TYPE V CAS PROTEINS AND APPLICATIONS THEREOF

Abstract

Type V Cas proteins, for example Type V Cas proteins referred to as ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, and ZRXE Type V Cas proteins; gRNAs for Type V Cas proteins; systems comprising Type V Cas proteins and gRNAs; nucleic acids encoding the Type V Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.

Claims

1-30. (canceled)

31. A fusion protein comprising: (a) a Type V Cas amino acid sequence comprising an amino acid sequence that is at least 90% identical to the full length of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO:85, SEQ ID NO: 86, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 115, or SEQ ID NO:116; and (b) one or more nuclear localization signals.

32. The fusion protein of claim 31, wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is at least 95% identical to the full length of SEQ ID NO:43.

33. The fusion protein of claim 31, wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is identical to SEQ ID NO:43.

34. The fusion protein of claim 31, wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is identical to SEQ ID NO:44.

35. The fusion protein of claim 31, which comprises a C-terminal nuclear localization signal.

36. The fusion protein of claim 31, which comprises an N-terminal nuclear localization signal.

37. The fusion protein of claim 31, which comprises a nuclear localization signal comprising the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:122), PKKKRKV (SEQ ID NO:123), PKKKRRV (SEQ ID NO:124), KRPAATKKAGQAKKKK (SEQ ID NO: 125), YGRKKRRQRRR (SEQ ID NO: 126), RKKRRQRRR (SEQ ID NO: 127), PAAKRVKLD (SEQ ID NO:128), RQRRNELKRSP (SEQ ID NO:129), VSRKRPRP (SEQ ID NO:130), PPKKARED (SEQ ID NO:131), PQPKKKPL (SEQ ID NO:132), SALIKKKKKMAP (SEQ ID NO: 133), PKQKKRK (SEQ ID NO: 134), RKLKKKIKKL (SEQ ID NO: 135), REKKKFLKRR (SEQ ID NO: 136), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:137), RKCLQAGMNLEARKTKK (SEQ ID NO: 138), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 139), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 140), or SSDDEATADSQHAAPPKKKRKV (SEQ ID NO:178).

38. The fusion protein of claim 31, which comprises a nuclear localization signal comprising the amino acid sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO: 180).

39. The fusion protein of claim 31, wherein the fusion protein comprises a Type V Cas amino acid sequence that is identical to SEQ ID NO:44 and a C-terminal nuclear localization signal comprising the amino acid sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO:180).

40. A system comprising the fusion protein of claim 31 and a guide RNA (gRNA) comprising a spacer positioned 3 to a crRNA scaffold and capable of forming a complex with the fusion protein and directing the fusion protein to a target DNA.

41. The system of claim 40, wherein the nucleotide sequence of the spacer is complementary to a target mammalian genomic sequence that is downstream of a NTTV, VTTV, NCTV, or TTTT protospacer adjacent motif (PAM) sequence.

42. The system of claim 40, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 151 or SEQ ID NO:211.

43. The system of claim 42, wherein the crRNA scaffold comprises a nucleotide sequence that is identical to SEQ ID NO: 151 or SEQ ID NO:211.

44. The system of claim 40, which is a ribonucleoprotein (RNP) comprising the fusion protein complexed to the gRNA.

45. A nucleic acid encoding the fusion protein of claim 31.

46. The nucleic acid of claim 45, wherein the nucleotide sequence encoding the fusion protein is codon optimized for expression in human cells.

47. The nucleic acid of claim 45, which is an adeno-associated virus (AAV) genome.

48. An adeno-associated virus (AAV) particle comprising the nucleic acid of claim 47.

49. An ex vivo human cell comprising the fusion protein of claim 31.

50. The ex vivo human cell of claim 49, which is a hematopoietic stem cell (HSC), pluripotent stem cell or an induced pluripotent stem cell (iPS).

51. An ex vivo human cell comprising the system of claim 40.

52. The ex vivo human cell of claim 51, which is a hematopoietic stem cell (HSC), pluripotent stem cell or an induced pluripotent stem cell (iPS).

53. A method for altering a cell comprising contacting the cell with the system of claim 40.

54. A guide RNA (gRNA) comprising a spacer and a crRNA scaffold, wherein: (a) the spacer is positioned 3 to the crRNA scaffold; and (b) the nucleotide sequence of the crRNA scaffold comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 144-163 or at least 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 211-213.

Description

5. BRIEF DESCRIPTION OF THE FIGURES

[0040] FIGS. 1A-1E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZWGD Type V-A Cas protein (FIG. 1A), ZJHK Type V-A Cas protein (FIG. 1B), ZIKV Type V-A Cas protein (FIG. 1C), ZZFT Type V-A Cas protein (FIG. 1D) and YYAN Type V-A Cas protein (FIG. 1E) are shown. Figures disclose SEQ ID NOS 390-394, respectively, in order of appearance.

[0041] FIGS. 2A-2E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZZGY Type V-A Cas protein (FIG. 2A), ZKBG Type V-A Cas protein (FIG. 2B), ZZKD Type V-A Cas protein (FIG. 2C), ZXPB Type V-A Cas protein (FIG. 2D) or ZPPX Type V-A Cas protein (FIG. 2E). Figures disclose SEQ ID NOS 395-399, respectively, in order of appearance.

[0042] FIGS. 3A-3E illustrate in silico predicted PAM specificities for ZWGD, ZJHK, ZIKV, ZZFT and YYAN Type V-A Cas proteins. PAM sequence logos for ZWGD (FIG. 3A), ZJHK (FIG. 3B), ZIKV (FIG. 3C), ZZFT (FIG. 3D) and YYAN (FIG. 3E) Type V-A Cas proteins are shown.

[0043] FIGS. 4A-4E illustrate in silico predicted PAM specificities for ZZGY, ZKBG, ZZKD, ZXPB and ZPPX Type V-A Cas proteins. PAM sequence logos for ZZGY (FIG. 4A), ZKGB (FIG. 4B), ZZKD (FIG. 4C), ZXPB (FIG. 4D) and ZPPX (FIG. 4E) Type V-A Cas proteins are shown.

[0044] FIG. 5 illustrates activity of Type V-A Cas proteins against an EGFP reporter in mammalian cells. The activity of the selected Type V-A Cas proteins was evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells stably expressing EGFP. For each Cas protein, 2 different gRNAs targeting the same two positions of the EGFP coding sequence were evaluated. Loss of EGFP fluorescence, expressed as % of EGFP-negative cells, was measured by cytofluorimetry. Data presented as meanSEM of n2 biologically independent runs. Untreated U2OS cells (U2OS sample) are included as a measurement of the background loss of fluorescence.

[0045] FIGS. 6A-6C illustrate activity of ZZKD Type V-A Cas protein against benchmark endogenous genomic loci in mammalian cells. The activity of ZZKD Type V-A Cas protein was evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells. Several gRNAs targeting the TRAC (FIG. 6A), B2M (FIG. 6B) and PD1 (FIG. 6C) benchmark loci were evaluated. Editing activity was measured by Sanger chromatogram deconvolution 3 days after transfection. Data presented as meanSEM of n2 biologically independent runs.

[0046] FIGS. 7A-7E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZXHQ Type V-A Cas protein (FIG. 7A), ZQKH Type V-A Cas protein (FIG. 7B), ZRGM Type V-A Cas protein (FIG. 7C), ZTAE Type V-A Cas protein (FIG. 7D) and ZSQQ Type V-A Cas protein (FIG. 7E) are shown. Figures disclose SEQ ID NOS 400-404, respectively, in order of appearance.

[0047] FIGS. 8A-8E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZSYN Type V-A Cas protein (FIG. 8A), ZRBH Type V-A Cas protein (FIG. 8B), ZWPU Type V-A Cas protein (FIG. 8C), ZZQE Type V-A Cas protein (FIG. 8D) and ZRXE Type V-A Cas protein (FIG. 8E) are shown. Figures disclose SEQ ID NOS 405-409, respectively, in order of appearance.

[0048] FIG. 9 illustrates in silico prediction of ZZQE Type V-A Cas protein PAM specificity. PAM sequence logo for ZZQE Type V-A Cas protein is shown.

[0049] FIG. 10 shows activity of novel Type V-A Cas proteins in human cells. Evaluation of the activity of novel Type V-A Cas protein after transient electroporation in U2OS-EGFP cells. Two different guide RNAs were evaluated (target sequences are common for all proteins) and EGFP downregulation was measured by flow cytometry 5 days post-electroporation. A non-transfected control sample has been included to measure the assay background (NT Ctrl). 23nt spacers were used. Data represented as meanSD of n=2 independent biological replicates.

[0050] FIG. 11 shows activity of selected Type V-A Cas proteins towards endogenous genomic loci in human cells. The editing activity of ZZKD, ZRGM and ZZQE Type V-A Cas proteins was evaluated for the benchmark TRAC-g3, B2M-g2 and PD1-g2 genomic loci after transient transfection in HEK293T cells. Given the PAM compatibility among the different proteins the same spacers were used (23nt in length). For ZZKD activity on the TRAC locus, data represented as meanSD of n=3 independent biological replicates.

[0051] FIGS. 12A-12C show in vitro analysis of PAM preferences of ZZKD Type V-A Cas protein. A PAM sequence logo is shown in FIG. 12A and PAM heatmap is shown in FIG. 12B for ZZKD Type V-A Cas protein FIG. 12C shows validation of the PAM preferences by measurement of indel formation after transient transfection of HEK293T cells using crRNAs associated with PAMs shown to be preferentially cut by the PAM assay. The PAM associated with each guide is reported on the graph. Data represented as meanSD of n2 independent biological replicates.

[0052] FIGS. 13A-13D show analysis of PAM preferences of ZRGM and ZZQE Type V-A Cas proteins. A PAM sequence logo is shown in FIG. 13A and a PAM heatmap is shown in FIG. 13B for ZRGM Type V-A Cas protein. A PAM sequence logo is shown in FIG. 13C and a PAM heatmap is shown in FIG. 13D for ZZQE Type V-A Cas protein.

[0053] FIGS. 14A-14B illustrate in vitro determination of the double strand break profile of ZZKD Type V-A Cas protein. In vitro cleavage reactions using a PCR-generated target (TRAC-g3) and recombinant ZZKD Type V-A Cas protein were run on an agarose gel and the separated fragments were independently Sanger sequenced using a forward and a reverse primer to sequence both DNA strands. Based on the drop in the chromatographic signal in the two sequencing reactions (FIG. 14A) it was possible to determine that ZZKD type V-A Cas protein produces a 6 nucleotide staggered cut, as indicated by the solid lines in the scheme shown in FIG. 14B. Figure discloses SEQ ID NOS 410-411, 410, and 412, respectively, in order of appearance.

[0054] FIG. 15 shows an evaluation of alternative nuclear localization signal (NLS) designs to improve the activity of ZZKD Type V-A Cas protein. FIG. 15 plots indel formation at the TRAC locus (g3) after transient transfection of HEK293T cells with alternative versions of ZZKD Type V-A Cas proteins characterized by different nuclear localization signal sequences positioned either at the N- or the C-terminus of the protein, as indicated on the graph. The amino acid sequence of each evaluated NLS is reported in the figure. Data represented as meanSD of n2 independent biological replicates. Figure discloses SEQ ID NOS 179, 122, 180, and 125, respectively, in order of appearance.

[0055] FIGS. 16A-16C show alternative crRNA scaffolds for selected Type V-A Cas proteins. Schematic representation of the hairpin structure generated for visualization using the RNAfold webserver (www.unafold.org) of the crRNA trimmed scaffolds (not including the spacer sequence) for ZZKD Type V-A Cas protein (FIG. 16A) (SEQ ID NO:211), ZZQE Type V-A Cas protein (FIG. 16B) (SEQ ID NO:212) and ZRGM Type V-A Cas protein (FIG. 16C) (SEQ ID NO:213).

[0056] FIGS. 17A-17B show the activity of alternative crRNA scaffolds for selected Type V-A Cas proteins. FIG. 17A shows indel formation measured after transient transfection of HEK293T cells with alternative versions (full-length or trimmed) of the crRNAs targeting the TRAC-g3 locus for ZZKD, ZZQE and ZRGM Type V-A Cas proteins. FIG. 17B shows indel formation measured after transient transfection of HEK293T cells with alternative versions (full-length or trimmed) of ZZKD Type V-A Cas protein crRNAs targeting the BCL11A, TRAC, AAVS1 and B2M loci, as indicated on the graph. Data represented as meanSD of n=2 independent biological replicates.

[0057] FIGS. 18A-18B illustrate the effect of alternative spacer lengths on ZZKD Type V-A Cas protein editing activity. Indel formation in HEK293T cells after transient transfection of ZZKD Type V-A Cas protein in combination with families of crRNAs characterized by different spacer lengths (from 20nt to 24nt) targeting either the Match6 (FIG. 18A) or the TRAC locus (g3, FIG. 18B). Data represented as meanSD of n=2 independent biological replicates.

[0058] FIG. 19 shows a side-by-side comparison of ZZKD Type V-A Cas protein activity with AsCs12a Ultra. The figure shows a violin plot summarizing the editing activity of ZZKD Type V-A Cas protein and AsCas12a Ultra on a panel of endogenous genomic loci (TRAC, PD1, B2M, EMX1, AAVS1, BCL11a, PCSK9, Match6, VEGFA) after transient transfection of HEK292T cells, using crRNAs for the two nucleases that overlap on each locus. Each point on the graph represents the mean of n=2 independent runs except for B2M-g1_21nt for AsCas12a Ultra (n=1).

[0059] FIGS. 20A-20D show activity of ZZKD Type V-A Cas protein in subsaturating conditions. Titration curves obtained by measuring indel formation at the BCL11A-g4 (FIG. 20A), VEGFA-g1 (FIG. 20B), B2M-g1_21nt (FIG. 20C) and B2M-g2_21nt (FIG. 20D) target sites after a 2-fold serial dilution of the amount of ZZKD and crRNA plasmids transiently transfected in HEK293T cells. The activity of AsCas12a Ultra was measured in the same study conditions as a benchmark and is reported on each graph. Data represented as meanSD of n=2 independent biological replicates.

[0060] FIGS. 21A-21C show activity of ZZKD Type V-A Cas after direct ribonucleoprotein delivery in human cell lines. Indel formation after ZZKD Type V-A Cas RNP electroporation in U2OS cells to target either the TRAC-g3 locus (FIG. 21A) or the B2M-g2 locus (FIG. 21B). Cells were also transfected with plasmids expressing ZZKD and its crRNA as a positive control. IVT, in vitro transcribed crRNA; syn, unmodified chemically synthesized crRNA; AltR, chemically synthesized crRNA including commercially available AltR modifications from IDT. FIG. 21C shows the results of a titration study in U2OS cells delivering different amounts of recombinant ZZKD and cognate crRNA targeting the B2M-g2 locus by electroporation. The amount (pmol) of recombinant protein and crRNA used in each condition is indicated below each bar. Data represented as meanSD of n2 independent biological replicates, except for B2M-g2 IVT and panel (FIG. 21C) where only one replicate is available.

[0061] FIG. 22 shows activity of ZZKD Type V-A Cas after direct ribonucleoprotein delivery in primary human T cells. The figure shows percentage of TRAC-negative cells measured by flow cytometry after ZZKD Type V-A Cas RNP electroporation in commercial human primary T cells to target the TRAC-g3 locus.

6. DETAILED DESCRIPTION

[0062] In one aspect, the disclosure provides Type V Cas proteins, e.g., a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, and a ZRXE Type V Cas protein. Type V Cas proteins of the disclosure can be in the form of fusion proteins. Unless required otherwise by context, disclosures relating to Type V Cas proteins encompass Type V Cas proteins which are not fusion proteins and Type V Cas proteins which are in the form of fusion proteins (e.g., Type V Cas protein comprising one or more nuclear localization signals and/or one or more tags).

[0063] In some embodiments, a Type V Cas protein of the disclosure comprises an amino acid sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) sequence identity to a WED-1 domain, REC1 domain, REC2 domain, WED-II domain, PI domain, WED-III domain, RuvC-I domain, BH domain, RuvC-II domain, NUC domain, or RuvC-III domain of a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein.

[0064] In some embodiments, a Type V Cas protein of the disclosure is a chimeric Type V Cas protein, for example, comprising one or more domains from a ZWGD Type V Cas protein and/or a ZJHK Type V Cas protein and/or a ZIKV Type V Cas protein and/or a ZZFT Type V Cas protein and/or a YYAN Type V Cas protein and/or a ZZGY Type V Cas protein and/or a ZKBG Type V Cas protein and/or a ZZKD Type V Cas protein and/or a ZXPB Type V Cas protein and/or a ZPPX Type V Cas protein and/or a ZXHQ Type V Cas protein and/or a ZQKH Type V Cas protein and/or a ZRGM Type V Cas protein and/or a ZTAE Type V Cas protein and/or a ZSQQ Type V Cas protein and/or a ZSYN Type V Cas protein and/or a ZRBH Type V Cas protein and/or a ZWPU Type V Cas protein and/or a ZZQE Type V Cas protein and/or a ZRXE Type V Cas protein, and one or more domains from a different Type V Cas protein such as AsCas12a.

[0065] Exemplary features of Type V Cas proteins of the disclosure are described in Section 6.2.

[0066] In further aspects, the disclosure provides guide (gRNA) molecules and combinations of guide RNA molecules, for example combinations of two or more gRNAs. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure are further described in Section 6.3.

[0067] In further aspects, the disclosure provides systems comprising a Type V Cas protein of the disclosure and one or more gRNAs. Exemplary features of systems are described in Section 6.4.

[0068] In further aspects, the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type V Cas protein of the disclosure and, optionally, a gRNA, and provides nucleic acids encoding a gRNA, of the disclosure and, optionally, a Type V Cas protein. Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5.

[0069] In further aspects, the disclosure provides particles comprising the Type V Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6.

[0070] In another aspect, the disclosure provides cells and populations of cells containing or contacted with a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.6.

[0071] In another aspect, the disclosure provides pharmaceutical compositions comprising a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients. Exemplary features of pharmaceutical compositions are described in Section 6.7.

[0072] In another aspect, the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type V Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure. Features of exemplary methods of altering cells are described in Section 6.8.

6.1. Definitions

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. The following definitions are provided for the full understanding of terms used in this specification.

[0074] As used in the specification and claims, the singular form a, an, and the include plural references unless the context clearly dictates otherwise. For example, the term an agent includes a plurality of agents, including mixtures thereof.

[0075] Unless indicated otherwise, an or conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term and/or is used for the same purpose, which shall not be construed to imply that or is used with reference to mutually exclusive alternatives.

[0076] AsCas12a refers to a Cas12a protein having the following amino acid sequence:

TABLE-US-00001 (SEQIDNO:121) MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLD WENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLK QLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVP SLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKND ETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHI FISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQ KTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEME PSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPE KEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLY HISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYR PKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKD RRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTI QQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTG IAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPL TGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNET QFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVAL IRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRN

[0077] A Type V Cas protein refers to a wild-type or engineered Type V Cas protein. Engineered Type V Cas proteins can also be referred to as Type V Cas variants. For the avoidance of doubt, any disclosure pertaining to a Type V Cas or Type V Cas protein pertains to wild-type Type V Cas proteins and Type V Cas variants, unless the context dictates otherwise. A Type V Cas protein can have nuclease activity or be catalytically inactive (e.g., as in a dCas).

[0078] As used herein, the percentage identity between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100, and dividing by the length of the aligned region. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another, nor does it consider substitutions or deletions as matches. For calculation of the percent sequence identity (% sequence identity), two sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (available at www.ebi.ac.uk/jdispatcher/psa/emboss_needle) with the following parameters: Matrix: BLOSUM62 (for protein sequences) or DNAfull (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: false; End Gap Open: 10; and End Gap Extend: 0.5.

[0079] Guide RNA molecule (gRNA) refers to an RNA capable of forming a complex with a Type V Cas protein and which can direct the Type V Cas protein to a target DNA. gRNAs typically comprise a spacer of 15 to 30 nucleotides in length. gRNAs of the disclosure typically comprise a crRNA scaffold region at the 5 end of the molecule and a spacer at the 3 end of the molecule. Various non-limiting examples of crRNA scaffolds are described in Section 6.3.

[0080] An gRNA can in some embodiments comprise no uracil base at the 3 end of the gRNA sequence. Alternatively, a gRNA can comprise one or more uracil bases at the 3 end of the sgRNA sequence. For example, a gRNA can comprise 1 uracil (U) at the 3 end of the gRNA sequence, 2 uracil (UU) at the 3 end of the gRNA sequence, 3 uracil (UUU) at the 3 end of the gRNA sequence, 4 uracil (UUUU) at the 3 end of the gRNA sequence, 5 uracil (UUUUU) at the 3 end of the gRNA sequence, 6 uracil (UUUUUU) at the 3 end of the gRNA sequence, 7 uracil (UUUUUUU) at the 3 end of the gRNA sequence, or 8 uracil (UUUUUUUU) at the 3 end of the gRNA sequence. Different length stretches of uracil can be appended at the 3 end of a gRNA as terminators.

[0081] A gRNA can in some embodiments comprise a 5 guanine (G) at it's 5 end. A 5-G can promote efficient transcription from a U6 promoter.

[0082] Peptide, protein, and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications. A polypeptide may be attached to other molecules, for instance molecules required for function. Examples of molecules which may be attached to a polypeptide include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc. Non-limiting examples of polypeptides include peptide fragments, denatured/unstructured polypeptides, polypeptides having quaternary or aggregated structures, etc. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected/unintended purposes, or have unknown function. A polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used. The standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). The terms polypeptide sequence or amino acid sequence are an alphabetical representation of a polypeptide molecule.

[0083] Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, the term nucleotide sequence is the alphabetical representation of a polynucleotide molecule. The letters used in polynucleotide sequences described herein correspond to IUPAC notation. For example, the letter N in a nucleotide sequence represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA sequence; the letter R in a nucleotide sequence represents a nucleotide which can be A or G; the letter V in a nucleotide sequence represents a nucleotide which can be A, C, or G; and the letter Y in a nucleotide sequence represents a nucleotide which can be C or T.

[0084] Protospacer adjacent motif (PAM) refers to a DNA sequence upstream (e.g., immediately upstream) of a target sequence on the non-target strand recognized by a Type V Cas protein. A PAM sequence is located 5 of the target sequence on the non-target strand.

[0085] Spacer refers to a region of a gRNA molecule which is partially or fully complementary to a target sequence found in the + or strand of genomic DNA. When complexed with a Type V Cas protein, the gRNA directs the Type V Cas to the target sequence in the genomic DNA. A spacer of a Type V Cas gRNA is typically 15 to 30 nucleotides in length (e.g., 20-25 nucleotides). The nucleotide sequence of a spacer can be, but is not necessarily, fully complementary to the target sequence. For example, a spacer can contain one or more mismatches with a target sequence, e.g., the spacer can comprise one, two, or three mismatches with the target sequence.

6.2. Type V Cas Proteins

6.2.1. ZWGD Type V Cas Proteins

[0086] In one aspect, the disclosure provides ZWGD Type V Cas proteins. ZWGD Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZWGD Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1. In some embodiments, the ZWGD Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, a ZWGD Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 1.

[0087] Exemplary ZWGD Type V Cas protein sequences and nucleotide sequences encoding exemplary ZWGD Type V Cas proteins are set forth in Table 1A.

