG CTTCCGATCTTG CTCTTCCGATCT AGCCCTATGAA AGCCCTATGAA TAGGGTCCAGG AAGATTGC AAGATTGC CAAGAGAAA EMX1_8 ACACTCTTTCCC TCTGTCTGGCA ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GATGATACCC TACACGACGCT TCAGACGTGTG CTTCCGATCTAC CTTCCGATCTAC CTCTTCCGATCT ATTGCTACCCCT ATTGCTACCCCT ATCTGCTTCCTC TGGTGA TGGTGA GTGGTCAT EMX1_9 ACACTCTTTCCC GATCTGATCTTA ACACTCTTTCCC GTGACTGGAGT TACACGACGCT CCCCAGAAGC TACACGACGCT TCAGACGTGTG CTTCCGATCTC CTTCCGATCTC CTCTTCCGATCT GGTTCCGGTAC GGTTCCGGTAC CTGCTACTTGG TTCATGTC TTCATGTC CTGACCACA EMX1_10 CTCCTCCGACC GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT AGCAGAG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAA CTCTTCCGATCT TCCCTCAGCCA GGAGGTGCAGG TCCCTCAGCCA CTTTATTTCA AGCTAGA CTTTATTTCA EMX1_11 GGTGCTGTGGG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GGCATAG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTCC CTCTTCCGATCT ACAGGCGAACA TTGATTTGGAG ACAGGCGAACA GAACAGACA GGGTCTT GAACAGACA EMX1_12 CCCTTTCTTAAT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT AAATTACCCAGT TCAGACGTGTG TACACGACGCT TCAGACGTGTG TTC CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT AAAAAGATAGG GACTAAAACACT AAAAAGATAGG CAAACATAGGA GCCCAAG CAAACATAGGA AAA AAA EMX1_13 GCTTTTCTGGG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GACATAGCA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAC CTCTTCCGATCT AAGAATTCCAG TTCCCTTGTCAT AAGAATTCCAG GCAGTTAACCA CCCACA GCAGTTAACCA EMX1_14 CACAGGAATGT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT CTTGGGTCA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTCT CTCTTCCGATCT CTCTTCAATCCA TAGCCTGGGTC CTCTTCAATCCA TCGCCAGT ATGCACT TCGCCAGT EMX1_15 ACACTCTTTCCC GCACTTGTTGG ACACTCTTTCCC GTGACTGGAGT TACACGACGCT CCATTTGTA TACACGACGCT TCAGACGTGTG CTTCCGATCTTG CTTCCGATCTTG CTCTTCCGATCT AGGAGGCAAAA AGGAGGCAAAA TTTTGAATATGT GGGAATA GGGAATA TTTAAATTCTCC ACA EMX1_16 ACACTCTTTCCC GCACAGAGGGT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT TGTTTGCTT TACACGACGCT TCAGACGTGTG CTTCCGATCTAA CTTCCGATCTAA CTCTTCCGATCT GGCTAGCCCAG GGCTAGCCCAG TTCATCCTTTTG AGTCTCC AGTCTCC TGGGGTTC EMX1_17 GGAATCAATCAA GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT TGAAGTTGAAG TCAGACGTGTG TACACGACGCT TCAGACGTGTG A CTCTTCCGATCT CTTCCGATCTG CTCTTCCGATCT TTTGCAATTTGC CAATCTGAAGAA TTTGCAATTTGC TTAGTTATTGAA CAAAGAGCA TTAGTTATTGAA EMX1_18 ACACTCTTTCCC TCAAGAGACTG ACACTCTTTCCC GTGACTGGAGT TACACGACGCT TTGTTTTAGATT TACACGACGCT TCAGACGTGTG CTTCCGATCTTG GTC CTTCCGATCTTG CTCTTCCGATCT ACATTTGATAGA ACATTTGATAGA CCCAGTCCAAT ACAGATGGGTA ACAGATGGGTA GGCTGTAGT EMX1_19 CCCTGCAAATT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GAGTACGTG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT GTCCCGAAGTG GGGGCCATTCT GTCCCGAAGTG CTGGAATTA TTATAGTT CTGGAATTA EMX1_20 GACAGTCCTGG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GCTAGGTGA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTGA CTCTTCCGATCT CTCTGGACTCA GAGTCAGGAGT CTCTGGACTCA GCTCCCATC GCCCAGT GCTCCCATC EMX1_21 ACACTCTTTCCC AGATGAATGCA ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GGGAGCTGT TACACGACGCT TCAGACGTGTG CTTCCGATCTCC CACCATTG CTTCCGATCTCC CTCTTCCGATCT TCTCATTTCTAC TCTCATTTCTAC TTCTGAATTAAA CACCATTG AATGGAAAGAA CTG EMX1_22 ACAATTTCAGTA GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GTAGCATTAAG TCAGACGTGTG TACACGACGCT TCAGACGTGTG GAAT CTCTTCCGATCT CTTCCGATCTGA CTCTTCCGATCT TTGTGACAAACT ATGCCAGTTCT TTGTGACAAACT GCCCTCTG GGGTTGT GCCCTCTG EMX1_23 ACACTCTTTCCC CAAAAATCAACT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT CAAGATGGATTA TACACGACGCT TCAGACGTGTG CTTCCGATCTAA AA CTTCCGATCTAA CTCTTCCGATCT TTTCTGAACCCA TTTCTGAACCCA GAGAACCTAGG AAGACAGG AAGACAGG GAAAACTCTTCTG EMX1_24 ACACTCTTTCCC CTTGTGGATCAT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GGGTACTGAG TACACGACGCT TCAGACGTGTG CTTCCGATCTCC CTTCCGATCTCC CTCTTCCGATCT AAGCTATTTAAC AAGCTATTTAAC TGGGCCTTGGT TGGTATGCAC TGGTATGCAC ATTAGAGCA EMX1_25 ACACTCTTTCCC TGCTTTTTCACT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT TGTCTAGTTTTC TACACGACGCT TCAGACGTGTG CTTCCGATCTTC TT CTTCCGATCTTC CTCTTCCGATCT AAGGGGGTATA AAGGGGGTATA AACAATTTCCCA TAAAAGGAAGA TAAAAGGAAGA CAAAGTCCA

TABLE-US-00014 TABLE10 FANCF 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) FANCF_1 CTGAAGGTGCT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GGTTTAGGG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT TGTCTGATTGAG ACATCCAGGGT TGTCTGATTGAG TCCCCACA TTCAAGTC TCCCCACA FANCF_2 ACACTCTTTCCC TGACATGCATTT ACACTCTTTCCC GTGACTGGAGT (on TACACGACGCT CGACCAAT TACACGACGCT TCAGACGTGTG target) CTTCCGATCTAT CTTCCGATCTAT CTCTTCCGATCT GGATGTGGCGC GGATGTGGCGC AGCATTGCAGA AGGTAG AGGTAG GAGGCGTAT FANCF_3 CCTCAGGGATG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GATGAAGTG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTCC CTCTTCCGATCT TCCCAGTGAGA CTTACCAGATG TCCCAGTGAGA CCAGTTTGA GAGGACA CCAGTTTGA FANCF_4 CCCTTACCAGAT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GGAGGACA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTGT CTCTTCCGATCT ACCTTGAGTTTT GACCCAGGTCC ACCTTGAGTTTT GCCCAGTG AGTGTTT GCCCAGTG FANCF_5 AGCTTTAAAATG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GGGAATCCA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTCT CTCTTCCGATCT TTCCCAGCACT CCAGTACAGGG TTCCCAGCACT GTTCTGTTG GCTTTTG GTTCTGTTG FANCF_6 ACACAGGGTGC GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT AGTGGTACA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAG CTCTTCCGATCT TGGGGAGTATC GTGCTTCTGCA TGGGGAGTATC CTTGCAATC GGTCATC CTTGCAATC FANCF_7 ACGCCAGCACT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT TTCTAAGGA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTG CTCTTCCGATCT CACAGATTGAT CCTGCTGCACT CACAGATTGAT GCCACTGGA CTCTGAGTA GCCACTGGA FANCF_8 ACACTCTTTCCC ACACCTCCGAG ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GCCTTCT TACACGACGCT TCAGACGTGTG CTTCCGATCTTT CTTCCGATCTTT CTCTTCCGATCT TCCTCAACCTTT TCCTCAACCTTT CAGGTCCTCCT TCTGCTG TCTGCTG CTCCCAGTT FANCF_9 ACACTCTTTCCC GCCAGGATTTC ACACTCTTTCCC GTGACTGGAGT TACACGACGCT CTCAAACAA TACACGACGCT TCAGACGTGTG CTTCCGATCTCC CTTCCGATCTCC CTCTTCCGATCT TGAATAACTAAA TGAATAACTAAA GCCAAGTTCCC TGACAACATGG TGACAACATGG ATAAGCAAA FANCF_10 GCTCTCAAATG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GCTCCAAAC TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTC CTCTTCCGATCT CAGAGTGGCCT CTCCATCTCATT CAGAGTGGCCT GCTTACAATC CCCATC GCTTACAATC FANCF_11 GCCGAGAATTA GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT CCACGACAT TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTC CTCTTCCGATCT GGCACACAGCT ACAGCGAGGAA GGCACACAGCT GTACGTAGG GGACAAT GTACGTAGG FANCF_12 ACACTCTTTCCC CTCCTCAGTGG ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GTGAAGTCC TACACGACGCT TCAGACGTGTG CTTCCGATCTG CTTCCGATCTG CTCTTCCGATCT GAGCTCTCAGT GAGCTCTCAGT ACGGAGAGGTC TGGACTGG TGGACTGG ACATGAAGG FANCF_13 TGAAAAGCAGT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT CTAGGACACAA TCAGACGTGTG TACACGACGCT TCAGACGTGTG A CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT CAACTCTGCCAT GCAGGCTAGGT CAACTCTGCCAT GTGCCTTA TTAGAGC GTGCCTTA FANCF_14 CACATATGAAAT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT ATTAAATTTGAA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CCA CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT GGGAATATAGA AACCATGTTACC GGGAATATAGA AAAATCAAGAGA TTTTGACC AAAATCAAGAGA TGG TGG FANCF_15 CGTCTTCGCTCT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT TTGGTTTT TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTG CTCTTCCGATCT CACCCTGTAGA TGGCACATAGT CACCCTGTAGA TCTCTCTCACG CGTAACCTC TCTCTCTCACG

TABLE-US-00015 TABLE11 RNF2 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) RNF2_1 CCATAGCACTTC GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT (on CCTTCCAA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT target) CTTCCGATCTGC TTCCGATCTATTT CTTCCGATCTGC CAACATACAGAA CCAGCAATGTCT CAACATACAGAA GTCAGGAA CAGG GTCAGGAA

TABLE-US-00016 TABLE12 HBB 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) HBB_1 GGCAGAGAGAG GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT (on TCAGTGCCTA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT target) CTTCCGATCTCA TTCCGATCTGTC CTTCCGATCTCA GGGCTGGGCAT TCCACATGCCCA GGGCTGGGCAT AAAAGT GTTTC AAAAGT HBB_2 ACACTCTTTCCC GTGGGTGTCCTG ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC GGTTGTT TACACGACGCTC CAGACGTGTGCT TTCCGATCTCCT TTCCGATCTCCT CTTCCGATCTCA ACAGCCTGCGA ACAGCCTGCGA CCTGGAGGCTA GGAATA GGAATA GGCACT HBB_3 CCCACACAGGTT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT TTCTCCTC CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTCT TTCCGATCTCTT CTTCCGATCTCT AGGCCTTCACCT CCCTAGACCTGC AGGCCTTCACCT GGAACC CTCCT GGAACC HBB_4 ACACTCTTTCCC CAGAAAATAAAG ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC CAGCTGACTCAC TACACGACGCTC CAGACGTGTGCT TTCCGATCTTTG TTCCGATCTTTG CTTCCGATCTCC TGTAACAGCCAC TGTAACAGCCAC TGGCAAAAGTGT TCACCA TCACCA TTGGAT HBB_5 TTTGCATTCCTTT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT TAGCTTCTTTT CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTAG TTCCGATCTATG CTTCCGATCTAG CTACCACGGTGA GCTGTTATTCAG CTACCACGGTGA CAGTAACA GGAAA CAGTAACA HBB_6 ACACTCTTTCCC AAATGGTAAAAA ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC GAAACTCAAATG TACACGACGCTC CAGACGTGTGCT TTCCGATCTTCC C TTCCGATCTTCC CTTCCGATCTGG ACTTTGTTAGTC ACTTTGTTAGTC ATACCACTGGGC AGGAGATTC AGGAGATTC TTCTGA HBB_7 TTCAAATCTGGA GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT AAATAATCTATCA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CC CTTCCGATCTAT TTCCGATCTTTT CTTCCGATCTAT TTCCAGGCTATG CATACCCTTTCC TTCCAGGCTATG CTTCCA CGTTC CTTCCA