TABLE-US-00002 TABLE1A ZWGDTypeVCasSequences SEQID Name Sequence NO. Wildtype VSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFKKILD 1 aminoacid QVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKSLDHK sequence DKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTTNETSV (withoutN- AFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLDYFNRT terminal LSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQILSESK methionine) TESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRLYLERK NIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQKQQYEK DGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEGQDAESH ASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEFYQGLKNI IPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYYVGIINSAT NAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPVPADLREK YDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSDVNEFLSE LTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKAVFDQKNA EEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLCKDRRYMS DKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLVLIDQNGRII KQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENIKELKAGYL SNWVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFMRKDLDSSA IGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYENVEKTKSLIE KFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNKEEWQSKNYIL VEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRTTELNGTDADT KKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIMLKHLKEGLPEK RICDISTEEWVQFVEELNK Wildtype MVSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFKKIL 2 aminoacid DQVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKSLDH sequence(with KDKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTTNETS N-terminal VAFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLDYFNR methionine) TLSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQILSES KTESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRLYLER KNIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQKQQYE KDGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEGQDAES HASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEFYQGLK NIIPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYYVGIINS ATNAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPVPADLR EKYDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSDVNEFL SELTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKAVFDQK NAEEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLCKDRRY MSDKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLVLIDQNG RIIKQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENIKELKAG YLSNVVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFMRKDLDS SAIGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYENVEKTKS LIEKFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNKEEWQSKN YILVEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRTTELNGTDA DTKKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIMLKHLKEGLP EKRICDISTEEWVQFVEELNK Expression MGVSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFK 3 construct(with KILDQVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKS N-terminal LDHKDKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTT methionine, NETSVAFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLD V5-tagandC- YFNRTLSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQI terminalNLS) LSESKTESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRL aasequence YLERKNIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQK QQYEKDGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEG QDAESHASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEF YQGLKNIIPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYY VGIINSATNAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPV PADLREKYDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSD VNEFLSELTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKA VFDQKNAEEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLC KDRRYMSDKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLV LIDQNGRIIKQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENI KELKAGYLSNVVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFM RKDLDSSAIGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYE NVEKTKSLIEKFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNK EEWQSKNYILVEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRT TELNGTDADTKKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIML KHLKEGLPEKRICDISTEEWVQFVEELNKSRKRTADGSEFESPKKKRKVGSGKPIPNPL LGLDST Wildtype ATGGTGTCCGAAAAAGAAAATACACCAACTTTTAATAGTCTAACCAATCTCTATAGTG 4 coding TTTCAAAGACTCTTAGATTTGAACTTAGGCCACAATATTCAACTCTAGATCACATTAA sequence(with AGATGACCAAATTGTTGACAAAGGTGAAGAACTAAAAAACCACTACAAAACTTTCAA N-terminal GAAAATTCTTGATCAGGTCTTTTCAAGGATCATCAACGATAGCCTAGATAAAACCTA methionine TCTTGATCAAAAATATATTTCCACCTACCAAGATCTTGTATTCAAGCATCGAGACCGA andstop CTAACAGACAAAGACCGTGCAGAACTAAAGGCCTTAAAAGAAACACTCAAAAAGCA codon) GATCGACAAAAGCCTCGATCATAAAGATAAAAAAGCTATCTTCAGTGATCCCGTAAA TTTTCTCATCGACAATGAATCGGATTTTGCTGACTTAATTGGTGATAATCGTCCTAGT ATTGAAGCTTTCAACCGTCAAAAAGGTTATCTTTCCGGATATCTCCAAAATCGCGCA AATATCTTCGATCACACCACAAATGAAACTTCAGTCGCGTTTCGTATTGTCGAGGAA AACCTCGCTATCTTTTTAAATAATCGCCTCACATTACAGCATTTTTTCGAGAAAGTTG CAGATAAAGATGGGCTATTAAAATTTTTACAAGAGACACTTTCTCAGTTAGGTTTTAA GTTGAAACTCGAAGACCTTCTTTCCCTTGATTATTTTAATCGTACCCTATCTCAACCC GGCATCGATCAGTATAACCTCCTAATCTCTGGCAAGGCGCTAGAAGATGGAAAGAA AATGCAGGGAATTAATGAGGTCCTCAATCAATATCTCCAACAACATCAAGAAGAGAA GCTACATAAAATCAAACTCAAGCAACTCTATAAGCAGATCCTCTCAGAGTCAAAAAC TGAATCATTTACCCTTGATTTTGTGGAAGATAATAAAGGGCTTGCTGCCATGCTCCT ACAGTTTATCGATTTTGTAAACAAGCTGATTGAAGAGAAAATGCTTCTCCTTGATATG ATTCAGGGGCTAAAAGATAGCTCAGTTTCATCAGAATTTCTTTCACGACTCTATCTT GAACGCAAAAACATCAAGCGTCTTTCGAATTTTATCTATAAAGATTATGGCTATATTG AGCAATCCTTGGAAGAGAACTTTCTCTCGACAATTGAAGGCAAGATTACCAAGAAG GCACTCGAGGAACATCGCAAACAGGATGCTTTCACAATCCATGAAATCTTAGTTGC CCTACAAAAGCAACAATATGAAAAGGATGGAGCTCTAGAGTCCGCAGATCATCTTTT ACTTCCTGGTGTTGTTGACTTCCTCTACCAGAATTTGGATTGCAAACACTCCACTCT ACTTGAAAAAGTCGGGTCAGAAAAACAGCCACTACTCGACCTCTTCAACGAAAAAC AATTATTGGAAGGTCAAGACGCAGAATCTCATGCTTCCAAATATTCTGATCGTCCAT TCAACGACCACGAAATAAAGGTTGTTAAAACTGCTTTGGATTTTTATAAAAATCTACA GAGTAATTTTGCGATCTTTCAAATCCCGGATGAAAACCTTAAACTAGATTCCGAATTT TATTCCGAGTTTGATGAATTTTATCAAGGTCTCAAGAATATTATTCCAGTCTATAACA AGTCCAGAAATTTCCTCACTAAAAAACCATTCTCAACCGAAAAGACCAAGCTCATTT TTAACAACCCGCAACTACTTGACGGATGGAGTAAATCAAAAGAGTCAGATTGTTTAG GCACGATTTTTATTAAAGACGGCAAATATTATGTTGGCATTATTAATAGTGCTACGAA TGCTAAAAATACTTTATTTGAGCCTAACAATTTTGCAAACTTCGACCAAAAACAATAT TTTGAAAAGATGAACCTTTTCTTCCTTTCGGACTTGAAGCGAGATTTTCCTAAGAAAT ATTTTTCTGAAAAGTGGCATAATCAACACCCAGTTCCAGCCGATCTTCGTGAAAAGT ATGATTATTATCGAATCGACGAACATAAGGATGAGCGCAAAAATGATCTAAAATATC ATCATCAACTTATCGCCTATTATCAAGACTGTCTTAAAAAAGACACGGAATGGCAGA TTTATCAATTCAAATATAAGGCCCCTGAAGAATATTCAGATGTCAATGAATTCTTATC CGAGCTTACTCCAAATACCTACAAAATGGAGTTCAATAAAATCCCAGCTGAATATAT CAAAAAGCTTGTTGATGATGGAAAATTATATTTCTTCCAAATTTATTCCAAAGATTTTT CTGAGTTTGCAAAAGGTAAACCAAATCTCCATACTCTCTATCTAAAAGCGGTCTTTG ATCAGAAAAATGCGGAAGAGTTCAACTATAATTATAAAATTTCTGGTAGTGCCGAAA TCTTCTATCGTCCAGCCAGCATTGAAACTCGTGTCACTCATCCAAAAAATCAACCAA TCAAGAATAAGAATAAAAATAATCCAAAGGCTGAATCTGTCTTCCAGTATGATCTTTG TAAAGATCGTCGCTATATGTCAGATAAATTCTTTTTGCATCTTCCGATCGAATTAAAT CGTATTCCGTTACTCGCTAACGACTCCTCGGTAAATAGTATGGTCAATCAAGTCGTT AGTTCTCGTAATCAGAATTATTTCCTTGGTATTGACCGTGGCGAGAGGCATCTAATT TATCTAGTCCTGATCGATCAAAACGGTAGAATCATTAAACAGCAAACCTTAAATCAG ATCACTAGTTCATACCAAGAAAAAGCCAATAACCAAACGGTTGAAGTTATTACGGAT TATCATGATCTCTTGAATGACAAAGAAAAACTGCGAAAGAAGAATCTCCAAGAGTGG CAATCCGTCGAAAATATCAAGGAGTTAAAGGCTGGGTACCTAAGTAATGTGGTGAA TGAAATCGGTAAGATTATCGTTGAATATCAGCCAGTTATTATGCTGGAAAATCTTAAT ACTGGATTTAAAAACTCACGAATTAAAATTGAGAAACAGGTGTACCAGAAATTTGAG AAGGCGCTCATTGATAAGTTTAACTACTTTATGAGAAAAGATCTCGACTCTTCAGCT ATTGGTGGTCTCTATCACGCTTTGCAGTTGACTAAGGAATACTCTAAGCAGTACAAC GGCAAGCAGAATGGTATCATCTACTATATTCCTGCAAGCTACACTAGTAATATTGAT CCAACTACTGGTTTCATCTCGGCCTTTATACAGACTAGATACGAAAACGTCGAGAAA ACAAAATCCTTAATCGAAAAGTTTAATGATATCACTTATGATGCAGAAGAATCTCTCT TCTGCTTCTCCGCAGATTACAAGAAATTTAGTCCAGAGGCCAAGCTTTGGCAGCAG ACGATTTGGCAGATTTATACTAATGGCGATCGTATTTATACATTTAAGAACAAAGAAG AGTGGCAGAGCAAAAACTACATCCTCGTTGAGGAGTTCAAAGATCTCTTTGCTAAAT ATCACATCGATTATTGCAGGGACCTTAAGGCGCAGATTCTGTCACAAACTGACGCG AGCTTCTTCAAGCAGTTCCTCTTCTTGTTGCGACTAACCTTGCAGATGCGAAATAGT CGCACTACCGAATTAAATGGAACTGATGCTGATACTAAAAAACGTGAGAATGATTAT ATTATTTCTCCAGTTAAGAATCAGTATGGCAAGTTCTATGATTCCCGCAAGGATTAT GTGGACTGGCCAGAAAATGCAGATGCAAATGGCGCATACAATATTGCCAGAAAAGG TCTCATCATGCTAAAACACCTAAAAGAAGGTCTTCCCGAAAAACGTATCTGTGATAT ATCGACTGAAGAATGGGTACAGTTTGTCGAAGAACTAAATAAATAG Codon GTGTCTGAAAAGGAAAACACCCCTACCTTCAACTCTCTGACCAACCTGTACAGCGTT 5 optimized TCTAAAACCCTGCGGTTCGAGCTGCGGCCTCAGTACAGCACCCTGGACCACATCAA coding GGACGATCAGATCGTGGACAAGGGAGAGGAGCTAAAGAACCACTACAAGACATTC sequence(no AAAAAAATCCTGGACCAGGTGTTCTCTCGGATCATCAACGACTCTCTGGATAAAACT N-terminal TACCTGGATCAGAAGTACATCTCCACCTACCAGGATCTGGTGTTCAAGCACAGAGA methionine,no TAGACTGACAGATAAGGACAGAGCCGAACTGAAGGCCCTGAAGGAGACACTGAAG stopcodon) AAGCAGATCGACAAAAGCCTGGATCACAAAGACAAGAAGGCTATCTTCTCCGACCC TGTGAACTTCCTGATCGACAATGAGAGCGACTTCGCCGACCTGATTGGAGACAACC GGCCCAGCATCGAGGCCTTTAACCGCCAGAAGGGATATCTGTCCGGCTACCTGCA GAATAGAGCCAACATCTTCGATCATACAACCAACGAAACCAGCGTTGCTTTCAGAAT CGTGGAAGAGAACCTCGCCATCTTCCTCAACAACCGCCTGACCCTGCAGCATTTCT TCGAGAAAGTGGCCGACAAAGACGGACTGCTGAAGTTCCTGCAGGAGACACTGAG CCAGCTGGGCTTCAAGCTGAAGCTGGAGGATCTGCTGAGCCTGGATTACTTTAACC GGACACTGAGCCAGCCTGGCATCGACCAATACAACCTGCTGATCAGCGGAAAGGC CCTGGAAGATGGCAAGAAGATGCAGGGCATCAATGAAGTGCTGAACCAGTACCTG CAGCAGCACCAGGAGGAAAAGCTGCACAAAATCAAGCTGAAGCAGCTGTATAAGCA AATCCTGAGCGAAAGCAAGACAGAGAGCTTCACGCTGGACTTCGTGGAGGACAAC AAGGGCCTGGCCGCCATGCTGCTGCAGTTTATCGATTTCGTGAACAAGTTAATAGA AGAGAAGATGCTGCTGCTGGATATGATCCAGGGACTGAAAGACAGCAGTGTGTCCA GCGAGTTCTTGAGCCGGCTTTACCTGGAAAGAAAGAACATCAAGCGGCTGAGCAAC TTCATCTACAAGGACTATGGCTATATCGAGCAGTCCCTGGAAGAAAACTTCCTGAG CACCATCGAGGGCAAGATCACTAAGAAGGCCCTGGAAGAGCATAGAAAACAGGAC GCCTTTACCATTCACGAGATCCTGGTCGCACTGCAGAAACAACAGTACGAAAAGGA CGGCGCCCTAGAGAGCGCCGACCACCTGCTGCTTCCAGGCGTGGTGGATTTCCTC TACCAAAACCTGGACTGTAAGCACAGCACGCTGCTGGAAAAGGTGGGCAGCGAGA AGCAGCCCCTGCTGGATCTTTTCAACGAAAAGCAGCTGCTTGAGGGCCAGGACGC CGAGTCCCACGCCTCTAAGTACAGCGATCGGCCTTTCAACGACCACGAGATCAAG GTGGTGAAAACCGCCCTGGACTTCTACAAGAACCTGCAATCTAACTTTGCTATCTTC CAGATCCCCGACGAAAACCTGAAGCTGGATAGCGAGTTTTACAGCGAGTTTGATGA GTTCTACCAGGGCCTGAAAAATATTATTCCTGTGTACAACAAAAGCCGGAACTTCCT GACAAAAAAGCCGTTCAGCACCGAAAAGACCAAACTGATCTTCAACAACCCCCAGC TGCTCGATGGCTGGAGCAAGAGCAAGGAAAGCGACTGTCTGGGGACCATCTTCAT CAAAGACGGCAAGTACTATGTGGGAATCATCAACAGCGCCACCAACGCTAAGAATA CACTGTTCGAGCCTAACAACTTCGCCAATTTCGACCAAAAACAATACTTCGAGAAGA TGAACCTGTTCTTCCTGAGCGATCTGAAGCGAGACTTCCCCAAGAAGTATTTCTCC GAGAAGTGGCACAACCAGCACCCCGTGCCCGCTGACCTTAGAGAAAAGTACGACT ACTACCGGATCGACGAGCATAAGGATGAGAGAAAGAATGACCTGAAATACCACCAC CAGTTAATCGCCTACTACCAAGACTGCCTGAAAAAGGATACAGAGTGGCAGATCTA CCAGTTCAAGTACAAGGCCCCTGAGGAGTACAGCGACGTGAACGAGTTCCTGAGT GAACTGACCCCTAATACCTACAAGATGGAGTTCAACAAGATTCCTGCCGAGTACATT AAGAAGCTGGTGGATGACGGCAAGCTGTACTTTTTTCAGATATACTCCAAAGACTTT AGCGAATTTGCCAAGGGCAAGCCAAACCTGCACACCCTCTACCTGAAGGCCGTGTT CGACCAGAAGAACGCCGAGGAGTTCAACTACAACTATAAAATATCTGGATCTGCTG AAATCTTTTACAGACCTGCTTCTATCGAGACAAGAGTGACCCACCCTAAGAATCAGC CTATCAAGAACAAGAACAAGAACAATCCTAAGGCTGAAAGCGTGTTCCAGTACGAC CTGTGCAAGGACCGGCGGTACATGTCCGACAAGTTCTTCCTGCACCTTCCCATCGA ACTTAACAGAATCCCTCTGCTGGCTAACGATTCCTCCGTGAATAGCATGGTCAACCA GGTGGTGAGCAGCAGAAACCAGAACTACTTCCTGGGCATCGATAGAGGCGAGAGA CACCTGATCTACCTGGTGCTGATCGACCAGAACGGTAGAATCATCAAGCAACAGAC CCTGAATCAGATTACAAGCAGCTACCAAGAAAAGGCCAACAACCAGACAGTGGAGG TGATCACAGACTACCACGACCTGCTGAACGACAAGGAAAAGCTCAGAAAGAAGAAT CTTCAGGAGTGGCAGTCCGTGGAGAATATCAAAGAGCTGAAGGCCGGCTACCTGA GCAACGTGGTCAACGAGATCGGCAAGATCATCGTGGAGTACCAGCCTGTGATCAT GCTGGAAAACCTCAACACCGGATTTAAAAACTCAAGAATCAAGATTGAGAAGCAGG TGTACCAGAAGTTCGAGAAGGCCTTAATCGATAAGTTCAATTACTTCATGCGGAAGG ATCTGGACTCTAGCGCCATCGGCGGCCTGTACCACGCCCTGCAGCTGACCAAAGA GTATAGCAAGCAGTACAACGGCAAGCAGAACGGCATCATCTACTACATCCCAGCTT CTTACACCTCTAATATCGACCCCACCACCGGCTTTATTAGCGCCTTCATCCAGACCA GATACGAGAACGTGGAAAAGACCAAGTCTCTGATCGAGAAATTTAATGACATCACCT ACGACGCCGAAGAGTCGCTGTTCTGCTTCAGCGCCGATTACAAGAAATTTTCACCT GAAGCTAAGCTGTGGCAGCAAACCATCTGGCAGATCTATACCAACGGCGACAGAAT CTACACCTTCAAGAACAAGGAAGAGTGGCAAAGCAAGAACTACATTCTGGTGGAGG AGTTTAAGGACCTGTTCGCCAAATACCACATCGACTATTGCAGGGACCTGAAAGCC CAGATCCTGAGCCAGACCGACGCATCTTTTTTCAAGCAGTTTCTCTTCCTGCTGAGA CTGACACTGCAAATGAGAAATAGTCGTACCACAGAGCTGAACGGCACCGACGCCG ACACCAAGAAAAGAGAGAATGACTACATCATCTCTCCAGTGAAAAATCAGTACGGC AAATTCTATGATTCCCGCAAGGACTACGTGGACTGGCCTGAGAACGCCGACGCCAA TGGCGCCTACAACATCGCCAGAAAGGGCCTGATCATGCTGAAGCACCTGAAGGAA GGACTGCCTGAGAAGAGGATCTGCGACATCAGCACAGAAGAATGGGTTCAGTTTGT GGAAGAACTGAACAAG Expression ATGggcGTGTCTGAAAAGGAAAACACCCCTACCTTCAACTCTCTGACCAACCTGTAC 6 construct(with AGCGTTTCTAAAACCCTGCGGTTCGAGCTGCGGCCTCAGTACAGCACCCTGGACC N-terminal ACATCAAGGACGATCAGATCGTGGACAAGGGAGAGGAGCTAAAGAACCACTACAA methionine GACATTCAAAAAAATCCTGGACCAGGTGTTCTCTCGGATCATCAACGACTCTCTGGA andstop TAAAACTTACCTGGATCAGAAGTACATCTCCACCTACCAGGATCTGGTGTTCAAGCA codon, CAGAGATAGACTGACAGATAAGGACAGAGCCGAACTGAAGGCCCTGAAGGAGACA includesV5- CTGAAGAAGCAGATCGACAAAAGCCTGGATCACAAAGACAAGAAGGCTATCTTCTC tagandC- CGACCCTGTGAACTTCCTGATCGACAATGAGAGCGACTTCGCCGACCTGATTGGAG terminalNLS) ACAACCGGCCCAGCATCGAGGCCTTTAACCGCCAGAAGGGATATCTGTCCGGCTA CCTGCAGAATAGAGCCAACATCTTCGATCATACAACCAACGAAACCAGCGTTGCTTT CAGAATCGTGGAAGAGAACCTCGCCATCTTCCTCAACAACCGCCTGACCCTGCAGC ATTTCTTCGAGAAAGTGGCCGACAAAGACGGACTGCTGAAGTTCCTGCAGGAGACA CTGAGCCAGCTGGGCTTCAAGCTGAAGCTGGAGGATCTGCTGAGCCTGGATTACTT TAACCGGACACTGAGCCAGCCTGGCATCGACCAATACAACCTGCTGATCAGCGGA AAGGCCCTGGAAGATGGCAAGAAGATGCAGGGCATCAATGAAGTGCTGAACCAGT ACCTGCAGCAGCACCAGGAGGAAAAGCTGCACAAAATCAAGCTGAAGCAGCTGTAT AAGCAAATCCTGAGCGAAAGCAAGACAGAGAGCTTCACGCTGGACTTCGTGGAGG ACAACAAGGGCCTGGCCGCCATGCTGCTGCAGTTTATCGATTTCGTGAACAAGTTA ATAGAAGAGAAGATGCTGCTGCTGGATATGATCCAGGGACTGAAAGACAGCAGTGT GTCCAGCGAGTTCTTGAGCCGGCTTTACCTGGAAAGAAAGAACATCAAGCGGCTGA GCAACTTCATCTACAAGGACTATGGCTATATCGAGCAGTCCCTGGAAGAAAACTTC CTGAGCACCATCGAGGGCAAGATCACTAAGAAGGCCCTGGAAGAGCATAGAAAAC AGGACGCCTTTACCATTCACGAGATCCTGGTCGCACTGCAGAAACAACAGTACGAA AAGGACGGCGCCCTAGAGAGCGCCGACCACCTGCTGCTTCCAGGCGTGGTGGATT TCCTCTACCAAAACCTGGACTGTAAGCACAGCACGCTGCTGGAAAAGGTGGGCAG CGAGAAGCAGCCCCTGCTGGATCTTTTCAACGAAAAGCAGCTGCTTGAGGGCCAG GACGCCGAGTCCCACGCCTCTAAGTACAGCGATCGGCCTTTCAACGACCACGAGA TCAAGGTGGTGAAAACCGCCCTGGACTTCTACAAGAACCTGCAATCTAACTTTGCTA TCTTCCAGATCCCCGACGAAAACCTGAAGCTGGATAGCGAGTTTTACAGCGAGTTT GATGAGTTCTACCAGGGCCTGAAAAATATTATTCCTGTGTACAACAAAAGCCGGAAC TTCCTGACAAAAAAGCCGTTCAGCACCGAAAAGACCAAACTGATCTTCAACAACCC CCAGCTGCTCGATGGCTGGAGCAAGAGCAAGGAAAGCGACTGTCTGGGGACCATC TTCATCAAAGACGGCAAGTACTATGTGGGAATCATCAACAGCGCCACCAACGCTAA GAATACACTGTTCGAGCCTAACAACTTCGCCAATTTCGACCAAAAACAATACTTCGA GAAGATGAACCTGTTCTTCCTGAGCGATCTGAAGCGAGACTTCCCCAAGAAGTATT TCTCCGAGAAGTGGCACAACCAGCACCCCGTGCCCGCTGACCTTAGAGAAAAGTA CGACTACTACCGGATCGACGAGCATAAGGATGAGAGAAAGAATGACCTGAAATACC ACCACCAGTTAATCGCCTACTACCAAGACTGCCTGAAAAAGGATACAGAGTGGCAG ATCTACCAGTTCAAGTACAAGGCCCCTGAGGAGTACAGCGACGTGAACGAGTTCCT GAGTGAACTGACCCCTAATACCTACAAGATGGAGTTCAACAAGATTCCTGCCGAGT ACATTAAGAAGCTGGTGGATGACGGCAAGCTGTACTTTTTTCAGATATACTCCAAAG ACTTTAGCGAATTTGCCAAGGGCAAGCCAAACCTGCACACCCTCTACCTGAAGGCC GTGTTCGACCAGAAGAACGCCGAGGAGTTCAACTACAACTATAAAATATCTGGATCT GCTGAAATCTTTTACAGACCTGCTTCTATCGAGACAAGAGTGACCCACCCTAAGAAT CAGCCTATCAAGAACAAGAACAAGAACAATCCTAAGGCTGAAAGCGTGTTCCAGTA CGACCTGTGCAAGGACCGGCGGTACATGTCCGACAAGTTCTTCCTGCACCTTCCCA TCGAACTTAACAGAATCCCTCTGCTGGCTAACGATTCCTCCGTGAATAGCATGGTCA ACCAGGTGGTGAGCAGCAGAAACCAGAACTACTTCCTGGGCATCGATAGAGGCGA GAGACACCTGATCTACCTGGTGCTGATCGACCAGAACGGTAGAATCATCAAGCAAC AGACCCTGAATCAGATTACAAGCAGCTACCAAGAAAAGGCCAACAACCAGACAGTG GAGGTGATCACAGACTACCACGACCTGCTGAACGACAAGGAAAAGCTCAGAAAGAA GAATCTTCAGGAGTGGCAGTCCGTGGAGAATATCAAAGAGCTGAAGGCCGGCTAC CTGAGCAACGTGGTCAACGAGATCGGCAAGATCATCGTGGAGTACCAGCCTGTGA TCATGCTGGAAAACCTCAACACCGGATTTAAAAACTCAAGAATCAAGATTGAGAAGC AGGTGTACCAGAAGTTCGAGAAGGCCTTAATCGATAAGTTCAATTACTTCATGCGGA AGGATCTGGACTCTAGCGCCATCGGCGGCCTGTACCACGCCCTGCAGCTGACCAA AGAGTATAGCAAGCAGTACAACGGCAAGCAGAACGGCATCATCTACTACATCCCAG CTTCTTACACCTCTAATATCGACCCCACCACCGGCTTTATTAGCGCCTTCATCCAGA CCAGATACGAGAACGTGGAAAAGACCAAGTCTCTGATCGAGAAATTTAATGACATC ACCTACGACGCCGAAGAGTCGCTGTTCTGCTTCAGCGCCGATTACAAGAAATTTTC ACCTGAAGCTAAGCTGTGGCAGCAAACCATCTGGCAGATCTATACCAACGGCGACA GAATCTACACCTTCAAGAACAAGGAAGAGTGGCAAAGCAAGAACTACATTCTGGTG GAGGAGTTTAAGGACCTGTTCGCCAAATACCACATCGACTATTGCAGGGACCTGAA AGCCCAGATCCTGAGCCAGACCGACGCATCTTTTTTCAAGCAGTTTCTCTTCCTGCT GAGACTGACACTGCAAATGAGAAATAGTCGTACCACAGAGCTGAACGGCACCGAC GCCGACACCAAGAAAAGAGAGAATGACTACATCATCTCTCCAGTGAAAAATCAGTA CGGCAAATTCTATGATTCCCGCAAGGACTACGTGGACTGGCCTGAGAACGCCGAC GCCAATGGCGCCTACAACATCGCCAGAAAGGGCCTGATCATGCTGAAGCACCTGA AGGAAGGACTGCCTGAGAAGAGGATCTGCGACATCAGCACAGAAGAATGGGTTCA GTTTGTGGAAGAACTGAACAAGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAA AGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTG GGCCTGGACAGCACCTGA

[0088] In some embodiments a ZWGD Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, a ZWGD Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D891 substitution, wherein the position of the D891 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E990 substitution, wherein the position of the E990 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1200 substitution, wherein the position of the R1200 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1248 substitution, wherein the position of the D1248 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZWGD Type V Cas protein is catalytically inactive, for example due to a R1200 substitution in combination with a D891 substitution, a E990 substitution, and/or D1248 substitution.

6.2.2. ZJHK Type V Cas Proteins

[0089] In one aspect, the disclosure provides ZJHK Type V Cas proteins. ZJHK Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZJHK Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7. In some embodiments, the ZJHK Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In some embodiments, a ZJHK Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:7.

[0090] Exemplary ZJHK Type V Cas protein sequences and nucleotide sequences encoding exemplary ZJHK Type V Cas proteins are set forth in Table 1B.

TABLE-US-00003 TABLE1B ZJHKTypeVCasSequences SEQID Name Sequence NO. Wildtype KSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILDKE 7 aminoacid YQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHPLV sequence DKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTEEK (withoutN- STAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTALS terminal QAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEKEH methionine) LIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQSLS NVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQSFY SIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNERNS LKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIYFY NKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAITN SLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEPNE YFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTKWI QFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKIEQ LEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKAEI FRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYKE CLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNPD VNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDKE RKTWKNIETIKDLKEGYISQWVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQV YQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCVF YVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFSF NYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDYR NEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNGE FFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDFY KISNAEWFMFRQKEK Wildtype MKSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILDK 8 aminoacid EYQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHPL sequence(with VDKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTEE N-terminal KSTAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTAL methionine) SQAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEKE HLIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQSL SNVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQSF YSIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNERN SLKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIYF YNKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAIT NSLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEPN EYFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTKW IQFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKIE QLEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKAE IFRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYKE CLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNPD VNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDKE RKTWKNIETIKDLKEGYISQWVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQV YQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCVF YVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFSF NYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDYR NEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNGE FFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDFY KISNAEWFMFRQKEK Expression MGKSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILD 9 construct(with KEYQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHP N-terminal LVDKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTE methionine, EKSTAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTA V5-tagandC- LSQAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEK terminalNLS) EHLIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQ aasequence SLSNVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQS FYSIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNER NSLKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIY FYNKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAI TNSLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEP NEYFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTK WIQFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKI EQLEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKA EIFRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYK ECLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNP DVNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDK ERKTWKNIETIKDLKEGYISQVVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQ VYQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCV FYVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFS FNYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDY RNEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNG EFFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDF YKISNAEWFMFRQKEKSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAAAGTATTTATGAAAATTTTATTGGATTGGAGTCAAAAAATTTGACGCTG 10 coding CGCTTTGCGTTGAATCCAGAAGCTAAGACACAAGAAAATTTGAAGTTGTACTG sequence(with GGACAAATTGCGTGATGAGGAGAGAGATAGGGCGTATCCAATTGTAAAAAAG N-terminal ATATTGGATAAGGAATATCAGCAGCTGATTTCGGAAGGACTGAAATTATTAGA methionine GAATCAGAATGTGTTGGATTGGACAGAATTAGCAGAGTATATACGGACAAGTG andstop ATTTAAGTAAGAAGAAAAAAGAAGATAAACGCTTAAGAAAATTAATAGCACAAA codon) ATTTAAAAGCGCATCCGTTAGTTGACAAACTGAAAGTAAAAAATGCATTTGGTA AAAATGGCTATCTTGAAACTTTACCGTTGGGAAAAGAAGAGAAAGAGGCAGTA AAAGTTTTTGCCGGTTTTGGCGGCTTTTTCAATAACTACAATAAAAACAGGGAA AATTATTTTTCAACCGAGGAAAAAAGCACTGCAATCGCAAACCGAATTGTAAAT GAAAATTTTTCAAAACATTTTTCAAATGTAGAAATAGTTACCAAAATTCAAAAGG AAGTGCCAGAATTAATTCAAATCGTGGAAGCACAATTCAAGGGATATGATACT ATCTTTACAGTAAATGGTTATAATACGGCATTGTCACAGGCAGGGATTGATAC ATATAATGAGATGGTTGCAATCTGGAATAAAGAAGCAAATTTGTATGCGCAAA AGGCAGGAAAACTTCCAGATGGACATCCGTTAAAGAAAAAGAGAAATTACTTA TTGTCGGCATTGTTTAAACAGATTGGGAGTGAAAAGGAGCATTTGATTCAAAT TGATAGATTTGATGGAGATGAAGAGGTGATTGAGGCATTGACGGGTGTGAAA AAAATGCTTCAAGAGGCAGATGTATTTGAAAAATTGAATATGCTTGTGGAGGA TATGGAGAATTGGGATTATAGTAAAATATATTTGTCAGCACAGAGTTTATCCAA TGTTTCTGTGTTCCTAAATAATTTATATGAGGATGAACGGGAGAACTCATGGAA TTATCTTGATAATGTCCTAAGAGAAAAATGGCAAATAGAATTACAGGGAAAGAA AAAGGGGACAGATCTGGAAGAAGCGATTCGGAAGAAAAAACAAAGTTTCTATT CAATAGAAGAACTTCAAGAGGCAGTGAATGCCATAGAAGAAACAGATAAATGT TATAATGTATCTAAATGGCTTCTAGGAGCAATGAAAAGCGAAAGGGTAATAGA AGAAAAAAAGAAGGATGTGGAAGATTTTTGCACACAGTGGAAAAATGAAAGAA ACTCGCTGAAAGAGACAGATATAACTGCACTGAAAGAATATCTGGAGCAATGG ATTTTTTTGGCAAGATATTGCAAATCTTTTTATGCAAATGGAATTGAAAAAAAAG AAAAAGATGAAGCATTTTATCATATTTTAGAAGATGTGTTGTATGTTTTGGATG AAGTAATATATTTTTATAATAAAGTTCGAAATTATGTAACGAAGAAGCCATATTC TCTTGAAAAAATGCATTTAAAATTTGGTCATAATGAACTGGCAAATGGATGGTC TGTTAACAAAGAAGAGAACTATGGTACGGCAATATTGAGGCGAAATGGCAAAT ACTATTTGGCAATTACAAATTCATTGAATAAAAAGATGAGTATTCCCACTCAAT TAGAAAGTACAGGAAATAATTATGAAAAGATGGTATTGAATGTATTCCCAAATG TATTTCGGATGATACCAAAATGTACTACAGGAAGAAATGATGTGAAAAGTTGTT TTGAAAGAAAAGAGCCAAATGAGTATTTCTTTATTGATACACCGGAATTTGTTA ACCCATTTAAAGTTACGCGCGAGGAATATGAGTTAAATAAGATAACTTATGATG GTGTTAAAAAGTGGCAATCTGATTATTCAAAAAATACGCAGGATGAAAAAGGA TACAAAGAGGCAGTGACAAAATGGATTCAGTTTTGTATGCGCTTTTTACAATCT TATAAGAGTACAGCAATATATGATTATTCAACTTTACAGCAACCGGAGAAATAT GAGACGGTGGATTCTTTTTATCATGACGTTGAAAAAATATTATATGAATGTCAT TTTGAGTACGTTCCGGCTAATAAAATAGAGCAGTTGGAAGAAGAAGGAAGAAT TTTTCTGTTTCAGATTTACAACAAAGATTTTTCGGAAAACAGACGCCCGGACA GCAAAAAGAATTTGCATACACTTTATTGGGAGGCATTGTTTTCAGAAGAAAATC GGAAAGCAAAAGTGATACAATTAAATGGCAAAGCTGAAATATTTCGGAGAGAA AAAAGCATTGAACATCCGATTGTTCATAAAGCTGGGGAAGTGTTAGTGAATAA ACGAACGAAAGACGGGGAACCAATACCAGATGATATTTATAAAGATTTGAGCA ACTATTTTAACGGAAGAAATGTAACATCTGAAAAGGAAGAGTATAAGGAATGT CTGGATAAAGTGTATACTTCGACCAAAAAATATGATATTACAAAGGATAAACGT TTTACTGAAACCAAATATGAATTTCATGTTCCGATTACCTTGAACTATCAGGCG GACGGTGTTAAATATTTGAATCAGAAAATACTTCATGTGCTGAGGGATAATCC AGATGTGAATATTATAGGTCTAGATAGAGGCGAGCGTAATCTGATTTCCTACG TAGTATTGAACCGAGAAGGCAAGATTGTTAACAATCAGCAGGGGAGTTTCAAT ATTGTGGGTAAGATGGACTATCAGAAGAAACTGTATCAAAAAGAAAAGAATCG TGACAAAGAACGAAAAACTTGGAAAAATATCGAAACAATAAAGGATTTGAAGG AAGGATATATTTCACAAGTCGTTCATGAATTGACCGATATGGCGATTCGCAAT AATGCAATTATTGTGATGGAAGATCTGAATTTTGGATTTAAAAGGGGACGCAC CAAAGTGGAACGGCAGGTATATCAGAAGTTTGAGCTGGCGCTTCTGAAGAAA TTGCATTATCTGGTTACGGATAAAACAGAAGGTGAGGCTATGCTTAAGCCTGG CGGTGTCCTTCAAGGTTATCAGCTTGCAAGAGAAGTAAAAACCCTAAAAGAAA TCGGAAAGCAATGCGGATGTGTATTTTATGTTCCACCGGGATATACTTCTAAA ATCGATCCAACAACCGGATTTGTTGATGTGTTTAACATGTCAGGTGTTACGAA TCGTGAAAAGAAAAAAGCATTTTTTGAAAAGTTCGATAATATGTTCTATGATGA AAAGCGGGATATGTTTGGATTTTCATTTAACTATGAGAAGTTTACAACATATCA AAGTTCTTATAGAAATGATTGGACTGTATATTCGAATGGAAGCAAATATGTGTG GAACTCTTTAAAAAGGACAGACGAGCTTATTGATGTTACAAAAGAATTGAAACT GCTCTTTGAAAAGTATGCAATTGATTACAGAAACGAAGCATTGTTTGAACAAAT CATGTCCCAAGATACGGATAAAAACAATGCTGACTTTTGGAATAAATTGTTCTG GTATTTTCGTGTTTTGCTCCGTCTGAGAAACAGTTCAGATGAATTAGATCAGAT TGTTTCACCGGTACTTAATCAAAACGGAGAATTTTTTGAAACACCGAAAAAAAT CACGGAGAAAAGTTATTTGTCTGATTATCCGATGGATGCGGATACCAATGGTG CGTATCACATCGCTTTAAAAGGGTTGTATCTCATACAGGAAAAAATTGCAGAT GAGAGCGTAGATTTGGATAACAAATTACCAAAAGATTTTTACAAGATCTCTAAT GCAGAGTGGTTTATGTTTAGGCAGAAGGAGAAGTAA Codon AAGAGCATCTACGAGAACTTCATCGGTCTTGAGAGCAAGAACCTGACACTGA 11 optimized GATTCGCCCTGAACCCTGAGGCTAAAACCCAGGAGAACCTGAAGCTGTACTG coding GGACAAACTGAGGGACGAAGAAAGAGATAGAGCCTACCCTATCGTGAAAAAA sequence(no ATCCTCGACAAGGAGTATCAGCAGCTCATCAGCGAGGGCCTGAAACTGCTGG N-terminal AAAATCAAAACGTGCTGGACTGGACCGAACTGGCCGAGTACATCAGAACCAG methionine,no CGATCTGTCTAAGAAGAAGAAGGAGGACAAGAGACTGCGCAAGCTGATCGCC stopcodon) CAGAACCTGAAAGCCCACCCCCTGGTCGACAAGCTGAAGGTGAAGAATGCCT TCGGCAAGAACGGCTACCTGGAAACCCTGCCATTAGGAAAGGAAGAAAAAGA GGCCGTGAAGGTGTTTGCCGGATTCGGAGGCTTTTTCAACAACTACAACAAG AATCGGGAGAACTACTTCAGTACCGAGGAGAAGTCCACCGCCATCGCCAACA GAATCGTGAACGAGAACTTCAGCAAGCACTTCAGCAACGTGGAAATCGTTACA AAGATCCAAAAAGAAGTGCCAGAGCTGATTCAAATCGTGGAAGCTCAGTTCAA GGGTTACGACACCATCTTTACCGTGAACGGCTACAACACCGCCCTGAGCCAG GCTGGCATCGACACATACAACGAAATGGTGGCCATCTGGAACAAGGAGGCAA ACCTGTACGCTCAAAAAGCCGGCAAGCTGCCAGACGGCCACCCGCTGAAGA AGAAGCGTAACTACCTGCTGAGCGCCCTCTTCAAACAGATCGGCAGCGAAAA AGAACACCTGATCCAGATCGACAGATTCGACGGCGACGAGGAAGTGATCGAA GCCCTGACTGGCGTGAAAAAGATGCTGCAGGAGGCCGACGTGTTCGAGAAG CTGAACATGCTGGTCGAGGACATGGAAAATTGGGATTACTCCAAGATCTACCT GTCTGCCCAGAGCCTGAGTAACGTGTCCGTGTTCCTGAACAACCTGTATGAA GATGAACGGGAGAACAGCTGGAACTACCTGGATAACGTGCTGAGAGAGAAGT GGCAGATTGAACTGCAGGGCAAAAAAAAGGGAACAGATCTGGAAGAGGCCAT TAGAAAGAAGAAGCAGAGCTTTTACTCTATCGAGGAACTTCAGGAGGCAGTG AACGCCATCGAGGAAACCGACAAGTGCTACAATGTGTCTAAATGGCTGCTGG GAGCCATGAAGAGCGAGAGAGTGATCGAGGAGAAGAAGAAAGACGTGGAGG ATTTCTGCACACAGTGGAAGAACGAGAGAAACAGCCTCAAGGAAACCGACAT CACCGCCCTGAAGGAGTACCTGGAGCAGTGGATCTTCCTGGCTAGGTACTGC AAGAGCTTCTACGCCAATGGCATCGAAAAGAAAGAGAAGGATGAGGCTTTTTA CCACATCCTGGAGGATGTGCTGTACGTGCTGGACGAAGTGATCTACTTCTAC AACAAGGTGCGGAACTACGTGACCAAAAAGCCTTACAGTCTGGAGAAGATGC ACCTGAAGTTCGGCCACAACGAGCTGGCCAACGGCTGGAGCGTGAACAAGG AAGAAAATTACGGCACCGCCATCCTGAGAAGAAACGGCAAGTACTACCTGGC CATCACCAACAGCCTGAACAAGAAAATGAGCATCCCTACCCAGCTGGAGAGC ACAGGAAATAATTATGAGAAGATGGTCCTGAACGTTTTTCCCAACGTGTTCCG GATGATCCCAAAGTGCACCACAGGCAGGAACGACGTGAAGTCATGCTTCGAG AGAAAGGAACCCAACGAGTACTTCTTCATCGACACCCCTGAGTTCGTGAACC CCTTTAAGGTCACACGGGAGGAGTACGAACTGAATAAGATCACCTACGACGG AGTTAAGAAGTGGCAGAGCGACTACAGCAAGAACACACAGGACGAAAAGGGC TATAAGGAAGCCGTGACCAAGTGGATTCAGTTTTGTATGCGGTTCCTGCAGTC TTATAAGAGCACCGCCATATATGACTACAGCACCCTGCAGCAACCTGAAAAAT ACGAAACAGTGGACAGCTTCTATCATGATGTGGAAAAGATCCTGTACGAGTGC CACTTCGAGTACGTGCCCGCTAACAAGATCGAGCAGCTTGAAGAAGAGGGAA GAATCTTCCTGTTCCAGATCTACAACAAGGATTTTTCTGAGAACAGACGGCCT GATAGCAAGAAAAACCTCCACACCCTGTACTGGGAGGCGCTGTTCTCCGAAG AGAATAGAAAGGCCAAGGTGATTCAGCTGAATGGCAAGGCCGAGATCTTCAG ACGGGAGAAATCAATCGAGCACCCTATCGTGCATAAGGCTGGCGAGGTGCTG GTGAACAAGCGGACCAAAGATGGCGAACCTATTCCTGACGACATCTACAAGG ACCTGAGCAACTATTTCAACGGCAGAAACGTTACCTCTGAGAAGGAAGAGTAC AAGGAGTGTCTGGACAAGGTGTACACCAGCACCAAAAAGTACGATATCACCA AGGACAAAAGATTCACCGAGACAAAGTACGAGTTCCACGTGCCTATCACCCT GAACTACCAGGCCGACGGCGTGAAGTACCTGAATCAGAAGATCCTGCACGTG CTGCGGGACAACCCTGATGTTAACATCATCGGCCTGGATAGAGGCGAAAGAA ACCTGATCTCTTATGTTGTGCTGAACAGAGAGGGCAAGATCGTGAACAATCAG CAGGGTTCTTTCAACATCGTGGGCAAAATGGACTACCAGAAAAAGCTGTACCA GAAGGAGAAAAACCGGGATAAAGAACGGAAAACGTGGAAAAACATCGAAACC ATCAAGGACCTGAAGGAGGGCTATATCAGCCAGGTGGTACACGAGCTGACCG ATATGGCCATCCGGAATAACGCGATCATCGTGATGGAAGATCTGAATTTCGGA TTCAAGCGGGGCCGGACCAAGGTGGAACGGCAGGTGTACCAGAAGTTTGAG CTGGCCCTGCTGAAGAAGCTGCACTACCTCGTGACCGACAAGACCGAGGGA GAAGCTATGCTGAAACCCGGCGGCGTGCTGCAAGGCTACCAGCTGGCTAGA GAAGTCAAGACCCTGAAAGAGATCGGCAAGCAGTGCGGCTGTGTGTTCTACG TGCCCCCTGGCTACACAAGCAAGATCGACCCTACAACCGGCTTCGTCGACGT GTTCAACATGTCTGGAGTTACAAACCGCGAGAAAAAGAAAGCCTTTTTCGAAA AATTTGATAACATGTTCTACGACGAGAAGAGAGACATGTTCGGCTTCAGCTTC AATTACGAAAAGTTTACTACCTACCAGAGCAGCTACAGAAACGACTGGACCGT GTACAGCAACGGCAGCAAGTATGTGTGGAACTCCCTTAAGAGAACAGACGAG TTAATTGACGTGACAAAGGAGCTCAAGCTGCTGTTCGAGAAGTACGCCATCG ATTACCGGAACGAAGCTCTGTTTGAGCAGATCATGAGCCAGGATACAGATAA GAACAACGCCGACTTCTGGAACAAACTGTTCTGGTACTTCCGGGTGCTGCTG CGGCTGAGAAATAGCAGCGACGAACTGGACCAAATCGTCAGCCCTGTGCTGA ATCAGAACGGAGAGTTCTTCGAAACCCCTAAGAAAATCACAGAGAAGTCCTAC CTGTCTGATTACCCTATGGACGCCGATACAAACGGCGCCTACCACATCGCCC TGAAGGGCCTGTACCTGATCCAGGAGAAGATCGCTGACGAATCTGTGGACCT GGACAACAAGCTGCCTAAGGACTTCTACAAGATCAGCAACGCCGAGTGGTTC ATGTTTAGACAGAAAGAAAAA Expression ATGggcAAGAGCATCTACGAGAACTTCATCGGTCTTGAGAGCAAGAACCTGAC 12 construct(with ACTGAGATTCGCCCTGAACCCTGAGGCTAAAACCCAGGAGAACCTGAAGCTG N-terminal TACTGGGACAAACTGAGGGACGAAGAAAGAGATAGAGCCTACCCTATCGTGA methionine AAAAAATCCTCGACAAGGAGTATCAGCAGCTCATCAGCGAGGGCCTGAAACT andstop GCTGGAAAATCAAAACGTGCTGGACTGGACCGAACTGGCCGAGTACATCAGA codon, ACCAGCGATCTGTCTAAGAAGAAGAAGGAGGACAAGAGACTGCGCAAGCTGA includesV5- TCGCCCAGAACCTGAAAGCCCACCCCCTGGTCGACAAGCTGAAGGTGAAGAA tagandC- TGCCTTCGGCAAGAACGGCTACCTGGAAACCCTGCCATTAGGAAAGGAAGAA terminalNLS) AAAGAGGCCGTGAAGGTGTTTGCCGGATTCGGAGGCTTTTTCAACAACTACA ACAAGAATCGGGAGAACTACTTCAGTACCGAGGAGAAGTCCACCGCCATCGC CAACAGAATCGTGAACGAGAACTTCAGCAAGCACTTCAGCAACGTGGAAATC GTTACAAAGATCCAAAAAGAAGTGCCAGAGCTGATTCAAATCGTGGAAGCTCA GTTCAAGGGTTACGACACCATCTTTACCGTGAACGGCTACAACACCGCCCTG AGCCAGGCTGGCATCGACACATACAACGAAATGGTGGCCATCTGGAACAAGG AGGCAAACCTGTACGCTCAAAAAGCCGGCAAGCTGCCAGACGGCCACCCGC TGAAGAAGAAGCGTAACTACCTGCTGAGCGCCCTCTTCAAACAGATCGGCAG CGAAAAAGAACACCTGATCCAGATCGACAGATTCGACGGCGACGAGGAAGTG ATCGAAGCCCTGACTGGCGTGAAAAAGATGCTGCAGGAGGCCGACGTGTTC GAGAAGCTGAACATGCTGGTCGAGGACATGGAAAATTGGGATTACTCCAAGA TCTACCTGTCTGCCCAGAGCCTGAGTAACGTGTCCGTGTTCCTGAACAACCT GTATGAAGATGAACGGGAGAACAGCTGGAACTACCTGGATAACGTGCTGAGA GAGAAGTGGCAGATTGAACTGCAGGGCAAAAAAAAGGGAACAGATCTGGAAG AGGCCATTAGAAAGAAGAAGCAGAGCTTTTACTCTATCGAGGAACTTCAGGAG GCAGTGAACGCCATCGAGGAAACCGACAAGTGCTACAATGTGTCTAAATGGC TGCTGGGAGCCATGAAGAGCGAGAGAGTGATCGAGGAGAAGAAGAAAGACG TGGAGGATTTCTGCACACAGTGGAAGAACGAGAGAAACAGCCTCAAGGAAAC CGACATCACCGCCCTGAAGGAGTACCTGGAGCAGTGGATCTTCCTGGCTAGG TACTGCAAGAGCTTCTACGCCAATGGCATCGAAAAGAAAGAGAAGGATGAGG CTTTTTACCACATCCTGGAGGATGTGCTGTACGTGCTGGACGAAGTGATCTAC TTCTACAACAAGGTGCGGAACTACGTGACCAAAAAGCCTTACAGTCTGGAGAA GATGCACCTGAAGTTCGGCCACAACGAGCTGGCCAACGGCTGGAGCGTGAA CAAGGAAGAAAATTACGGCACCGCCATCCTGAGAAGAAACGGCAAGTACTAC CTGGCCATCACCAACAGCCTGAACAAGAAAATGAGCATCCCTACCCAGCTGG AGAGCACAGGAAATAATTATGAGAAGATGGTCCTGAACGTTTTTCCCAACGTG TTCCGGATGATCCCAAAGTGCACCACAGGCAGGAACGACGTGAAGTCATGCT TCGAGAGAAAGGAACCCAACGAGTACTTCTTCATCGACACCCCTGAGTTCGT GAACCCCTTTAAGGTCACACGGGAGGAGTACGAACTGAATAAGATCACCTAC GACGGAGTTAAGAAGTGGCAGAGCGACTACAGCAAGAACACACAGGACGAAA AGGGCTATAAGGAAGCCGTGACCAAGTGGATTCAGTTTTGTATGCGGTTCCT GCAGTCTTATAAGAGCACCGCCATATATGACTACAGCACCCTGCAGCAACCT GAAAAATACGAAACAGTGGACAGCTTCTATCATGATGTGGAAAAGATCCTGTA CGAGTGCCACTTCGAGTACGTGCCCGCTAACAAGATCGAGCAGCTTGAAGAA GAGGGAAGAATCTTCCTGTTCCAGATCTACAACAAGGATTTTTCTGAGAACAG ACGGCCTGATAGCAAGAAAAACCTCCACACCCTGTACTGGGAGGCGCTGTTC TCCGAAGAGAATAGAAAGGCCAAGGTGATTCAGCTGAATGGCAAGGCCGAGA TCTTCAGACGGGAGAAATCAATCGAGCACCCTATCGTGCATAAGGCTGGCGA GGTGCTGGTGAACAAGCGGACCAAAGATGGCGAACCTATTCCTGACGACATC TACAAGGACCTGAGCAACTATTTCAACGGCAGAAACGTTACCTCTGAGAAGGA AGAGTACAAGGAGTGTCTGGACAAGGTGTACACCAGCACCAAAAAGTACGAT ATCACCAAGGACAAAAGATTCACCGAGACAAAGTACGAGTTCCACGTGCCTAT CACCCTGAACTACCAGGCCGACGGCGTGAAGTACCTGAATCAGAAGATCCTG CACGTGCTGCGGGACAACCCTGATGTTAACATCATCGGCCTGGATAGAGGCG AAAGAAACCTGATCTCTTATGTTGTGCTGAACAGAGAGGGCAAGATCGTGAAC AATCAGCAGGGTTCTTTCAACATCGTGGGCAAAATGGACTACCAGAAAAAGCT GTACCAGAAGGAGAAAAACCGGGATAAAGAACGGAAAACGTGGAAAAACATC GAAACCATCAAGGACCTGAAGGAGGGCTATATCAGCCAGGTGGTACACGAGC TGACCGATATGGCCATCCGGAATAACGCGATCATCGTGATGGAAGATCTGAA TTTCGGATTCAAGCGGGGCCGGACCAAGGTGGAACGGCAGGTGTACCAGAA GTTTGAGCTGGCCCTGCTGAAGAAGCTGCACTACCTCGTGACCGACAAGACC GAGGGAGAAGCTATGCTGAAACCCGGCGGCGTGCTGCAAGGCTACCAGCTG GCTAGAGAAGTCAAGACCCTGAAAGAGATCGGCAAGCAGTGCGGCTGTGTGT TCTACGTGCCCCCTGGCTACACAAGCAAGATCGACCCTACAACCGGCTTCGT CGACGTGTTCAACATGTCTGGAGTTACAAACCGCGAGAAAAAGAAAGCCTTTT TCGAAAAATTTGATAACATGTTCTACGACGAGAAGAGAGACATGTTCGGCTTC AGCTTCAATTACGAAAAGTTTACTACCTACCAGAGCAGCTACAGAAACGACTG GACCGTGTACAGCAACGGCAGCAAGTATGTGTGGAACTCCCTTAAGAGAACA GACGAGTTAATTGACGTGACAAAGGAGCTCAAGCTGCTGTTCGAGAAGTACG CCATCGATTACCGGAACGAAGCTCTGTTTGAGCAGATCATGAGCCAGGATAC AGATAAGAACAACGCCGACTTCTGGAACAAACTGTTCTGGTACTTCCGGGTG CTGCTGCGGCTGAGAAATAGCAGCGACGAACTGGACCAAATCGTCAGCCCTG TGCTGAATCAGAACGGAGAGTTCTTCGAAACCCCTAAGAAAATCACAGAGAAG TCCTACCTGTCTGATTACCCTATGGACGCCGATACAAACGGCGCCTACCACAT CGCCCTGAAGGGCCTGTACCTGATCCAGGAGAAGATCGCTGACGAATCTGTG GACCTGGACAACAAGCTGCCTAAGGACTTCTACAAGATCAGCAACGCCGAGT GGTTCATGTTTAGACAGAAAGAAAAAtctagaAAGCGGACAGCAGACGGCTCCG AATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCA ATCCCCTGCTGGGCCTGGACAGCACCTGA