TABLE-US-00017 TABLE13 EK2 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) HEK2_1 ACACTCTTTCCC TTTTCTTGTGAA ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC ACAGAAATGTCA TACACGACGCTC CAGACGTGTGCT TTCCGATCTCGT TTCCGATCTCGT CTTCCGATCTAA ACTATGCAAGCC ACTATGCAAGCC TGCTCCCACACC ACATTG ACATTG ATTTTT HEK2_2 ACACTCTTTCCC TTCCCAAGTGAG ACACTCTTTCCC GTGACTGGAGTT (on TACACGACGCTC AAGCCAGT TACACGACGCTC CAGACGTGTGCT target) TTCCGATCTAGG TTCCGATCTAGG CTTCCGATCTAA ACGTCTGCCCAA ACGTCTGCCCAA AATTGTCCAGCC TATGT TATGT CCATCT HEK2_3 ATTTACAAAACTT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT AGGAGAATCAAA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT GG CTTCCGATCTCA TTCCGATCTTCA CTTCCGATCTCA GCTGCTGTTATC AAGGAAAAGCAA GCTGCTGTTATC CTTCCTC CGTGA CTTCCTC

TABLE-US-00018 TABLE14 HEK3 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) HEK3_1 GCAGTTGCTTG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT ACTAGAGGTAG TCAGACGTGTG TACACGACGCT TCAGACGTGTG C CTCTTCCGATCT CTTCCGATCTTC CTCTTCCGATCT AGTGATGTGGG CAGATTCCTGGT AGTGATGTGGG AGGTTCCTG CCAAAG AGGTTCCTG HEK3_2 AAGGCATGGAT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT (on GAGAGAAGC TCAGACGTGTG TACACGACGCT TCAGACGTGTG target) CTCTTCCGATCT CTTCCGATCTAA CTCTTCCGATCT CTCCCTAGGTG ACGCCCATGCA CTCCCTAGGTG CTGGCTTC ATTAGTC CTGGCTTC HEK3_3 CTCAGGAGGCT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GAGGTAGGA TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAG CTCTTCCGATCT ACGTGTCTGCG GAAGATGAGGC ACGTGTCTGCG GTTAGCAG TGCAGTG GTTAGCAG HEK3_4 TTATGCGGCAAA GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT ACAAAATG TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTGA CTCTTCCGATCT TCGTCGCTGAC TCTCATCCCCTG TCGTCGCTGAC AATTTCTGA TTGACC AATTTCTGA HEK3_5 TGTTATCAACTG GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT GGGGTTGC TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTAG CTCTTCCGATCT TCCTTCATGGAC AGGGGCATCTC TCCTTCATGGAC TGGTAGGC GTGTAGA TGGTAGGC HEK3_6 ACACTCTTTCCC AAGCTATGATGT ACACTCTTTCCC GTGACTGGAGT TACACGACGCT GATGTGACTGG TACACGACGCT TCAGACGTGTG CTTCCGATCTTG CTTCCGATCTTG CTCTTCCGATCT TGTGCATGGTTC TGTGCATGGTTC CATGGTGTCTCA ATCTCC ATCTCC CCCCTGTA HEK3_7 GCCATGATCCT GTGACTGGAGT ACACTCTTTCCC GTGACTGGAGT CGTGATTTT TCAGACGTGTG TACACGACGCT TCAGACGTGTG CTCTTCCGATCT CTTCCGATCTTC CTCTTCCGATCT ACTTACCGAAG TCATGCTGTCTT ACTTACCGAAG GCAGGGACT GGATAAACA GCAGGGACT

TABLE-US-00019 TABLE15 HEK4 1stPCR 2ndPCR ID Forward(5to3) Reverse(5to3) Forward(5to3) Reverse(5to3) HEK4_1 ACACTCTTTCCC GACGTCCAAAAC ACACTCTTTCCC GTGACTGGAGTT (on TACACGACGCTC CAGACTCC TACACGACGCTC CAGACGTGTGCT target) TTCCGATCTCTC TTCCGATCTCTC CTTCCGATCTAC CCTTCAAGATGG CCTTCAAGATGG TCCTTCTGGGGC CTGAC CTGAC CTTTT HEK4_2 TCCCCAATGTTT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT TCTTGTGA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTGA TTCCGATCTTAG CTTCCGATCTGA TTACACAGAGGA AAGCGGACCCC TTACACAGAGGA GGCACCA ACATAG GGCACCA HEK4_3 TGAGAGAACATG GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT GTGCTTTG CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTAG TTCCGATCTGAA CTTCCGATCTAG GCTGTGGTAGG TGTGGACAGCAT GCTGTGGTAGG GACTCAC TGCAT GACTCAC HEK4_4 ACACTCTTTCCC AACCAACATGGT ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC GGGACACT TACACGACGCTC CAGACGTGTGCT TTCCGATCTCCA TTCCGATCTCCA CTTCCGATCTAG GAAGAGTGTGGT GAAGAGTGTGGT GCTGTGGTGAAG GCAGT GCAGT AGGATG HEK4_5 GGAGTTAGGCGT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT AGCTTCAGG CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTCC TTCCGATCTAAT CTTCCGATCTCC TGGCACAGACCT CCAATCAATGGG TGGCACAGACCT TCCTAA AGCAT TCCTAA HEK4_6 ACACTCTTTCCC GCTGGTCATGCA ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC GTGTCTGT TACACGACGCTC CAGACGTGTGCT TTCCGATCTAAA TTCCGATCTAAA CTTCCGATCTCC GCCCAGCTCTGC GCCCAGCTCTGC CCATTTCTGCCT TGATA TGATA GATTT HEK4_7 ACACTCTTTCCC TGGGCTCAACCC ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC AGGTGT TACACGACGCTC CAGACGTGTGCT TTCCGATCTGGG TTCCGATCTGGG CTTCCGATCTCC CATGGCTTCTGA CATGGCTTCTGA GGATGATTCTCC GACT GACT TACTTCC HEK4_8 ACACTCTTTCCC AGTTGTGGGGTT ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC TTCTGCTG TACACGACGCTC CAGACGTGTGCT TTCCGATCTGCC TTCCGATCTGCC CTTCCGATCTAT AACTAGAGGCAG AACTAGAGGCAG TCTGGAGGCAAC ACAGG ACAGG TCCTCA HEK4_9 GGCAAAACCCAT GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT TCCAGAAG CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTTG TTCCGATCTACC CTTCCGATCTTG TTAGGAGCTCCC ACGTCAGGACTT TTAGGAGCTCCC CATCAC GTGTG CATCAC HEK4_10 ATGTTAGCCGGG GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT ATGGTCTA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTTC TTCCGATCTGAT CTTCCGATCTTC CAGGGTATCAGG CTCTTGACTTGG CAGGGTATCAGG AAAGGTT TGATCCA AAAGGTT HEK4_11 ACACTCTTTCCC CACAGCCCATCT ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC CTCCACTC TACACGACGCTC CAGACGTGTGCT TTCCGATCTAAA TTCCGATCTAAA CTTCCGATCTTG TCCTCAGCACAC TCCTCAGCACAC GGCTCCAACCTC GACAA GACAA TTCTAA HEK4_12 CCCTGGTGAGCA GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT AACACAC CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTCA TTCCGATCTCCC CTTCCGATCTCA GGTCCTGTGCCA ACGTGGTATTCA GGTCCTGTGCCA CCTC CCTCT CCTC HEK4_13 GCCATCTAATCA GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT CAGCCACA CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTGC TTCCGATCTCTC CTTCCGATCTGC ATCTTGTCCCTT CTGGGTGCTCAG ATCTTGTCCCTT CTCAGC ACTTC CTCAGC HEK4_14 ACACTCTTTCCC CACCATGCCTGG ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC CTAATTTT TACACGACGCTC CAGACGTGTGCT TTCCGATCTGTT TTCCGATCTGTT CTTCCGATCTTT GAGAAGCAGCAA GAGAAGCAGCAA AGTAGGGACGG GGTGA GGTGA GGTTTCA HEK4_15 CAGAACCCAAGG GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT CTCTTGAC CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTAT TTCCGATCTTCC CTTCCGATCTAT TTTGCTCAGACC AAGATGCCTTCT TTTGCTCAGACC CAGCAT GCTCT CAGCAT HEK4_16 ACACTCTTTCCC TTTCTCACGATG ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC ACATTTTGG TACACGACGCTC CAGACGTGTGCT TTCCGATCTAAC TTCCGATCTAAC CTTCCGATCTCG AGAGCCCTGCA AGAGCCCTGCA GAGGAGGTAGAT GAACAT GAACAT TGGAGA HEK4_17 ACACTCTTTCCC TGTTCCTAGAGC ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC AACCTTCACA TACACGACGCTC CAGACGTGTGCT TTCCGATCTCAT TTCCGATCTCAT CTTCCGATCTGG GTATGCAGCTGC GTATGCAGCTGC AGAGCCAGAGT TTTTGA TTTTGA GGCTAAA HEK4_18 CTGAAAGAGGGA GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT GGGGAGAC CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTCT TTCCGATCTCTC CTTCCGATCTCT TCGCCAGGTCTT GGGAGAGAGGA TCGCCAGGTCTT CTGTTC AAGGAC CTGTTC HEK4_19 ACACTCTTTCCC GACGCATCCCAC ACACTCTTTCCC GTGACTGGAGTT TACACGACGCTC CTCCTC TACACGACGCTC CAGACGTGTGCT TTCCGATCTCCC TTCCGATCTCCC CTTCCGATCTCT GGCCGATTTAAC GGCCGATTTAAC GGGGCACGAAA TTTTA TTTTA TGTCC HEK4_20 CCAGGAACAGA GTGACTGGAGTT ACACTCTTTCCC GTGACTGGAGTT GGGACCAT CAGACGTGTGCT TACACGACGCTC CAGACGTGTGCT CTTCCGATCTCC TTCCGATCTCCA CTTCCGATCTCC TGGTTCCAGTCA GGTCCAGAGACA TGGTTCCAGTCA CCTCTC AGACG CCTCTC

[0216] FIG. 7 is a Venn diagram showing the number of sites with DNA cleavage scores 2.5 or higher identified by Digenome-seq of Cas9 nuclease- and Base editor-treated genomic DNA.

[0217] As can be seen from the above results, seven BE3 deaminases plus USER cleaved human genomic DNA in vitro at just 1-24 (83) sites, far fewer than did Cas9 nucleases with the same set of sgRNAs (7030 sites) in a multiplex Digenome-seq analysis (Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target-specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome research 26, 406-415 (2016)) (FIG. 7). This means that BE3 has far fewer potential, not necessarily genuine, off-target sites than does Cas9. Sequence logos, obtained by comparing Digenome-identified sites, showed that both the PAM-distal and PAM-proximal regions contributed to the specificities of BE3 deaminases (FIG. 6c, d).