[0091] In some embodiments a ZJHK Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, a ZJHK Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D900 substitution, wherein the position of the D900 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E987 substitution, wherein the position of the E987 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1203 substitution, wherein the position of the R1203 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1244 substitution, wherein the position of the D1244 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 121 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZJHK Type V Cas protein is catalytically inactive, for example due to a R1203 substitution in combination with a D900 substitution, a E987 substitution, and/or D1244 substitution.

6.2.3. ZIKV Type V Cas Proteins

[0092] In one aspect, the disclosure provides ZIKV Type V Cas proteins. ZIKV Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZIKV Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 13. In some embodiments, the ZIKV Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, a ZIKV Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 13.

[0093] Exemplary ZIKV Type V Cas protein sequences and nucleotide sequences encoding exemplary ACEE Type V Cas proteins are set forth in Table 1C.

TABLE-US-00004 TABLE1C ZIKVTypeVCasSequences SEQID Name Sequence NO. Wildtype NIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKENRA 13 aminoacid FIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEISTQL sequence KGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSIGH (withoutN- RIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQSG terminal IDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDNQLL methionine) ECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYISNLI NEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFKEKIN LVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDKDDD FYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKDYGT KKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPRKFIY SKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKIGVN RDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSGKIYL FKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLNLDK KTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNRALK YCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKKKNS RKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFESNVY QKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYIPAW MTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFTDRA QGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDKDIKA QIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQNEKM PMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL Wildtype MNIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKENR 14 aminoacid AFIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEISTQ sequence(with LKGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSIG N-terminal HRIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQS methionine) GIDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDNQL LECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYISN LINEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFKEK INLVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDKDD DFYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKDYG TKKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPRKFI YSKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKIGV NRDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSGKI YLFKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLNL DKKTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNRA LKYCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKKK NSRKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFESN VYQKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYIP AWMTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFTD RAQGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDKDI KAQIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQNE KMPMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL Expression MGNIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKEN 15 construct(with RAFIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEIST N-terminal QLKGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSI methionine, GHRIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQ V5-tagandC- SGIDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDN terminalNLS) QLLECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYI aasequence SNLINEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFK EKINLVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDK DDDFYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKD YGTKKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPR KFIYSKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKI GVNRDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSG KIYLFKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLN LDKKTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNR ALKYCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKK KNSRKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFES NVYQKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYI PAWMTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFT DRAQGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDK DIKAQIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQ NEKMPMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL SRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAACATTTACGAAAATTTTACTAATATGTATCAGGTAAATAAGACTATAAGAA 16 coding TGGGGTTAAAGCCAATATGTAAAACTGGTGAAAATATTGCTAAATTTCTTGAGG sequence(with AAGATAAGGAAACAAGTGATAAATACAAGATAGCTAAAGAAGTAATTGATAAG N-terminal GAAAATAGAGCTTTTATAGAGGATAGATTAAAGGATTTTTCAATTTCAGGGTTG methionine GATGAATATTTGGAATTGCTTAAACAAAAAAAGAATTTAACCAAAAATCAAAAT andstop AAAATGAAAAAGGAAATTTCAACACAGTTAACAAAAATACAAAATAAAATGAGA codon) GATGAAATTTCAACACAGTTAAAAGGCTTCCCTCAATTTGATAATAAATATAAA TTCAAATATATTACAGATAAAGAAGATATAGAAATTTTAAAATATTTTAAAGATA AGAAATTTATTACTTTCTTTGAAGAATTTAATACTAATAGAAAAAATGTCTACTC TAAAGAAAATATTTCAACTTCTATTGGACACAGAATTGTTCACGAAAATCTTCC AAAATTTATTTCAAATTTTAGGATTTTAAATAAAGCAATAGAGGCGTTTGGAATA AGTAAAATAAATGAAGATTTTAAGAATAATGGAATTAATGTTACAGTTGAAGAA CTTAATAAAATAGATTATTTTAACAAGGTTTTAACTCAATCAGGAATAGATTTGT ATAATAATTTGATAGGTATTTTAAATCAAAATATAAATCTATATAATCAACAACA GAAAGTAAAAAAGAATAAAATTGGAAAGTTAGAAATATTATATAAGCAAATTTTA AGTAAAACAGATAAAGTATCGTTTATTGAAGAATTTACTGAAGATAACCAACTT TTGGAATGTATTGATGAATATTTTAAAGAAAAATATAGTTTGATAACTGTAGATT TAAATAATTTACTTGAAAATATTGATACTTATAATTTGAATGGTATCTTTATTAAA AGTGATAAGTCCTTGGGAAATATATCTAATTATTTATATAAAGATTGGTGGTAT ATATCAAATCTTATAAACGAAGAATACGATTATAAACATAAGAATAAGGTAAGA GATGATAAGTATTATGAAACAAGAAAAAAAGCTATAGATAAGATTAAATATTTTT CCATAGGACATATTGATGAATTGTTAAAAGATAAAAATGTTCCTATGGTAGAAA ACTATTTCAAAGAAAAGATAAATTTAGTAGTAAAAGAATTTAATGCTTATTTAAA CAAATTTAATGAATATAAGTTTATAAATGAGCTAAAAACTGATGAAATTGCTGT CGAAATAATAAAAAATTTATGTGATTCAATAAAGAATGTACAGGGGATAGTAAA GCCTTTAATAATTACTGGAAATGATAAAGACGATGATTTTTATGTGGAAATCAA TTATATATGGGACGAGCTTAATAAGTTTGATAAAATATATAATATGGTTAGAAAT TATCTTACAAAAAAGGATTACATAGAGGAAAAAATTAGAATGATGTTTTCAAAG AGCAGTTTTATGGATGGTTGGGGAAAAGATTATGGAACAAAAAAAGCACATAT AGTTTATCATGATAAAAATTATTATTTAGTAATAGTAGACAAGAAATTAAAATTA GAGGATATAGATAAATTATATAAACCAGGTGGAGATACTGTACATTATGTATAT AATTACCAATCAACAGAAAATGGAAATATTCCTAGAAAATTCATATATTCTAAG GGTAAAAGATTTGCACCATCTGTAGAAAAATATAATTTACCAATAGAAGATGTT ATCGAAGTGTATAACAATGAATATCATACAACAGATTACGAAAAGAAAAATCCT GAAATTTACAAGAAATCATTAACATCCTTAATTGATTATTTTAAAATAGGGGTAA ATAGGGATATGGATTTTGAAAAATTTGATTTTAGATTAAAAGATTCAAACGAAT ACAAAAATATAAAAGAATTTTATGATAATTTGGAAACTTGTTGCTATAAGTTACA AGAAGAAAAAGTTAATTTTAATGTACTTGAAGAGCTTTCATATAGTGGAAAAAT TTATTTATTTAAAATATACAATAAGGATTTTTCTGAAAATAGCAAAGGAATACCT AATCTTCATACTTTATATTTTAAAATGCTATTTGACAAAGAAAACCTTGAAAATC CGATTTATAAACTTAGTGGAAAGGCTAAAATGTTTTTTAGAAAGGGTAGTCTTA ATTTAGACAAAAAAACTGTTGATTATGATAAAAAGCCAATAGATAAGAAAGAAA ATGACAAAAAAATTAAAAATAGAAGATATAAAGTTGATAGTTTTACATTACATAT GTCAATTATTACGAACTTTCAGTCATATGAAAATAAAAATGTAAATGAAACTGT AAATAGGGCTTTAAAATATTGTGATGATGTTTATGCCATAGGTATAGACAGAG GAATAAGAAATTTATTATATGCTTGTGTAGTAAATTCAAAGGGAGAAATAGTAA AACAAGTTCCTTTAAATATTATAAATAATAAAGATTATCACAATTTACTTGCAGA AAGAGAAGAGAAGAAAAAGAATAGTAGGAAAAATTGGAAAATCATTGATAATA TAAGGAATTTAAAGGAAGGCTATTTAAGTCAGGCCATACATATAATAACTGACC TTATGGTTGAATATAATGCTGTACTTGTTTTAGAGAATTTGAATTTTAGATTTAA AGAAAAACAAATGAAATTTGAAAGTAATGTTTATCAAAAATTTGAAAAGATGCT TATTGATAAATTGAATTTCTTAGTTGATAAAAAGCTTGATAAGAACGCCAATGG TGGATTGTTTAATGCGTATCAATTAACAGAAAAATTTACAAACTTTAAAGATATG AAAAATCAAAATGGTATAATATTTTATATTCCTGCTTGGATGACAAGCAAAATT GACCCAGTTACAGGATTTACAAATTTATTCTATATTAAATATGAGAGTATTGAA AAGGCTAAAGAGTTTTTTGGTAAGTTTAAATCAATAAAATTTAATAAGGTAGAC AACTATTTTGAATTTGAATTTGATTATAATGATTTTACTGACAGAGCTCAAGGTA CAAGGTCTAAATGGACAGTTTGTAGTTTTGGCCCTAGAATTGAAGGTTTTAGA AATCCTGAAAAAAATAATAGTTGGGATGGTAGAGAAATAGATATAACAGAGAA AATTAAAAAATTACTTGATGATTATAATGTATOTTTAGATAAAGATATTAAAGCT CAAATTATGGATATAAATACTAAGGATTTCTTTGAAAAATTTATTAAATATTTTAA ACTTGTATTGCAAATGAGAAACAGTAAAACAGGTACAGATATTGATTATATCAT TTCTCCGGTTAAAAATAAGCAAAATGAATTTTTTGACAGTAGAAAGCAAAATGA AAAAATGCCTATGGATGCAGATGCAAATGGTGCTTATAATATTGCTAGAAAAG GCTTAATGTTTATTGATATAATAAAAGAAACTGAAGATAAAGATTTAAAGATGC CTAAATTGTTCATTAAAAATAAAGATTGGTTAAATTATGTACAAAAGAGTGATTT GTAA Codon AATATCTATGAGAACTTCACCAACATGTACCAGGTGAACAAGACAATCCGCAT 17 optimized GGGCCTGAAGCCTATCTGTAAAACCGGAGAAAACATCGCCAAGTTCCTGGAG coding GAGGACAAGGAAACCAGCGACAAGTACAAGATCGCCAAGGAGGTCATCGACA sequence(no AGGAGAACAGAGCCTTTATCGAGGACAGACTGAAGGACTTCAGCATCAGCGG N-terminal CCTGGACGAGTACCTGGAACTGCTGAAGCAGAAGAAAAACCTGACAAAGAAC methionine,no CAGAACAAGATGAAAAAGGAAATCTCCACCCAGCTGACAAAGATCCAGAACAA stopcodon) GATGCGGGACGAGATATCGACACAGCTGAAGGGCTTCCCTCAGTTCGATAAC AAATACAAGTTCAAATATATCACAGACAAGGAGGACATCGAAATCCTCAAGTA CTTCAAGGATAAGAAGTTCATTACATTCTTTGAGGAATTTAATACCAATCGGAA AAACGTGTACAGCAAGGAAAACATCAGCACCTCTATCGGCCATAGAATCGTG CACGAGAACCTGCCAAAGTTCATCAGCAACTTCAGAATCCTGAATAAGGCCAT CGAGGCCTTCGGCATCTCTAAAATCAATGAGGACTTCAAGAACAATGGCATCA ACGTGACCGTAGAAGAACTGAACAAGATCGACTACTTCAACAAGGTCCTGACA CAGAGCGGCATTGACCTGTACAACAACCTGATTGGCATCCTGAACCAGAACA TCAACCTGTACAATCAGCAGCAGAAGGTGAAGAAGAACAAAATCGGAAAGCT GGAAATCCTGTACAAGCAAATCTTGTCCAAAACCGACAAGGTGTCTTTCATTG AGGAGTTCACCGAGGACAACCAGCTGCTGGAGTGCATCGACGAGTACTTTAA AGAGAAATACAGCCTGATCACCGTGGACCTGAACAACCTGCTTGAAAATATCG ACACCTACAATCTCAACGGCATCTTCATCAAATCTGATAAAAGCCTGGGCAAC ATCAGCAACTACCTGTACAAGGATTGGTGGTACATCAGCAACCTGATCAACGA AGAATACGACTACAAGCACAAGAACAAGGTCAGAGATGATAAGTACTACGAGA CAAGAAAGAAGGCCATCGACAAGATCAAGTACTTCTCTATCGGACACATCGAT GAGCTGCTGAAGGACAAGAACGTTCCAATGGTGGAAAACTACTTCAAGGAGA AGATCAACCTGGTCGTGAAGGAGTTCAATGCTTATCTGAACAAGTTCAATGAA TATAAATTCATCAACGAGCTGAAAACAGACGAGATCGCCGTGGAAATCATCAA GAACCTGTGCGACAGCATCAAGAACGTGCAGGGCATCGTGAAGCCCCTGATC ATCACCGGCAACGACAAGGATGATGATTTTTACGTGGAGATCAACTACATCTG GGATGAGCTTAACAAGTTCGACAAAATCTACAACATGGTCAGGAATTACCTAA CCAAGAAGGACTACATCGAGGAAAAGATCAGAATGATGTTTTCCAAGAGCAG CTTTATGGACGGCTGGGGCAAGGACTACGGCACCAAGAAGGCCCACATCGT GTACCACGACAAGAACTACTACCTGGTGATCGTGGACAAGAAGCTGAAACTG GAAGATATCGACAAACTATACAAGCCAGGCGGCGACACAGTTCACTACGTGT ACAACTACCAGTCTACCGAGAACGGAAACATCCCTCGGAAGTTCATCTACTCT AAGGGCAAGCGGTTCGCCCCTAGCGTGGAAAAATATAACCTGCCTATTGAAG ATGTGATTGAGGTGTACAACAACGAGTACCACACCACCGACTATGAGAAAAAG AACCCTGAGATATACAAAAAGTCCCTGACCAGCCTGATCGACTATTTCAAGAT CGGCGTGAACAGAGATATGGACTTCGAGAAGTTTGATTTTCGGCTAAAGGACT CCAACGAATACAAGAACATCAAGGAGTTCTACGATAACCTGGAGACATGCTGC TACAAGCTGCAGGAGGAAAAGGTGAACTTCAACGTGCTGGAGGAACTGAGCT ACAGCGGAAAGATCTACCTGTTCAAGATCTACAACAAAGATTTCAGCGAGAAT AGCAAAGGCATCCCTAACCTGCATACCCTGTACTTCAAAATGCTGTTCGACAA AGAGAACCTGGAGAACCCCATCTACAAGCTGTCTGGAAAAGCTAAGATGTTTT TCAGAAAGGGCAGCCTGAACCTGGACAAAAAAACCGTTGACTATGACAAAAAA CCTATCGATAAGAAGGAAAACGACAAAAAAATCAAGAATAGGCGGTACAAGGT GGACAGCTTCACCCTGCACATGAGCATCATCACCAACTTCCAGAGCTACGAG AACAAGAACGTTAATGAGACTGTGAACCGGGCCCTGAAGTACTGCGACGACG TGTACGCCATCGGCATCGACCGCGGAATCCGGAACCTGCTGTACGCTTGTGT GGTGAACAGCAAGGGCGAGATCGTGAAGCAAGTGCCCCTCAACATCATTAAC AATAAGGATTACCACAACCTGCTGGCCGAGAGAGAAGAAAAGAAGAAAAACA GCAGAAAGAATTGGAAGATCATAGACAACATCAGAAACCTGAAGGAAGGCTA CCTGAGCCAGGCCATCCACATCATCACCGACCTGATGGTGGAATACAACGCC GTGCTGGTGCTGGAGAACCTGAATTTCAGATTCAAGGAGAAGCAGATGAAGT TTGAAAGCAATGTGTACCAAAAATTCGAAAAAATGCTGATCGACAAGCTGAAT TTCCTGGTCGATAAAAAACTGGACAAGAATGCCAATGGCGGACTGTTTAACGC CTATCAGCTGACAGAGAAGTTCACCAACTTTAAGGATATGAAGAATCAGAACG GCATCATCTTCTACATCCCCGCCTGGATGACAAGCAAGATCGATCCCGTGAC CGGCTTCACAAACCTGTTTTATATCAAATACGAGAGCATCGAGAAGGCAAAGG AGTTCTTCGGCAAGTTTAAGTCTATCAAGTTCAATAAGGTGGACAATTATTTCG AGTTCGAGTTCGACTACAACGACTTTACCGACAGAGCTCAAGGCACCAGAAG CAAGTGGACCGTGTGTAGCTTCGGTCCTCGGATCGAGGGCTTCAGAAACCCC GAGAAAAACAATTCCTGGGACGGCAGAGAAATCGACATCACAGAGAAGATCA AGAAGCTGCTGGATGACTACAATGTGAGCCTGGACAAAGACATCAAAGCCCA GATCATGGACATCAACACCAAGGATTTCTTCGAGAAGTTCATCAAGTACTTCA AGCTGGTGCTGCAGATGAGAAACAGCAAGACCGGCACCGACATCGATTACAT TATCTCCCCTGTGAAGAACAAGCAGAACGAGTTTTTCGACTCCAGAAAGCAGA ACGAGAAGATGCCTATGGACGCTGATGCCAACGGCGCCTACAACATCGCTAG AAAGGGGCTGATGTTCATCGATATCATCAAGGAAACAGAGGACAAGGACCTG AAAATGCCTAAGCTGTTCATAAAGAACAAGGATTGGCTGAACTATGTGCAGAA ATCAGATCTG Expression ATGggcAATATCTATGAGAACTTCACCAACATGTACCAGGTGAACAAGACAATC 18 construct(with CGCATGGGCCTGAAGCCTATCTGTAAAACCGGAGAAAACATCGCCAAGTTCC N-terminal TGGAGGAGGACAAGGAAACCAGCGACAAGTACAAGATCGCCAAGGAGGTCA methionine TCGACAAGGAGAACAGAGCCTTTATCGAGGACAGACTGAAGGACTTCAGCAT andstop CAGCGGCCTGGACGAGTACCTGGAACTGCTGAAGCAGAAGAAAAACCTGACA codon, AAGAACCAGAACAAGATGAAAAAGGAAATCTCCACCCAGCTGACAAAGATCCA includesV5- GAACAAGATGCGGGACGAGATATCGACACAGCTGAAGGGCTTCCCTCAGTTC tagandC- GATAACAAATACAAGTTCAAATATATCACAGACAAGGAGGACATCGAAATCCT terminalNLS) CAAGTACTTCAAGGATAAGAAGTTCATTACATTCTTTGAGGAATTTAATACCAA TCGGAAAAACGTGTACAGCAAGGAAAACATCAGCACCTCTATCGGCCATAGA ATCGTGCACGAGAACCTGCCAAAGTTCATCAGCAACTTCAGAATCCTGAATAA GGCCATCGAGGCCTTCGGCATCTCTAAAATCAATGAGGACTTCAAGAACAATG GCATCAACGTGACCGTAGAAGAACTGAACAAGATCGACTACTTCAACAAGGTC CTGACACAGAGCGGCATTGACCTGTACAACAACCTGATTGGCATCCTGAACC AGAACATCAACCTGTACAATCAGCAGCAGAAGGTGAAGAAGAACAAAATCGG AAAGCTGGAAATCCTGTACAAGCAAATCTTGTCCAAAACCGACAAGGTGTCTT TCATTGAGGAGTTCACCGAGGACAACCAGCTGCTGGAGTGCATCGACGAGTA CTTTAAAGAGAAATACAGCCTGATCACCGTGGACCTGAACAACCTGCTTGAAA ATATCGACACCTACAATCTCAACGGCATCTTCATCAAATCTGATAAAAGCCTG GGCAACATCAGCAACTACCTGTACAAGGATTGGTGGTACATCAGCAACCTGAT CAACGAAGAATACGACTACAAGCACAAGAACAAGGTCAGAGATGATAAGTACT ACGAGACAAGAAAGAAGGCCATCGACAAGATCAAGTACTTCTCTATCGGACA CATCGATGAGCTGCTGAAGGACAAGAACGTTCCAATGGTGGAAAACTACTTCA AGGAGAAGATCAACCTGGTCGTGAAGGAGTTCAATGCTTATCTGAACAAGTTC AATGAATATAAATTCATCAACGAGCTGAAAACAGACGAGATCGCCGTGGAAAT CATCAAGAACCTGTGCGACAGCATCAAGAACGTGCAGGGCATCGTGAAGCCC CTGATCATCACCGGCAACGACAAGGATGATGATTTTTACGTGGAGATCAACTA CATCTGGGATGAGCTTAACAAGTTCGACAAAATCTACAACATGGTCAGGAATT ACCTAACCAAGAAGGACTACATCGAGGAAAAGATCAGAATGATGTTTTCCAAG AGCAGCTTTATGGACGGCTGGGGCAAGGACTACGGCACCAAGAAGGCCCAC ATCGTGTACCACGACAAGAACTACTACCTGGTGATCGTGGACAAGAAGCTGA AACTGGAAGATATCGACAAACTATACAAGCCAGGCGGCGACACAGTTCACTA CGTGTACAACTACCAGTCTACCGAGAACGGAAACATCCCTCGGAAGTTCATCT ACTCTAAGGGCAAGCGGTTCGCCCCTAGCGTGGAAAAATATAACCTGCCTATT GAAGATGTGATTGAGGTGTACAACAACGAGTACCACACCACCGACTATGAGA AAAAGAACCCTGAGATATACAAAAAGTCCCTGACCAGCCTGATCGACTATTTC AAGATCGGCGTGAACAGAGATATGGACTTCGAGAAGTTTGATTTTCGGCTAAA GGACTCCAACGAATACAAGAACATCAAGGAGTTCTACGATAACCTGGAGACAT GCTGCTACAAGCTGCAGGAGGAAAAGGTGAACTTCAACGTGCTGGAGGAACT GAGCTACAGCGGAAAGATCTACCTGTTCAAGATCTACAACAAAGATTTCAGCG AGAATAGCAAAGGCATCCCTAACCTGCATACCCTGTACTTCAAAATGCTGTTC GACAAAGAGAACCTGGAGAACCCCATCTACAAGCTGTCTGGAAAAGCTAAGA TGTTTTTCAGAAAGGGCAGCCTGAACCTGGACAAAAAAACCGTTGACTATGAC AAAAAACCTATCGATAAGAAGGAAAACGACAAAAAAATCAAGAATAGGCGGTA CAAGGTGGACAGCTTCACCCTGCACATGAGCATCATCACCAACTTCCAGAGC TACGAGAACAAGAACGTTAATGAGACTGTGAACCGGGCCCTGAAGTACTGCG ACGACGTGTACGCCATCGGCATCGACCGCGGAATCCGGAACCTGCTGTACG CTTGTGTGGTGAACAGCAAGGGCGAGATCGTGAAGCAAGTGCCCCTCAACAT CATTAACAATAAGGATTACCACAACCTGCTGGCCGAGAGAGAAGAAAAGAAG AAAAACAGCAGAAAGAATTGGAAGATCATAGACAACATCAGAAACCTGAAGGA AGGCTACCTGAGCCAGGCCATCCACATCATCACCGACCTGATGGTGGAATAC AACGCCGTGCTGGTGCTGGAGAACCTGAATTTCAGATTCAAGGAGAAGCAGA TGAAGTTTGAAAGCAATGTGTACCAAAAATTCGAAAAAATGCTGATCGACAAG CTGAATTTCCTGGTCGATAAAAAACTGGACAAGAATGCCAATGGCGGACTGTT TAACGCCTATCAGCTGACAGAGAAGTTCACCAACTTTAAGGATATGAAGAATC AGAACGGCATCATCTTCTACATCCCCGCCTGGATGACAAGCAAGATCGATCC CGTGACCGGCTTCACAAACCTGTTTTATATCAAATACGAGAGCATCGAGAAGG CAAAGGAGTTCTTCGGCAAGTTTAAGTCTATCAAGTTCAATAAGGTGGACAAT TATTTCGAGTTCGAGTTCGACTACAACGACTTTACCGACAGAGCTCAAGGCAC CAGAAGCAAGTGGACCGTGTGTAGCTTCGGTCCTCGGATCGAGGGCTTCAGA AACCCCGAGAAAAACAATTCCTGGGACGGCAGAGAAATCGACATCACAGAGA AGATCAAGAAGCTGCTGGATGACTACAATGTGAGCCTGGACAAAGACATCAA AGCCCAGATCATGGACATCAACACCAAGGATTTCTTCGAGAAGTTCATCAAGT ACTTCAAGCTGGTGCTGCAGATGAGAAACAGCAAGACCGGCACCGACATCGA TTACATTATCTCCCCTGTGAAGAACAAGCAGAACGAGTTTTTCGACTCCAGAA AGCAGAACGAGAAGATGCCTATGGACGCTGATGCCAACGGCGCCTACAACAT CGCTAGAAAGGGGCTGATGTTCATCGATATCATCAAGGAAACAGAGGACAAG GACCTGAAAATGCCTAAGCTGTTCATAAAGAACAAGGATTGGCTGAACTATGT GCAGAAATCAGATCTGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAG CCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCT GGGCCTGGACAGCACCTGA

[0094] In some embodiments a ZIKV Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, a ZIKV Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D814 substitution, wherein the position of the D814 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 14 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E899 substitution, wherein the position of the E899 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 14 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1111 substitution, wherein the position of the R1111 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 14 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1148 substitution, wherein the position of the D1148 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 14 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZIKV Type V Cas protein is catalytically inactive, for example due to a R1111 substitution in combination with a D814 substitution, a E899 substitution, and/or D1148 substitution.