[0218] The inventors further improved the computer program (termed Digenome 2.0) to identify potential off-target sites more comprehensively. The inventors counted the number of positions whose DNA cleavage scores were over a cutoff value that ranged from 0.0001 to 10 and the number of PAM (5-NGN-3 or 5-NNG-3)-containing sites with 10 or fewer mismatches, compared to the on-target site, among the positions with scores over the cutoff value (FIG. 8). FIG. 8 is a graph showing the number of total sites (.square-solid.) and the number of PAM-containing sites with ten or fewer mismatches () for a range of DNA cleavage scores. Such result was obtained by performing whole genome sequencing (WGS) for intact human genomic DNA (left) and human genomic DNA (right) cleaved by BE3 and USER. Cutoff score of 0.1 was selected, because WGS data obtained using intact genomic DNA, which had not been treated with BE3 and USER and thus served as a negative control, did not yield any false-positive sites with this cutoff score 0.1 (FIG. 8). Based on these results, in determining off-target sites by Digenome 2.0, sites with DNA cleavage score of 0.1 or more and 10 or less mismatch and having PAM (5-NGN-3 or 5-NNG-3) are determined as a off-target sites. In determining off-target sites by Digenome 2.0, sites with DNA cleavage score of 2.5 or more are determined as off-target sites. On the other hand, in the off-target localization by Digenome 1.0, a site with a DNA cleavage score of 2.5 or more is determined as off-target site candidates.

[0219] With Digenome 2.0, it was able to identify many additional BE3- and Cas9-associated DNA cleavage sites, including two sites that had been missed in the previous study ((Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target-specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res (2016)) but had been captured by both HTGTS and GUIDE-seq using EMX1-specific Cas9. FIG. 9 is a Venn diagram showing the number of PAM-containing homologous sites with DNA cleavage scores over 0.1 or higher identified by Digenome-seq of Cas9 nuclease- and Base editor-treated genomic DNA. BE3 deaminases induced base conversions in vitro at 1-67 (189) sites, whereas Cas9 nucleases cleaved genomic DNA at 30-241 (9030) sites.

Example 5. Fraction of Homologous Sites Captured by Digenome-Seq

[0220] The inventors examined the BE3- and Cas9-associated sites as shown in FIGS. 7 and 9. FIG. 10 shows fractions of homologous sites captured by Digenome-seq, wherein bars represent the number of homologous sites that differ from on-target sites by up to 6 nt, squares (BE3) and triangles (Cas9) represent the fraction of Digenome-seq captured sites for a range of mismatch numbers. As shown in FIG. 10, regardless of the number of mismatches, fewer homologous sites were identified by Digenome-seq when BE3 was used than when Cas9 was used.

[0221] FIGS. 11a and 11b are graphs showing the significant correlation between the number of BE3- and Cas9-associated sites identified by Digenome 1.0 (11a) and Digenome 2.0 (11b). As shown in FIGS. 11a and 11b, there was a statistically significant correlation [R.sup.2=0.97 (Score >2.5, Digenome 1.0) or 0.86 (Digenome 2.0)] between the number of Cas9- and BE3-associated sites. These results suggest that sgRNAs were the primary determinants of both Cas9 and BE3 specificities.

[0222] FIGS. 12a and 12b show the correlation between the number of BE3-associated sites identified by Digenome 1.0 (12a) or Digenome 2.0 (12b) and the number of sites with 6 or fewer mismatches. As shown in FIGS. 12a and 12b, a strong correlation [R.sup.2=0.94 (Digenome 1.0) or 0.95 (Digenome 2.0)] was observed between the number of BE3-associated, Digenome-captured sites and the number of homologous sites with 6 mismatches in the human genome (defined as orthogonality). Of particular interest are those associated with BE3 alone or Cas9 alone. Interestingly, 69% (=18/26) of sites associated with BE3 alone had missing or extra nucleotides, compared to their respective on-target sites, producing, respectively, an RNA or DNA bulge at the DNA-gRNA interface (Table 1). By contrast, these bulge-type off-target sites were rare among Cas9-associated sites. Just 4% (=25/647) of sites associated with Cas9 had missing or extra nucleotides.

[0223] FIG. 13 shows examples of Digenome-captured off-target sites associated only with Cas9, which contain no cytosines at positions 4-9. Thirteen % (=73/548) of sites associated with Cas9 alone had no cytosines at positions 4-8 (numbered 1-20 in the 5 to 3 direction), the window of BE3-mediated deamination.

[0224] To validate off-target effects at BE3-associated sites identified by Digenome-seq, the inventors performed targeted deep sequencing and measured BE3-induced substitution frequencies and Cas9-induced indel frequencies in HEK293T cells. The results are shown in 6e to 6h as above and Table 16 as below.