6.2.4. ZZFT Type V Cas Proteins

[0095] In one aspect, the disclosure provides ZZFT Type V Cas proteins. ZZFT Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZFT Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 19. In some embodiments, the ZZFT Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19. In some embodiments, a ZZFT Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 19.

[0096] Exemplary ZZFT Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZFT Type V Cas proteins are set forth in Table 1D.

TABLE-US-00005 TABLE1D ZZFTTypeVCasSequences SEQID Name Sequence NO. Wildtype EISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILDNC 19 aminoacid HKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTKQD sequence VTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSEEAK (withoutN- STAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSINNYN terminal NILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFNQILS methionine) DREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIKGGE DLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSVSLHS IANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDEELRT NSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINPLYNK VRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGINKCL SKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQLPIETI LDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKASSEY NDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGLPNLH TIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFTEKKT SIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIKHIIGID RGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADANAQARR DWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENNVYQKF EQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQSGFVFY IPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIRIDYNK FSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFATKDIDI SGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGNFFDS RNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLDYVQR Wildtype MEISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILDN 20 aminoacid CHKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTKQ sequence(with DVTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSEEA N-terminal KSTAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSINNY methionine) NNILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFNQIL SDREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIKGG EDLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSVSLH SIANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDEELR TNSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINPLYN KVRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGINKC LSKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQLPIE TILDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKASSE YNDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGLPNL HTIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFTEKK TSIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIKHIIGI DRGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADANAQAR RDWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENNVYQK FEQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQSGFVF YIPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIRIDYN KFSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFATKDID ISGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGNFFDS RNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLDYVQR Expression MGEISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILD 21 construct(with NCHKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTK N-terminal QDVTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSE methionine, EAKSTAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSIN V5-tagandC- NYNNILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFN terminalNLS) QILSDREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIK aasequence GGEDLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSV SLHSIANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDE ELRTNSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINP LYNKVRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGI NKCLSKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQ LPIETILDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKA SSEYNDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGL PNLHTIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFT EKKTSIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIK HIIGIDRGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADAN AQARRDWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENN VYQKFEQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQS GFVFYIPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIR IDYNKFSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFAT KDIDISGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGN FFDSRNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLD YVQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGGAAATTTCGAACCGATTCACAAACAAGTATCAAGTAAGCAAGACCCTCCG 22 coding CTTTCGCCTTGAGCCAACCGGAGGTACTGATGATTTACTTTGCCAAGCACAAA sequence(with TCATCGAGGGAGACGAGCGCCGCAATAAAGAGGCTATAACAATGAAACAGAT N-terminal TTTGGACAATTGTCACAAACAGATAATTGAGCGCGTATTGTCCGACTTTAATTT methionine TAAAGAGCATTCTCTTGAAGAGTTTTTCAAAGTGTATACCAGAAACGATGATGA andstop CCGCGAAAAGGACATTGAAAATCTCCAAGCAAAAATGCGCAAAGAAATAGCC codon) GCCGCCTTCACCAAACAGGATGTTACGAAACTTTTCTCAAGCAAATTCAAGGA TTTTGTTGAAAGAGGCTTGATTAAATATGCATCAAACGAGAAGGAACGCAACA TCGTTTCCCGCTTCAAAGGTTTTGCCACTTACTTTACAGGGTTCAATACCAATA GACTGAATATGTACTCAGAAGAAGCAAAATCCACAGCTATATCATTCAGATTAA TTAATCAAAACTTGATAAAGTTCATAGACAACATCCTTGTATATAAAAAAGTGT CTCAAACGTTGCCTTCAGATGTGCTATCAAACATTTATATAGACTTTAAGGCAA TCATCAACACATCAAGTCTTGAAGAATTCTTCTCCATAAACAACTACAATAACA TACTCACCCAGAAACAGATTGAGATTTTCAATGCAGTTATCGGAGGTAAAAAA GACAAGGATGAAAAAATAATAACCAAAGGATTCAACCAATATATAAACGAATAC AACCAGACCAATAAAAACATCCGTCTGCCTAAGATGATGCGGTTATTCAATCA AATCCTAAGCGACAGAGAAGGTGTTTCTGCAAGACCAGAGCCATTCAATAACG CGAACGAGACAATCAGTTCCGTCCGTGATTGTTTTACAAACGAAATATCAAAA CAAATAACGATATTGTCTGAAACAACATCCAAAATTGAATCATTCGACATTGAT AGAATTTACATTAAGGGCGGAGAAGATCTGAGAGCATTATCCAACAGTATATA TGGATATTTCAATTATATCCATGACCGTATCGCAGACAAATGGAAACACAACAA TCCTCAGGGCAAAAAGAGCCCCGAAAGCTACCAAAAAAACCTCAACGCATAT CTGAAAGGCATAAAAAGCGTCTCTTTACACAGTATTGCAAACATCTGTGGTGA CAACAAAGTTATTGAGTATTTCAGGAATCTTGGTGCAGAAAACACTGTTGATTT CCAAAGAGAGAACGTTGTATCATTAATCGACAACAAATACAACTGCGCTTCAA ATCTTTTATCCGACGCCCAAATTACGGATGAAGAACTTCGCACAAACAGTCGC TCAATTAAAGACTTGCTTGACGCCGTCAAGAGTGCCCAACGATTTTTCCGTCT ACTGTGCGGTTCTGGCAACGAACCAGACAAAGACCACTCTTTTTATGACGAGT ATACACCAGCATTTGAAGCACTTGAGAATTCAATAAATCCCCTATATAACAAAG TCAGGAGTTTTGTAACCAAAAAAGATTTCTCCACCGATAAATTCAAATTGAATT TCGACAGCAGCAGCTTTCTATCCGGTTGGGCAAAGAAATCAGAATATGAGAA GAGTTCTGCATTTATATTTATTCGCGACAATCAATATTACTTAGGAATAAACAA ATGCCTTAGCAAAGAAGACATTGCCTACCTTGAGGACTCAACAAGCTCATCAG ATACAAAAAGAGTGGTATATATGTTCCAAAAAGTGGACGCCACGAATATTCCC AGAATATTCATCCGTTCCAAAGGTTCCAATTTAGCTCCTGCTGTCAACGAATTC CAACTGCCGATAGAAACCATTCTTGACATTTATGACAATAAGTTCTTCACTACC AGTTATCAGAAAAAAGACCGGACTAAATGGAAAGAATCATTGACCAAACTCAT TGACTATTACAAGCTTGGATTCAGCCAGCACAAGTCATACGCAGATTTCGACT TAAAATGGAAAGCATCCAGTGAATATAACGACATAAATGACTTTCTTGCAGAC GTACAGAGATTCTGCTACAGAATCGAATTTATAAATATCAATTGGGACAAGCT GATAGAATTCACAGAAGATGGCAAATTTTACCTATTCCGCATTGCAAATAAAGA TTTATCAGGCAATAGCACAGGTCTGCCCAATTTGCACACGATTTATTGGAAAA TGCTTTTTGACGAAAGCAACCTCAAAGATATTGTCTATAAATTGTCGGGCAATG CGGAAGTCTTTATGCGCTATAATTCATTAAAAAATCCAATTGTGCATAAAGCGG GAGTGGAGATTAAAAACAAATGCCCTTTTACTGAAAAAAAGACAAGCATATTTG ACTACGACATTATAAAAGACCGTCGCTATACAAAAGATCAGCTTGAACTGCAT GTTCCAATCCTAATGAACTTCAAAAGCCCATCGGCAGCAAAAGGCAAAGCTTT CAACAAAGAATGCTTGGAATACATAAGAAATAATGGTATAAAGCATATTATAGG AATAGACCGAGGTGAACGGAATCTACTTTATATGGTTATAACAGACCTTGACG GCAACATCGTTGAGCAAAAGTCTTTGAACCAAATTGCGAGCAATCCGAAATTG CCTCTTTTCAGACAAGACTACAACAAGCTGCTGAAGACAAAGGCTGATGCAAA CGCACAAGCACGTCGTGATTGGGAAACAATAGACACCGTAAAGGAGATAAAA TTCGGCTTCTTGAGTCAGATTGTACATGAGATAGCAATGGCTATCATAAAATAC GATGCAATTGTTGTTTTGGAGAATCTGAACAGAGGGTTTATGCAGAAACGAGG TCTTGAAAACAACGTCTATCAGAAATTCGAACAAATGCTGCTTGACAAGTTGA GCTACTATGTCGACAAAACGAAACATCCGGAAGAGGCCGGAGGAGCTTTGCA CGCATATCAGCTCTCTGACACTTACGCGAACTTCAATTCTCTGTCGAAGAATG CGATGGTGCGACAGTCGGGTTTTGTTTTCTATATTCCTGCATGGCTTACAAGC AAAATAGACCCCGTCACAGGATTCGCCTCCTTTTTGAAATTTCACAGAGATGA CAGTATGGCAACAATCAAATCTACAATTTCAAAGTTTGATTGTTTCAAATACGA CAAGGAATGCGACATGTTCCACATCCGCATTGACTATAACAAGTTTAGCACAA GCTGCAGCGGAGGTCAACGCAAATGGGACTTGTTCACTTTTGGCGATCGAAT CTTGGCAGAACGCAATACAATGCAAAACAGCAGATATGTTTACCAAACAGTCA ATTTAACTTCTGAATTCAAAAACTTATTTGCCACAAAGGATATCGACATTTCAG GCAACCTGAAGGACTCTATATGCAAAATTGAGGATGTTGGCTTTTTCAGAAAA CTAAGCCAACTCTTGTCACTCACGCTTCAATTACGCAACAGCAATGCTGAAAC AGGAGAAGACTTCTTGATTTCCCCAGTAGCTGACAAAGATGGCAATTTCTTCG ATTCAAGAAACTGTCCCGACTCTCTCCCAAAAGACGCAGATGCCAATGGCGC ATACAACATTGCTAGGAAGGGATTAATGCTTGTCGAGCAATTGAAGAGATGCA AAGATGTATCAAAATTCAAGCCCGCGATAAAAAACGAGGACTGGTTAGACTAT GTTCAACGCTGA Codon GAAATCAGTAATCGGTTTACAAACAAGTACCAGGTGTCTAAGACCCTGCGGTT 23 optimized CAGACTGGAGCCTACAGGCGGGACCGATGACCTGCTGTGCCAGGCCCAGAT coding CATCGAGGGCGATGAGCGGCGCAACAAAGAAGCCATCACCATGAAACAGATC sequence(no CTCGACAACTGTCACAAGCAGATCATCGAAAGAGTGCTGTCCGACTTCAACTT N-terminal CAAAGAGCACTCCCTGGAAGAGTTCTTTAAGGTGTACACACGGAACGACGAT methionine,no GACAGAGAGAAGGATATCGAGAACCTGCAGGCAAAGATGCGCAAGGAAATCG stopcodon) CCGCCGCCTTTACTAAGCAAGACGTGACAAAACTGTTTTCTTCCAAGTTTAAA GACTTTGTCGAAAGGGGTCTGATCAAGTACGCCAGCAACGAGAAGGAGCGGA ATATCGTGTCCCGGTTCAAGGGCTTTGCCACATACTTCACCGGCTTCAACACA AACCGCCTGAACATGTACAGCGAGGAAGCCAAATCTACGGCCATTAGCTTCC GGCTGATCAACCAGAACCTCATCAAATTCATCGACAATATCCTGGTGTACAAG AAGGTGTCTCAGACCCTCCCTTCTGATGTCCTGAGCAACATCTACATCGACTT CAAGGCCATCATCAATACCAGCAGCCTGGAGGAGTTCTTCTCCATCAACAACT ACAACAACATCCTGACCCAGAAGCAGATCGAGATCTTCAACGCTGTGATCGG CGGAAAGAAGGATAAGGATGAGAAAATTATCACAAAGGGCTTCAACCAGTACA TCAATGAATATAATCAGACCAACAAGAATATCAGACTGCCAAAGATGATGAGA CTGTTCAATCAGATACTGAGCGACCGGGAAGGCGTGTCAGCTAGACCTGAGC CCTTCAACAACGCCAACGAGACAATCAGCTCCGTGAGAGACTGTTTTACAAAC GAAATCAGCAAGCAGATCACCATCCTGTCTGAAACCACCAGTAAGATCGAGA GCTTCGACATCGATAGAATCTACATCAAGGGCGGAGAGGACCTGCGGGCCCT GAGCAACAGCATCTACGGCTACTTCAACTACATCCACGATAGAATCGCTGATA AGTGGAAGCACAACAATCCTCAGGGCAAGAAGAGCCCCGAGAGCTACCAAAA GAATCTGAACGCCTACCTGAAGGGCATAAAGAGCGTGAGCCTGCATTCTATC GCCAACATCTGTGGCGACAACAAGGTGATCGAATATTTTAGAAATCTCGGCGC CGAGAACACAGTGGATTTTCAGAGAGAAAACGTGGTGTCCCTAATTGACAACA AATACAACTGTGCCTCAAACCTGCTGTCCGACGCCCAAATCACCGACGAGGA GCTGAGGACCAACAGCAGAAGCATCAAGGATCTGCTCGACGCCGTGAAGAGT GCCCAGAGATTCTTCAGACTGCTGTGCGGTTCTGGCAATGAGCCTGATAAAG ACCACAGCTTTTATGACGAGTACACCCCTGCTTTCGAGGCCCTGGAAAACAG CATCAACCCCCTGTACAACAAGGTCCGCAGCTTCGTGACCAAAAAGGACTTC AGCACAGACAAGTTCAAACTGAACTTCGACAGCAGCAGCTTCCTGAGCGGAT GGGCCAAGAAAAGCGAGTACGAGAAGAGCAGCGCTTTCATCTTCATCAGGGA TAATCAGTACTACCTGGGAATTAATAAGTGCCTGAGTAAAGAGGACATCGCCT ACCTGGAGGACAGCACCTCTAGCAGCGACACAAAGAGAGTGGTGTACATGTT TCAGAAGGTGGATGCCACCAATATCCCAAGAATCTTCATCAGATCCAAGGGCA GCAACCTGGCCCCTGCTGTGAACGAGTTCCAGCTGCCTATCGAAACCATCCT GGATATCTACGACAACAAGTTCTTCACCACCAGTTACCAGAAGAAGGATAGAA CCAAATGGAAGGAAAGCCTGACCAAGCTGATCGACTACTACAAGCTGGGCTT TAGCCAGCACAAGTCCTATGCCGATTTCGATTTAAAGTGGAAAGCCAGCTCAG AATACAATGACATCAATGATTTCCTGGCCGACGTGCAGAGATTCTGCTACAGA ATTGAGTTCATCAATATCAATTGGGACAAGCTCATCGAGTTCACAGAGGACGG CAAGTTCTACCTGTTTAGAATCGCCAACAAAGACCTGTCTGGCAACAGCACTG GCCTGCCCAATCTGCACACCATCTACTGGAAGATGCTGTTCGACGAGAGCAA CCTGAAGGACATCGTGTACAAGCTGAGCGGCAACGCTGAGGTGTTTATGCGC TACAACAGCCTGAAGAACCCCATTGTGCACAAGGCCGGAGTGGAAATCAAGA ATAAGTGTCCTTTCACCGAGAAGAAAACCAGCATCTTTGACTACGACATTATC AAGGACCGCAGATACACCAAGGACCAGCTGGAACTGCATGTGCCTATCCTGA TGAACTTCAAGTCTCCATCTGCCGCTAAAGGCAAAGCCTTTAACAAGGAGTGC CTGGAATACATCAGAAACAACGGCATCAAGCACATCATCGGCATCGACAGAG GAGAGCGGAATCTGCTTTACATGGTGATCACAGACCTGGACGGCAACATCGT GGAACAGAAGTCTCTGAACCAGATCGCCTCCAATCCAAAGCTGCCTCTGTTCA GACAGGACTACAACAAGCTGCTGAAAACCAAAGCTGACGCCAACGCACAAGC CAGAAGAGACTGGGAGACAATAGACACCGTGAAGGAGATTAAGTTCGGCTTC CTGAGCCAGATCGTGCACGAGATCGCTATGGCCATCATCAAGTACGACGCCA TTGTGGTCCTGGAAAACCTGAACAGAGGCTTCATGCAAAAACGGGGCCTGGA AAACAACGTGTATCAGAAGTTCGAGCAAATGCTCCTCGATAAACTGAGCTACT ATGTCGACAAGACCAAACACCCTGAGGAAGCTGGCGGAGCCCTGCACGCCT ATCAGTTAAGCGATACCTACGCCAACTTCAATTCCTTGAGCAAGAACGCTATG GTGAGACAGTCTGGCTTCGTGTTCTACATCCCCGCCTGGCTGACCAGCAAGA TCGATCCTGTGACCGGCTTCGCCTCTTTCCTGAAGTTCCACAGAGATGATAGC ATGGCCACCATCAAGAGCACCATCTCCAAATTCGACTGCTTCAAGTACGACAA GGAATGCGACATGTTCCACATCAGAATAGATTACAACAAATTTAGCACTTCAT GCAGCGGTGGCCAGCGGAAGTGGGATCTGTTCACATTCGGAGACAGAATCCT GGCCGAGAGAAACACCATGCAGAACAGTAGATACGTTTACCAGACAGTTAAC CTGACCTCTGAGTTCAAGAACCTGTTCGCCACAAAGGATATCGATATAAGCGG GAACCTGAAGGATAGCATCTGCAAGATCGAGGACGTGGGCTTCTTCCGGAAG CTGAGCCAGCTGCTGAGCCTGACACTACAGCTTCGGAACAGCAACGCTGAAA CCGGAGAAGATTTCCTGATCAGCCCTGTGGCCGACAAGGACGGCAACTTCTT TGACAGCAGAAACTGCCCCGACAGCCTGCCAAAGGATGCAGACGCGAATGG CGCTTATAACATTGCCAGGAAGGGCCTGATGCTGGTGGAGCAACTGAAGCGG TGCAAGGACGTGAGCAAGTTCAAGCCTGCTATCAAGAACGAGGACTGGCTGG ACTACGTGCAGCGG Expression ATGggcGAAATCAGTAATCGGTTTACAAACAAGTACCAGGTGTCTAAGACCCTG 24 construct(with CGGTTCAGACTGGAGCCTACAGGCGGGACCGATGACCTGCTGTGCCAGGCC N-terminal CAGATCATCGAGGGCGATGAGCGGCGCAACAAAGAAGCCATCACCATGAAAC methionine AGATCCTCGACAACTGTCACAAGCAGATCATCGAAAGAGTGCTGTCCGACTTC andstop AACTTCAAAGAGCACTCCCTGGAAGAGTTCTTTAAGGTGTACACACGGAACGA codon, CGATGACAGAGAGAAGGATATCGAGAACCTGCAGGCAAAGATGCGCAAGGAA includesV5- ATCGCCGCCGCCTTTACTAAGCAAGACGTGACAAAACTGTTTTCTTCCAAGTT tagandC- TAAAGACTTTGTCGAAAGGGGTCTGATCAAGTACGCCAGCAACGAGAAGGAG terminalNLS) CGGAATATCGTGTCCCGGTTCAAGGGCTTTGCCACATACTTCACCGGCTTCAA CACAAACCGCCTGAACATGTACAGCGAGGAAGCCAAATCTACGGCCATTAGC TTCCGGCTGATCAACCAGAACCTCATCAAATTCATCGACAATATCCTGGTGTA CAAGAAGGTGTCTCAGACCCTCCCTTCTGATGTCCTGAGCAACATCTACATCG ACTTCAAGGCCATCATCAATACCAGCAGCCTGGAGGAGTTCTTCTCCATCAAC AACTACAACAACATCCTGACCCAGAAGCAGATCGAGATCTTCAACGCTGTGAT CGGCGGAAAGAAGGATAAGGATGAGAAAATTATCACAAAGGGCTTCAACCAG TACATCAATGAATATAATCAGACCAACAAGAATATCAGACTGCCAAAGATGAT GAGACTGTTCAATCAGATACTGAGCGACCGGGAAGGCGTGTCAGCTAGACCT GAGCCCTTCAACAACGCCAACGAGACAATCAGCTCCGTGAGAGACTGTTTTA CAAACGAAATCAGCAAGCAGATCACCATCCTGTCTGAAACCACCAGTAAGATC GAGAGCTTCGACATCGATAGAATCTACATCAAGGGCGGAGAGGACCTGCGG GCCCTGAGCAACAGCATCTACGGCTACTTCAACTACATCCACGATAGAATCGC TGATAAGTGGAAGCACAACAATCCTCAGGGCAAGAAGAGCCCCGAGAGCTAC CAAAAGAATCTGAACGCCTACCTGAAGGGCATAAAGAGCGTGAGCCTGCATT CTATCGCCAACATCTGTGGCGACAACAAGGTGATCGAATATTTTAGAAATCTC GGCGCCGAGAACACAGTGGATTTTCAGAGAGAAAACGTGGTGTCCCTAATTG ACAACAAATACAACTGTGCCTCAAACCTGCTGTCCGACGCCCAAATCACCGAC GAGGAGCTGAGGACCAACAGCAGAAGCATCAAGGATCTGCTCGACGCCGTG AAGAGTGCCCAGAGATTCTTCAGACTGCTGTGCGGTTCTGGCAATGAGCCTG ATAAAGACCACAGCTTTTATGACGAGTACACCCCTGCTTTCGAGGCCCTGGAA AACAGCATCAACCCCCTGTACAACAAGGTCCGCAGCTTCGTGACCAAAAAGG ACTTCAGCACAGACAAGTTCAAACTGAACTTCGACAGCAGCAGCTTCCTGAGC GGATGGGCCAAGAAAAGCGAGTACGAGAAGAGCAGCGCTTTCATCTTCATCA GGGATAATCAGTACTACCTGGGAATTAATAAGTGCCTGAGTAAAGAGGACATC GCCTACCTGGAGGACAGCACCTCTAGCAGCGACACAAAGAGAGTGGTGTACA TGTTTCAGAAGGTGGATGCCACCAATATCCCAAGAATCTTCATCAGATCCAAG GGCAGCAACCTGGCCCCTGCTGTGAACGAGTTCCAGCTGCCTATCGAAACCA TCCTGGATATCTACGACAACAAGTTCTTCACCACCAGTTACCAGAAGAAGGAT AGAACCAAATGGAAGGAAAGCCTGACCAAGCTGATCGACTACTACAAGCTGG GCTTTAGCCAGCACAAGTCCTATGCCGATTTCGATTTAAAGTGGAAAGCCAGC TCAGAATACAATGACATCAATGATTTCCTGGCCGACGTGCAGAGATTCTGCTA CAGAATTGAGTTCATCAATATCAATTGGGACAAGCTCATCGAGTTCACAGAGG ACGGCAAGTTCTACCTGTTTAGAATCGCCAACAAAGACCTGTCTGGCAACAGC ACTGGCCTGCCCAATCTGCACACCATCTACTGGAAGATGCTGTTCGACGAGA GCAACCTGAAGGACATCGTGTACAAGCTGAGCGGCAACGCTGAGGTGTTTAT GCGCTACAACAGCCTGAAGAACCCCATTGTGCACAAGGCCGGAGTGGAAATC AAGAATAAGTGTCCTTTCACCGAGAAGAAAACCAGCATCTTTGACTACGACAT TATCAAGGACCGCAGATACACCAAGGACCAGCTGGAACTGCATGTGCCTATC CTGATGAACTTCAAGTCTCCATCTGCCGCTAAAGGCAAAGCCTTTAACAAGGA GTGCCTGGAATACATCAGAAACAACGGCATCAAGCACATCATCGGCATCGAC AGAGGAGAGCGGAATCTGCTTTACATGGTGATCACAGACCTGGACGGCAACA TCGTGGAACAGAAGTCTCTGAACCAGATCGCCTCCAATCCAAAGCTGCCTCT GTTCAGACAGGACTACAACAAGCTGCTGAAAACCAAAGCTGACGCCAACGCA CAAGCCAGAAGAGACTGGGAGACAATAGACACCGTGAAGGAGATTAAGTTCG GCTTCCTGAGCCAGATCGTGCACGAGATCGCTATGGCCATCATCAAGTACGA CGCCATTGTGGTCCTGGAAAACCTGAACAGAGGCTTCATGCAAAAACGGGGC CTGGAAAACAACGTGTATCAGAAGTTCGAGCAAATGCTCCTCGATAAACTGAG CTACTATGTCGACAAGACCAAACACCCTGAGGAAGCTGGCGGAGCCCTGCAC GCCTATCAGTTAAGCGATACCTACGCCAACTTCAATTCCTTGAGCAAGAACGC TATGGTGAGACAGTCTGGCTTCGTGTTCTACATCCCCGCCTGGCTGACCAGC AAGATCGATCCTGTGACCGGCTTCGCCTCTTTCCTGAAGTTCCACAGAGATGA TAGCATGGCCACCATCAAGAGCACCATCTCCAAATTCGACTGCTTCAAGTACG ACAAGGAATGCGACATGTTCCACATCAGAATAGATTACAACAAATTTAGCACTT CATGCAGCGGTGGCCAGCGGAAGTGGGATCTGTTCACATTCGGAGACAGAAT CCTGGCCGAGAGAAACACCATGCAGAACAGTAGATACGTTTACCAGACAGTT AACCTGACCTCTGAGTTCAAGAACCTGTTCGCCACAAAGGATATCGATATAAG CGGGAACCTGAAGGATAGCATCTGCAAGATCGAGGACGTGGGCTTCTTCCGG AAGCTGAGCCAGCTGCTGAGCCTGACACTACAGCTTCGGAACAGCAACGCTG AAACCGGAGAAGATTTCCTGATCAGCCCTGTGGCCGACAAGGACGGCAACTT CTTTGACAGCAGAAACTGCCCCGACAGCCTGCCAAAGGATGCAGACGCGAAT GGCGCTTATAACATTGCCAGGAAGGGCCTGATGCTGGTGGAGCAACTGAAGC GGTGCAAGGACGTGAGCAAGTTCAAGCCTGCTATCAAGAACGAGGACTGGCT GGACTACGTGCAGCGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAA GCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGC TGGGCCTGGACAGCACCTGA

[0097] In some embodiments a ZZFT Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, a ZZFT Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D856 substitution, wherein the position of the D856 substitution is defined with respect to the amino acid numbering of SEQ ID NO:20 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E949 substitution, wherein the position of the E949 substitution is defined with respect to the amino acid numbering of SEQ ID NO:20 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1166 substitution, wherein the position of the R1166 substitution is defined with respect to the amino acid numbering of SEQ ID NO:20 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1203 substitution, wherein the position of the D1203 substitution is defined with respect to the amino acid numbering of SEQ ID NO:20 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZZFT Type V Cas protein is catalytically inactive, for example due to a R1166 substitution in combination with a D856 substitution, a E949 substitution, and/or D1203 substitution.

6.2.5. YYAN Type V Cas Proteins

[0098] In one aspect, the disclosure provides YYAN Type V Cas proteins. YYAN Type V Cas proteins can be further classified as Type V-A Cas proteins. The YYAN Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:25. In some embodiments, the YYAN Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25. In some embodiments, a YYAN Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:25.

[0099] Exemplary YYAN Type V Cas protein sequences and nucleotide sequences encoding exemplary YYAN Type V Cas proteins are set forth in Table 1E.

TABLE-US-00006 TABLE1E YYANTypeVCasSequences SEQID Name Sequence NO. Wildtype KINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYHKH 25 aminoacid FIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKLLD sequence KKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLIEE (withoutN- NLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNGID terminal VYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPAKF methionine) NDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKVSN DVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSGKEE KIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAVQEF KKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKINFDN AELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKKMIY KLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESISKYE DWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFRLAN KDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTHPKN VPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERINDLV RQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKMLET REHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKHGR EKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNGFIF YVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRYDD FGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDVKR QIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSLPC DADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKA Wildtype MKINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYHK 26 aminoacid HFIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKLL sequence(with DKKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLIE N-terminal ENLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNGI methionine) DVYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPAK FNDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKVS NDVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSGK EEKIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAVQ EFKKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKINF DNAELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKK MIYKLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESIS KYEDWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFR LANKDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTH PKNVPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERIN DLVRQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKM LETREHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKH GREKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNG FIFYVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRY DDFGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDV KRQIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSL PCDADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKA Expression MGKINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYH 27 construct(with KHFIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKL N-terminal LDKKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLI methionine, EENLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNG V5-tagandC- IDVYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPA terminalNLS) KFNDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKV aasequence SNDVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSG KEEKIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAV QEFKKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKIN FDNAELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKK MIYKLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESIS KYEDWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFR LANKDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTH PKNVPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERIN DLVRQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKM LETREHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKH GREKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNG FIFYVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRY DDFGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDV KRQIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSL PCDADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKASRKRTAD GSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAAATTAACGCTTTTATCAACTGTTATTCGATGTCCAAGACGTTGCGATTCAA 28 coding GCTTGCGCCCGAATACGAGACGGAAAAGAACCTTTTGGAAAAGGGATTTCTTGAT sequence(with CGCGACAAATTGCGCGCGGACGATTATGATTTAATGAAAAAAGTTATCGATAAAT N-terminal ATCACAAACATTTTATCGATAAAGCGTTGGAAGGTTTCAAATTCGATTTATTGCAA methionine GAGTATGCCGAAGCGTTTTATTCGCAATCGGCCGATGACGACGGCAAAAAACTT andstop GAAGAAATCAAAAAGAAAATGTGCAAGGAGTTGGCGACTTGTTTTTCGAAACAAG codon) ACGAGTTTAAATTACTCGATAAAAAAGAACTGGTCGAAAAACTAATCCCTGCTGCC GAATTTATTGAAGACGAAGAAAAAGATATTGCGAAGAGATTCAAGGGGTTTACGA CCTATTTCACGGGATTCAACGAAAACAGGCAAAACTTATACGCCGCAGAACTGAA ACACGGGACGATTGCGTTCAGATTGATTGAAGAAAATTTGCCTGCATTTTTGTACA ACTGCAAAAAGGGAGTAAAAATATTCGAGGGACTCGACGCAGTCGATGCAGAAA CGCTTAATAATGAACTTGGAGAGATTCTTTCAATCGAAAACGTAAAAGATGTATTA AGCGTAGAGTATTACAATAAAACGCTCACGCAAAACGGCATAGACGTTTACAACC GGATTATAGGCGGCTATACACAGGAAGACGGGACGAAAATCAAAGGTGTCAACG AGTACGTCAATTTGTATAACCAGACGCACGACAAAAAACTTCCGTCGCTCGCAAA ACTCAAAAAACAGATTTTAAGCGACAGTTATTCGTTGTCGTTTTTGCCCGCAAAAT TCAACGACGATTCCGAATTGCTTTTATCGCTTAAAAAGTTTTATTCGACGGTAAAC GAAGAGACCGGTTTAAGCGTAGAAAAGGCGATACAGGAAATGCGCGACGTTTTT TCACACATCGATGACTGTGATTTGCATAACGTTTTTATCGACGCAAAATTTATAAA CAAGGTTTCAAACGACGTTTTCGGGAATTGGAGCGTTTTGATTGACGGCATAAAT GCGGAATATGAGAAACTCAATCCGTTCAACGGGAAAAACCTCGACAATTATGAGG AAAAACGCAAAGCGTTTTTAAACAAGATCGAAAGCTATTCTGTTGACGCGTTGCA GGCATATTCGGGTAAAGAAGAAAAAATCGCCGACTACGTTCAAAAACGTGCGGTC GAACTTTACGATAGTGTCGCATGCGCATATGAGAATATGAGTAATAAGGTAATAAA TGCGCGAGAAGGGAAGGTTAAACTTTATCAGGACGATGAAAAAACCGAAATAATC AAAACGTTTTTGGACGCGGTACAGGAATTCAAAAAGTTTGCCGAGATGTTTTGCT ATGACGGCACCGACGGCGATACGACGTTTTACGGCGAATTTGCGAATTATTACG GACAAATTGCCGAAATTATACCGCTTTACAATAAATGCAGGAACTATTTGACGAAA AAGCCGTATTCCGAAGACAAAATCAAAATAAACTTTGACAACGCTGAGCTTTTGCA TGGATGGGACGCAAACAAAGAAAAGAATTATCTGACTGTATTATTATTTAAAAACG GCAGTTATTATCTCGGTATTCTGGATAAAAAGCATAAGAACGTTTTGATCAAAGAC GTGCCCGAAAAGACGCAGGAGGAGCCGTGTTTCAAGAAAATGATTTACAAATTAC TCCCTGATCCGAAACGAAATATGCCTAGAATAATATTACATGCAAAAAGTAACAAG AAGTTGTTTGAGCCTAGTGATGAGATATATAGGATATATGAAACAGAATCGTTTAA AACTGACATTGACGACTGCCATAGGTTGATTGATTTTTATAAAGAAAGTATAAGCA AGTACGAGGACTGGAAGACGTTCGGGTTCAAGTTCAAAGAAACGAGCGAGTATA AAAACATAGGGCAATTTTATAACGAAGTTAAAGAGCAGGGATATAAGATTTCATTC ACGGATATACCCGAAAGTTACGTCAAAGACTTGGTAAACGACGGGAAACTGTATT TATTCAGGCTTGCTAATAAAGATTTTTCTCCGTACAGCAAGGGCAAAAAGAATTTG CATACGATGTATTTCGAGGGAATATTTGATCCTGAAAACATAAAAGAAAAGGTTTA TGCGCTTAACGGCGGCGGCGAGTTGTTTTTCAGATGCGCGAGCTTGAATTACGA CAAACCGACGCATCCGAAAAACGTACCGATTAAAAACAAAACGTATGATTTCCGC ACCGATAATGCGAAAAAAGAAACAAGCACGTTTGAATACGACCTCATAAAAGATA AGCGATATACGAAAGATCAATACACGTTGCATTGTCCGGTGACGCTTAATTTTAA GGAAAGAGGAATCGAAAGAATAAACGATCTCGTAAGGCAATCGTTGCGTGAAAGT GACGACAACTACGTAATCGGCATTGATCGGGGCGAAAGAAACTTAATTTACATCA GTGTTATCGACGGAAAAGGAAAGATTGTCGAGCAATTCTCGATGAACAATTTGTT AAGCGGTAACGACGTGTCGATAGATTTCCACAAAATGCTCGAAACGCGGGAGCA CGAGCGCGACGCGTCCAGAAAAAACTGGAATACAATCGACAATATCAAAGACTTG AAGCAAGGATATTTAAGTTATGTCGTAAAGAAAATTTGCGACCTTGTCGTAAAATA CGACGCGATTGTCGCAATGGAAGACTTAAACGTCGGGTTCAAGCACGGACGAGA AAAGTTCGAGCGACAGGTATATCAGAAATTTGAAAAAGCACTTGTCGACAAAATG AGTTATATCGTAAACAAGAACGCGTCGCCGCATTCCGACGGAGGTTTGTTCAGG GCATACCAGCTGACCAATAAAAAGTATAATGAAAACGAAAAACAAAACGGTTTTAT TTTCTATGTCAGAGCGTGGAATACCAGTAAGATCGATCCGACGACCGGGTTTGTA AACATGCTTCCGTTAAAATATCAGAGCAAAGAAAAATCAAAAGAATTTTTCGATAA ATTTGAAGATATTTTTTACGATGAAAACAAGGATATGTTCGGTTTTACATTCAGATA TGACGATTTCGGTATAAATATCGATCATAAAAACGAATGGACGGCTTATTCAAACG GCGAACGAATAATCACCGTACGAAATTCGTTCGGCAAGTGGGATAAAGCGAAGA TCGTATTGACGCCGGCATTTAAGAAACTGTTTGACGACTATAACGTGGATTGTCG CGGCGACGTCAAACGACAGATTATGAACGTTGACGACAAAGACTTTTTCGTTAGG TTATATAAGCTTTTGTCGTATACGATGCAGTTGAGAAACTCCGACGATGTTGACGA CTATATTTTGTCGCCCGTCGTTAATGCGGAAGGGAAGTTCTTTGACAGTCGCAAT TCGGACGGCAGTTTGCCTTGCGACGCGGACGCAAACGGAGCGTATCATATTGCC AAAAAGGCAATGTGGGCAATCGGGAAGATAAAAGAAGCGGACGAAGAAAGTTTT AAAAAGACAAGTCTTGCAATCGACAACAAGACGTGGCTTGAATTCGTTCAAAAGG CATAA Codon AAGATCAACGCTTTTATCAACTGTTACAGCATGAGCAAGACCCTGAGATTCAAGC 29 optimized TGGCCCCTGAGTACGAAACCGAGAAGAACCTGCTGGAAAAGGGCTTTCTGGACC coding GGGACAAGCTGAGAGCCGACGACTACGACCTGATGAAGAAGGTGATAGACAAGT sequence(no ACCACAAGCACTTCATCGACAAGGCCCTGGAAGGCTTCAAGTTTGACCTGCTGC N-terminal AAGAATACGCTGAGGCCTTTTACAGCCAGAGCGCCGACGACGACGGCAAGAAGC methionine,no TCGAAGAGATCAAGAAGAAGATGTGCAAGGAGCTGGCCACATGCTTCAGCAAGC stopcodon) AAGACGAGTTCAAGCTACTGGATAAGAAAGAGCTGGTGGAAAAGCTGATCCCAG CCGCTGAGTTCATCGAGGACGAGGAAAAAGACATTGCCAAGAGATTCAAAGGCT TTACAACCTACTTTACCGGCTTCAATGAAAACAGACAGAATCTGTACGCCGCCGA GCTGAAGCACGGAACAATCGCCTTCAGACTGATCGAGGAGAACTTGCCTGCCTT CCTGTACAATTGCAAGAAGGGTGTTAAGATCTTCGAGGGCCTGGACGCTGTGGA TGCTGAGACTCTCAACAACGAGCTGGGCGAGATCCTGAGCATCGAAAACGTGAA GGACGTGCTGTCCGTGGAGTACTACAACAAAACCCTGACCCAAAACGGCATCGA TGTGTACAATAGAATCATCGGCGGCTACACCCAGGAGGATGGCACCAAGATCAA GGGAGTGAACGAGTACGTGAACCTGTATAACCAGACACACGACAAGAAACTGCC TTCTCTGGCTAAGCTGAAGAAGCAAATCCTGTCTGACTCCTATTCTCTGTCATTCC TGCCCGCCAAGTTTAACGACGACTCTGAGCTCCTGCTCAGCCTGAAGAAGTTTTA CAGCACCGTGAACGAGGAAACAGGACTGAGCGTGGAGAAAGCTATCCAGGAGAT GAGAGATGTGTTCAGCCACATTGACGACTGCGACCTTCACAACGTCTTTATCGAT GCCAAGTTCATCAACAAGGTGAGCAACGACGTGTTCGGCAACTGGTCGGTCCTG ATCGATGGCATCAATGCCGAGTACGAGAAGCTGAACCCCTTCAACGGCAAGAAC CTGGACAACTACGAGGAAAAAAGAAAGGCCTTTCTGAACAAAATCGAGAGCTATA GCGTGGACGCCCTGCAGGCCTACAGCGGCAAGGAAGAGAAGATCGCCGATTAT GTGCAGAAACGGGCCGTTGAACTGTACGACAGCGTGGCTTGTGCTTACGAAAAC ATGAGCAACAAAGTGATCAACGCCCGGGAAGGCAAGGTGAAGCTGTACCAGGAC GACGAAAAGACCGAGATTATCAAGACCTTCCTGGATGCTGTTCAGGAGTTCAAGA AGTTCGCCGAAATGTTCTGCTACGATGGAACAGATGGAGATACCACCTTCTACGG CGAGTTCGCCAATTATTACGGCCAGATCGCCGAGATAATCCCCCTGTACAACAAG TGCAGAAACTATCTGACAAAGAAACCTTACAGCGAGGACAAGATTAAGATCAACT TCGATAACGCGGAACTGCTGCATGGATGGGACGCCAACAAGGAAAAGAACTACC TGACAGTCCTGCTGTTCAAAAATGGATCATATTACCTGGGCATCCTGGATAAAAA GCATAAGAACGTGCTGATTAAGGACGTTCCTGAAAAGACACAGGAAGAGCCCTG TTTCAAAAAAATGATCTACAAGCTGCTGCCTGATCCCAAGCGGAATATGCCTAGG ATCATCTTGCACGCCAAAAGCAATAAAAAACTGTTCGAGCCTAGCGATGAGATCT ACAGAATCTATGAGACAGAGAGCTTCAAGACCGACATCGACGATTGCCACAGACT GATCGATTTCTACAAGGAATCCATCAGCAAGTACGAGGACTGGAAAACCTTTGGA TTTAAATTCAAAGAAACCAGCGAGTACAAGAACATCGGACAGTTCTACAACGAGG TGAAGGAACAGGGCTACAAGATTAGCTTCACCGACATCCCTGAGAGCTACGTGA AGGATCTGGTGAATGATGGCAAGCTGTATCTGTTTAGACTCGCCAACAAGGATTT CTCTCCATACTCCAAGGGCAAAAAGAACCTGCACACCATGTACTTCGAGGGAATC TTCGACCCCGAAAACATCAAGGAGAAAGTGTACGCCCTGAACGGCGGCGGCGA GCTGTTCTTCCGCTGTGCCTCTCTGAACTACGACAAGCCTACCCACCCCAAGAAC GTGCCTATCAAGAACAAGACCTACGATTTTAGAACCGATAACGCTAAGAAAGAAA CCAGTACATTCGAGTACGACCTGATCAAAGATAAACGGTACACAAAGGACCAGTA CACACTGCACTGCCCTGTGACACTGAATTTCAAGGAGCGTGGAATCGAACGCAT CAACGACCTGGTGCGGCAGAGCCTGCGGGAAAGCGACGACAACTACGTCATCG GCATCGACAGAGGGGAGAGAAATCTGATCTACATCTCTGTGATCGACGGCAAGG GCAAGATCGTCGAGCAGTTCAGCATGAACAACCTGCTGTCCGGCAACGACGTCA GCATCGACTTCCACAAGATGCTGGAAACCAGAGAGCACGAGCGGGACGCCTCCA GAAAGAACTGGAACACCATCGACAACATCAAGGACCTGAAGCAGGGCTACCTGA GTTACGTGGTGAAAAAGATCTGCGACCTGGTCGTGAAGTATGATGCCATCGTGG CTATGGAGGATCTGAACGTGGGCTTTAAACACGGCAGAGAGAAGTTCGAGAGAC AGGTGTACCAGAAGTTTGAGAAAGCCCTGGTGGACAAGATGAGCTACATCGTGA ATAAAAATGCTAGTCCTCACAGCGATGGCGGCCTGTTCAGAGCTTATCAGCTGAC CAACAAGAAATACAACGAGAATGAAAAGCAGAACGGATTCATCTTTTACGTGAGA GCCTGGAATACCAGCAAGATCGACCCAACAACAGGCTTCGTGAACATGTTGCCA CTGAAATACCAATCTAAGGAAAAGTCCAAGGAGTTCTTCGACAAGTTCGAGGATA TCTTCTATGATGAAAACAAAGACATGTTCGGCTTCACCTTCCGGTACGACGACTT CGGCATCAACATCGACCACAAGAATGAATGGACCGCCTACAGCAATGGTGAGCG GATCATCACCGTGCGGAACAGCTTCGGCAAATGGGATAAAGCGAAGATCGTGCT GACCCCTGCTTTTAAGAAGCTGTTCGATGATTACAACGTGGACTGCAGAGGCGA CGTGAAGCGACAGATTATGAACGTGGACGACAAAGATTTCTTCGTGCGGCTGTA CAAGCTGCTGAGCTACACCATGCAGCTGAGAAACAGCGACGACGTGGACGATTA CATCCTGAGCCCCGTGGTGAATGCCGAAGGCAAGTTCTTCGACAGCAGAAACTC TGACGGCTCTCTGCCTTGTGACGCCGATGCCAACGGCGCCTACCACATCGCCAA GAAGGCCATGTGGGCCATCGGCAAGATCAAGGAAGCCGATGAGGAATCTTTTAA GAAAACCTCCCTCGCCATCGACAACAAAACCTGGCTGGAGTTCGTGCAGAAAGC C Expression ATGggcAAGATCAACGCTTTTATCAACTGTTACAGCATGAGCAAGACCCTGAGATT 30 construct(with CAAGCTGGCCCCTGAGTACGAAACCGAGAAGAACCTGCTGGAAAAGGGCTTTCT N-terminal GGACCGGGACAAGCTGAGAGCCGACGACTACGACCTGATGAAGAAGGTGATAG methionine ACAAGTACCACAAGCACTTCATCGACAAGGCCCTGGAAGGCTTCAAGTTTGACCT andstop GCTGCAAGAATACGCTGAGGCCTTTTACAGCCAGAGCGCCGACGACGACGGCAA codon, GAAGCTCGAAGAGATCAAGAAGAAGATGTGCAAGGAGCTGGCCACATGCTTCAG includesV5- CAAGCAAGACGAGTTCAAGCTACTGGATAAGAAAGAGCTGGTGGAAAAGCTGAT tagandC- CCCAGCCGCTGAGTTCATCGAGGACGAGGAAAAAGACATTGCCAAGAGATTCAA terminalNLS) AGGCTTTACAACCTACTTTACCGGCTTCAATGAAAACAGACAGAATCTGTACGCC GCCGAGCTGAAGCACGGAACAATCGCCTTCAGACTGATCGAGGAGAACTTGCCT GCCTTCCTGTACAATTGCAAGAAGGGTGTTAAGATCTTCGAGGGCCTGGACGCT GTGGATGCTGAGACTCTCAACAACGAGCTGGGCGAGATCCTGAGCATCGAAAAC GTGAAGGACGTGCTGTCCGTGGAGTACTACAACAAAACCCTGACCCAAAACGGC ATCGATGTGTACAATAGAATCATCGGCGGCTACACCCAGGAGGATGGCACCAAG ATCAAGGGAGTGAACGAGTACGTGAACCTGTATAACCAGACACACGACAAGAAA CTGCCTTCTCTGGCTAAGCTGAAGAAGCAAATCCTGTCTGACTCCTATTCTCTGT CATTCCTGCCCGCCAAGTTTAACGACGACTCTGAGCTCCTGCTCAGCCTGAAGAA GTTTTACAGCACCGTGAACGAGGAAACAGGACTGAGCGTGGAGAAAGCTATCCA GGAGATGAGAGATGTGTTCAGCCACATTGACGACTGCGACCTTCACAACGTCTTT ATCGATGCCAAGTTCATCAACAAGGTGAGCAACGACGTGTTCGGCAACTGGTCG GTCCTGATCGATGGCATCAATGCCGAGTACGAGAAGCTGAACCCCTTCAACGGC AAGAACCTGGACAACTACGAGGAAAAAAGAAAGGCCTTTCTGAACAAAATCGAGA GCTATAGCGTGGACGCCCTGCAGGCCTACAGCGGCAAGGAAGAGAAGATCGCC GATTATGTGCAGAAACGGGCCGTTGAACTGTACGACAGCGTGGCTTGTGCTTAC GAAAACATGAGCAACAAAGTGATCAACGCCCGGGAAGGCAAGGTGAAGCTGTAC CAGGACGACGAAAAGACCGAGATTATCAAGACCTTCCTGGATGCTGTTCAGGAG TTCAAGAAGTTCGCCGAAATGTTCTGCTACGATGGAACAGATGGAGATACCACCT TCTACGGCGAGTTCGCCAATTATTACGGCCAGATCGCCGAGATAATCCCCCTGTA CAACAAGTGCAGAAACTATCTGACAAAGAAACCTTACAGCGAGGACAAGATTAAG ATCAACTTCGATAACGCGGAACTGCTGCATGGATGGGACGCCAACAAGGAAAAG AACTACCTGACAGTCCTGCTGTTCAAAAATGGATCATATTACCTGGGCATCCTGG ATAAAAAGCATAAGAACGTGCTGATTAAGGACGTTCCTGAAAAGACACAGGAAGA GCCCTGTTTCAAAAAAATGATCTACAAGCTGCTGCCTGATCCCAAGCGGAATATG CCTAGGATCATCTTGCACGCCAAAAGCAATAAAAAACTGTTCGAGCCTAGCGATG AGATCTACAGAATCTATGAGACAGAGAGCTTCAAGACCGACATCGACGATTGCCA CAGACTGATCGATTTCTACAAGGAATCCATCAGCAAGTACGAGGACTGGAAAACC TTTGGATTTAAATTCAAAGAAACCAGCGAGTACAAGAACATCGGACAGTTCTACAA CGAGGTGAAGGAACAGGGCTACAAGATTAGCTTCACCGACATCCCTGAGAGCTA CGTGAAGGATCTGGTGAATGATGGCAAGCTGTATCTGTTTAGACTCGCCAACAAG GATTTCTCTCCATACTCCAAGGGCAAAAAGAACCTGCACACCATGTACTTCGAGG GAATCTTCGACCCCGAAAACATCAAGGAGAAAGTGTACGCCCTGAACGGCGGCG GCGAGCTGTTCTTCCGCTGTGCCTCTCTGAACTACGACAAGCCTACCCACCCCA AGAACGTGCCTATCAAGAACAAGACCTACGATTTTAGAACCGATAACGCTAAGAA AGAAACCAGTACATTCGAGTACGACCTGATCAAAGATAAACGGTACACAAAGGAC CAGTACACACTGCACTGCCCTGTGACACTGAATTTCAAGGAGCGTGGAATCGAA CGCATCAACGACCTGGTGCGGCAGAGCCTGCGGGAAAGCGACGACAACTACGT CATCGGCATCGACAGAGGGGAGAGAAATCTGATCTACATCTCTGTGATCGACGG CAAGGGCAAGATCGTCGAGCAGTTCAGCATGAACAACCTGCTGTCCGGCAACGA CGTCAGCATCGACTTCCACAAGATGCTGGAAACCAGAGAGCACGAGCGGGACG CCTCCAGAAAGAACTGGAACACCATCGACAACATCAAGGACCTGAAGCAGGGCT ACCTGAGTTACGTGGTGAAAAAGATCTGCGACCTGGTCGTGAAGTATGATGCCAT CGTGGCTATGGAGGATCTGAACGTGGGCTTTAAACACGGCAGAGAGAAGTTCGA GAGACAGGTGTACCAGAAGTTTGAGAAAGCCCTGGTGGACAAGATGAGCTACAT CGTGAATAAAAATGCTAGTCCTCACAGCGATGGCGGCCTGTTCAGAGCTTATCAG CTGACCAACAAGAAATACAACGAGAATGAAAAGCAGAACGGATTCATCTTTTACG TGAGAGCCTGGAATACCAGCAAGATCGACCCAACAACAGGCTTCGTGAACATGT TGCCACTGAAATACCAATCTAAGGAAAAGTCCAAGGAGTTCTTCGACAAGTTCGA GGATATCTTCTATGATGAAAACAAAGACATGTTCGGCTTCACCTTCCGGTACGAC GACTTCGGCATCAACATCGACCACAAGAATGAATGGACCGCCTACAGCAATGGT GAGCGGATCATCACCGTGCGGAACAGCTTCGGCAAATGGGATAAAGCGAAGATC GTGCTGACCCCTGCTTTTAAGAAGCTGTTCGATGATTACAACGTGGACTGCAGAG GCGACGTGAAGCGACAGATTATGAACGTGGACGACAAAGATTTCTTCGTGCGGC TGTACAAGCTGCTGAGCTACACCATGCAGCTGAGAAACAGCGACGACGTGGACG ATTACATCCTGAGCCCCGTGGTGAATGCCGAAGGCAAGTTCTTCGACAGCAGAA ACTCTGACGGCTCTCTGCCTTGTGACGCCGATGCCAACGGCGCCTACCACATCG CCAAGAAGGCCATGTGGGCCATCGGCAAGATCAAGGAAGCCGATGAGGAATCTT TTAAGAAAACCTCCCTCGCCATCGACAACAAAACCTGGCTGGAGTTCGTGCAGAA AGCCtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGA GAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCAC CTGA

[0100] In some embodiments a YYAN Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, a YYAN Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D838 substitution, wherein the position of the D838 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E928 substitution, wherein the position of the E928 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1135 substitution, wherein the position of the R1135 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1170 substitution, wherein the position of the D1170 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a YYAN Type V Cas protein is catalytically inactive, for example due to a R1135 substitution in combination with a D838 substitution, a E928 substitution, and/or D1170 substitution.

6.2.6. ZZGY Type V Cas Proteins

[0101] In one aspect, the disclosure provides ZZGY Type V Cas proteins. ZZGY Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZGY Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:31. In some embodiments, the ZZGY Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31. In some embodiments, a ZZGY Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:31.

[0102] Exemplary ZZGY Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZGY Type V Cas proteins are set forth in Table 1F.