TABLE-US-00020 TABLE 16 Mutation frequencies of Cas9 and BE3 in on-target and off-target sites captured by Digenome-seq Base editing efficiency (%) EMX1 G A G T C C G A G C On- C--> Untreated 0.04 0.06 0.15 target Other (+)BE1 8.49 4.72 0.08 (EMX1_4) bases (+)BE2 11.08 10.72 0.09 (+)BE3 49.17 45.06 0.10 G A G T C t a A G C EMX1_1 C--> Untreated 0.04 0.05 other (+)BE1 3.13 0.05 bases (+)BE2 0.75 0.05 (+)BE3 15.57 0.07 G A a T C C a A G C EMX1_2 C--> Untreated 0.08 0.08 0.07 other (+)BE1 0.65 0.31 0.06 bases (+)BE2 0.32 0.32 0.07 (+)BE3 0.84 0.81 0.07 a A G T C t G A G C EMX1_3 C--> Untreated 0.02 0.07 other (+)BE1 0.02 0.07 bases (+)BE2 0.02 0.05 (+)BE3 0.13 0.07 G A a T C C a A G EMX1_5 C--> Untreated 0.06 0.10 other (+)BE1 0.63 0.24 bases (+)BE2 0.32 0.34 (+)BE3 0.96 0.96 G A G T C C t A G C EMX1_6 C--> Untreated 0.02 0.04 0.04 other (+)BE1 0.06 0.07 0.07 bases (+)BE2 0.07 0.08 0.05 (+)BE3 2.43 2.40 0.04 G A G T C C a A G C EMX1_7 C--> Untreated 0.03 0.06 0.06 other (+)BE1 0.07 0.10 0.07 bases (+)BE2 0.03 0.06 0.09 (+)BE3 0.05 0.09 0.07 G t G T C C t A G EMX1_8 C--> Untreated 0.05 0.03 other (+)BE1 0.64 0.57 bases (+)BE2 0.54 0.39 (+)BE3 0.37 0.34 a A G T C C G A G g EMX1_9 C--> Untreated 0.05 0.16 other (+)BE1 0.06 0.18 bases (+)BE2 0.06 0.17 (+)BE3 0.09 0.25 G A G g C C G A G C EMX1_10 C--> Untreated 0.14 0.10 0.13 other (+)BE1 0.44 0.24 0.16 bases (+)BE2 0.51 0.48 0.15 (+)BE3 3.45 3.70 0.17 a g t T C C a A G C EMX1_11 C--> Untreated 0.06 0.05 0.07 other (+)BE1 1.19 0.44 0.08 bases (+)BE2 0.46 0.43 0.05 (+)BE3 0.74 0.62 0.06 G A G T C C a c a C EMX1_12 C--> Untreated 0.08 0.26 0.11 0.11 other (+)BE1 0.08 0.24 0.11 0.11 bases (+)BE2 0.08 0.23 0.10 0.10 (+)BE3 0.17 0.33 0.17 0.10 G A G T C C a A G EMX1_13 C--> Untreated 0.08 0.12 other (+)BE1 0.07 0.11 bases (+)BE2 0.07 0.11 (+)BE3 0.08 0.13 G A G T C C t A G EMX1_14 C--> Untreated 0.06 0.13 other (+)BE1 0.09 0.17 bases (+)BE2 0.05 0.10 (+)BE3 0.05 0.13 G A a T C C a A G C EMX1_15 C--> Untreated 0.04 0.07 0.05 other (+)BE1 0.03 0.08 0.06 bases (+)BE2 0.04 0.07 0.06 (+)BE3 0.14 0.18 0.05 G t a c C a G A G EMX1_16 C--> Untreated 0.06 0.06 other (+)BE1 0.05 0.05 bases (+)BE2 0.05 0.05 (+)BE3 0.05 0.05 G A G T C C c A G C EMX1_17 C--> Untreated 0.10 0.19 0.09 0.07 other (+)BE1 0.13 0.17 0.09 0.05 bases (+)BE2 0.10 0.20 0.06 0.03 (+)BE3 0.11 0.20 0.07 0.07 a A G T C C a A G t EMX1_18 C--> Untreated 0.05 0.09 other (+)BE1 0.08 0.09 bases (+)BE2 0.08 0.10 (+)BE3 0.09 0.11 a A G T C C a t G C EMX1_19 C--> Untreated 0.03 0.07 0.10 other (+)BE1 0.17 0.10 0.12 bases (+)BE2 0.09 0.14 0.08 (+)BE3 0.24 0.30 0.12 G A G T C C t A G EMX1_20 C--> Untreated 0.05 0.12 other (+)BE1 0.28 0.24 bases (+)BE2 0.39 0.42 (+)BE3 0.50 0.57 G A G T C C c t C EMX1_21 C--> Untreated 0.16 0.08 0.07 0.03 other (+)BE1 0.15 0.10 0.06 0.04 bases (+)BE2 0.20 0.13 0.11 0.05 (+)BE3 0.20 0.12 0.10 0.06 a c G T C t G A G C EMX1_22 C--> Untreated 0.14 0.04 0.11 other (+)BE1 0.17 0.36 0.10 bases (+)BE2 0.13 0.14 0.11 (+)BE3 0.15 0.62 0.12 G A G T t C c A G a EMX1_23 C--> Untreated 0.06 0.08 other (+)BE1 0.09 0.13 bases (+)BE2 0.06 0.10 (+)BE3 0.06 0.09 G G A G T C C t A a EMX1_24 C--> Untreated 0.05 0.18 other (+)BE1 0.04 0.18 bases (+)BE2 0.05 0.19 (+)BE3 0.05 0.22 c A G T C C a A a C EMX1_25 C--> Untreated 0.11 0.05 0.11 0.11 other (+)BE1 0.08 0.10 0.10 0.10 bases (+)BE2 0.10 0.06 0.10 0.11 (+)BE3 0.11 0.07 0.13 0.11 A G A A G A A G A A On- C--> Untreated target other (+)BE1 (EMX1_4) bases (+)BE2 (+)BE3 A G A A G A A G A A EMX1_1 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G A g EMX1_2 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A c A A G A A G A A EMX1_3 C--> Untreated 0.06 other (+)BE1 0.04 bases (+)BE2 0.05 (+)BE3 0.05 A G A A G A A G A A EMX1_5 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G g A G A A G A A EMX1_6 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G t A G A g G A A EMX1_7 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G A A EMX1_8 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A g G A A G A A EMX1_9 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A a g A EMX1_10 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G c A EMX1_11 C--> Untreated 0.06 other (+)BE1 0.07 bases (+)BE2 0.07 (+)BE3 0.07 A G A A G A A G A A EMX1_12 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G t g EMX1_13 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G g A EMX1_14 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G g A G A A G A A EMX1_15 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G A g EMX1_16 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A a A A G A A G A A EMX1_17 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 G A A G A A G A A EMX1_18 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A g G A A EMX1_19 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A a A A EMX1_20 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G g A G A A G A A EMX1_21 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G A A EMX1_22 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G A A G A A G A A EMX1_23 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 G A G A A G A A G c A EMX1_24 C--> Untreated 0.11 other (+)BE1 0.12 bases (+)BE2 0.11 (+)BE3 0.12 A G A A G A g G A A EMX1_25 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 G G G On- C--> Untreated 0.15 61.59 Validated Validated target other (+)BE1 (EMX1_4) bases (+)BE2 (+)BE3 G A G EMX1_1 C--> Untreated 0.29 38.25 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A A G EMX1_2 C--> Untreated 0.00 0.01 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G EMX1_3 C--> Untreated 0.10 3.45 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G EMX1_5 C--> Untreated 0.01 0.01 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G A G EMX1_6 C--> Untreated 0.00 8.63 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G EMX1_7 C--> Untreated 0.01 0.01 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G EMX1_8 C--> Untreated 0.08 0.08 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_9 C--> Untreated 0.01 0.23 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 C G G EMX1_10 C--> Untreated 0.00 7.94 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G EMX1_11 C--> Untreated 0.00 0.01 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G A EMX1_12 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_13 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_14 C--> Untreated 0.01 0.01 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G A EMX1_15 C--> Untreated 0.46 0.89 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_16 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A A G EMX1_17 C--> Untreated 0.01 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_18 C--> Untreated 0.01 0.01 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G EMX1_19 C--> Untreated 0.01 0.02 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G EMX1_20 C--> Untreated 0.27 0.25 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G EMX1_21 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G EMX1_22 C--> Untreated 0.02 0.17 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G A G EMX1_23 C--> Untreated 0.01 0.01 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G G EMX1_24 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G EMX1_25 C--> Untreated 1.06 1.04 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 FANCF G G A A T C C C T T On- C--> Untreated 0.06 0.10 0.04 target other (+)BE1 0.81 0.39 0.42 (FANCF_2) bases (+)BE2 2.11 2.06 1.97 (+)BE3 10.26 9.44 9.28 t G A A T C C C a T FNACF_1 C--> Untreated 0.07 0.10 0.08 other (+)BE1 0.07 0.10 0.09 bases (+)BE2 0.10 0.10 0.12 (+)BE3 0.16 0.16 0.18 G G A g T C C C T c FNACF_3 C--> Untreated 0.06 0.09 0.05 0.08 other (+)BE1 0.06 0.09 0.06 0.08 bases (+)BE2 0.06 0.10 0.05 0.08 (+)BE3 0.20 0.23 0.18 0.16 G G A g T C C C T c FNACF_4 C--> Untreated 0.06 0.05 0.05 0.05 other (+)BE1 0.06 0.05 0.05 0.02 bases (+)BE2 0.07 0.06 0.03 0.05 (+)BE3 0.11 0.09 0.12 0.05 G G A A T C C C T T FNACF_5 C--> Untreated 0.09 0.07 0.05 other (+)BE1 0.07 0.06 0.04 bases (+)BE2 0.08 0.05 0.06 (+)BE3 0.10 0.07 0.05 G G A g T C C C T c FNACF_6 C--> Untreated 0.04 0.04 0.04 0.02 other (+)BE1 0.05 0.05 0.02 0.02 bases (+)BE2 0.04 0.05 0.05 0.03 (+)BE3 0.13 0.09 0.09 0.05 G G A A c C C C g T FNACF_7 C--> Untreated 0.03 0.07 0.07 0.06 other (+)BE1 0.05 0.06 0.04 0.07 bases (+)BE2 0.04 0.08 0.05 0.08 (+)BE3 1.06 1.07 1.07 1.02 G t c t c C C C T T FNACF_8 C--> Untreated 0.02 0.03 0.04 0.02 0.05 other (+)BE1 0.02 0.02 0.03 0.04 0.05 bases (+)BE2 0.01 0.02 0.02 0.05 0.05 (+)BE3 0.02 0.02 0.04 0.04 0.08 a a A A T C C C T T FNACF_9 C--> Untreated 0.07 0.02 0.04 other (+)BE1 0.08 0.03 0.04 bases (+)BE2 0.08 0.02 0.03 (+)BE3 0.10 0.04 0.05 t G t A T t t C T T FNACF_10 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 G G A A T a t C T T FNACF_11 C--> Untreated 0.03 other (+)BE1 0.03 bases (+)BE2 0.03 (+)BE3 0.04 G a g t g C C C T g FNACF_12 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 a c c A T C C C T c FNACF_13 C--> Untreated 0.07 0.06 0.04 0.04 0.04 0.06 other (+)BE1 0.14 0.07 0.07 0.04 0.05 0.06 bases (+)BE2 0.11 0.07 0.04 0.03 0.04 0.06 (+)BE3 0.13 0.08 0.15 0.15 0.14 0.13 t G A A T C C t a a FNACF_14 C--> Untreated 0.09 0.05 other (+)BE1 0.09 0.04 bases (+)BE2 0.07 0.05 (+)BE3 0.10 0.08 c t c t g t C C T T FNACF_15 C--> Untreated 0.03 0.04 0.05 0.02 other (+)BE1 0.03 0.02 0.04 0.02 bases (+)BE2 0.04 0.03 0.04 0.03 (+)BE3 0.03 0.02 0.07 0.03 C T G C A G C A C C On- C--> Untreated 0.03 0.13 0.13 0.05 0.04 target other (+)BE1 0.07 0.12 0.13 0.06 0.03 (FANCF_2) bases (+)BE2 0.39 0.14 0.09 0.07 0.02 (+)BE3 4.12 0.18 0.12 0.05 0.04 C T c C A G C A C C FNACF_1 C--> Untreated 0.03 0.04 0.04 0.07 0.04 0.07 other (+)BE1 0.03 0.05 0.05 0.09 0.03 0.06 bases (+)BE2 0.03 0.02 0.06 0.07 0.02 0.08 (+)BE3 0.06 0.05 0.07 0.09 0.03 0.07 C T a C A G C A C C FNACF_3 C--> Untreated 0.03 0.06 0.07 0.06 0.14 other (+)BE1 0.03 0.06 0.06 0.07 0.13 bases (+)BE2 0.04 0.05 0.07 0.07 0.15 (+)BE3 0.06 0.08 0.06 0.07 0.18 C T a C A G C A C C FNACF_4 C--> Untreated 0.06 0.06 0.03 0.03 0.06 other (+)BE1 0.07 0.07 0.03 0.03 0.05 bases (+)BE2 0.06 0.07 0.02 0.04 0.06 (+)BE3 0.06 0.07 0.04 0.04 0.04 C T a C A G C A t C FNACF_5 C--> Untreated 0.03 0.07 0.03 0.03 other (+)BE1 0.03 0.07 0.03 0.03 bases (+)BE2 0.03 0.06 0.05 0.03 (+)BE3 0.03 0.07 0.03 0.02 C T G C A G C A C C FNACF_6 C--> Untreated 0.04 0.09 0.06 0.02 0.04 other (+)BE1 0.04 0.12 0.04 0.05 0.05 bases (+)BE2 0.06 0.11 0.06 0.05 0.05 (+)BE3 0.06 0.12 0.06 0.05 0.03 C T G C A G C A C C FNACF_7 C--> Untreated 0.03 0.20 0.05 0.03 0.07 other (+)BE1 0.01 0.21 0.05 0.02 0.05 bases (+)BE2 0.02 0.23 0.06 0.02 0.05 (+)BE3 0.71 0.22 0.07 0.03 0.07 C T G C A G C A C C FNACF_8 C--> Untreated 0.03 0.11 0.03 0.02 0.03 other (+)BE1 0.03 0.08 0.04 0.02 0.04 bases (+)BE2 0.02 0.09 0.03 0.02 0.04 (+)BE3 0.03 0.10 0.03 0.02 0.03 C c G C A G C A C C FNACF_9 C--> Untreated 0.05 0.06 0.05 0.04 0.05 0.06 other (+)BE1 0.04 0.07 0.04 0.04 0.05 0.04 bases (+)BE2 0.03 0.07 0.04 0.04 0.04 0.06 (+)BE3 0.05 0.06 0.06 0.04 0.05 0.03 C T G C c t C A g g FNACF_10 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C T G C A G C c C C FNACF_11 C--> Untreated 0.05 0.22 0.03 0.05 0.05 0.10 other (+)BE1 0.04 0.23 0.03 0.06 0.05 0.09 bases (+)BE2 0.04 0.21 0.03 0.06 0.07 0.09 (+)BE3 0.03 0.21 0.02 0.06 0.05 0.09 a a G C c t C A g C FNACF_12 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C T G C A G C A C C FNACF_13 C--> Untreated 0.05 0.10 0.03 0.08 0.04 other (+)BE1 0.04 0.10 0.04 0.06 0.05 bases (+)BE2 0.04 0.12 0.04 0.05 0.05 (+)BE3 0.09 0.10 0.04 0.06 0.04 C T G C A G C A C C FNACF_14 C--> Untreated 0.04 0.09 0.06 0.08 0.06 other (+)BE1 0.05 0.10 0.06 0.10 0.07 bases (+)BE2 0.04 0.07 0.06 0.09 0.06 (+)BE3 0.03 0.11 0.07 0.10 0.07 C T G C A G C A C C FNACF_15 C--> Untreated 0.02 0.06 0.02 0.01 0.03 other (+)BE1 0.02 0.06 0.03 0.02 0.04 bases (+)BE2 0.02 0.05 0.03 0.02 0.04 (+)BE3 0.02 0.05 0.03 0.02 0.04 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 T G G On- C--> Untreated 0.01 44.48 Validated Validated target other (+)BE1 (FANCF_2) bases (+)BE2 (+)BE3 A G G FNACF_1 C--> Untreated 0.00 0.02 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_3 C--> Untreated 0.01 0.37 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_4 C--> Untreated 0.01 0.22 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 C T G FNACF_5 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G A FNACF_6 C--> Untreated 0.00 0.28 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_7 C--> Untreated 0.01 12.06 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_8 C--> Untreated 0.03 0.05 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T A G FNACF_9 C--> Untreated 0.00 0.08 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 C T G FNACF_10 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_11 C--> Untreated 0.02 0.03 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G FNACF_12 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_13 C--> Untreated 0.01 0.03 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G FNACF_14 