TABLE-US-00007 TABLE1F ZZGYTypeVCasSequences SEQ ID Name Sequence NO. Wildtype SKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHREFI 31 aminoacid EKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVFGS sequence YTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYENRK (withoutN- NLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLNIEL terminal KFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLYKQI methionine) LSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFVQG KNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFSISF LSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFYRFA QSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFENPNLA SGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRYLLFK GFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVKKYQK EYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYEDVDNL TYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKNLEN GIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENIYAF VNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPSKFN DKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVDYH NKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIVVMENLNQGF KRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIGN QCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKFSI DYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEYQK GQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDTDK VEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQITL RD Wildtype MSKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHRE 32 aminoacid FIEKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVFG sequence(with SYTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYENR N-terminal KNLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLNI methionine) ELKFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLYK QILSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFVQ GKNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFSI SFLSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFYR FAQSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFENP NLASGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRYL LFKGFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVKK YQKEYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYEDV DNLTYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKNL ENGIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENIY AFVNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPSK FNDKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVDY HNKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIVVMENLNQ GFKRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIG NQCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKF SIDYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEY QKGQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDT DKVEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQ ITLRD Expression MGSKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHR 33 construct(with EFIEKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVF N-terminal GSYTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYEN methionine, RKNLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLN V5-tagandC- IELKFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLY terminalNLS) KQILSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFV aasequence QGKNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFS ISFLSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFY RFAQSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFEN PNLASGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRY LLFKGFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVK KYQKEYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYED VDNLTYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKN LENGIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENI YAFVNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPS KFNDKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVD YHNKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIVVMENLNQ GFKRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIG NQCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKF SIDYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEY QKGQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDT DKVEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQ ITLRDSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGTCTAAATTATCAACTTTTAATGAACATTTTCAAAAAACGTTAACTTTAAGAAAC 34 coding GAACTAGTTCCTGTAGGAAAAACTCTTGAAAATATCATATCTTCAAATGTATTGATA sequence(with AATGATGAGAAAAGAAGTGAAGATTATAAAAAGGCTAAAGAGATCATAGATTCTTA N-terminal TCATCGAGAGTTTATAGAGAAATCACTTTCATCAGTAAATGTTGATTGGAATGATC methionine TGTACTCGTATTTATCCAAAAAAGAACCAGAAGACTATGCTCAAAAGCAGAAGTTC andstop CTCGAAGAGTTAGAAAATATTCTCCTTGAAAAGAGAAAAATTATTGTTAAACAGTT codon) TGAGCAATACGTTTTCGGATCATATACAGATTCAAAAGGTAAAAAAACAAAAGATC TAAAATTTGAGAATCTTTTTAAATCAGAGTTGTTTGATTATCTTTTGCCAAATTTCC TAAAAAATGATGAAGATAAAAAAGTAATAGGTAGTTTTAATAAATTTACATCGTATT TTACAGGTTTTTACGAAAATCGAAAGAATTTATATAAATCAGAGCCATTGCCAACA GCTGTGGCTTATAGAATAGTTAACGAAAACTTTCCTAAATTCATTTCTAATAAAAAT ATCTTTCGCGTGTGGAAAGATAATGTTCCTCAGTTTATAGAAATAGCGAAAACTAA ACTAAGAGAAGAAGGCATTTCTGATTTAAATATAGAATTAAAATTTGATTTAACTAA TTTCAATTCATGCTTAAATCAAACTGGAATTGATACTTACAATGACTTGATAGGTCA ACTCAACTTTGCAATTAACCTTGAATGTCAGAAAGACAAGAATTTATGTGACCTTT TAAGGAAGAAAAGAAGCCTTAAAATGGTACCTCTGTATAAACAGATTTTATCTGAT AATGATTCTTCATTCAGTATTGATGAATTTGATAATGATGAATCGGCAATAAAAGAT GTAATTTCTTTTTATAAGAAAATGATTGGTGAAAATTGTCCTCAACGAACACTATCT GAATTGCTACATGGTTTGTCATCTCACGATCTTGAAAAGATATTTGTTCAAGGTAA AAACTTAAATTCGGTTTCTAAAAATTTATTTGGAGGGAAGAACTGGTCTTTACTAA GGGATGCAGTTATAGAAGAAAAGTCAAAAGAAAAAGTCTTCAAAAAGGTTATAAA GTCAAATTCTACCGCAGATGAATTAGACAAAGTTCTTTCCAAGGAAGAATTTTCAA TTTCATTCTTATCAAAAGTGAGCGGTAAAGATTTATCAGTAGAAATTGATAAATTTG TAAAAAAACAAGACGAACTACTTGTTGAAAATAATATACAAAATTGGCCAAGTTCT CTTAAGAACAGCGAAGAGAAAAATCTCATAAAAGCTCCTTTAGATTTCTTACTTAA TTTTTATAGATTTGCACAATCATTCTCTTCAAATAATATTGATAAGGATATGTCATTT TATGCTGACTTTGATGAATCTCTATCGTCTTTAGAAAATGTAATAGGTCTTTATAAC AAAGTCAGAAACTATGCAACTAAGAAACCTTATACACTCGAAAAGATCAAATTGAA TTTTGAAAATCCAAATTTAGCTTCTGGATGGAGTGAAAGCAAAGAAAATGATTGTT TATCAATTATCTTATTAAAAGAGAAAAAATATTTTTTAGGAATTTTCAACAAAAATAA TAAACCTAATTTTTCTGAAGGCATTTCTCATTCACTTTCTTCAAATGGTTGCTACAG AAAAATGAGGTATTTATTATTCAAGGGATTCAATAAAATGCTTCCTAAATGTGCTTT TACAGGAGAAGTTAAAGATCATTTTAAAGAATCATCGGATGATTTTTCTCTTTTTAA CAAGGATACTTTTATCTCTCCTCTTGTAATTACCAAAGAGATCTTTGATTTAGCATG TAGTAAAGAAAAGGTAAAAAAATATCAAAAAGAATATGAAAAGATCAATCGTGCTG AATATAGACAATCATTGGTTAAGTGGATTACTTTTGGTCTTAAATTTTTGTCATCAT ATAAAACTACAACTCAATTTGATTTATCAAATTTAAAAAGACCTGAAGAATACTGCG ATCTAAAGGAATTTTATGAAGATGTAGATAATCTTACATACAAGATAGAATTTTTAA ATATAAAAGAAGAAGATGTAGATGCATTGGTTGAAAAAGGTCAACTGTATTTATTT GAAATTCGAAATAAAGATTTTGCAAAAAATGCAAGTGGCACTCCTAATCTACATAC TCTCTATTTTAAAAGTATTTTCGATTCGAAAAATTTAGAGAATGGCATTGTCAAGCT TAATGGTGAAGCAGAGATATTTTATAGAAAGAAAAGCTTGAAGAAAGATGACATAA CTGTTCATCGAGAAGGCAGTTATCTTGTAAATAAGGTGTGTGTCGATCCTAATTCT GGAAAAACAGAACAGATTCCTGACAAAATTTATGAAAATATTTATGCTTTCGTAAA TGGTAAATCAAGAGATTTATCTAAGGAGGATGAAGTATATTATGCAAAAGCCACAA TAAAAAAAGCTACCCATGAGATCGTAAAAGATAGACGCTTTACTGTAGATAAATTC TTTTTCCACTGCCCTATTACTATTAACTATAAATCTAAAGATAAACCTTCAAAATTC AATGACAAGGTTTTAGATTTCTTAAGAAATAATAAAGACATCAACATTATAGGCATA GATCGAGGAGAGAGAAATCTTATTTATGTAACTGTAATTAATCAAAATGGCGAAAT TATTGATTGCAAATCATTTAATACTATCAAACATCAGTCTTCAACAGTGAATTACGA TGTTGATTATCACAACAAATTACAAGAAAGAGAAAAAAATAGAAAAGAAGAAAAGA GATCTTGGAATAGTATTACTAAAATTGCAGATCTCAAAGAAGGCTATCTTTCTGCT GTAATTCATGAAGTTTCATTAATGATGGTTAAGTACAATGCCATTGTCGTTATGGA AAATTTGAATCAAGGTTTTAAGAGAATTAGAGGAGGAATTGCTGAAAGATCCGTAT ACCAAAAATTTGAAAAGATGCTGATAGATAAACTGAATTATTTTGTTATAAAAAATG AAAATTGGACAAATCCTGGTGGGGTCCTCAATGGATATCAGTTAACTAACAAAGT GTCTACAATCAAAGATATCGGTAATCAGTGTGGATTTTTATTTTACGTTCCTGCAA CTTATACCTCAAAGATTGATCCTTCTACAGGCTTTGTTAATTTAATTAATTTCAATA AATATAAAAATTCAGAAGATCGAAGAAAACTCATTTGTAGCTTTGACAAGATATGC TTTGTACAGAATGAGAATTTATTTAAATTTTCTATAGATTATGGAAAATTATGCCCA GATAGCAAAATTGCTATAAAAAAATGGGATGTTTTCTCCTACGGAACAAGAATTAT TAAGGAAAATCTAACAACTGGTCATATAGAAGAAAATCCTGAATACGATCCGACA GAAGAGCTTAAATCTCTGCTTTCCTCAAGAGGAATTGAGTATCAAAAAGGTCAAAA TTTACTAGAAACAATACCTACTAGTGATATGACTAGAGAATTTTGGAATTCTCTTTT CAAGATTTTTAAAGCAATTTTACAAATGAGAAACAGTCTAACTAATTCACCAATAGA CAGGCTTTTATCTCCAGTTAAAGGAAAAGATGGAACCTTCTTTGATACAGATAAAG TAGAAGGTACTAAGTTTGAAAAGTTAAAAGACGCTGATGCAAACGGAGCATATAA CATTGCGTTAAAAGGATTGTTAGTCCTCGAGAAAAATGATTCTGTAGAGTCCAATA AGGATCTAAAAAATGTTAAGAAAATTAGTCTTGAGGATTGGTTAAAGTTTGTCCAA ATCACATTAAGAGATTAA 35 Codon AGCAAATTGTCGACCTTCAATGAGCACTTTCAGAAAACCCTGACCCTGCGGAATG optimized AGCTGGTGCCCGTGGGCAAGACACTGGAGAACATCATCAGCTCTAACGTGCTGA coding TCAACGACGAGAAGCGGTCCGAGGACTACAAAAAGGCCAAGGAAATCATTGACA sequence(no GCTATCACCGGGAGTTCATCGAGAAAAGCCTGAGCTCTGTGAATGTGGACTGGA N-terminal ATGATCTGTACAGCTACCTGAGCAAGAAAGAACCCGAGGACTATGCCCAGAAAC methionine,no AGAAGTTCCTGGAGGAGTTAGAGAACATCCTGCTGGAAAAGAGAAAGATCATCGT stopcodon) GAAGCAGTTCGAGCAGTACGTGTTCGGTTCCTATACCGACAGCAAGGGAAAAAA GACCAAGGACCTGAAATTCGAAAACCTGTTTAAGTCCGAACTCTTTGACTACCTG CTGCCTAACTTCTTGAAAAACGACGAGGATAAGAAGGTGATTGGCTCCTTCAATA AGTTCACCAGCTATTTCACCGGCTTTTACGAGAACAGAAAAAACCTGTACAAGAG CGAGCCTCTGCCTACCGCCGTCGCCTACAGAATCGTGAACGAGAACTTCCCCAA GTTTATCTCTAACAAGAACATCTTTAGAGTGTGGAAGGACAACGTCCCTCAATTCA TCGAGATCGCAAAGACCAAACTGAGAGAAGAAGGCATCTCTGATCTGAACATCGA GCTGAAGTTTGATTTGACAAATTTCAACTCCTGCCTGAATCAGACCGGCATCGAT ACCTACAACGACCTGATCGGCCAGCTGAACTTTGCTATCAACCTCGAATGTCAGA AGGACAAGAACCTTTGTGACCTGCTGCGCAAGAAGCGGAGCCTTAAGATGGTGC CACTGTACAAGCAAATCCTGTCCGACAACGATAGCAGCTTCAGCATCGACGAGTT CGACAATGATGAAAGCGCCATCAAGGACGTTATCAGCTTCTACAAGAAGATGATC GGCGAGAACTGCCCTCAGCGGACCCTGTCTGAGCTGCTGCACGGCCTGTCTAG CCACGATCTGGAGAAAATTTTCGTGCAAGGGAAGAACCTGAACAGCGTGTCCAA GAACCTGTTCGGCGGCAAGAACTGGTCCCTGCTGCGGGACGCCGTGATCGAGG AAAAAAGCAAAGAGAAGGTGTTCAAGAAGGTGATCAAGAGCAACAGCACCGCTG ATGAGCTGGATAAGGTGCTGTCTAAGGAGGAGTTCAGCATCTCTTTCCTATCCAA GGTGTCCGGCAAGGATCTGAGCGTGGAAATCGACAAGTTCGTCAAAAAACAGGA CGAGCTTCTGGTGGAGAACAATATCCAGAACTGGCCTTCTTCTCTCAAGAATAGC GAAGAAAAGAACCTGATCAAGGCCCCTCTGGACTTTTTGTTGAATTTCTACAGGT TCGCCCAGAGCTTCAGCAGCAACAACATCGATAAAGATATGTCCTTCTACGCTGA TTTTGACGAGTCTCTGTCAAGCCTGGAAAATGTGATAGGCCTGTACAACAAAGTG CGGAACTACGCCACCAAGAAACCTTACACACTGGAAAAGATCAAGCTAAACTTCG AGAACCCTAACCTGGCCTCTGGATGGAGTGAGAGCAAGGAAAACGATTGCCTGA GTATCATCCTGCTGAAGGAGAAGAAATACTTCCTGGGCATCTTCAACAAGAACAA CAAGCCCAACTTTTCAGAGGGCATCAGCCACAGCCTGTCAAGCAACGGCTGTTA CCGGAAGATGAGATACCTGCTGTTCAAGGGATTCAACAAGATGCTGCCTAAGTG CGCCTTCACAGGAGAGGTGAAGGACCACTTCAAGGAAAGCTCCGATGACTTCAG CCTGTTCAACAAGGACACCTTCATCAGCCCCCTGGTGATCACCAAGGAAATTTTC GATCTGGCTTGCAGCAAGGAAAAAGTGAAGAAGTACCAAAAAGAATACGAGAAAA TCAACAGAGCCGAGTACCGGCAGTCTCTGGTGAAGTGGATCACCTTTGGCCTGA AGTTTCTGTCTAGCTACAAAACCACCACCCAGTTCGACCTGAGCAATTTGAAGCG CCCCGAGGAATACTGCGACCTGAAAGAATTTTACGAGGACGTGGATAACTTAACC TACAAGATTGAGTTCCTGAACATTAAAGAGGAGGACGTGGACGCTCTGGTCGAG AAAGGCCAGCTGTACCTGTTTGAGATTAGAAACAAGGACTTCGCCAAGAATGCCA GCGGCACGCCCAACCTGCATACACTGTATTTCAAGAGCATCTTCGATAGCAAGAA CCTGGAAAATGGCATCGTGAAACTGAACGGCGAGGCCGAAATTTTCTACAGAAA GAAGAGCCTGAAGAAGGATGATATCACCGTGCACAGAGAGGGAAGCTACCTCGT CAACAAAGTCTGCGTGGACCCTAATTCCGGCAAGACAGAGCAGATCCCAGATAA GATCTACGAGAACATCTACGCCTTCGTCAACGGCAAGTCACGGGACCTGAGCAA GGAGGACGAGGTGTACTACGCCAAAGCCACCATCAAGAAGGCTACCCACGAGAT CGTGAAGGATCGAAGATTCACCGTCGACAAGTTCTTCTTCCACTGCCCCATCACT ATCAACTACAAGAGCAAAGACAAGCCAAGCAAGTTTAACGACAAAGTGCTGGACT TCCTGAGAAATAACAAGGACATCAATATCATCGGCATCGACAGAGGCGAAAGAAA CTTGATCTACGTGACCGTGATCAACCAGAACGGAGAGATCATCGACTGTAAGAG CTTCAATACCATTAAGCACCAGAGCAGCACAGTGAACTACGACGTGGACTACCAC AACAAGCTGCAGGAGCGGGAAAAGAACAGAAAGGAAGAAAAGAGATCTTGGAAC AGCATCACCAAGATCGCCGATCTGAAAGAGGGCTACCTGTCTGCCGTGATTCAC GAGGTTAGCCTGATGATGGTGAAGTACAACGCCATAGTTGTGATGGAAAACCTGA ACCAGGGCTTCAAGAGAATCCGGGGCGGCATCGCCGAACGGAGCGTGTACCAA AAGTTTGAAAAGATGCTCATCGACAAGCTGAACTACTTCGTGATCAAGAACGAGA ACTGGACCAATCCTGGCGGAGTGCTGAATGGATACCAGCTGACAAACAAGGTGT CCACAATCAAGGATATTGGAAATCAGTGCGGCTTCCTGTTCTACGTGCCCGCCAC TTATACATCTAAAATCGATCCTAGCACTGGATTTGTGAACCTGATCAACTTCAACA AGTACAAGAACAGCGAGGACAGAAGGAAGCTGATCTGTAGCTTCGACAAGATCT GCTTTGTGCAGAATGAGAACCTGTTCAAGTTCTCTATCGATTACGGCAAACTGTG CCCTGACAGCAAGATCGCCATCAAAAAGTGGGACGTATTCTCCTATGGCACCAG GATCATCAAGGAAAACCTGACAACAGGCCACATCGAAGAAAATCCAGAGTACGA CCCTACAGAGGAACTGAAATCCCTGCTTTCCAGCAGAGGCATCGAGTACCAGAA GGGCCAAAACCTGCTAGAAACCATCCCTACCAGCGACATGACCAGAGAGTTCTG GAATAGCCTGTTCAAGATCTTCAAGGCCATCCTGCAGATGAGAAACTCTCTGACA AACTCTCCTATCGACCGGCTGCTAAGCCCTGTGAAGGGGAAAGATGGAACCTTC TTCGACACCGACAAGGTGGAAGGCACAAAATTTGAGAAACTGAAGGACGCTGAC GCTAACGGCGCCTACAACATCGCCCTGAAGGGCCTGCTGGTGCTGGAAAAAAAC GACTCTGTCGAGAGCAACAAGGACCTCAAGAACGTGAAGAAAATCTCACTGGAG GACTGGCTGAAATTCGTGCAGATCACACTTAGAGAC Expression ATGggcAGCAAATTGTCGACCTTCAATGAGCACTTTCAGAAAACCCTGACCCTGCG 36 construct(with GAATGAGCTGGTGCCCGTGGGCAAGACACTGGAGAACATCATCAGCTCTAACGT N-terminal GCTGATCAACGACGAGAAGCGGTCCGAGGACTACAAAAAGGCCAAGGAAATCAT methionine TGACAGCTATCACCGGGAGTTCATCGAGAAAAGCCTGAGCTCTGTGAATGTGGA andstop CTGGAATGATCTGTACAGCTACCTGAGCAAGAAAGAACCCGAGGACTATGCCCA codon, GAAACAGAAGTTCCTGGAGGAGTTAGAGAACATCCTGCTGGAAAAGAGAAAGAT includesV5- CATCGTGAAGCAGTTCGAGCAGTACGTGTTCGGTTCCTATACCGACAGCAAGGG tagandC- AAAAAAGACCAAGGACCTGAAATTCGAAAACCTGTTTAAGTCCGAACTCTTTGACT terminalNLS) ACCTGCTGCCTAACTTCTTGAAAAACGACGAGGATAAGAAGGTGATTGGCTCCTT CAATAAGTTCACCAGCTATTTCACCGGCTTTTACGAGAACAGAAAAAACCTGTACA AGAGCGAGCCTCTGCCTACCGCCGTCGCCTACAGAATCGTGAACGAGAACTTCC CCAAGTTTATCTCTAACAAGAACATCTTTAGAGTGTGGAAGGACAACGTCCCTCA ATTCATCGAGATCGCAAAGACCAAACTGAGAGAAGAAGGCATCTCTGATCTGAAC ATCGAGCTGAAGTTTGATTTGACAAATTTCAACTCCTGCCTGAATCAGACCGGCA TCGATACCTACAACGACCTGATCGGCCAGCTGAACTTTGCTATCAACCTCGAATG TCAGAAGGACAAGAACCTTTGTGACCTGCTGCGCAAGAAGCGGAGCCTTAAGAT GGTGCCACTGTACAAGCAAATCCTGTCCGACAACGATAGCAGCTTCAGCATCGA CGAGTTCGACAATGATGAAAGCGCCATCAAGGACGTTATCAGCTTCTACAAGAAG ATGATCGGCGAGAACTGCCCTCAGCGGACCCTGTCTGAGCTGCTGCACGGCCT GTCTAGCCACGATCTGGAGAAAATTTTCGTGCAAGGGAAGAACCTGAACAGCGT GTCCAAGAACCTGTTCGGCGGCAAGAACTGGTCCCTGCTGCGGGACGCCGTGA TCGAGGAAAAAAGCAAAGAGAAGGTGTTCAAGAAGGTGATCAAGAGCAACAGCA CCGCTGATGAGCTGGATAAGGTGCTGTCTAAGGAGGAGTTCAGCATCTCTTTCCT ATCCAAGGTGTCCGGCAAGGATCTGAGCGTGGAAATCGACAAGTTCGTCAAAAA ACAGGACGAGCTTCTGGTGGAGAACAATATCCAGAACTGGCCTTCTTCTCTCAAG AATAGCGAAGAAAAGAACCTGATCAAGGCCCCTCTGGACTTTTTGTTGAATTTCTA CAGGTTCGCCCAGAGCTTCAGCAGCAACAACATCGATAAAGATATGTCCTTCTAC GCTGATTTTGACGAGTCTCTGTCAAGCCTGGAAAATGTGATAGGCCTGTACAACA AAGTGCGGAACTACGCCACCAAGAAACCTTACACACTGGAAAAGATCAAGCTAAA CTTCGAGAACCCTAACCTGGCCTCTGGATGGAGTGAGAGCAAGGAAAACGATTG CCTGAGTATCATCCTGCTGAAGGAGAAGAAATACTTCCTGGGCATCTTCAACAAG AACAACAAGCCCAACTTTTCAGAGGGCATCAGCCACAGCCTGTCAAGCAACGGC TGTTACCGGAAGATGAGATACCTGCTGTTCAAGGGATTCAACAAGATGCTGCCTA AGTGCGCCTTCACAGGAGAGGTGAAGGACCACTTCAAGGAAAGCTCCGATGACT TCAGCCTGTTCAACAAGGACACCTTCATCAGCCCCCTGGTGATCACCAAGGAAAT TTTCGATCTGGCTTGCAGCAAGGAAAAAGTGAAGAAGTACCAAAAAGAATACGAG AAAATCAACAGAGCCGAGTACCGGCAGTCTCTGGTGAAGTGGATCACCTTTGGC CTGAAGTTTCTGTCTAGCTACAAAACCACCACCCAGTTCGACCTGAGCAATTTGA AGCGCCCCGAGGAATACTGCGACCTGAAAGAATTTTACGAGGACGTGGATAACT TAACCTACAAGATTGAGTTCCTGAACATTAAAGAGGAGGACGTGGACGCTCTGGT CGAGAAAGGCCAGCTGTACCTGTTTGAGATTAGAAACAAGGACTTCGCCAAGAAT GCCAGCGGCACGCCCAACCTGCATACACTGTATTTCAAGAGCATCTTCGATAGCA AGAACCTGGAAAATGGCATCGTGAAACTGAACGGCGAGGCCGAAATTTTCTACA GAAAGAAGAGCCTGAAGAAGGATGATATCACCGTGCACAGAGAGGGAAGCTACC TCGTCAACAAAGTCTGCGTGGACCCTAATTCCGGCAAGACAGAGCAGATCCCAG ATAAGATCTACGAGAACATCTACGCCTTCGTCAACGGCAAGTCACGGGACCTGA GCAAGGAGGACGAGGTGTACTACGCCAAAGCCACCATCAAGAAGGCTACCCACG AGATCGTGAAGGATCGAAGATTCACCGTCGACAAGTTCTTCTTCCACTGCCCCAT CACTATCAACTACAAGAGCAAAGACAAGCCAAGCAAGTTTAACGACAAAGTGCTG GACTTCCTGAGAAATAACAAGGACATCAATATCATCGGCATCGACAGAGGCGAAA GAAACTTGATCTACGTGACCGTGATCAACCAGAACGGAGAGATCATCGACTGTAA GAGCTTCAATACCATTAAGCACCAGAGCAGCACAGTGAACTACGACGTGGACTA CCACAACAAGCTGCAGGAGCGGGAAAAGAACAGAAAGGAAGAAAAGAGATCTTG GAACAGCATCACCAAGATCGCCGATCTGAAAGAGGGCTACCTGTCTGCCGTGAT TCACGAGGTTAGCCTGATGATGGTGAAGTACAACGCCATAGTTGTGATGGAAAAC CTGAACCAGGGCTTCAAGAGAATCCGGGGCGGCATCGCCGAACGGAGCGTGTA CCAAAAGTTTGAAAAGATGCTCATCGACAAGCTGAACTACTTCGTGATCAAGAAC GAGAACTGGACCAATCCTGGCGGAGTGCTGAATGGATACCAGCTGACAAACAAG GTGTCCACAATCAAGGATATTGGAAATCAGTGCGGCTTCCTGTTCTACGTGCCCG CCACTTATACATCTAAAATCGATCCTAGCACTGGATTTGTGAACCTGATCAACTTC AACAAGTACAAGAACAGCGAGGACAGAAGGAAGCTGATCTGTAGCTTCGACAAG ATCTGCTTTGTGCAGAATGAGAACCTGTTCAAGTTCTCTATCGATTACGGCAAACT GTGCCCTGACAGCAAGATCGCCATCAAAAAGTGGGACGTATTCTCCTATGGCAC CAGGATCATCAAGGAAAACCTGACAACAGGCCACATCGAAGAAAATCCAGAGTA CGACCCTACAGAGGAACTGAAATCCCTGCTTTCCAGCAGAGGCATCGAGTACCA GAAGGGCCAAAACCTGCTAGAAACCATCCCTACCAGCGACATGACCAGAGAGTT CTGGAATAGCCTGTTCAAGATCTTCAAGGCCATCCTGCAGATGAGAAACTCTCTG ACAAACTCTCCTATCGACCGGCTGCTAAGCCCTGTGAAGGGGAAAGATGGAACC TTCTTCGACACCGACAAGGTGGAAGGCACAAAATTTGAGAAACTGAAGGACGCT GACGCTAACGGCGCCTACAACATCGCCCTGAAGGGCCTGCTGGTGCTGGAAAAA AACGACTCTGTCGAGAGCAACAAGGACCTCAAGAACGTGAAGAAAATCTCACTG GAGGACTGGCTGAAATTCGTGCAGATCACACTTAGAGACtctagaAAGCGGACAGC AGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAAC CTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0103] In some embodiments a ZZGY Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, a ZZGY Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D905 substitution, wherein the position of the D905 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E998 substitution, wherein the position of the E998 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1214 substitution, wherein the position of the R1214 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1254 substitution, wherein the position of the D1254 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZZGY Type V Cas protein is catalytically inactive, for example due to a R1214 substitution in combination with a D905 substitution, a E998 substitution, and/or D1254 substitution.

6.2.7. ZKBG Type V Cas Proteins

[0104] In one aspect, the disclosure provides ZKBG Type V Cas proteins. ZKBG Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZKBG Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:37. In some embodiments, the ZKBG Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:37. In some embodiments, a ZKBG Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:37.

[0105] Exemplary ZKBG Type V Cas protein sequences and nucleotide sequences encoding exemplary ZKBG Type V Cas proteins are set forth in Table 1G.

TABLE-US-00008 TABLE1G ZKBGTypeVCasSequences SEQ ID Name Sequence NO. Wildtype KRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYHKE 37 aminoacid FIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRFG sequence NLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRIV (withoutN- NQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLCQ terminal ADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSLS methionine) FTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQR Wildtype MKRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYHK 38 aminoacid EFIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRFG sequence(with NLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRIV N-terminal NQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLCQ methionine) ADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSLS FTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQR Expression MGKRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYH 39 construct(with KEFIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRF N-terminal GNLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRI methionine, VNQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLC V5-tagandC- QADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSL terminalNLS) SFTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF aasequence GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQRSRKRTADGSEFESPKKKR KVGSGKPIPNPLLGLDST Wildtype ATGAAACGCCTAATTGACTTTACAAACATCTATCAGCGATCAAAGACTTTGAGGTT 40 coding TCGATTGGAGCCTATCGGTAAAACGGCCGACTATATTAAGAATTCTCAGTCCCTC sequence(with GAAACTGATGCGCGTTTGGCAAAAGAGAGCAAGAAGGTAAAAGAGCTTGCTGAT N-terminal GAATATCACAAAGAGTTTATTGGAGATGTCCTGTCTTCGTTGGAATTGCCTTTAAG methionine CAAAATCAACGAGTTATGGGATATATATATATATATATATATGTCCAATGATACAGA andstop CCGCGAGATAAAATTCAAAAAACTGCAAGAGAACCTGCGAAAGGTGATTGCAGA codon) GGCTTTTAGTAAGGACAAACGGTTTGGTAATTTATTCAAAAAGGAGATAATCACAG ACATTCTGCCGGAATTCTTGCAAGATAAGGATGATGATATTAAGATCGTAAATAGA TTCAAAGGATTTACCACATATTTTTACGCCTTTCATAAGAATAGGGAAAATATGTAT GTCTCGGAAGAGAAATCGACTGCAATACCATATCGAATTGTGAATCAAAATCTCG TCAAGTATTTTGACAACTACAAGACGTTCAAAGAGAAGGTAATGCCTCTTCTGAAA GACAAGAATATAGTCGAAAGCATAGAGAGAGACTTCAAAGACATCTTGAACGAAA AATCAATAGAGGATGTTTTTGGCCTTGCCAACTTCACTCATACTTTATGTCAGGCT GACATCGAGAAATACAATACGTTGATAGGTGGCCTTGTCGTCAAAAACGAAAAAA AAGAGATTAAAGGTATTAATCAGTACATTAACGAACATAACCAAACGAGTAAAAAA GGGAATGGAATTCCGAAACTAAAGCCGTTGTTCAATCAGATTTTGAGCGATAGAA AATCGTTATCGTTTACCTTAGACGATATCAAAAAAACGTCGGAGGCTATTCGCAC CATTAAGGATGAGTATGAAAATCTCCGAGACAAGTTGGCGACCATCGAAAGGCTT ATTAAGTCTATCAAGGAGTATGATCTTGCAGGTATTTACATCAAGATGGGAGAGG ATACTTCGACAATATCGCAGCATTGGTTTGGTGCGTATTATAAAATCATCGAAGCG ATAGCAGATGCATGGGAACGACGAAATCCGAAGAAAAACAGAGAATCCAAGGCA TATAGCAAGTATGTATCGTCCCTAAAAAGCATCAGTCTCCAAGAAATAGATGATCT CAAAATCGGAGAGCCTATAGAGAACTACTTCGCAACTTTTGGCACGACTTGTTCA GACCGAACAAGTGGAGTTTCTTCGCTCAATAGGATAAAAGCTGCTTATACCGAGT TCGTGAACAAATTTCCTGAAGGATTTGAAGATGGCGATGACTGTAACGATGCCTA CTTTAAGGCTAATGTGGAAGTCGTCAAAAATCTGCTGGATTCAATTAAAGATTTTC AGCGTTTTGTGAAGCCTTTGCTTGGCAATGAGGACGAAAGAGACAAAGACGAGG CATTCTATGGAGAGTTTGTCCCGACATACACAGATATGGATAACATCATAACCCCT CTATACAACCGTGTACGCAATTTTGCCACCAAGAAACCATACTCTACAGACAAGA TAAAAATCAACTTTGAAAACGTAGTATTGCTAAAAGGATGGGACAAAAACAAGGA GTCAGACTACGCATCCATCATATTGATGAAAGACGGACAATACTTTTTAGGGGTA CTCCGTAATGGTTCAAAAAGTACTCTTAAAACCATATTGCCTAACACAGGTGATTG CTATCAAAAAATGGTTTATAAGTATTTTAAGGATATAAAATCAAATCTTCCCCGGTG TACGACCCAGAGGAAAGACGTGAAAGCGCACTTTGCCGAATCGAGCGACGATTA CACTCTTTTAGATACAAAGGCCTTTGTTTCGGCACTGACTATCAGCAGAGAAGTG TTCGAACTATACAATGCCCCCGATAAGGAGAAAAAATTCAAAAAGGAATATTTGAA GAACACAAACGATAGTATAGGCTACGCCAATGCTGTATCCGTATGTAAACGCTTC TGTTTGGAGTTCCTAAAAAAATATCGCAGCACTGCCATATATGATCTTTCGGATGT TGAAACTTCAGTCGATTCGTTTGACGATTTGTCCTCATTCTATCAAGAGATAGACA AAAGGCTGTACAGCATCTCATTCGAAAATGTATCTGTCGATTCCGTCAATGAGCTT GTAGACAATGGCAATATGCTTCTATTCCGTATCGCGAATAAAGATTTTTCGCCTAA CAGCAAGGGCCGTCCCAATCTTCATACTATATATTGGCGAATGCTTTTCGACCCG GCCAACCTGAAGGATGTTGTATATCAGCTCAATGGTAATGCCGAAATATTCTTCC GTAAGGCAAGCGTTACGAGGACGGAGCCTACACATCCGGCTAACGTTGCCATCA AAAACAAGAGCGAATATAACAAACAGAATAAGCCGTATAGTACATTCAAGTACGG TTTAATCAAGGATAGGCGCTACACTACCGACCAGTTCGAGTTTCATGTACCCATC ACAATGAACTTCAAGCAACCAGAGTCGTCTAAACTACAGGACAAGCTCAACAAGC AAGTGCTTGACTTCTTGAAACAGGACGGCGTACGCCATATTATAGGCATTGATCG GGGCGAACGTAATCTGCTATACTTGGTGATGGTAGATATGGAGGGCAAAATCAAA AAACAAATATCACTCAACGAGATAGCCGGTAATCCGAAGAATCCCGAGTTCAAAC AAGACTTCCTTGCACTACTGCACGAGCGCGAAGGTGACCGTTTGGAGTCACGTC GCAGTTGGAACACCATTCAGAGCATTAAAGAACTCAAAGAAGGTTACATGAGCTT GGTGGTTCATGAAATAGCGAATATGATGCTTGAGAATGATGCTATAGTAGTGCTC GAAAATCTGAATCGCTCGTTTATGCAAAAGCGCGGCGGCATAGAAAAGTCTGTAT ACCAAAAGTTCGAAAAGATGCTTATCGACAAGTTGGGATACATCGTGGATAAGAC TAAAGATGTGTCCGACAACGGAGGCGCACTACATGCTGTACAGCTTGCTGATAC GTTTGAAAACTTCAATAAGACCCAAAAAGGAGCTATTCGTCAATGTGGATTCATAT TCTATATTCCTGCATGGCGTACCAGCAAGATTGACCCCGTTACCGGCTTTGTGCC AATGCTTAGGTGTCAATATGAAAGCATCGTAGAATCCAAAAAATTCTTCGGAAAGT TCGACAGTATATACTACGATGCGACAGGAAAGTATTTTGTCTTCCAAACTGACTTT ACCAAATTCAATACCGAGAGCAAAGGAGGAATCCAAAAATGGGATATATGCACCT ATGGAGACAGAATATATGCTCCTCGCACCAAAGACCGGAATAATAACCCTGTTTC GGAACGTGTAAACCTTACTGAGGAGATGAAATCACTGTTTGTATCGCATAATATCA ATATTCAAGGCGATATCAAAGCCGGAATTATGCAGCAGACAGACAAGGAGTTCTT CGAGTCACTGCATCGATTGCTTCGACTTACGTTGCAAATACGCAATAGCAAAAAA TCTACAGGCAAAGACTATGAAGACTATATCATATCGCCGGTGATGGGCAAGGAC GGTCGTTTCTTTGATTCGCGTAACGCGGATGCTACGCAACCTAAGGATGCAGATG CCAATGGCGCGTACAATATTGCACGCAAAGGCTTGATGCTGCTTCGCCAGATTCA AGCCCAAGAGAAGCAAGACCTATCCAACGGAAAATGGCTTGAATTTGCCCAAAG GTGA Codon AAGCGGCTCATCGACTTCACCAACATCTACCAGCGTTCTAAGACCCTGAGATTCA 41 optimized GACTGGAACCTATCGGCAAGACCGCGGACTACATCAAAAACAGCCAGTCCCTGG coding AAACAGACGCCAGACTGGCCAAGGAATCCAAGAAAGTGAAGGAACTGGCCGATG sequence(no AGTACCACAAAGAGTTTATCGGCGACGTGCTGAGCAGCCTGGAGCTGCCCCTGA N-terminal GCAAAATCAACGAGCTGTGGGACATCTATATCTACATCTACATGAGCAACGACAC methionine,no CGATCGGGAAATCAAATTTAAGAAGCTCCAGGAGAACCTGCGGAAGGTGATCGC stopcodon) CGAGGCCTTTAGCAAGGATAAGAGATTCGGCAACCTGTTCAAGAAAGAAATCATC ACAGATATCCTGCCCGAGTTCCTGCAAGATAAAGATGACGATATCAAAATCGTGA ACCGGTTCAAGGGTTTTACAACCTACTTCTACGCCTTCCACAAGAATCGGGAAAA CATGTACGTGTCTGAAGAGAAGAGCACAGCCATCCCCTACAGAATCGTGAATCAA AACCTGGTGAAATACTTCGATAACTACAAGACTTTTAAGGAGAAGGTGATGCCTC TGCTGAAGGACAAGAACATCGTCGAAAGCATCGAGCGCGACTTCAAGGACATCC TGAACGAGAAAAGCATCGAGGACGTGTTCGGCCTGGCCAATTTCACCCACACCC TGTGCCAGGCTGACATCGAGAAGTACAACACCTTGATAGGCGGACTGGTGGTGA AGAACGAAAAGAAGGAGATCAAGGGCATCAACCAGTATATTAACGAGCACAACCA GACCTCTAAGAAGGGCAACGGCATCCCAAAGCTGAAGCCTCTGTTTAACCAGAT CCTGAGCGACAGAAAATCTCTCAGCTTCACCCTGGATGATATCAAGAAAACCAGC GAGGCCATCAGAACAATTAAGGACGAGTATGAGAACCTGAGAGATAAGCTGGCC ACAATCGAACGGCTGATCAAGAGCATCAAGGAATACGACCTGGCCGGCATCTAC ATCAAGATGGGCGAGGACACCTCTACCATCTCCCAGCACTGGTTCGGTGCCTAT TACAAGATTATCGAAGCCATCGCCGACGCCTGGGAGAGAAGAAACCCAAAGAAA AACAGAGAGAGCAAGGCCTACAGCAAGTACGTGAGCAGCCTTAAGAGCATCAGC CTGCAGGAGATCGACGACCTGAAGATCGGCGAGCCTATCGAGAATTACTTCGCC ACCTTTGGAACAACATGTAGCGACCGGACATCTGGCGTGAGCTCTCTGAACCGG ATCAAAGCCGCCTACACCGAGTTCGTGAACAAGTTCCCCGAGGGCTTTGAGGAT GGCGATGATTGCAACGACGCTTACTTCAAAGCCAATGTGGAGGTGGTGAAGAAC TTGCTGGATAGCATAAAAGACTTCCAGAGATTTGTGAAGCCTCTACTGGGCAATG AGGACGAGCGGGACAAAGATGAGGCCTTCTACGGCGAGTTCGTTCCTACCTACA CAGATATGGACAACATCATCACGCCTCTGTATAATAGAGTCAGAAACTTCGCTAC CAAGAAGCCTTACAGTACAGACAAGATCAAAATAAACTTCGAAAACGTGGTACTG CTGAAGGGCTGGGATAAGAACAAGGAGAGCGACTATGCCAGCATCATCCTGATG AAGGACGGCCAGTACTTTCTGGGAGTGCTGAGAAACGGATCTAAGAGCACTCTG AAAACCATCCTGCCTAACACCGGTGACTGCTACCAGAAAATGGTGTACAAGTATT TCAAGGATATCAAGTCTAACCTGCCCAGATGCACCACCCAGAGAAAGGACGTGA AGGCACATTTCGCTGAAAGCAGCGATGATTACACCCTGCTTGATACAAAAGCCTT CGTGAGCGCTCTGACGATCTCCAGAGAGGTGTTCGAACTGTACAACGCTCCTGA TAAGGAAAAGAAATTCAAGAAGGAATACCTGAAGAACACCAACGACTCCATCGGC TACGCCAATGCAGTGAGCGTGTGCAAGAGATTCTGCCTGGAGTTCCTGAAAAAG TACCGGAGCACCGCCATCTACGACCTGAGCGATGTTGAAACCTCTGTGGACAGT TTCGACGACCTGAGCAGCTTCTACCAGGAGATCGATAAGAGACTGTACAGCATCA GCTTCGAAAACGTGAGCGTGGACAGCGTGAACGAGCTGGTGGATAACGGCAATA TGCTGCTGTTCAGAATCGCCAACAAGGATTTCTCTCCTAATAGCAAGGGCAGACC TAATCTGCACACAATTTACTGGAGAATGCTGTTCGACCCTGCTAATCTCAAGGAC GTCGTGTACCAACTGAACGGCAATGCCGAAATCTTCTTCCGGAAGGCCAGCGTT ACAAGGACAGAACCAACACACCCCGCCAATGTGGCCATCAAGAACAAGAGCGAG TACAACAAGCAGAACAAACCTTACAGCACCTTCAAGTACGGCCTCATCAAGGACC GGCGATACACCACCGATCAGTTCGAGTTCCACGTGCCTATCACCATGAACTTCAA GCAACCTGAGTCATCTAAGCTGCAGGACAAACTGAATAAGCAAGTGCTGGACTTC CTGAAGCAAGACGGCGTGCGGCACATCATCGGCATCGACCGGGGAGAAAGAAA CCTGCTGTACCTGGTGATGGTCGACATGGAAGGAAAAATCAAGAAGCAGATCAG CCTGAATGAAATCGCCGGAAACCCAAAGAACCCTGAGTTTAAGCAGGACTTCTTA GCTCTGCTGCATGAGAGAGAGGGCGATAGACTGGAGTCCAGAAGAAGTTGGAAC ACCATCCAGAGCATCAAGGAGCTGAAAGAAGGCTACATGTCCCTGGTGGTGCAC GAGATCGCTAACATGATGCTGGAGAATGATGCCATCGTGGTCTTGGAAAACCTTA ACAGATCCTTTATGCAGAAGAGAGGCGGCATTGAGAAAAGCGTGTACCAGAAGT TTGAGAAAATGCTGATCGACAAGCTGGGCTACATCGTGGACAAAACAAAAGATGT GTCAGATAATGGCGGAGCCCTGCACGCCGTGCAGCTGGCTGACACCTTCGAGAA CTTTAACAAGACCCAGAAAGGCGCCATCCGGCAGTGCGGCTTCATCTTTTATATC CCCGCCTGGCGGACAAGCAAAATTGACCCGGTAACCGGCTTTGTGCCCATGCTG AGATGTCAGTACGAATCTATCGTGGAATCCAAGAAGTTCTTTGGCAAATTCGACT CTATCTACTACGACGCCACCGGAAAGTACTTCGTGTTCCAGACCGACTTTACCAA GTTCAACACCGAGTCTAAGGGGGGCATCCAGAAGTGGGACATCTGTACCTACGG AGACAGAATCTACGCCCCTAGAACCAAAGACAGAAATAACAACCCTGTGTCCGAA AGAGTGAACCTGACAGAAGAAATGAAGAGCCTGTTCGTAAGCCACAATATCAACA TCCAGGGCGACATCAAGGCCGGCATTATGCAGCAGACAGACAAGGAGTTCTTCG AGTCGCTGCACAGACTGCTGAGACTGACCCTGCAGATCCGGAACAGCAAGAAAA GCACCGGCAAGGACTACGAGGACTACATTATCAGTCCTGTGATGGGCAAGGACG GAAGATTCTTCGACAGCCGGAACGCCGACGCCACCCAGCCCAAGGACGCCGAC GCAAACGGCGCCTACAACATTGCCAGAAAAGGCCTGATGCTGCTGCGCCAGATC CAGGCCCAGGAGAAGCAGGACCTGTCTAATGGGAAGTGGCTGGAGTTCGCCCA GCGG Expression ATGggcAAGCGGCTCATCGACTTCACCAACATCTACCAGCGTTCTAAGACCCTGA 42 construct(with GATTCAGACTGGAACCTATCGGCAAGACCGCGGACTACATCAAAAACAGCCAGT N-terminal CCCTGGAAACAGACGCCAGACTGGCCAAGGAATCCAAGAAAGTGAAGGAACTGG methionine CCGATGAGTACCACAAAGAGTTTATCGGCGACGTGCTGAGCAGCCTGGAGCTGC andstop CCCTGAGCAAAATCAACGAGCTGTGGGACATCTATATCTACATCTACATGAGCAA codon, CGACACCGATCGGGAAATCAAATTTAAGAAGCTCCAGGAGAACCTGCGGAAGGT includesV5- GATCGCCGAGGCCTTTAGCAAGGATAAGAGATTCGGCAACCTGTTCAAGAAAGA tagandC- AATCATCACAGATATCCTGCCCGAGTTCCTGCAAGATAAAGATGACGATATCAAA terminalNLS) ATCGTGAACCGGTTCAAGGGTTTTACAACCTACTTCTACGCCTTCCACAAGAATC GGGAAAACATGTACGTGTCTGAAGAGAAGAGCACAGCCATCCCCTACAGAATCG TGAATCAAAACCTGGTGAAATACTTCGATAACTACAAGACTTTTAAGGAGAAGGT GATGCCTCTGCTGAAGGACAAGAACATCGTCGAAAGCATCGAGCGCGACTTCAA GGACATCCTGAACGAGAAAAGCATCGAGGACGTGTTCGGCCTGGCCAATTTCAC CCACACCCTGTGCCAGGCTGACATCGAGAAGTACAACACCTTGATAGGCGGACT GGTGGTGAAGAACGAAAAGAAGGAGATCAAGGGCATCAACCAGTATATTAACGA GCACAACCAGACCTCTAAGAAGGGCAACGGCATCCCAAAGCTGAAGCCTCTGTT TAACCAGATCCTGAGCGACAGAAAATCTCTCAGCTTCACCCTGGATGATATCAAG AAAACCAGCGAGGCCATCAGAACAATTAAGGACGAGTATGAGAACCTGAGAGAT AAGCTGGCCACAATCGAACGGCTGATCAAGAGCATCAAGGAATACGACCTGGCC GGCATCTACATCAAGATGGGCGAGGACACCTCTACCATCTCCCAGCACTGGTTC GGTGCCTATTACAAGATTATCGAAGCCATCGCCGACGCCTGGGAGAGAAGAAAC CCAAAGAAAAACAGAGAGAGCAAGGCCTACAGCAAGTACGTGAGCAGCCTTAAG AGCATCAGCCTGCAGGAGATCGACGACCTGAAGATCGGCGAGCCTATCGAGAAT TACTTCGCCACCTTTGGAACAACATGTAGCGACCGGACATCTGGCGTGAGCTCT CTGAACCGGATCAAAGCCGCCTACACCGAGTTCGTGAACAAGTTCCCCGAGGGC TTTGAGGATGGCGATGATTGCAACGACGCTTACTTCAAAGCCAATGTGGAGGTG GTGAAGAACTTGCTGGATAGCATAAAAGACTTCCAGAGATTTGTGAAGCCTCTAC TGGGCAATGAGGACGAGCGGGACAAAGATGAGGCCTTCTACGGCGAGTTCGTTC CTACCTACACAGATATGGACAACATCATCACGCCTCTGTATAATAGAGTCAGAAA CTTCGCTACCAAGAAGCCTTACAGTACAGACAAGATCAAAATAAACTTCGAAAAC GTGGTACTGCTGAAGGGCTGGGATAAGAACAAGGAGAGCGACTATGCCAGCATC ATCCTGATGAAGGACGGCCAGTACTTTCTGGGAGTGCTGAGAAACGGATCTAAG AGCACTCTGAAAACCATCCTGCCTAACACCGGTGACTGCTACCAGAAAATGGTGT ACAAGTATTTCAAGGATATCAAGTCTAACCTGCCCAGATGCACCACCCAGAGAAA GGACGTGAAGGCACATTTCGCTGAAAGCAGCGATGATTACACCCTGCTTGATACA AAAGCCTTCGTGAGCGCTCTGACGATCTCCAGAGAGGTGTTCGAACTGTACAAC GCTCCTGATAAGGAAAAGAAATTCAAGAAGGAATACCTGAAGAACACCAACGACT CCATCGGCTACGCCAATGCAGTGAGCGTGTGCAAGAGATTCTGCCTGGAGTTCC TGAAAAAGTACCGGAGCACCGCCATCTACGACCTGAGCGATGTTGAAACCTCTG TGGACAGTTTCGACGACCTGAGCAGCTTCTACCAGGAGATCGATAAGAGACTGT ACAGCATCAGCTTCGAAAACGTGAGCGTGGACAGCGTGAACGAGCTGGTGGATA ACGGCAATATGCTGCTGTTCAGAATCGCCAACAAGGATTTCTCTCCTAATAGCAA GGGCAGACCTAATCTGCACACAATTTACTGGAGAATGCTGTTCGACCCTGCTAAT CTCAAGGACGTCGTGTACCAACTGAACGGCAATGCCGAAATCTTCTTCCGGAAG GCCAGCGTTACAAGGACAGAACCAACACACCCCGCCAATGTGGCCATCAAGAAC AAGAGCGAGTACAACAAGCAGAACAAACCTTACAGCACCTTCAAGTACGGCCTCA TCAAGGACCGGCGATACACCACCGATCAGTTCGAGTTCCACGTGCCTATCACCA TGAACTTCAAGCAACCTGAGTCATCTAAGCTGCAGGACAAACTGAATAAGCAAGT GCTGGACTTCCTGAAGCAAGACGGCGTGCGGCACATCATCGGCATCGACCGGG GAGAAAGAAACCTGCTGTACCTGGTGATGGTCGACATGGAAGGAAAAATCAAGA AGCAGATCAGCCTGAATGAAATCGCCGGAAACCCAAAGAACCCTGAGTTTAAGC AGGACTTCTTAGCTCTGCTGCATGAGAGAGAGGGCGATAGACTGGAGTCCAGAA GAAGTTGGAACACCATCCAGAGCATCAAGGAGCTGAAAGAAGGCTACATGTCCC TGGTGGTGCACGAGATCGCTAACATGATGCTGGAGAATGATGCCATCGTGGTCT TGGAAAACCTTAACAGATCCTTTATGCAGAAGAGAGGCGGCATTGAGAAAAGCGT GTACCAGAAGTTTGAGAAAATGCTGATCGACAAGCTGGGCTACATCGTGGACAAA ACAAAAGATGTGTCAGATAATGGCGGAGCCCTGCACGCCGTGCAGCTGGCTGAC ACCTTCGAGAACTTTAACAAGACCCAGAAAGGCGCCATCCGGCAGTGCGGCTTC ATCTTTTATATCCCCGCCTGGCGGACAAGCAAAATTGACCCGGTAACCGGCTTTG TGCCCATGCTGAGATGTCAGTACGAATCTATCGTGGAATCCAAGAAGTTCTTTGG CAAATTCGACTCTATCTACTACGACGCCACCGGAAAGTACTTCGTGTTCCAGACC GACTTTACCAAGTTCAACACCGAGTCTAAGGGGGGCATCCAGAAGTGGGACATC TGTACCTACGGAGACAGAATCTACGCCCCTAGAACCAAAGACAGAAATAACAACC CTGTGTCCGAAAGAGTGAACCTGACAGAAGAAATGAAGAGCCTGTTCGTAAGCC ACAATATCAACATCCAGGGCGACATCAAGGCCGGCATTATGCAGCAGACAGACA AGGAGTTCTTCGAGTCGCTGCACAGACTGCTGAGACTGACCCTGCAGATCCGGA ACAGCAAGAAAAGCACCGGCAAGGACTACGAGGACTACATTATCAGTCCTGTGA TGGGCAAGGACGGAAGATTCTTCGACAGCCGGAACGCCGACGCCACCCAGCCC AAGGACGCCGACGCAAACGGCGCCTACAACATTGCCAGAAAAGGCCTGATGCTG CTGCGCCAGATCCAGGCCCAGGAGAAGCAGGACCTGTCTAATGGGAAGTGGCT GGAGTTCGCCCAGCGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGC CCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGG CCTGGACAGCACCTGA