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G FNACF_15 C--> Untreated 0.02 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+1BE3 RNF2 G T C A T C T T A G On- C--> Untreated 0.07 target other (+)BE1 2.90 (RNF2_1) bases (+)BE2 3.89 (+)BE3 31.12 T C A T T A C C T G On- C--> Untreated 0.06 0.03 0.07 target other (+)BE1 0.08 0.03 0.07 (RNF2_1) bases (+)BE2 0.62 0.05 0.08 (+)BE3 3.45 0.16 0.08 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 A G G On- C--> Untreated 0.03 66.13 Validated Validated target other (+)BE1 (RNF2_1) bases (+)BE2 (+)BE3 HBB C T T G C C C C A C On- C--> Untreated 0.05 0.08 0.03 0.05 0.04 0.04 target other (+)BE1 0.04 0.08 0.14 0.17 0.08 0.05 (HBB_1) bases (+)BE2 0.08 0.56 0.80 0.83 0.80 0.07 (+)BE3 0.10 3.01 4.51 4.88 4.64 0.14 t T g c t C C C A C HBB_2 C--> Untreated 0.07 0.06 0.04 0.05 0.04 other (+)BE1 0.07 0.09 0.07 0.07 0.04 bases (+)BE2 0.14 0.24 0.22 0.22 0.05 (+)BE3 0.42 0.89 0.84 0.86 0.07 g c T G C C C C A C HBB_3 C--> Untreated 0.07 0.06 0.06 0.11 0.03 0.07 other (+)BE1 0.08 0.06 0.06 0.10 0.03 0.05 bases (+)BE2 0.09 0.13 0.15 0.17 0.09 0.05 (+)BE3 0.09 0.80 0.86 0.87 0.75 0.07 g T g G C C C C A C HBB_4 C--> Untreated 0.07 0.13 0.06 0.09 0.04 other (+)BE1 0.09 0.14 0.07 0.08 0.05 bases (+)BE2 0.09 0.15 0.08 0.12 0.04 (+)BE3 0.14 0.20 0.13 0.16 0.07 a T T G C C C C A C HBB_5 C--> Untreated 0.12 0.19 0.73 0.40 0.16 other (+)BE1 0.16 0.20 0.76 0.47 0.19 bases (+)BE2 0.14 0.16 0.77 0.51 0.17 (+)BE3 0.36 0.42 0.95 0.73 0.20 a c T c t C C C A C HBB_6 C--> Untreated 0.11 0.12 0.08 0.11 0.20 0.08 0.05 other (+)BE1 0.10 0.16 0.10 0.09 0.20 0.10 0.04 bases (+)BE2 0.08 0.16 0.11 0.11 0.21 0.10 0.05 (+)BE3 0.10 0.14 0.13 0.13 0.22 0.09 0.05 t c a G C C C C A C HBB_7 C--> Untreated 0.03 0.07 0.07 0.09 0.05 0.05 other (+)BE1 0.14 0.09 0.09 0.11 0.06 0.06 bases (+)BE2 0.27 0.09 0.22 0.25 0.19 0.05 (+)BE3 2.82 0.80 2.89 4.01 4.20 0.14 A G G G C A G T A A On- C--> Untreated 0.08 target other (+)BE1 0.07 (HBB_1) bases (+)BE2 0.06 (+)BE3 0.08 A G G G C A G T A A HBB_2 C--> Untreated 0.06 other (+)BE1 0.07 bases (+)BE2 0.06 (+)BE3 0.06 A G G G C A G c A A HBB_3 C--> Untreated 0.14 0.09 other (+)BE1 0.10 0.08 bases (+)BE2 0.12 0.09 (+)BE3 0.11 0.09 A G G G C A G g A A HBB_4 C--> Untreated 0.06 other (+)BE1 0.08 bases (+)BE2 0.07 (+)BE3 0.08 g G G G C A G T g A HBB_5 C--> Untreated 0.20 other (+)BE1 0.25 bases (+)BE2 0.28 (+)BE3 0.21 A a G G C A G T A A HBB_6 C--> Untreated 0.17 other (+)BE1 0.14 bases (+)BE2 0.20 (+)BE3 0.17 A G G G C A G T A A HBB_7 C--> Untreated 0.08 other (+)BE1 0.14 bases (+)BE2 0.09 (+)BE3 0.09 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 C G G On- C--> Untreated 0.02 38.35 Validated Validated target other (+)BE1 (HBB_1) bases (+)BE2 (+)BE3 A C G HBB_2 C--> Untreated 0.02 0.01 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G HBB_3 C--> Untreated 0.01 3.57 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HBB_4 C--> Untreated 0.00 0.70 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 C G G HBB_5 C--> Untreated 0.00 0.35 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HBB_6 C--> Untreated 0.02 0.01 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G HBB_7 C--> Untreated 0.00 20.92 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 HEK2 G A A C A C A A A G On- C--> Untreated 0.04 0.05 target other (+)BE1 0.65 10.29 (HEK2_2) bases (+)BE2 7.32 14.69 (+)BE3 11.74 33.30 G A A C A C A A t G HEK2_1 C--> Untreated 0.10 0.09 other (+)BE1 0.10 0.10 bases (+)BE2 0.13 0.12 (+)BE3 0.17 0.21 a A c t c C A A A G HEK2_3 C--> Untreated 0.09 0.09 0.34 other (+)BE1 0.08 0.07 0.37 bases (+)BE2 0.09 0.07 0.38 (+)BE3 0.09 0.07 0.38 C A T A G A C T G C On- C--> Untreated 0.04 0.16 target other (+)BE1 0.04 0.18 (HEK2_2) bases (+)BE2 0.03 0.17 (+)BE3 0.07 0.18 C A T A G A t T G C HEK2_1 C--> Untreated 0.11 0.18 other (+)BE1 0.13 0.21 bases (+)BE2 0.11 0.16 (+)BE3 0.11 0.19 C A T A t A C T G C HEK2_3 C--> Untreated 0.25 0.09 other (+)BE1 0.24 0.08 bases (+)BE2 0.19 0.08 (+)BE3 0.24 0.07 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 G G G On- C--> Untreated 0.00 43.28 Validated Validated target other (+)BE1 (HEK2_2) bases (+)BE2 (+)BE3 C G G HEK2_1 C--> Untreated 0.00 1.01 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK2_3 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 HEK3 G G C C C A G A C T On- C--> Untreated 0.13 0.46 0.42 0.14 target other (+)BE1 0.38 6.45 8.56 0.59 (HEK3_2) bases (+)BE2 0.37 6.27 8.17 0.41 (+)BE3 1.00 24.71 31.39 0.76 a G C t C A G A C T HEK3_1 C--> Untreated 0.12 0.04 0.04 other (+)BE1 0.12 0.04 0.07 bases (+)BE2 0.13 0.05 0.08 (+)BE3 0.13 0.09 0.05 G t g g C c c A g a HEK3_3 C--> Untreated 0.07 0.06 0.07 other (+)BE1 0.08 0.05 0.10 bases (+)BE2 0.08 0.05 0.06 (+)BE3 0.07 0.05 0.07 c a C C C A G A C T HEK3_4 C--> Untreated 0.08 0.07 0.07 0.05 0.01 other (+)BE1 0.09 0.06 0.08 0.06 0.03 bases (+)BE2 0.09 0.07 0.07 0.06 0.02 (+)BE3 0.08 0.05 0.08 0.06 0.02 c G g C C c a A C T HEK3_5 C--> Untreated 0.16 0.08 0.13 0.10 0.06 other (+)BE1 0.19 0.11 0.14 0.07 0.06 bases (+)BE2 0.16 0.08 0.13 0.09 0.05 (+)BE3 0.16 0.08 0.13 0.09 0.05 a G a C C A G A C T HEK3_6 C--> Untreated 0.08 0.10 0.06 other (+)BE1 0.09 0.12 0.06 bases (+)BE2 0.08 0.12 0.06 (+)BE3 0.10 0.11 0.05 G G C C a c t c a T HEK3_7 C--> Untreated 0.45 0.15 0.05 0.19 other (+)BE1 0.45 0.16 0.08 0.19 bases (+)BE2 0.47 0.17 0.09 0.19 (+)BE3 0.44 0.16 0.08 0.19 G A G C A C G T G A On- C--> Untreated 0.10 0.07 target other (+)BE1 0.14 0.08 (HEK3_2) bases (+)BE2 0.20 0.06 (+)BE3 0.09 0.10 G A G C A a G T G A HEK3_1 C--> Untreated 0.14 other (+)BE1 0.13 bases (+)BE2 0.17 (+)BE3 0.13 G A G C A C G T G t HEK3_3 C--> Untreated 0.12 0.13 other (+)BE1 0.09 0.11 bases (+)BE2 0.11 0.12 (+)BE3 0.10 0.10 G A G C A C G T G c HEK3_4 C--> Untreated 0.14 0.06 0.04 other (+)BE1 0.13 0.04 0.04 bases (+)BE2 0.10 0.05 0.05 (+)BE3 0.13 0.05 0.05 G A G C A a G T G A HEK3_5 C--> Untreated 0.19 other (+)BE1 0.21 bases (+)BE2 0.16 (+)BE3 0.20 G A G C A a G a G A HEK3_6 C--> Untreated 0.20 other (+)BE1 0.19 bases (+)BE2 0.19 (+)BE3 0.16 G g c C A C a T a c HEK3_7 C--> Untreated 0.29 0.26 0.06 other (+)BE1 0.30 0.28 0.06 bases (+)BE2 0.31 0.24 0.06 (+)BE3 0.29 0.26 0.06 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 T G G On- C--> Untreated 0.00 60.16 Validated Validated target other (+)BE1 (HEK3_2) bases (+)BE2 (+)BE3 G G G HEK3_1 C--> Untreated 0.00 2.93 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK3_3 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK3_4 C--> Untreated 0.00 4.16 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK3_5 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK3_6 C--> Untreated 0.00 0.02 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK3_7 C--> Untreated 0.00 0.00 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 HEK4 G G C A C T G C G G On- C--> Untreated 0.16 0.11 0.20 target other (+)BE1 0.17 6.18 0.25 (HEK4_1) bases (+)BE2 0.65 10.35 0.84 (+)BE3 2.34 41.18 0.80 G G C A C T G C t G HEK4_2 C--> Untreated 0.11 0.05 0.15 other (+)BE1 0.13 0.38 0.14 bases (+)BE2 0.16 0.46 0.13 (+)BE3 0.31 5.93 0.22 G G C A C T G C a HEK4_3 C--> Untreated 0.08 0.05 0.07 other (+)BE1 0.10 0.22 0.09 bases (+)BE2 0.11 0.22 0.07 (+)BE3 0.09 0.39 0.08 G G C t C T G C G G HEK4_4 C--> Untreated 0.04 0.05 0.34 other (+)BE1 0.05 0.26 0.35 bases (+)BE2 0.06 0.19 0.35 (+)BE3 0.07 2.07 0.34 a G C A C T G C a G HEK4_5 C--> Untreated 0.08 0.07 0.11 other (+)BE1 0.09 0.11 0.11 bases (+)BE2 0.09 0.07 0.10 (+)BE3 0.10 0.52 0.20 G G C A C T G C G G HEK4_6 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 t G C A C T G C G G HEK4_7 C--> Untreated 0.21 0.12 0.36 other (+)BE1 0.15 0.53 0.31 bases (+)BE2 0.19 1.25 0.32 (+)BE3 0.37 10.75 0.41 G G C A C T g G G HEK4_8 C--> Untreated 0.09 0.05 other (+)BE1 0.07 0.15 bases (+)BE2 0.08 0.17 (+)BE3 0.07 0.18 G G C A C T G t G G HEK4_9 C--> Untreated 0.09 0.03 other (+)BE1 0.08 0.04 bases (+)BE2 0.12 0.03 (+)BE3 0.12 0.02 t G C t C T G C G G HEK4_10 C--> Untreated 0.08 0.17 0.06 other (+)BE1 0.07 0.17 0.06 bases (+)BE2 0.08 0.18 0.07 (+)BE3 0.08 0.19 0.07 a G C A C T G C a G HEK4_11 C--> Untreated 0.16 0.05 0.13 other (+)BE1 0.12 0.47 0.12 bases (+)BE2 0.13 0.64 0.14 (+)BE3 0.19 1.83 0.18 G G C A C T G a G G HEK4_12 C--> Untreated 0.10 0.03 other (+)BE1 0.07 0.65 bases (+)BE2 0.10 0.47 (+)BE3 0.09 0.99 G G C A C T G g G G HEK4_13 C--> Untreated 0.13 0.15 other (+)BE1 0.12 0.14 bases (+)BE2 0.10 0.13 (+)BE3 0.11 0.12 a G g A C T G C G G HEK4_14 C--> Untreated 0.06 0.28 other (+)BE1 0.50 0.37 bases (+)BE2 0.63 0.38 (+)BE3 5.20 0.50 G G C A C T G C a a HEK4_15 C--> Untreated 0.11 0.06 0.12 other (+)BE1 0.10 0.08 0.07 bases (+)BE2 0.08 0.08 0.08 (+)BE3 0.10 0.26 0.09 G G C A C T G g G G HEK4_16 C--> Untreated 0.17 0.16 other (+)BE1 0.14 1.01 bases (+)BE2 0.17 0.58 (+)BE3 0.38 3.41 G c C A C T G C a G HEK4_17 C--> Untreated 0.14 0.05 0.07 0.20 other (+)BE1 0.10 0.06 0.24 0.13 bases (+)BE2 0.09 0.10 0.27 0.14 (+)BE3 0.12 0.34 3.12 0.22 G c C A C T G C G a HEK4_18 C--> Untreated 0.14 0.07 0.06 60.77 other (+)BE1 0.10 0.05 0.08 61.73 bases (+)BE2 0.12 0.03 0.05 60.63 (+)BE3 0.10 0.08 0.12 60.98 G G C A C T G G G HEK4_19 C--> Untreated 0.06 0.06 other (+)BE1 0.07 0.04 bases (+)BE2 0.08 0.06 (+)BE3 0.08 0.05 a G C t C T G C G G HEK4_20 C--> Untreated 0.24 0.02 0.20 other (+)BE1 0.21 0.03 0.20 bases (+)BE2 0.21 0.02 0.17 (+)BE3 0.23 0.02 0.22 C T G G A G G T G G On- C--> Untreated 0.07 target other (+)BE1 0.07 (HEK4_1) bases (+)BE2 0.06 (+)BE3 0.07 C T G G g G G T G G HEK4_2 C--> Untreated 0.98 other (+)BE1 0.98 bases (+)BE2 0.93 (+)BE3 1.07 C T G G A G G T t G HEK4_3 C--> Untreated 0.05 other (+)BE1 0.05 bases (+)BE2 0.05 (+)BE3 0.03 C T G G A G G g G G HEK4_4 C--> Untreated 0.13 other (+)BE1 0.13 bases (+)BE2 0.15 (+)BE3 0.17 a T G G A G G a G G HEK4_5 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C a G G g a G g a G HEK4_6 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C c G G A G G a G G HEK4_7 C--> Untreated 0.14 0.09 other (+)BE1 0.13 0.08 bases (+)BE2 0.11 0.14 (+)BE3 0.12 0.07 C T G a A G G T a G HEK4_8 C--> Untreated 0.08 other (+)BE1 0.05 bases (+)BE2 0.07 (+)BE3 0.06 C T G c A G G T G G HEK4_9 C--> Untreated 0.02 0.04 other (+)BE1 0.04 0.03 bases (+)BE2 0.04 0.03 (+)BE3 0.04 0.05 C a G G A G G a G G HEK4_10 C--> Untreated 0.06 other (+)BE1 0.05 bases (+)BE2 0.07 (+)BE3 0.07 C T G G g a G T G G HEK4_11 C--> Untreated 0.07 other (+)BE1 0.07 bases (+)BE2 0.08 (+)BE3 0.08 g T G G A G G T G G HEK4_12 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C T G G A G a c G G HEK4_13 C--> Untreated 0.13 0.23 other (+)BE1 0.11 0.18 bases (+)BE2 0.09 0.15 (+)BE3 0.12 0.18 C T G G g G G T G G HEK4_14 C--> Untreated 0.05 other (+)BE1 0.03 bases (+)BE2 0.04 (+)BE3 0.04 C T G G A a G T G a HEK4_15 C--> Untreated 0.03 other (+)BE1 0.02 bases (+)BE2 0.02 (+)BE3 0.03 t T G G A G G T G G HEK4_16 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 C T a G A G G T G G HEK4_17 C--> Untreated 0.03 other (+)BE1 0.04 bases (+)BE2 0.04 (+)BE3 0.04 C T G G A G G a G G HEK4_18 C--> Untreated 0.04 other (+)BE1 0.03 bases (+)BE2 0.05 (+)BE3 0.04 C T G G A G G c G G HEK4_19 C--> Untreated 0.05 0.12 other (+)BE1 0.06 0.11 bases (+)BE2 0.04 0.10 (+)BE3 0.07 0.09 C a G G A G t T G G HEK4_20 C--> Untreated 0.12 other (+)BE1 0.08 bases (+)BE2 0.08 (+)BE3 0.11 Indel frequency (%) () (+) Validation Base editing efficiency (%) RGEN RGEN BE3 Cas9 G G G On- C--> Untreated 0.00 59.38 target other (+)BE1 Validated Validated (HEK4_1) bases (+)BE2 (+)BE3 T G G HEK4_2 C--> Untreated 0.02 35.65 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK4_3 C--> Untreated 0.00 0.00 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK4_4 C--> Untreated 0.07 29.61 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 C G G HEK4_5 C--> Untreated 0.00 0.08 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_6 C--> Untreated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK4_7 C--> Untreated 0.02 35.87 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_8 C--> Untreated 0.04 0.04 Validated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_9 C--> Untreated 0.02 25.09 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_10 C--> Untreated 2.67 3.08 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_11 C--> Untreated 0.04 8.97 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_12 C--> Untreated 0.08 10.38 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_13 C--> Untreated 0.11 0.69 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK4_14 C--> Untreated 0.38 46.26 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 T G G HEK4_15 C--> Untreated 0.01 0.14 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_16 C--> Untreated 0.12 25.87 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_17 C--> Untreated 0.01 2.93 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_18 C--> Untreated 0.16 0.37 Validated Validated other (+)BE1 bases (+)BE2 (+)BE3 G G G HEK4_19 C--> Untreated 0.10 0.11 Invalidated Invalidated other (+)BE1 bases (+)BE2 (+)BE3 A G G HEK4_20 C--> Untreated 0.02 0.07 Invalidated Validated other (+)BE1 bases (+)BE2 (+)BE3

[0225] The inventors analyzed a total of 75 sites identified using 7 sgRNAs and observed BE3-induced point mutations at 50 sites, including all 7 on-target sites, with frequencies above noise levels caused by sequencing errors (typically in the range of 0.1-2%), resulting in a validation rate of 67%. It is possible that BE3 can still induce mutagenesis at the other BE3-associated, Digenome-positive sites with frequencies below background noise levels. Importantly, we were able to identify BE3 off-target sites at which base editing was detected with a frequency of 0.1%, demonstrating that Digenome-seq is a highly sensitive method. Cas9 nucleases detectably induced indels at 70% (=44/63) of the sites associated with both Cas9 and BE3 but failed to do so at each of the 12 sites associated with BE3 alone (Tables 2-8).

[0226] FIGS. 14a-14c show base editing efficiencies at Digenome-captured sites associated only with 3 different Cas9 nucleases. As shown in FIGS. 14a-14c, BE3 did not detectably cause substitutions at 24 Digenome-positive sites associated with 3 different Cas9 nucleases alone. Furthermore, FIGS. 15a-15c show base editing efficiencies of 3 different BE3 deaminases at Digenome-negative sites. As shown in FIGS. 15a-15c, the 3 BE3 deaminases did not induce base editing at 28 Digenome-negative sites with 3 mismatches, identified using Cas-OFFinder (Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics (2014)) (FIGS. 15a-15c). Frequencies of BE3-induced substitutions were well-correlated with those of Cas9-mediated indels [R.sup.2=0.92 (EMX1) or 0.89 (HBB)] (FIG. 6e, f). Nevertheless, there were many off-target sites validated by BE3 but not by Cas9. 64% (=7/11) of these validated, BE3-exclusive off-target sites had a missing nt, compared to their respective on-target sites. These results show that Cas9 and BE3 off-target sites largely overlap with each other but that there are off-target sites exclusively associated with Cas9 alone or BE3 alone (FIG. 10).

Example 6. Reducing BE3 Off-Target Effects Via Modified sgRNAs

[0227] To reduce BE3 off-target effects, the inventors replaced conventional sgRNAs (termed gX.sub.19 or GX.sub.19; g and G represent, respectively, a mismatched and matched guanine) with truncated sgRNAs (termed gX.sub.18 or gX.sub.17) or extended sgRNAs containing one or two extra guanines at the 5 terminus (termed gX.sub.20 or ggX.sub.20) and measured on-target and off-target base-editing frequencies in HEK293T cells. The results are shown in FIGS. 16-17 and Table 3.