[0106] In some embodiments a ZKBG Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, a ZKBG Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D885 substitution, wherein the position of the D885 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E978 substitution, wherein the position of the E978 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1194 substitution, wherein the position of the R1194 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1234 substitution, wherein the position of the D1234 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZKBG Type V Cas protein is catalytically inactive, for example due to a R1194 substitution in combination with a D885 substitution, a E978 substitution, and/or D1234 substitution.

6.2.8. ZZKD Type V Cas Proteins

[0107] In one aspect, the disclosure provides ZZKD Type V Cas proteins. ZZKD Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZKD Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:43. In some embodiments, the ZZKD Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:43. In some embodiments, a ZZKD Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:43.

[0108] Exemplary ZZKD Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZKD Type V Cas proteins are set forth in Table 1H.

TABLE-US-00009 TABLE1H ZZKDTypeVCasSequences SEQID Name Sequence NO. Wildtype AEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLMDEY 43 aminoacid YRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLESRETY sequence SSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKSTAIA (withoutN- YRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLSQRG terminal IELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLSFLA methionine) DAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITGLSN SLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAGLSE LRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMKELE AVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIKLTF DSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYSKM DYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALIDF FKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLVRE GKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRKKSI PEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPGSGR INRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKANNVAK SVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNAIVVM EDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLTEKF ESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDIQFC DDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQELDLT EEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETDYML SPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDKVDL KVTNLDWMKFAQR Wildtype MAEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLMDE 44 aminoacid YYRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLESRET sequence(with YSSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKSTAI N-terminal AYRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLSQR methionine) GIELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLSFL ADAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITGLS NSLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAGLS ELRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMKEL EAVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIKLT FDSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYSKM DYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALIDF FKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLVRE GKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRKKSI PEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPGSGR INRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKANNVAK SVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNAIVVM EDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLTEKF ESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDIQFC DDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQELDLT EEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETDYML SPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDKVDL KVTNLDWMKFAQR Expression MGAEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLM 45 construct(with DEYYRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLES N-terminal RETYSSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKS methionine, TAIAYRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLS V5-tagandC- QRGIELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLS terminalNLS) FLADAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITG aasequence LSNSLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAG LSELRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMK ELEAVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIK LTFDSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYS KMDYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALI DFFKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLV REGKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRK KSIPEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPG SGRINRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKAN NVAKSVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNA IVVMEDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLT EKFESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDI QFCDDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQE LDLTEEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETD YMLSPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDK VDLKVTNLDWMKFAQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGGCTGAGATGTTTAAAGATTTTACGAATTTGTATCCTGTTTCAAAAACCTTGC 46 coding GTTTTGAATTAATTCCTGAAGGGGAAACATTGCATTATCTTGAAAAAAATGGCGT sequence(with TCTGGAAAACGATGAGAAGCGAAACGAAGATTATAAGAAGTTGAAAAAACTGAT N-terminal GGATGAATATTACCGTGCATACATCGATGAAGCTTTATCTAATGTTCATCTTTCA methionine GATTTGGATAGATATGCAGAATTATATTCAATTCAGAATAAATCGGATGAAGAAA andstop ATGTAGAATTCGAAAATGTTCAACTGAGATTGAGAACACAAATTGTTGGATTCTT codon) AGAATCCAGAGAAACCTATTCTTCACTTTTCAAAAAAGAACTGATTGAGAAGGAA CTTCCTAAATTCTTTATTCGGAGAGAAGAGGAGCTTAATTTAATCAAATCATTTAA AGGTTTTACAACGATGTGCACCGGCTTCTGGGAAAATCGGAAAAATATGTTTTCT GCCGAAGAAAAATCTACAGCAATAGCATATCGTGTAGTCCATGAAAACCTACCTA AGTTTATGAATAATATAAGAATTTTTCGTTTGTTCATTGATGAAAAGTTGGACTGT TCTGAAAAATTGCTGGAAAAAGCCGGAGTGAATTCTCTGAGTGAAGTGTTTGAA CTTGATTATTTTAACAATACATTATCCCAACGTGGCATTGAATTGTATAACTGTAT ATTGGGCGGATTTACCGAGGATGAAAAGCATAAGATTCAAGGCGTAAACGAATT GATTAATTTGTACAATCAGCAGACAAAAGAGAAGAAGATTCCACAGTTGCAGCC GCTGTACAAGCAGATTCTCAGCGATACCAAGAGCCTTTCATTTCTTGCAGATGC ATTTGAAAACGACGGGGGGGTCTTAGCGACTGTAAAAGCATTATATGATGAATTT CATGAAGAGATTTTGAGCGAAAGGGGATTAATCTCTACGACATTACAGAATATTG AAAAGTATGATTCAAAAGGCATCTTCGTAAAAAACGATTTAACGATTACCGGTTT ATCAAATAGTTTGTTCGGCGACTGGAAGGCTATTAATGGTAGTTTAAATTCGTGG TATGAGGAGAACGTGCCTCGAAAAGAAAGAACTGAAGAGAAACATGTAGAGGTA AGAAAAGCCTATTTTAAAAAGTTAAAATCAATAAGCCTGGAATTTATCGAGGAGG CCGGATTGTCGGAACTCCGTTGCAAATATAAAGCCCTTCTTTTAGAAAAAGCAGA GGCTGTTTGCGATGCGTACAAAAATGCAGAAGAGCTTTTTAGTGAAGCTTATAAT GAAAATACTAACCTTATTGCCGATGGAAAGTCTGTGGAAAAAATAAAAGCGCTAT TGGATTCTATGAAAGAGCTTGAAGCGGTGATTCTTATGCTTTCCGGAACCGGAG AGGAAGCAGAACGGGATGAATTGTTTTACGGCGAATTTGAAAAACATAGGTTCG TATTGAATCTCTTAGACAACGTATTTAATAAAACGAGAAATTACGTAACAAAGAAA CCATATAAGACTGAGAAGATTAAATTAACATTTGATTCCCCAACGCTGCTAGACG GGTGGGATCGTAATAAAGAAACATCAAACAAGTCCGTGATACTTATGAAAGATG GCTATTATTACCTTGGAATTATGAACAAGGCAAATAACAAAGCCTTTGAGAATTT GAAAGACACAGGCGGGAAATGCTATAGCAAGATGGATTACAAACTTTTGCCTGG ACCAAACAAGATGTTGCCGAAGGTGTTTTTTGCAAAGAAAAACATCGACTATTAT GCACCAAGCGAAGACTTGCTACAGAAATATAAAGAGGGAACACATAAAAAAGGA AAGAAATTTAATCTAGAGGATTGTCACGCGTTAATAGACTTTTTTAAAGACTCAAT TGCAAAGCATCCAGAATGGAACGAGTTTGGATTTGATTTTTCAGATACGAAATCA TATCGAGATATTAGTGATTTCTATAAGGAGGTTTCAGAGCAGGGATACAAAATCA GTTATCGAAATGTATCTGTTAATTACATAGATTCTCTAGTAAGAGAAGGGAAATT GTATTTGTTCAAAATTTATAATAAAGATTTTTCACCGTACAGCAAAGGCAGACCAA ATCTTCATACGATGTATTGGAAAGCGTTATTCGCTAATAAGAATTTTGAAAATCG CATATATAAGTTAAATGGCCAGGCAGAAATGTTCTATCGAAAAAAGAGCATTCCG GAAGACAAGAGGGTGATTCACTCGGCAAAAGAACCAATCGATCAGAGAAGAAAT ACGGATGAAAAGAGCCTCTTTGATTATGACATTATTAAAGATCGGCGATATACTG TGGACAAATTCCAATTTAATGTTCCGATTACGATGAATTACACTGCACCGGGTTC CGGCCGAATTAACAGAAAAATGCGGGAAGCGATTAAGAACTGTGAAAATATGCA TATTATCGGAATAGATAGAGGCGAACGTCATTTGCTGTATGTGACGGTTATCGAT ATGCAGGGAAACATTAAAGAACAGTTTTCATTAAATCGAATCCTGAGTGAGTACA AGGCAAACAATGTGGCTAAAAGTGTCGAAACGGACTACAAAACACTCCTGACAA AAAAAGAAATTGAACGACAGGATGCAAGAAAGCAGTGGAAGAGCATTGAAAATA TTAAGGAATTAAAAGACGGCTACATGAGCCAGGTTGTGCATGTGATTGCCGAAC TCATGATAAAGTACAATGCGATTGTGGTTATGGAGGATTTGAATTTCGGATTCAA GCGAGGAAGACAGAAGGTTGAGAGACAGGTTTACCAGAAGTTTGAGAAGGCAT TAATTGATAAATTGAACTATTTGGTTGATAAAACAGCCTCTGAAATGGAGAACAC CGGTCTGTATGCGGCATTGCAGCTTACAGAAAAATTTGAGAGCTTTAAGAAAAT GGGCAAACAAAATGGTGGATTATTTTATGTAAACGCATGGAATACCAGTAAAATG GATCCAACAACCGGTTTTGTGAACCTTCTCTATCCTAAATATGAGAGCATTGAAA AAAGCAAAGCGTATATTGAGAAATTCAAGGATATTCAGTTTTGTGATGATGACGA ATATGGAAAGTACCTTGCAATATCTTTTGATTATAACGATTTCACGGAGAAGGCA AAGGGCGCAAAAACGGAATGGACCATTTGCTCTTATGGAAAGAGATTGTATAAT CACAGAAATAAAGATGGGTATTGGGAAGAGCAGGAATTGGATCTTACAGAAGAG TATTTCAATCTGTTTGAAGAATTTGGAATTAATGCAGCGTCTAATATTAAAGAACA AGTCATCGCACAGAATTCTGCAGACTTTTTTAGACGGTTTATGTGGCTTTTGAAA ATGACCTTACAGATTAGAAACAGTGAAACAAATGGGGAGACGGATTATATGCTTT CTCCGGTAAAAAATGAAGACGGAAAATTCTTTAATTCAGATGAAGTCAAGGATGA CACGCTTCCGGAAAATGCGGATGCGAATGGTGCATACAACATCGCTAGAAAAG GATTACTGCTTGTGGAAAGAATTAAAGACTGTCCGGACGAAGAACTTGATAAGG TTGATTTGAAGGTAACAAATTTAGATTGGATGAAATTTGCACAGAGGTAA Codon GCCGAAATGTTCAAGGACTTCACCAACCTGTACCCAGTGTCCAAAACCCTCCGG 47 optimized TTCGAATTGATCCCCGAGGGCGAAACACTGCACTACCTAGAAAAGAACGGAGTG coding CTGGAAAACGACGAGAAGAGAAATGAGGATTACAAGAAGCTGAAGAAACTCATG sequence(no GATGAATACTACCGGGCCTACATCGACGAGGCCTTATCTAATGTCCACCTGTCC N-terminal GATCTGGACCGGTACGCCGAACTGTATTCTATCCAGAACAAGAGCGATGAGGA methionine,no GAACGTGGAGTTCGAGAATGTGCAGCTGCGCCTGAGAACCCAGATCGTGGGCT stopcodon) TCCTGGAAAGCAGAGAAACCTACAGCAGCCTGTTCAAGAAGGAGCTGATCGAAA AAGAACTGCCTAAGTTTTTCATCAGAAGAGAGGAAGAGCTGAACCTGATAAAGA GCTTTAAGGGCTTTACCACTATGTGCACCGGCTTCTGGGAAAATCGGAAGAACA TGTTCAGCGCCGAGGAAAAGTCCACAGCCATCGCCTATAGAGTGGTCCATGAAA ACCTGCCCAAGTTCATGAACAACATTAGAATCTTCCGGCTGTTTATCGACGAGAA GCTGGATTGTAGCGAGAAGCTGCTGGAGAAGGCCGGCGTGAACAGCCTGAGC GAGGTGTTCGAGCTTGACTATTTCAATAACACCCTGAGCCAGAGAGGCATCGAG CTGTACAACTGCATCCTGGGCGGATTCACCGAGGATGAAAAACACAAGATCCAG GGAGTGAACGAGTTGATCAACCTGTACAACCAGCAGACAAAGGAGAAGAAAATT CCTCAGCTGCAACCTCTGTACAAACAGATCCTGTCTGACACGAAGTCGCTGTCC TTTCTGGCTGATGCCTTTGAAAACGACGGAGGAGTGCTGGCTACAGTGAAGGCT TTATATGATGAGTTTCACGAGGAAATCCTGAGCGAGAGAGGCCTGATCAGCACA ACCCTGCAGAACATTGAGAAGTACGATAGTAAGGGCATCTTTGTTAAGAACGAT CTCACCATTACAGGCCTGTCCAACAGCCTGTTTGGAGATTGGAAGGCCATCAAT GGAAGCCTGAACAGCTGGTACGAGGAGAACGTGCCCCGGAAGGAGCGAACAG AAGAGAAACACGTGGAAGTGAGAAAGGCTTATTTTAAGAAGCTGAAGTCTATCA GCCTGGAGTTCATCGAGGAGGCCGGACTGAGCGAGCTGCGGTGCAAGTACAA GGCCCTGCTGCTGGAGAAAGCCGAGGCTGTGTGCGACGCGTACAAGAACGCC GAGGAGCTGTTTAGCGAGGCCTATAATGAGAACACCAATCTGATCGCCGATGG CAAATCTGTGGAAAAAATCAAAGCCCTGCTGGACAGCATGAAGGAGCTGGAGG CCGTGATCCTGATGCTGAGCGGCACAGGCGAGGAGGCCGAGCGGGACGAACT GTTTTATGGCGAGTTCGAAAAACATAGATTCGTGCTGAATCTGCTGGACAACGT GTTCAACAAGACCAGAAACTACGTGACCAAGAAGCCTTACAAGACCGAGAAGAT CAAGCTCACCTTCGACAGCCCTACCCTTCTGGATGGCTGGGACCGTAACAAGG AGACAAGCAACAAGAGCGTGATCCTGATGAAGGATGGCTACTACTACCTGGGC ATCATGAACAAAGCCAACAACAAGGCCTTCGAGAACCTGAAGGACACAGGAGG CAAATGCTACAGCAAGATGGACTACAAGCTGCTGCCTGGCCCTAACAAGATGCT GCCTAAGGTGTTCTTTGCCAAAAAGAACATCGACTACTACGCCCCTAGCGAGGA CCTGCTGCAGAAGTACAAGGAGGGCACCCACAAGAAAGGGAAGAAGTTCAATC TTGAGGACTGTCACGCCCTGATCGACTTCTTCAAGGACAGCATCGCTAAACACC CCGAGTGGAACGAGTTCGGCTTCGACTTTTCTGACACCAAGTCTTATAGAGACA TCTCGGATTTCTACAAGGAGGTCAGCGAACAGGGCTACAAGATTAGCTACCGGA ACGTGAGTGTTAACTACATCGACAGTCTGGTGCGGGAAGGTAAGCTGTACCTGT TCAAGATCTACAACAAGGACTTCAGCCCATACTCCAAAGGACGTCCCAACCTGC ACACCATGTACTGGAAAGCCCTGTTCGCCAATAAAAACTTCGAAAACCGGATCT ACAAGCTGAACGGCCAGGCCGAAATGTTCTACAGAAAGAAATCTATCCCTGAAG ATAAGCGGGTGATCCACAGCGCCAAAGAACCTATCGATCAGAGAAGAAACACC GACGAAAAGTCTCTGTTTGACTACGACATCATCAAGGACAGACGGTACACCGTG GACAAGTTCCAGTTCAACGTGCCAATCACAATGAACTACACCGCCCCTGGCAGC GGCAGAATCAACAGAAAGATGCGGGAAGCTATCAAGAATTGCGAGAATATGCAC ATCATCGGCATCGACCGGGGAGAGCGGCACCTGCTGTACGTGACCGTGATCGA CATGCAGGGCAACATCAAAGAACAGTTCTCTCTCAACCGCATCCTGTCTGAGTA CAAGGCCAATAACGTCGCCAAGAGCGTGGAGACAGACTACAAAACACTGCTGA CGAAAAAAGAGATCGAGAGACAGGACGCTAGAAAGCAATGGAAGAGCATCGAA AACATCAAAGAGCTGAAAGACGGCTATATGAGCCAGGTGGTGCACGTGATAGC AGAGCTGATGATCAAGTACAACGCCATAGTTGTGATGGAGGACCTGAATTTCGG CTTCAAGAGAGGCCGGCAAAAGGTGGAGAGACAGGTGTACCAGAAATTCGAGA AGGCCCTGATCGATAAGCTGAATTACCTGGTGGATAAGACAGCTTCCGAGATGG AAAACACCGGCCTGTACGCCGCCCTGCAGCTGACAGAGAAGTTCGAATCCTTC AAGAAGATGGGCAAACAGAACGGCGGCTTGTTCTACGTGAACGCCTGGAACAC CAGCAAGATGGACCCTACCACCGGATTCGTGAACCTGCTGTACCCTAAGTACGA ATCTATCGAAAAGAGCAAGGCCTATATCGAGAAATTCAAGGATATCCAGTTTTGT GACGACGATGAATACGGCAAATACCTGGCAATTTCTTTCGACTACAACGACTTC ACAGAAAAGGCCAAGGGCGCCAAGACCGAGTGGACCATCTGCAGCTACGGCAA AAGACTGTACAACCACAGAAATAAGGACGGCTACTGGGAGGAGCAGGAGCTGG ATCTGACCGAGGAGTACTTCAACCTGTTCGAAGAGTTCGGCATCAACGCTGCCA GCAACATCAAGGAACAAGTGATCGCTCAGAACAGCGCCGATTTCTTCAGAAGAT TCATGTGGCTGCTGAAGATGACCCTGCAGATCAGGAACTCTGAAACTAACGGCG AAACCGATTACATGCTGAGCCCTGTGAAGAACGAGGACGGCAAATTCTTCAACT CTGACGAGGTGAAGGACGACACCCTGCCCGAGAATGCCGACGCCAACGGCGC CTACAACATCGCAAGAAAGGGCCTGCTGCTGGTCGAACGTATCAAGGATTGCC CCGACGAAGAACTAGACAAGGTGGACCTGAAGGTCACCAACCTGGACTGGATG AAATTCGCCCAAAGA Expression ATGggcGCCGAAATGTTCAAGGACTTCACCAACCTGTACCCAGTGTCCAAAACCC 48 construct(with TCCGGTTCGAATTGATCCCCGAGGGCGAAACACTGCACTACCTAGAAAAGAACG N-terminal GAGTGCTGGAAAACGACGAGAAGAGAAATGAGGATTACAAGAAGCTGAAGAAA methionine CTCATGGATGAATACTACCGGGCCTACATCGACGAGGCCTTATCTAATGTCCAC andstop CTGTCCGATCTGGACCGGTACGCCGAACTGTATTCTATCCAGAACAAGAGCGAT codon, GAGGAGAACGTGGAGTTCGAGAATGTGCAGCTGCGCCTGAGAACCCAGATCGT includesV5- GGGCTTCCTGGAAAGCAGAGAAACCTACAGCAGCCTGTTCAAGAAGGAGCTGA tagandC- TCGAAAAAGAACTGCCTAAGTTTTTCATCAGAAGAGAGGAAGAGCTGAACCTGA terminalNLS) TAAAGAGCTTTAAGGGCTTTACCACTATGTGCACCGGCTTCTGGGAAAATCGGA AGAACATGTTCAGCGCCGAGGAAAAGTCCACAGCCATCGCCTATAGAGTGGTC CATGAAAACCTGCCCAAGTTCATGAACAACATTAGAATCTTCCGGCTGTTTATCG ACGAGAAGCTGGATTGTAGCGAGAAGCTGCTGGAGAAGGCCGGCGTGAACAG CCTGAGCGAGGTGTTCGAGCTTGACTATTTCAATAACACCCTGAGCCAGAGAGG CATCGAGCTGTACAACTGCATCCTGGGCGGATTCACCGAGGATGAAAAACACAA GATCCAGGGAGTGAACGAGTTGATCAACCTGTACAACCAGCAGACAAAGGAGA AGAAAATTCCTCAGCTGCAACCTCTGTACAAACAGATCCTGTCTGACACGAAGT CGCTGTCCTTTCTGGCTGATGCCTTTGAAAACGACGGAGGAGTGCTGGCTACA GTGAAGGCTTTATATGATGAGTTTCACGAGGAAATCCTGAGCGAGAGAGGCCTG ATCAGCACAACCCTGCAGAACATTGAGAAGTACGATAGTAAGGGCATCTTTGTT AAGAACGATCTCACCATTACAGGCCTGTCCAACAGCCTGTTTGGAGATTGGAAG GCCATCAATGGAAGCCTGAACAGCTGGTACGAGGAGAACGTGCCCCGGAAGGA GCGAACAGAAGAGAAACACGTGGAAGTGAGAAAGGCTTATTTTAAGAAGCTGAA GTCTATCAGCCTGGAGTTCATCGAGGAGGCCGGACTGAGCGAGCTGCGGTGCA AGTACAAGGCCCTGCTGCTGGAGAAAGCCGAGGCTGTGTGCGACGCGTACAAG AACGCCGAGGAGCTGTTTAGCGAGGCCTATAATGAGAACACCAATCTGATCGCC GATGGCAAATCTGTGGAAAAAATCAAAGCCCTGCTGGACAGCATGAAGGAGCT GGAGGCCGTGATCCTGATGCTGAGCGGCACAGGCGAGGAGGCCGAGCGGGAC GAACTGTTTTATGGCGAGTTCGAAAAACATAGATTCGTGCTGAATCTGCTGGAC AACGTGTTCAACAAGACCAGAAACTACGTGACCAAGAAGCCTTACAAGACCGAG AAGATCAAGCTCACCTTCGACAGCCCTACCCTTCTGGATGGCTGGGACCGTAAC AAGGAGACAAGCAACAAGAGCGTGATCCTGATGAAGGATGGCTACTACTACCTG GGCATCATGAACAAAGCCAACAACAAGGCCTTCGAGAACCTGAAGGACACAGG AGGCAAATGCTACAGCAAGATGGACTACAAGCTGCTGCCTGGCCCTAACAAGAT GCTGCCTAAGGTGTTCTTTGCCAAAAAGAACATCGACTACTACGCCCCTAGCGA GGACCTGCTGCAGAAGTACAAGGAGGGCACCCACAAGAAAGGGAAGAAGTTCA ATCTTGAGGACTGTCACGCCCTGATCGACTTCTTCAAGGACAGCATCGCTAAAC ACCCCGAGTGGAACGAGTTCGGCTTCGACTTTTCTGACACCAAGTCTTATAGAG ACATCTCGGATTTCTACAAGGAGGTCAGCGAACAGGGCTACAAGATTAGCTACC GGAACGTGAGTGTTAACTACATCGACAGTCTGGTGCGGGAAGGTAAGCTGTAC CTGTTCAAGATCTACAACAAGGACTTCAGCCCATACTCCAAAGGACGTCCCAAC CTGCACACCATGTACTGGAAAGCCCTGTTCGCCAATAAAAACTTCGAAAACCGG ATCTACAAGCTGAACGGCCAGGCCGAAATGTTCTACAGAAAGAAATCTATCCCT GAAGATAAGCGGGTGATCCACAGCGCCAAAGAACCTATCGATCAGAGAAGAAA CACCGACGAAAAGTCTCTGTTTGACTACGACATCATCAAGGACAGACGGTACAC CGTGGACAAGTTCCAGTTCAACGTGCCAATCACAATGAACTACACCGCCCCTGG CAGCGGCAGAATCAACAGAAAGATGCGGGAAGCTATCAAGAATTGCGAGAATAT GCACATCATCGGCATCGACCGGGGAGAGCGGCACCTGCTGTACGTGACCGTGA TCGACATGCAGGGCAACATCAAAGAACAGTTCTCTCTCAACCGCATCCTGTCTG AGTACAAGGCCAATAACGTCGCCAAGAGCGTGGAGACAGACTACAAAACACTG CTGACGAAAAAAGAGATCGAGAGACAGGACGCTAGAAAGCAATGGAAGAGCAT CGAAAACATCAAAGAGCTGAAAGACGGCTATATGAGCCAGGTGGTGCACGTGA TAGCAGAGCTGATGATCAAGTACAACGCCATAGTTGTGATGGAGGACCTGAATT TCGGCTTCAAGAGAGGCCGGCAAAAGGTGGAGAGACAGGTGTACCAGAAATTC GAGAAGGCCCTGATCGATAAGCTGAATTACCTGGTGGATAAGACAGCTTCCGAG ATGGAAAACACCGGCCTGTACGCCGCCCTGCAGCTGACAGAGAAGTTCGAATC CTTCAAGAAGATGGGCAAACAGAACGGCGGCTTGTTCTACGTGAACGCCTGGA ACACCAGCAAGATGGACCCTACCACCGGATTCGTGAACCTGCTGTACCCTAAGT ACGAATCTATCGAAAAGAGCAAGGCCTATATCGAGAAATTCAAGGATATCCAGTT TTGTGACGACGATGAATACGGCAAATACCTGGCAATTTCTTTCGACTACAACGA CTTCACAGAAAAGGCCAAGGGCGCCAAGACCGAGTGGACCATCTGCAGCTACG GCAAAAGACTGTACAACCACAGAAATAAGGACGGCTACTGGGAGGAGCAGGAG CTGGATCTGACCGAGGAGTACTTCAACCTGTTCGAAGAGTTCGGCATCAACGCT GCCAGCAACATCAAGGAACAAGTGATCGCTCAGAACAGCGCCGATTTCTTCAGA AGATTCATGTGGCTGCTGAAGATGACCCTGCAGATCAGGAACTCTGAAACTAAC GGCGAAACCGATTACATGCTGAGCCCTGTGAAGAACGAGGACGGCAAATTCTT CAACTCTGACGAGGTGAAGGACGACACCCTGCCCGAGAATGCCGACGCCAACG GCGCCTACAACATCGCAAGAAAGGGCCTGCTGCTGGTCGAACGTATCAAGGAT TGCCCCGACGAAGAACTAGACAAGGTGGACCTGAAGGTCACCAACCTGGACTG GATGAAATTCGCCCAAAGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAA AGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGC TGGGCCTGGACAGCACCTGA

[0109] In some embodiments a ZZKD Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 43, SEQ ID NO:44, or SEQ ID NO:45. In some embodiments, a ZZKD Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO:45. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D828 substitution, wherein the position of the D828 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E925 substitution, wherein the position of the E925 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1138 substitution, wherein the position of the R1138 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1176 substitution, wherein the position of the D1176 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZZKD Type V Cas protein is catalytically inactive, for example due to a R1138 substitution in combination with a D828 substitution, a E925 substitution, and/or D1176 substitution.