TABLE-US-00021 TABLE 17 Analysis of BE3 off-target effect via modified sgRNAs EMX1 A G T C C G A G C A G A A G A A G A A G G G 0.04 0.06 0.15 49.01 46.36 0.10 54.78 50.04 0.14 49.17 45.06 0.10 48.68 37.61 0.09 48.71 37.70 0.14 A G T C t a A G C A G A A G A A G A A G A G 0.04 0.05 1.26 0.05 8.50 0.06 15.57 0.07 0.06 0.05 0.07 0.05 A a T C C a A G C A G A A G A A G A g A A G 0.06 0.08 0.07 0.40 0.36 0.05 0.80 0.75 0.07 0.84 0.81 0.07 0.22 0.23 0.08 0.16 0.17 0.06 A G T C t G A G C A c A A G A A G A A T G G 0.02 0.07 0.06 0.03 0.06 0.06 0.03 0.07 0.05 0.13 0.07 0.05 0.02 0.07 0.05 0.02 0.08 0.04 A a T C C a A G A G A A G A A G A A T G G 0.11 0.05 1.09 1.11 2.31 2.27 0.96 0.96 0.06 0.11 0.08 0.12 A G T C C t A G C A G g A G A A G A A G A G 0.02 0.04 0.04 0.34 0.35 0.05 1.69 1.71 0.05 2.43 2.40 0.04 0.02 0.02 0.05 0.02 0.04 0.04 A G T C C a A G C A G t A G A g G A A G G G 0.03 0.06 0.06 0.03 0.06 0.08 0.04 0.08 0.08 0.07 0.10 0.07 0.03 0.05 0.09 0.03 0.05 0.08 t G T C C t A G A G A A G A A G A A G G G 0.05 0.03 0.10 0.09 0.36 0.35 0.64 0.57 0.60 0.56 0.62 0.80 A G T C C G A G g A G A g G A A G A A A G G 0.05 0.16 0.04 0.14 0.05 0.17 0.06 0.18 0.05 0.18 0.05 0.16 A G g C C G A G C A G A A G A A a g A C G G 0.14 0.10 0.13 0.26 0.21 0.12 0.44 0.39 0.19 3.45 3.70 0.17 0.17 0.10 0.15 0.18 0.09 0.16 g t T C C a A G C A G A A G A A G c A T G G 0.06 0.05 0.07 0.06 0.27 0.28 0.07 0.07 0.74 0.70 0.07 0.08 0.74 0.62 0.06 0.07 0.06 0.05 0.07 0.06 0.07 0.06 0.07 0.07 A G T C C a c a C A G A A G A A G A A A G A 0.06 0.26 0.11 0.11 0.07 0.21 0.12 0.11 0.09 0.23 0.09 0.11 0.17 0.33 0.17 0.10 0.07 0.25 0.10 0.13 0.08 0.23 0.11 0.11 A G T C C a A G A G A A G A A G t g A G G 0.06 0.12 0.06 0.11 0.07 0.11 0.08 0.13 0.08 0.13 0.07 0.13 A G T C C t A G A G A A G A A G g A A G G 0.06 0.13 0.07 0.19 0.07 0.17 0.05 0.13 0.06 0.12 0.05 0.14 A a T C C a A G C A G g A G A A G A A G G A 0.04 0.07 0.05 0.09 0.15 0.04 0.54 0.60 0.08 0.14 0.18 0.05 0.04 0.07 0.05 0.01 0.07 0.06 t a c C a G A G A G A A G A A G A g A G G 0.06 0.06 0.05 0.05 0.06 0.05 0.05 0.05 0.06 0.04 0.06 0.05 A G T C C c A G C A a A A G A A G A A A A G 0.10 0.19 0.09 0.07 0.10 0.16 0.10 0.07 0.19 0.24 0.13 0.05 0.11 0.20 0.07 0.07 0.12 0.24 0.09 0.06 0.12 0.20 0.07 0.06 A G T C C a A G t G A A G A A G A A A G G 0.06 0.09 0.05 0.09 0.05 0.08 0.09 0.11 0.05 0.08 0.05 0.09 A G T C C a t G C A G A A G A g G A A G G G 0.03 0.07 0.10 0.05 0.07 0.09 0.03 0.08 0.12 0.24 0.30 0.12 0.03 0.08 0.10 0.05 0.07 0.09 A G T C C t A G A G A A G A A a A A G G G 0.05 0.12 0.21 0.26 0.43 0.50 0.50 0.57 0.06 0.12 0.05 0.12 A G T C C c t C A G g A G A A G A A A G G 0.16 0.08 0.07 0.03 0.12 0.07 0.06 0.04 0.15 0.11 0.08 0.04 0.24 0.17 0.16 0.06 0.12 0.09 0.07 0.04 0.14 0.08 0.06 0.02 c G T C t G A G C A G A A G A A G A A T G G 0.14 0.04 0.11 0.14 0.16 0.13 0.15 0.20 0.16 0.15 0.62 0.12 0.22 1.24 0.12 0.13 4.49 0.11 A G T t C c A G a A G A A G A A G A A G A G 0.06 0.08 0.06 0.09 0.09 0.13 0.06 0.09 0.07 0.11 0.07 0.09 A G T C C t A a A G A A G A A G c A G G G 0.05 0.18 0.11 0.06 0.20 0.16 0.07 0.19 0.12 0.05 0.22 0.12 0.07 0.19 0.15 0.04 0.18 0.12 A G T C C a A a C A G A A G A g G A A T G G 0.05 0.11 0.11 0.08 0.12 0.09 0.05 0.10 0.10 0.07 0.13 0.11 0.05 0.14 0.13 0.07 0.13 0.12 FANCF G A A T C C C T T C T G C A G C A C C T G G 0.06 0.10 0.04 0.03 0.13 0.13 0.05 0.04 9.20 8.19 7.94 4.25 0.12 0.12 0.06 0.04 8.12 7.31 6.89 3.01 0.13 0.12 0.05 0.03 10.26 9.44 9.28 4.12 0.18 0.12 0.05 0.04 9.74 8.81 8.16 3.14 0.15 0.14 0.06 0.02 3.36 2.80 2.77 1.14 0.12 0.12 0.05 0.04 G A A T C C C a T C T c C A G C A C C A G G 0.07 0.10 0.08 0.03 0.04 0.04 0.07 0.04 0.07 0.06 0.11 0.07 0.03 0.03 0.05 0.11 0.03 0.06 0.09 0.10 0.09 0.04 0.02 0.05 0.09 0.03 0.09 0.16 0.16 0.18 0.06 0.05 0.07 0.09 0.03 0.07 0.80 0.79 0.79 0.25 0.13 0.08 0.10 0.03 0.06 0.08 0.10 0.09 0.02 0.03 0.06 0.09 0.02 0.07 G A g T C C C T c C T a C A G C A C C A G G 0.06 0.09 0.05 0.08 0.03 0.06 0.07 0.06 0.14 0.06 0.08 0.04 0.07 0.04 0.08 0.05 0.07 0.15 0.10 0.13 0.08 0.10 0.05 0.07 0.06 0.07 0.15 0.20 0.23 0.18 0.16 0.08 0.08 0.06 0.07 0.18 0.05 0.09 0.05 0.08 0.03 0.05 0.06 0.08 0.17 0.05 0.08 0.04 0.11 0.05 0.06 0.07 0.08 0.15 G A g T C C C T c C T a C A G C A C C A G G 0.06 0.05 0.05 0.05 0.06 0.06 0.03 0.03 0.06 0.05 0.05 0.05 0.04 0.04 0.07 0.04 0.03 0.04 0.08 0.07 0.06 0.06 0.08 0.06 0.02 0.02 0.06 0.11 0.09 0.12 0.05 0.08 0.07 0.04 0.04 0.04 0.07 0.07 0.06 0.05 0.06 0.07 0.04 0.03 0.07 0.06 0.05 0.04 0.06 0.04 0.07 0.03 0.02 0.06 G A A T C C C T T C T a C A G C A t C C T G 0.09 0.07 0.05 0.03 0.07 0.03 0.03 0.07 0.07 0.04 0.04 0.06 0.03 0.02 0.07 0.05 0.06 0.04 0.05 0.04 0.03 0.10 0.07 0.05 0.03 0.07 0.03 0.02 0.08 0.06 0.06 0.03 0.07 0.04 0.03 0.09 0.05 0.05 0.05 0.08 0.04 0.02 G A g T C C C T c C T G C A G C A C C T G A 0.04 0.04 0.04 0.02 0.04 0.09 0.06 0.02 0.04 0.03 0.04 0.04 0.02 0.04 0.12 0.05 0.03 0.06 0.05 0.06 0.05 0.03 0.06 0.11 0.07 0.06 0.04 0.13 0.09 0.09 0.05 0.06 0.12 0.06 0.05 0.03 0.06 0.05 0.04 0.03 0.04 0.06 0.07 0.04 0.05 0.05 0.05 0.04 0.04 0.05 0.14 0.05 0.05 0.04 G A A c C C C g T C T G C A G C A C C A G G 0.03 0.07 0.07 0.06 0.03 0.20 0.05 0.03 0.07 0.27 0.29 0.28 0.32 0.10 0.21 0.05 0.02 0.07 1.46 1.50 1.49 1.48 0.80 0.20 0.04 0.04 0.06 1.06 1.07 1.07 1.02 0.71 0.22 0.07 0.03 0.07 0.04 0.07 0.05 0.09 0.01 0.17 0.04 0.04 0.06 0.04 0.06 0.04 0.09 0.01 0.17 0.05 0.03 0.06 t c t c C C C T T C T G C A G C A C C A G G 0.02 0.03 0.04 0.02 0.05 0.03 0.11 0.03 0.02 0.03 0.02 0.01 0.03 0.05 0.05 0.01 0.08 0.02 0.02 0.04 0.01 0.02 0.04 0.04 0.04 0.02 0.08 0.02 0.03 0.03 0.02 0.02 0.04 0.04 0.08 0.03 0.10 0.03 0.02 0.03 0.04 0.09 0.09 0.10 0.13 0.05 0.10 0.04 0.03 0.03 0.04 0.09 0.11 0.11 0.13 0.07 0.11 0.05 0.02 0.04 a A A T C C C T T C c G C A G C A C C T A G 0.07 0.02 0.04 0.05 0.06 0.05 0.04 0.05 0.06 0.08 0.03 0.04 0.03 0.06 0.06 0.05 0.05 0.04 0.09 0.03 0.04 0.04 0.05 0.05 0.04 0.06 0.04 0.10 0.04 0.05 0.05 0.06 0.06 0.04 0.05 0.03 0.10 0.06 0.07 0.06 0.05 0.07 0.03 0.06 0.04 0.06 0.06 0.06 0.05 0.06 0.04 0.03 0.05 0.06 G t A T t t C T T C T G C c t C A g g C T G G A A T a t C T T C T G C A G C c C C A G G 0.03 0.05 0.22 0.03 0.05 0.05 0.11 0.03 0.05 0.23 0.02 0.06 0.04 0.09 0.03 0.03 0.23 0.03 0.05 0.05 0.10 0.04 0.03 0.21 0.02 0.06 0.05 0.09 0.03 0.04 0.20 0.02 0.04 0.05 0.07 0.04 0.05 0.24 0.02 0.06 0.06 0.08 a g t g C C C T g a a G C c t C A g C T G G c c A T C C C T c C T G C A G C A C C A G G 0.07 0.06 0.04 0.04 0.04 0.06 0.05 0.10 0.03 0.06 0.04 0.13 0.07 0.05 0.04 0.05 0.05 0.02 0.08 0.04 0.03 0.08 0.10 0.08 0.04 0.04 0.04 0.06 0.04 0.09 0.04 0.07 0.04 0.13 0.08 0.15 0.15 0.14 0.13 0.09 0.10 0.04 0.06 0.04 0.14 0.12 1.03 0.99 0.94 0.40 0.14 0.09 0.04 0.05 0.05 0.15 0.15 2.04 1.96 1.94 0.75 0.26 0.10 0.04 0.06 0.04 G A A T C C t a a C T G C A G C A C C A G G 0.09 0.05 0.04 0.09 0.06 0.08 0.06 0.10 0.05 0.04 0.08 0.07 0.11 0.07 0.08 0.05 0.05 0.12 0.07 0.09 0.06 0.10 0.08 0.03 0.11 0.07 0.10 0.07 0.46 0.42 0.04 0.13 0.05 0.08 0.06 0.10 0.05 0.03 0.11 0.06 0.09 0.07 t c t g t C C T T C T G C A G C A C C T G G 0.04 0.05 0.02 0.02 0.06 0.02 0.01 0.03 0.03 0.04 0.03 0.02 0.07 0.01 0.02 0.04 0.03 0.06 0.03 0.02 0.05 0.02 0.03 0.04 0.02 0.07 0.03 0.02 0.05 0.03 0.02 0.04 0.04 0.05 0.03 0.03 0.05 0.02 0.02 0.02 0.02 0.05 0.04 0.03 0.06 0.04 0.02 0.02 RNF2 T C A T C T T A G T C A T T A C C T G A G G 0.06 0.07 0.06 0.03 0.07 22.35 29.23 3.10 0.10 0.08 20.82 28.93 3.23 0.10 0.09 19.23 31.12 3.45 0.16 0.08 9.19 19.16 1.61 0.07 0.08 2.34 7.73 0.95 0.06 0.09 HBB T T G C C C C A C A G G G C A G T A A C G G 0.08 0.03 0.05 0.04 0.04 0.08 4.66 6.16 6.49 5.84 0.15 0.08 2.76 3.27 3.37 3.07 0.11 0.07 3.01 4.51 4.88 4.64 0.14 0.08 2.20 8.12 6.80 6.30 0.15 0.07 0.63 3.27 4.07 3.74 0.10 0.10 T g c t C C C A C A G G G C A G T A A A C G 0.07 0.06 0.04 0.05 0.04 0.06 0.06 0.08 0.04 0.06 0.04 0.09 0.08 0.09 0.07 0.08 0.03 0.05 0.42 0.89 0.84 0.86 0.07 0.06 0.07 0.12 0.10 0.11 0.05 0.06 0.07 0.08 0.05 0.06 0.05 0.08 c T G C C C C A C A G G G C A G c A A A G G 0.07 0.08 0.06 0.11 0.03 0.07 0.14 0.09 0.10 0.09 0.11 0.14 0.08 0.07 0.15 0.07 0.10 0.74 0.77 0.79 0.70 0.09 0.13 0.08 0.09 0.80 0.88 0.87 0.75 0.07 0.11 0.09 0.12 0.46 0.64 0.64 0.53 0.05 0.11 0.10 0.09 0.16 0.19 0.24 0.18 0.04 0.14 0.09 T g G C C C C A C A G G G C A G g A A T G G 0.07 0.13 0.06 0.09 0.04 0.06 0.10 0.11 0.06 0.10 0.05 0.04 0.08 0.12 0.07 0.09 0.04 0.06 0.14 0.20 0.13 0.16 0.07 0.08 0.10 0.24 0.17 0.20 0.08 0.06 0.84 1.61 1.58 1.53 0.16 0.05 T T G C C C C A C g G G G C A G T g A C G G 0.12 0.19 0.73 0.40 0.16 0.20 0.16 0.20 0.73 0.48 0.19 0.25 0.20 0.23 0.80 0.47 0.14 0.21 0.38 0.42 0.95 0.73 0.20 0.21 0.24 0.32 0.89 0.60 0.20 0.24 0.17 0.20 0.75 0.49 0.20 0.22 c T c t C C C A C A a G G C A G T A A G G G 0.11 0.12 0.11 0.20 0.08 0.05 0.17 0.09 0.14 0.09 0.24 0.09 0.05 0.19 0.12 0.13 0.13 0.23 0.14 0.04 0.22 0.10 0.14 0.13 0.22 0.09 0.05 0.17 0.12 0.15 0.14 0.26 0.11 0.06 0.22 0.10 0.16 0.11 0.24 0.10 0.04 0.19 c a G C C C C A C A G G G C A G T A A G G G 0.03 0.07 0.07 0.09 0.05 0.05 0.08 0.37 0.17 0.76 0.99 1.08 0.08 0.09 0.47 0.41 1.72 2.24 2.30 0.15 0.08 0.82 0.80 2.89 4.01 4.20 0.14 0.09 0.34 1.71 5.48 7.00 7.65 0.30 0.08 0.86 1.68 5.97 7.44 7.65 0.15 0.10 HEK2 A A C A C A A A G C A T A G A C T G C G G G 0.05 0.05 0.03 0.03 0.19 30.30 47.30 0.03 0.14 0.15 36.78 44.99 0.08 0.13 0.16 11.89 34.66 0.05 0.27 0.15 2.02 45.27 0.02 0.03 0.19 2.77 30.94 0.02 0.03 0.18 A A C A C A A t G C A T A G A t T G C C G G 0.11 0.09 0.09 0.16 0.12 0.09 0.14 0.18 0.17 0.14 0.13 0.19 0.19 0.22 0.12 0.18 0.12 0.10 0.11 0.20 0.11 0.09 0.13 0.20 A c t c C A A A G C A T A t A C T G C T G G 0.07 0.09 0.37 0.24 0.24 0.09 0.08 0.39 0.24 0.30 0.08 0.08 0.38 0.25 0.28 0.08 0.08 0.38 0.24 0.27 0.06 0.08 0.39 0.24 0.30 0.06 0.06 0.36 0.23 0.28 HEK3 G C C C A G A C T G A G C A C G T A A T G G 0.15 0.47 0.39 0.15 0.08 0.06 6.89 25.21 26.19 0.61 0.07 0.05 6.36 32.68 37.05 1.76 0.06 0.11 0.93 25.39 32.09 0.75 0.09 0.13 0.85 14.23 21.59 1.68 0.09 0.10 0.14 0.65 0.85 0.40 0.10 0.06 G C t C A G A C T G A G C A a G T G A G G G 0.13 0.04 0.06 0.12 0.13 0.05 0.05 0.14 0.12 0.04 0.04 0.15 0.14 0.09 0.04 0.17 0.14 0.04 0.06 0.13 0.11 0.04 0.05 0.12 t g g C c c A g a G A G C A C G T G t G G G 0.06 0.04 0.08 0.09 0.12 0.07 0.04 0.09 0.10 0.12 0.06 0.06 0.08 0.10 0.09 0.07 0.05 0.08 0.10 0.18 0.08 0.05 0.10 0.10 0.11 0.07 0.05 0.09 0.08 0.12 a C C C A G A C T G A G C A C G T G c T G G 0.06 0.08 0.04 0.02 0.13 0.05 0.04 0.06 0.09 0.06 0.03 0.11 0.06 0.04 0.07 0.07 0.06 0.03 0.12 0.06 0.06 0.06 0.10 0.06 0.02 0.11 0.06 0.05 0.07 0.10 0.05 0.02 0.12 0.04 0.04 0.07 0.08 0.05 0.01 0.10 0.07 0.05 G g C C c a A C T G A G C A a G T G A T G G 0.09 0.14 0.08 0.06 0.17 0.08 0.15 0.09 0.04 0.19 0.08 0.13 0.09 0.03 0.18 0.10 0.13 0.09 0.05 0.20 0.08 0.15 0.09 0.05 0.18 0.07 0.14 0.07 0.06 0.19 G a C C A G A C T G A G C A a G a G A G G G 0.09 0.09 0.06 0.17 0.08 0.11 0.05 0.19 0.09 0.11 0.04 0.19 0.08 0.11 0.05 0.18 0.10 0.11 0.03 0.14 0.11 0.11 0.06 0.15 G C G a c t c a T G g c C A C a T a c T G G 0.38 0.18 0.06 0.18 0.30 0.30 0.07 0.06 0.42 0.15 0.07 0.20 0.28 0.27 0.07 0.05 0.39 0.14 0.08 0.15 0.28 0.21 0.08 0.06 0.44 0.15 0.07 0.17 0.28 0.26 0.06 0.06 0.45 0.14 0.07 0.16 0.25 0.26 0.06 0.05 0.42 0.14 0.07 0.19 0.26 0.26 0.07 0.04 HEK4 G C A C T G C G G C T G G A G G T G G G G G 0.17 0.08 0.23 0.07 1.97 48.84 1.50 0.08 1.20 44.02 1.39 0.06 1.38 41.26 0.50 0.10 0.27 39.88 1.43 0.07 0.23 5.72 1.10 0.35 G C A C T G C t G C T G G g G G T G G T G G 0.14 0.04 0.11 0.91 0.17 0.39 0.13 0.93 0.21 1.86 0.15 1.11 0.27 6.55 0.25 0.99 0.16 0.11 0.14 0.90 0.15 0.06 0.10 0.93 G C A C T G C a C T G G A G G T t G T G G 0.09 0.05 0.07 0.05 0.06 0.10 0.09 0.04 0.10 0.26 0.09 0.06 0.09 0.27 0.09 0.04 0.08 0.05 0.06 0.05 0.08 0.04 0.07 0.05 G C t C T G C G G C T G G A G G g G G T G G 0.05 0.05 0.29 0.14 0.13 2.87 0.34 0.13 0.11 2.94 0.36 0.14 0.10 2.53 0.35 0.15 0.04 0.13 0.30 0.12 0.05 0.05 0.29 0.13 G C A C T G C a G a T G G A G G a G G C G G 0.09 0.03 0.11 0.11 0.03 0.09 0.08 0.07 0.14 0.15 0.58 0.17 0.08 0.03 0.08 0.06 0.03 0.10 G C A C T G C G G C a G G g a G g a G G G G G C A C T G C G G C c G G A G G a G G T G G 0.24 0.10 0.38 0.14 0.08 0.18 0.38 0.29 0.13 0.06 0.19 1.64 0.36 0.14 0.09 0.43 9.74 0.32 0.13 0.08 1.01 11.33 0.56 0.11 0.06 0.18 0.16 0.26 0.13 0.08 G C A C T g G G C T G a A G G T a G A G G 0.08 0.03 0.09 0.18 0.64 0.05 0.18 0.62 0.05 0.07 0.16 0.06 0.08 0.03 0.06 0.07 0.03 0.06 G C A C T G t G G C T G c A G G T G G A G G 0.11 0.03 0.04 0.03 0.10 0.04 0.02 0.04 0.12 0.03 0.03 0.03 0.12 0.02 0.04 0.04 0.10 0.03 0.03 0.03 0.06 0.02 0.03 0.03 G C t C T G C G G C a G G A G G a G G A G G 0.08 0.18 0.07 0.07 0.07 0.17 0.06 0.07 0.09 0.16 0.06 0.05 0.06 0.16 0.09 0.07 0.07 0.17 0.07 0.06 0.04 0.06 0.02 0.04 G C A C T G C a G C T G G g a G T G G A G G 0.16 0.05 0.15 0.08 0.11 0.17 0.10 0.08 0.15 0.35 0.16 0.08 0.19 1.78 0.27 0.11 0.13 0.33 0.12 0.08 0.14 0.07 0.10 0.09 G C A C T G a G G g T G G A G G T G G G G G 0.07 0.04 0.27 1.09 0.30 1.94 0.07 1.09 0.07 0.04 0.10 0.03 G C A C T G g G G C T G G A G a c G G G G G 0.12 0.13 0.12 0.21 0.10 0.15 0.10 0.14 0.12 0.15 0.12 0.20 0.12 0.19 0.11 0.19 0.12 0.14 0.13 0.19 0.12 0.13 0.10 0.18 G g A C T G C G G C T G G g G G T G G T G G 0.05 0.29 0.03 1.37 0.31 0.04 1.03 0.44 0.05 4.70 0.38 0.06 1.67 0.29 0.04 6.06 0.88 0.07 G C A C T G C a a C T G G A a G T G a T G G 0.11 0.06 0.11 0.02 0.10 0.10 0.08 0.02 0.08 0.16 0.08 0.03 0.10 0.32 0.09 0.02 0.08 0.06 0.06 0.01 0.10 0.04 0.09 0.02 G C A C T G g G G t T G G A G G T G G G G G 0.16 0.18 0.69 2.90 0.87 3.94 0.29 3.17 0.18 0.21 0.15 0.15 c C A C T G C a G C T a G A G G T G G A G G 0.11 0.05 0.05 0.16 0.04 0.11 0.10 0.69 0.17 0.04 0.11 0.16 1.46 0.17 0.04 0.11 0.29 3.27 0.28 0.04 0.13 0.14 0.69 0.15 0.04 0.12 0.12 0.23 0.18 0.03 c C A C T G C G a C T G G A G G a G G G G G 0.16 0.06 0.06 61.49 0.05 0.12 0.06 0.06 60.75 0.04 0.10 0.07 0.06 60.11 0.05 0.12 0.08 0.11 61.02 0.05 0.14 0.08 0.08 60.97 0.03 0.12 0.07 0.08 60.12 0.05 G C A C T G G G C T G G A G G c G G G G G 0.03 0.06 0.05 0.08 0.04 0.11 0.08 0.08 0.04 0.10 0.05 0.11 0.05 0.05 0.09 0.08 0.03 0.05 0.07 0.09 0.01 0.03 0.02 0.06 G C t C T G C G G C a G G A G t T G G A G G 0.22 0.03 0.22 0.10 0.25 0.02 0.20 0.10 0.23 0.02 0.21 0.10 0.22 0.02 0.20 0.09 0.23 0.02 0.16 0.09 0.25 0.02 0.23 0.10

[0228] FIG. 16a schematically shows a conventional sgRNA (gX19 sgRNA), a truncated sgRNA (gX18 or gX17 sgRNA) and an extended sgRNA (gX20 or ggX20 sgRNA). FIG. 16b shows base-editing frequencies at the HBB on- and off-target sites in HEK293T cells measured by targeted deep sequencing. Specificity ratios were calculated by dividing the base-editing frequency at the on-target site with that at off-target sites. The heatmap represents relative specificities of modified sgRNAs, compared to that of conventional sgRNA.

[0229] FIG. 17 shows the result of reducing BE3 off-target effects using modified sgRNAs, wherein 17a shows a schematic view of conventional sgRNAs (GX.sub.19 sgRNA) and modified sgRNAs (GX.sub.17 sgRNA, gX.sub.18 sgRNA, gX.sub.20 sgRNA, and ggX.sub.20 sgRNA), and 17b shows base editing efficiencies (frequencies) measured at the EMX1 on- and off-target sites by targeted deep sequencing in HEK293T cells.

[0230] As shown in FIGS. 16a, 16b, 17a, and 17b, truncated sgRNAs reduced off-target effects at many sites but exacerbated them at sites with mismatches at the 5 terminus (shown by asterisks in FIGS. 16b and 17b). Extended sgRNAs reduced off-target effects at almost every site without sacrificing on-target effects. Interestingly, some extended sgRNAs were more active at on-target sites than conventional sgRNAs (Table 17). Use of attenuated Cas9 variants or delivery of BE3 RNPs rather than plasmids may further improve the genome-wide specificity of base editing.

[0231] In summary, the results obtained using mismatched sgRNAs, Digenome-seq, and targeted deep sequencing showed that BE3 deaminases were highly specific, catalyzing C-to-U conversions in vitro and base editing in human cells at a limited number of sites in the human genome. It was also found that BE3 and Cas9 off-target sites were not always coincidental, justifying independent assessments of each tool. It is expect that the above results and methods will accelerate broad use of RNA-guided programmable deaminases in research and medicine.

Example 7. BE1 (rAPOBEC1-dCas9)-Mediated Double Strand Breaks (DSBs)

[0232] A PCR amplicon containing a target sequence (ENX1 on-target sequence; SEQ ID NO: 31) was incubated with BE1 (rAPOBEC1-dCas9; Example 2) and its sgRNA (sgRNA targeting SEQ ID NO: 31) in vitro to induce Cytidine to Uracil conversions. Uracil, which is induced by rAPOBEC1, was removed by USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs). Then, 51 nuclease (Catalog #M5761; Promega) was treated to cleave phosphodiester bonds in a single-strand DNA, producing a DSB at the cytosine-deaminated site (FIG. 22 (a)).

[0233] The above-obtained PCR amplicon was subjected to electrophoresis, to confirm that they are cleaved by the treatment of BE1/sgRNA, USER, and 51 Nuclease (FIG. 22 (b)).

[0234] From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

METHOD FOR IDENTIFYING DNA BASE EDITING BY MEANS OF CYTOSINE DEAMINASE

Inventors

Cpc classification

Classification Explorer

C12N2310/20

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/907

CHEMISTRY; METALLURGY

Classification Explorer

C12N9/78

CHEMISTRY; METALLURGY

Classification Explorer

C12N9/22

CHEMISTRY; METALLURGY

Classification Explorer

C12Y302/02027

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/11

CHEMISTRY; METALLURGY

Classification Explorer

C12Y305/04004

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/113

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/90

CHEMISTRY; METALLURGY

Classification Explorer

C12Q1/6806

CHEMISTRY; METALLURGY

Classification Explorer

C12Y305/04001

CHEMISTRY; METALLURGY

Classification Explorer

C12Q1/6869

CHEMISTRY; METALLURGY

Classification Explorer

G16B30/00

PHYSICS

International classification

Classification Explorer

C12N15/90

CHEMISTRY; METALLURGY

Classification Explorer

C12N9/78

CHEMISTRY; METALLURGY

Classification Explorer

G16B30/00

PHYSICS

Classification Explorer

C12Q1/6869

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/113

CHEMISTRY; METALLURGY

Classification Explorer

C12N9/22

CHEMISTRY; METALLURGY

Classification Explorer

C12Q1/6806

CHEMISTRY; METALLURGY

Abstract

Claims

Description