6.2.9. ZXPB Type V Cas Proteins

[0110] In one aspect, the disclosure provides ZXPB Type V Cas proteins. ZXPB Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZXPB Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:49. In some embodiments, the ZXPB Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:49. In some embodiments, a ZXPB Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:49.

[0111] Exemplary ZXPB Type V Cas protein sequences and nucleotide sequences encoding exemplary ZXPB Type V Cas proteins are set forth in Table 11.

TABLE-US-00010 TABLE11 ZXPBTypeVCasSequences SEQ ID Name Sequence NO. Wildtype KLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHKAYI 49 aminoacid DKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDSDE sequence FKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIAYR (withoutN- LIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMGRY terminal NMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYDDLR methionine) IPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRRLNK RKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFAKAE NARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEAGKD ERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDKNKEP DCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPKVFFS ASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFGFEFS DTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAYSKGT PNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKNKNSR NDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADDVHVI GIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSWQAIE GIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEHMLID KLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDPATG FVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKWTVC TYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFFANM QRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAYNIAR KGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADE Wildtype MKLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHKA 50 aminoacid YIDKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDSD sequence(with EFKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIAY N-terminal RLIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMGR methionine) YNMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYDDL RIPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRRLN KRKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFAKA ENARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEAGK DERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDKNKE PDCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPKVFF SASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFGFEF SDTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAYSKG TPNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKNKNS RNDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADDVHV IGIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSWQAIE GIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEHMLID KLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDPATG FVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKWTVC TYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFFANM QRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAYNIAR KGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADE Expression MGKLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHK 51 construct(with AYIDKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDS N-terminal DEFKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIA methionine, YRLIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMG V5-tagandC- RYNMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYD terminalNLS) DLRIPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRR aasequence LNKRKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFA KAENARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEA GKDERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDK NKEPDCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPK VFFSASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFG FEFSDTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAY SKGTPNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKN KNSRNDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADD VHVIGIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSW QAIEGIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEH MLIDKLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDP ATGFVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKW TVCTYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFF ANMQRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAY NIARKGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADESRKRTADGSEFESP KKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAATTAGAAGATTTTACCAACCTGTATTCGTTATCCAAGACTCTGCGTTTCGA 52 coding ACTGCGGCCGATCGGCAAGACACGTGAAAATATCGAAAACGGAGGCCTTTTGAG sequence(with GCAGGACGAGGATCGTGCTGAAAAATATGTACACATAAAAAAACTAATCGATGAA N-terminal TATCATAAAGCATATATCGATAAACAATTGTCGGGTTTAGTGCTGCAATACGCCGA methionine TATCGGTAAAGCCAATTCATTGGAGGAGTATTATCACTCCACAAGAAAGAGCAAA andstop GATTCGGACAAGGATAAGATTGTCAAAATCCAGGATAATCTGCGTAAACAAATTG codon) TCAAACGGTTGAAAGACTCAGACGAATTCAAGCGTATCGATAAAAAAGAGTTGAT TCAATCGGATCTGGCAGAGTTCATAAAACCAGCCGAAGACAGAGCTTTGATTGCC GAATTCAAAAACTTCACAACATATTTTACCGGATTCAATGAAAACAGACAGAACAT GTATTCGGACAAAGCTATATCTACGGCAATAGCTTATCGTCTGATACATGAGAATC TTCCGAAGTTCATAGACAACATAGAGACTTTCGATCGCATCGCCGGTATAACGGA ATTGTACGACCAAACCTCCTCCGATGCCGAAATTTTCCGTCTGGAACATTTTTCG GAAACACTGAGCCAAAAGCAGATCGATGCCTATAACTCCGTTATGGGCAGATATA ACATGCTTATCAATGAGTACAATCAGACGCATAAACAGTCGCGCCTACCTAAATT CAAAATGCTGTACAAACAGATTCTTAGCGACCGCGAACACCCCTCGTGGCTGCC CGAGCAGTTCGAGTCGGACACGGCTGTATTGACAGCCATTCGCGAATGTTACGA TGATCTGCGCATACCTATGGCCAATTTGAAAACGCTTTTAGAGGGGTTGGGCAAC TATGACCCGAGTGGAATATTTTTGCGTAATGACCAACATCTCTCTCAGATATCCAA ACGATTGACAGGTGATCGGAGTAGCATTGAACGTAGCGTAACAGAAGACCTTCT GACATCGAGGAGACTCAACAAGCGAAAAAGCCGCACAACCGACGAGGAGGAATC GAGAAAACTGTTCAAGCAAAAGGGTAGTCTGAGTATAGGCTATATAGCTGACACG GCCAAAATCGATGTCGAAAGATACTTTGCCAAACTCGGTGCAATAAATACGGTAA CGGAGCAGAGCGAGAATCTATTCGCCAAGGCTGAGAATGCCCGCACGACAGCG GATGAGCTGCTCGCAAATGATTACCCGGCAGGCAAGAGGCTCGTTCAGTCCAAC GACGACATAGCATTGCTGAAAAATCTGCTCGATGCTTTAATGGAGCTGCAATGGT TCGTCAAGCCGCTGCTTGGCACGGGGGACGAAGCCGGCAAAGACGAACGTTTC TATGGAGAATTTGCACAGATATGGGAGCAGCTGGATCGTATAACGCCTCTCTATA ACATGGTGCGCAACTATGTTACCCGCAAGCCGTATTCGACCGACAAATTCAAGCT CAACTTTGAGAGCGCAGCGCTTCTCGGCGGCTGGGACAAGAACAAGGAGCCGG ACTGTCTGTCGGTAATCTTACGCAAGGATGAGCAATATTATCTCGGCATAATCAAT AAGAATCACAAAAAGATATTCGAGAACGATATCTTGCCGTGCGAAGGGGAGTGTT ACGACAAAATGGTATATAAACTCCTGCCCGGCGCAAACAAGATGCTGCCGAAAGT ATTCTTCTCGGCTTCGCGTATCGCCGAATTTGCACCGAGCGACGAAGTAAAACG GATATACAATGATAAGACTTTCCAAAAAGGCGAAAAGTTCGACTTGAACGATTGTC GCACACTGATCGACTTCTACAAGGCTTCTATCGACAAACATGAGGAGTGGAACAA GTTTGGATTCGAATTCTCGGATACGAACAATTATGAAGACATAAGCGGATTCTTTC GCGAGGTCGACAGGCAAGGCTATAAAATGTCATTCCGCCCGGTCGCAGCATCGT ATATCGAAACCCTTGTTGAAGAGGGCAAACTCTATCTTTTCCAAATATATAATAAG GATTTTTCGGCATATAGCAAAGGTACTCCCAATATGCACACGCTGTATTGGAGGA TGCTCTTCGACGAGCGCAATCTATCGGATGTCGTATATCAGCTCAACGGCGGAG CAGAGTTGTTCTTCCGAAGAAAGAGTCTTCAAAACGGCCGTCCGACGCATCCGG CAAATATTCCTATCAAAAACAAAAACAGTCGGAATGACAAAAAAGAGAGCCTGTTC GACTACGATTTGATCAAAGACAGACGCTATACTGTGGACAAATTTCAGTTCCATGT CCCGATAACCCTCAATTTCAAGAGCGACGGGGGGGGCAGGATCAACGAGCGTGT AAGGGAATATCTCCGCTCGGCGGACGACGTTCACGTCATAGGCATCGACCGCGG AGAACGCAATCTGCTGTATCTGGTCGTGACGGATATGGACGGCAATATCTGCGA ACAATTCTCGCTCAACGAAATTTGTAATACTGATTATCATTCTTTGTTGGATGAAC GCGAACACAAACGTATGCAGGAGAGACAGAGCTGGCAGGCGATAGAGGGCATC AAGGAGTTGAAAGAAGGTTATCTGTCTCAGGTCGTACACCGAATCGCGACACTCA TGGTTAAATATCGCGCCATTGTCGTACTGGAAGATCTCAACTTCGGCTTCATGCG TAGCCGCCAGAAGGTAGAGAAGTCTGTATACCAGAAATTCGAACACATGCTCATA GATAAGCTCAATTATCTGGTCGACAAGAAAGCCAATCCGACAACGCCGGGCGGT CTGCTAAAAGCCTATCAGTTGACAGACAAATTCGAGAGCTTCCAGAAGCTCGGCA AACAGAGCGGATTTCTATTCTACGTTCCGGCATGGAATACATCGAAGATCGATCC AGCAACCGGATTCGTCAACATGCTCGATCTCGGATACGAGAGCATCGACAAAGC CAAAACACTGCTCTGCAAGTTCGACTCTATACGCTACAATGCGTGCAAAGACTGG TTCGAGTTCGCTCTCGATTACGACAAGTTCGGCAGCAAGGCCACCGGTACCCGC ACGAAATGGACTGTTTGCACCTACGGACAACGTATCGATACTTATCGCAACAAAG ATTCGCAGTGGGTCAGCCGCGACGTCGATTTGACAAATGAGCTGAAATCACTCTT CTCCGAACACGGCATAGACATTTACAGCAATCTGAAAGATGCAATAGTCGCACAA AACGACAAAGAATTTTTCGCGAACATGCAGCGGATATTGAAACTGACCATGCAAA TGCGAAACAGCAAAACGGGTACCGACACAGACTATATCGTCTCGCCCGTCGCCG ATGCCAACGGCAGATTCTTCGACAGCAGGCAGGCCGATGCGACCATGCCCAAAG ATGCGGATGCGAACGGAGCGTATAATATCGCACGTAAGGGCATTATGCTCGTAC AGCAGATCAAGCAGTCCGACGATCTGCGTACAATGAAGTTCGACATAAGCAACAA GAGCTGGCTGCGCTTCGCCCAACATACGAACCAGGCGGACGAGTAA Codon AAGCTGGAGGACTTCACCAATCTGTACTCTCTGAGCAAGACCCTGCGGTTTGAG 53 optimized CTGCGGCCTATCGGCAAGACAAGAGAAAACATCGAAAACGGCGGACTGCTGCGT coding CAAGACGAGGACAGAGCCGAAAAGTACGTGCATATTAAAAAGCTGATCGATGAAT sequence(no ACCACAAGGCTTATATCGACAAGCAACTGAGTGGCCTGGTCCTGCAATACGCCG N-terminal ATATCGGCAAGGCCAATTCTCTGGAGGAGTACTACCACAGCACTAGAAAAAGCAA methionine,no GGACTCTGACAAGGATAAGATAGTCAAGATCCAGGACAACCTGCGCAAGCAGAT stopcodon) CGTCAAGAGATTGAAGGACAGCGATGAGTTTAAGAGGATCGATAAGAAGGAACT GATCCAGTCTGACCTGGCAGAGTTCATCAAGCCAGCCGAGGACAGGGCCCTGAT AGCCGAGTTCAAGAACTTCACCACCTACTTCACAGGATTCAACGAAAATAGGCAG AACATGTACAGCGATAAGGCTATCAGCACCGCCATCGCCTACCGGCTGATCCAC GAGAACCTGCCTAAGTTCATCGACAACATCGAAACCTTCGACCGGATCGCGGGC ATCACAGAGCTGTATGACCAGACATCCAGCGACGCAGAGATCTTTAGACTGGAG CACTTCAGTGAGACACTGAGCCAGAAGCAGATCGATGCCTATAACAGCGTGATG GGCCGGTACAACATGCTGATCAACGAATATAACCAGACCCACAAGCAATCTCGG CTGCCTAAATTCAAAATGCTGTACAAGCAGATCCTGAGCGACCGGGAGCACCCC AGCTGGCTGCCGGAACAGTTCGAGAGCGACACCGCCGTGCTGACCGCCATCAG AGAGTGTTACGACGACCTGAGAATCCCTATGGCCAACTTAAAAACCCTGCTTGAG GGCCTGGGAAATTACGATCCCTCTGGCATCTTCCTGCGGAACGATCAGCACCTG TCTCAGATCAGCAAAAGACTCACCGGAGACAGATCCAGCATCGAACGGAGCGTG ACCGAGGACTTATTAACGAGCCGGAGACTGAACAAAAGAAAGAGCAGAACCACC GATGAAGAGGAAAGCAGAAAGCTGTTCAAGCAAAAAGGCAGCCTGAGCATCGGC TACATCGCCGACACAGCCAAGATCGACGTGGAGAGATACTTCGCCAAGCTGGGA GCCATTAATACCGTGACCGAGCAGTCTGAGAACCTCTTCGCTAAGGCCGAGAAC GCCAGAACCACTGCTGACGAGCTGCTGGCCAACGACTACCCTGCCGGCAAAAGA CTGGTGCAGAGCAACGACGACATCGCTCTGCTGAAGAACCTATTGGACGCCCTG ATGGAACTGCAATGGTTCGTGAAGCCCCTGCTGGGCACCGGCGACGAGGCCGG CAAAGACGAACGGTTCTATGGCGAGTTCGCTCAGATCTGGGAGCAGCTGGATAG AATCACCCCTCTGTACAACATGGTGCGGAATTACGTGACAAGAAAGCCCTACTCC ACAGACAAGTTCAAGCTGAACTTCGAATCTGCCGCCCTGCTGGGCGGATGGGAC AAGAACAAAGAACCTGACTGCCTGTCCGTGATTCTGAGAAAGGACGAGCAGTAC TACCTGGGCATCATCAACAAGAACCACAAGAAGATCTTCGAGAATGACATTCTGC CTTGCGAGGGCGAGTGCTACGACAAGATGGTCTACAAGCTGCTGCCTGGCGCTA ACAAAATGCTGCCTAAGGTGTTCTTTAGCGCCTCCAGAATCGCTGAGTTCGCCCC TTCTGATGAGGTGAAAAGAATTTACAACGATAAGACCTTCCAGAAGGGCGAGAAG TTCGATCTGAACGACTGCAGAACCCTCATCGATTTCTACAAGGCTTCTATCGATAA GCACGAGGAGTGGAATAAATTTGGCTTCGAGTTTAGCGACACCAACAACTACGA GGACATCAGCGGCTTCTTCCGGGAGGTGGACAGACAGGGCTACAAGATGAGCTT TAGACCCGTGGCCGCCAGCTACATCGAAACGTTGGTGGAAGAGGGCAAACTGTA CCTGTTCCAGATCTACAACAAAGATTTCAGCGCCTACAGCAAGGGCACCCCTAAT ATGCACACCCTGTACTGGAGAATGCTGTTTGACGAGCGGAACCTGAGCGACGTG GTGTACCAGCTGAACGGCGGAGCTGAACTGTTCTTTAGACGCAAGTCCCTCCAG AACGGCCGGCCTACACACCCTGCCAACATCCCTATCAAGAACAAGAACAGCAGA AACGATAAAAAGGAATCACTGTTCGACTACGATCTCATCAAGGATCGTAGATACA CAGTGGATAAGTTCCAGTTCCACGTGCCAATCACACTGAATTTCAAGAGCGATGG CGCTGGCAGAATTAACGAGAGAGTGCGGGAGTACCTGAGATCTGCCGATGACGT GCACGTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTACCTCGTGGTGA CCGATATGGACGGCAACATCTGCGAACAGTTTAGCCTGAACGAAATCTGTAATAC CGACTACCACAGCCTGTTGGATGAGAGAGAGCACAAAAGAATGCAGGAAAGACA GAGCTGGCAGGCCATCGAGGGAATCAAGGAGCTGAAGGAAGGCTACCTGTCCC AAGTGGTCCACAGAATCGCCACCCTGATGGTGAAGTACAGAGCGATCGTGGTGC TGGAGGACCTGAACTTCGGCTTCATGCGGAGCAGACAGAAAGTGGAAAAAAGCG TGTACCAGAAGTTCGAGCACATGCTGATCGACAAACTGAACTACCTGGTGGACAA GAAAGCCAACCCTACCACACCCGGCGGCCTGCTGAAGGCCTACCAGCTGACAG ACAAGTTCGAGAGCTTCCAGAAGCTGGGCAAGCAGTCTGGATTCCTGTTTTATGT GCCCGCCTGGAACACAAGCAAGATCGACCCTGCTACCGGATTCGTGAACATGCT GGATCTGGGCTATGAGAGCATCGACAAGGCCAAAACCCTGCTGTGCAAGTTTGA CTCCATCAGATACAACGCCTGCAAGGACTGGTTCGAGTTTGCCCTGGACTACGA CAAGTTCGGCAGCAAGGCCACAGGCACACGGACCAAGTGGACAGTGTGCACCT ACGGCCAGCGGATCGATACTTATAGAAACAAGGACAGCCAGTGGGTGTCTCGGG ACGTGGATCTGACCAATGAGCTGAAGAGCCTGTTTTCTGAACATGGCATCGACAT CTACAGCAACCTGAAAGACGCCATCGTGGCCCAAAATGACAAAGAGTTCTTCGC CAACATGCAGAGAATCCTGAAGCTGACCATGCAGATGAGAAATTCTAAAACTGGA ACAGATACAGACTACATTGTGTCCCCTGTTGCCGATGCTAACGGAAGATTCTTCG ACAGCAGACAAGCCGACGCCACCATGCCAAAGGACGCCGACGCCAACGGCGCC TACAACATCGCTAGAAAGGGCATCATGCTGGTTCAGCAGATCAAGCAGAGCGAT GACCTCCGCACCATGAAATTCGACATCAGCAACAAGAGCTGGCTGAGATTCGCC CAGCATACCAACCAGGCCGATGAG Expression ATGggcAAGCTGGAGGACTTCACCAATCTGTACTCTCTGAGCAAGACCCTGCGGT 54 construct(with TTGAGCTGCGGCCTATCGGCAAGACAAGAGAAAACATCGAAAACGGCGGACTGC N-terminal TGCGTCAAGACGAGGACAGAGCCGAAAAGTACGTGCATATTAAAAAGCTGATCG methionine ATGAATACCACAAGGCTTATATCGACAAGCAACTGAGTGGCCTGGTCCTGCAATA andstop CGCCGATATCGGCAAGGCCAATTCTCTGGAGGAGTACTACCACAGCACTAGAAA codon, AAGCAAGGACTCTGACAAGGATAAGATAGTCAAGATCCAGGACAACCTGCGCAA includesV5- GCAGATCGTCAAGAGATTGAAGGACAGCGATGAGTTTAAGAGGATCGATAAGAA tagandC- GGAACTGATCCAGTCTGACCTGGCAGAGTTCATCAAGCCAGCCGAGGACAGGGC terminalNLS) CCTGATAGCCGAGTTCAAGAACTTCACCACCTACTTCACAGGATTCAACGAAAAT AGGCAGAACATGTACAGCGATAAGGCTATCAGCACCGCCATCGCCTACCGGCTG ATCCACGAGAACCTGCCTAAGTTCATCGACAACATCGAAACCTTCGACCGGATCG CGGGCATCACAGAGCTGTATGACCAGACATCCAGCGACGCAGAGATCTTTAGAC TGGAGCACTTCAGTGAGACACTGAGCCAGAAGCAGATCGATGCCTATAACAGCG TGATGGGCCGGTACAACATGCTGATCAACGAATATAACCAGACCCACAAGCAATC TCGGCTGCCTAAATTCAAAATGCTGTACAAGCAGATCCTGAGCGACCGGGAGCA CCCCAGCTGGCTGCCGGAACAGTTCGAGAGCGACACCGCCGTGCTGACCGCCA TCAGAGAGTGTTACGACGACCTGAGAATCCCTATGGCCAACTTAAAAACCCTGCT TGAGGGCCTGGGAAATTACGATCCCTCTGGCATCTTCCTGCGGAACGATCAGCA CCTGTCTCAGATCAGCAAAAGACTCACCGGAGACAGATCCAGCATCGAACGGAG CGTGACCGAGGACTTATTAACGAGCCGGAGACTGAACAAAAGAAAGAGCAGAAC CACCGATGAAGAGGAAAGCAGAAAGCTGTTCAAGCAAAAAGGCAGCCTGAGCAT CGGCTACATCGCCGACACAGCCAAGATCGACGTGGAGAGATACTTCGCCAAGCT GGGAGCCATTAATACCGTGACCGAGCAGTCTGAGAACCTCTTCGCTAAGGCCGA GAACGCCAGAACCACTGCTGACGAGCTGCTGGCCAACGACTACCCTGCCGGCA AAAGACTGGTGCAGAGCAACGACGACATCGCTCTGCTGAAGAACCTATTGGACG CCCTGATGGAACTGCAATGGTTCGTGAAGCCCCTGCTGGGCACCGGCGACGAG GCCGGCAAAGACGAACGGTTCTATGGCGAGTTCGCTCAGATCTGGGAGCAGCTG GATAGAATCACCCCTCTGTACAACATGGTGCGGAATTACGTGACAAGAAAGCCCT ACTCCACAGACAAGTTCAAGCTGAACTTCGAATCTGCCGCCCTGCTGGGCGGAT GGGACAAGAACAAAGAACCTGACTGCCTGTCCGTGATTCTGAGAAAGGACGAGC AGTACTACCTGGGCATCATCAACAAGAACCACAAGAAGATCTTCGAGAATGACAT TCTGCCTTGCGAGGGCGAGTGCTACGACAAGATGGTCTACAAGCTGCTGCCTGG CGCTAACAAAATGCTGCCTAAGGTGTTCTTTAGCGCCTCCAGAATCGCTGAGTTC GCCCCTTCTGATGAGGTGAAAAGAATTTACAACGATAAGACCTTCCAGAAGGGCG AGAAGTTCGATCTGAACGACTGCAGAACCCTCATCGATTTCTACAAGGCTTCTAT CGATAAGCACGAGGAGTGGAATAAATTTGGCTTCGAGTTTAGCGACACCAACAAC TACGAGGACATCAGCGGCTTCTTCCGGGAGGTGGACAGACAGGGCTACAAGATG AGCTTTAGACCCGTGGCCGCCAGCTACATCGAAACGTTGGTGGAAGAGGGCAAA CTGTACCTGTTCCAGATCTACAACAAAGATTTCAGCGCCTACAGCAAGGGCACCC CTAATATGCACACCCTGTACTGGAGAATGCTGTTTGACGAGCGGAACCTGAGCG ACGTGGTGTACCAGCTGAACGGCGGAGCTGAACTGTTCTTTAGACGCAAGTCCC TCCAGAACGGCCGGCCTACACACCCTGCCAACATCCCTATCAAGAACAAGAACA GCAGAAACGATAAAAAGGAATCACTGTTCGACTACGATCTCATCAAGGATCGTAG ATACACAGTGGATAAGTTCCAGTTCCACGTGCCAATCACACTGAATTTCAAGAGC GATGGCGCTGGCAGAATTAACGAGAGAGTGCGGGAGTACCTGAGATCTGCCGAT GACGTGCACGTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTACCTCGT GGTGACCGATATGGACGGCAACATCTGCGAACAGTTTAGCCTGAACGAAATCTG TAATACCGACTACCACAGCCTGTTGGATGAGAGAGAGCACAAAAGAATGCAGGA AAGACAGAGCTGGCAGGCCATCGAGGGAATCAAGGAGCTGAAGGAAGGCTACC TGTCCCAAGTGGTCCACAGAATCGCCACCCTGATGGTGAAGTACAGAGCGATCG TGGTGCTGGAGGACCTGAACTTCGGCTTCATGCGGAGCAGACAGAAAGTGGAAA AAAGCGTGTACCAGAAGTTCGAGCACATGCTGATCGACAAACTGAACTACCTGGT GGACAAGAAAGCCAACCCTACCACACCCGGCGGCCTGCTGAAGGCCTACCAGC TGACAGACAAGTTCGAGAGCTTCCAGAAGCTGGGCAAGCAGTCTGGATTCCTGT TTTATGTGCCCGCCTGGAACACAAGCAAGATCGACCCTGCTACCGGATTCGTGA ACATGCTGGATCTGGGCTATGAGAGCATCGACAAGGCCAAAACCCTGCTGTGCA AGTTTGACTCCATCAGATACAACGCCTGCAAGGACTGGTTCGAGTTTGCCCTGGA CTACGACAAGTTCGGCAGCAAGGCCACAGGCACACGGACCAAGTGGACAGTGT GCACCTACGGCCAGCGGATCGATACTTATAGAAACAAGGACAGCCAGTGGGTGT CTCGGGACGTGGATCTGACCAATGAGCTGAAGAGCCTGTTTTCTGAACATGGCA TCGACATCTACAGCAACCTGAAAGACGCCATCGTGGCCCAAAATGACAAAGAGTT CTTCGCCAACATGCAGAGAATCCTGAAGCTGACCATGCAGATGAGAAATTCTAAA ACTGGAACAGATACAGACTACATTGTGTCCCCTGTTGCCGATGCTAACGGAAGAT TCTTCGACAGCAGACAAGCCGACGCCACCATGCCAAAGGACGCCGACGCCAAC GGCGCCTACAACATCGCTAGAAAGGGCATCATGCTGGTTCAGCAGATCAAGCAG AGCGATGACCTCCGCACCATGAAATTCGACATCAGCAACAAGAGCTGGCTGAGA TTCGCCCAGCATACCAACCAGGCCGATGAGtctagaAAGCGGACAGCAGACGGCTC CGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCA ATCCCCTGCTGGGCCTGGACAGCACCTGA

[0112] In some embodiments a ZXPB Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 49, SEQ ID NO:50, or SEQ ID NO:51. In some embodiments, a ZXPB Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 49, SEQ ID NO:50, or SEQ ID NO:51. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D821 substitution, wherein the position of the D821 substitution is defined with respect to the amino acid numbering of SEQ ID NO:50 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E906 substitution, wherein the position of the E906 substitution is defined with respect to the amino acid numbering of SEQ ID NO:50 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1116 substitution, wherein the position of the R1116 substitution is defined with respect to the amino acid numbering of SEQ ID NO:50 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1153 substitution, wherein the position of the D1153 substitution is defined with respect to the amino acid numbering of SEQ ID NO:50 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZXPB Type V Cas protein is catalytically inactive, for example due to a R1116 substitution in combination with a D821 substitution, a E906 substitution, and/or D1153 substitution.

6.2.10. ZPPX Type V Cas Proteins

[0113] In one aspect, the disclosure provides ZPPX Type V Cas proteins. ZPPX Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZPPX Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:55. In some embodiments, the ZPPX Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:55. In some embodiments, a ZPPX Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:55.

[0114] Exemplary ZPPX Type V Cas protein sequences and nucleotide sequences encoding exemplary ZPPX Type V Cas proteins are set forth in Table 1J.

TABLE-US-00011 TABLE1J ZPPXTypeVCasSequences SEQ ID Name Sequence NO. Wildtype MKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKAFI 55 aminoacid TMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFKS sequence GNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAKS (withoutN- TAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYFP terminal WVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDKV methionine) ALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISNDL GLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISYL DSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSEE ATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVRN WLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLRD FPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIRLE EAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSSEY NSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPNMH TLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQNEK STSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGIDRGE RHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWKKIE NIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEHMLID KLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKIDPVTG FVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKTQWTL CTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAKREHL EPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADANGAYNI ARKGLWWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFE Wildtype MKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKAFI 56 aminoacid TMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFKS sequence(with GNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAKS N-terminal TAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYFP methionine) WVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDKV ALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISNDL GLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISYL DSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSEE ATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVRN WLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLRD FPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIRLE EAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSSEY NSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPNMH TLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQNEK STSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGIDRGE RHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWKKIE NIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEHMLID KLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKIDPVTG FVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKTQWTL CTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAKREHL EPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADANGAYNI ARKGLWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFE Expression MGKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKA 57 construct(with FITMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFK N-terminal SGNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAK methionine, STAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYF V5-tagandC- PVVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDK terminalNLS) VALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISND aasequence LGLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISY LDSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSE EATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVR NWLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLR DFPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIR LEEAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSS EYNSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPN MHTLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQ NEKSTSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGID RGERHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWK KIENIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEH MLIDKLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKID PVTGFVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKT QWTLCTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAK REHLEPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADAN GAYNIARKGLWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFESRKRTADGSEF ESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAAGACCTGACAGGGCAATATAGCCTGTCGAAAACTTTACGATTTGAGTTAA 58 coding AACCTATCGGTAAAACTCTTGAGCACATTGAGCAAAAAGGACTCTTGACACAGGA sequence(with CGAACAAAGAGCAGAAGAGTACGAGCAAATGAAAGGTATCATCGACCGATATCA N-terminal CAAGGCATTTATTACCATGTGTTTGAGAAACTGCAAAATCAAGGTAAATAATACAG methionine ACGACGAATTAGACTCATTAGAAGAATACTCCTCATTACTTTCCAAAAGTAAAAGA andstop GATGCTGATGATGAGAACAAATTGGAAAAGATTAAGGAAAATCTTCGCAAGCAAA codon) TCGTCAATGCTTTCAAAAGCGGCAACACTTATGGCGACTTGTTCACAAAGGAACT GATTAAGAATCATCTGCCCGACTTCGTCACAGACGAGGAAGAAAAGCAAGTGGT GGAGCATTTCTGCAATTTTACCACATATTTTACGGGTTTCCACGACAACCGCAAAA ACATGTACTCAGATAAGGCTAAATCCACGGCAATAGCCTATCGCCTGATACATGA GAATTTCCCTCGGTTTTTTGACAATCTTCGCTCTTTTGCAAAGATTTCAGAAAGCG AGGTGGCAAATCGGTTCCCTGAGATAGAATCTGCTTTCTCTCTGTATCTCAACGT GGAACACATCGCCGACATGTTCCACGTTGACTATTTCCCAGTTGTTCTTACCCAA GAACAAATTGATGTGTATAATAATATTATTGGAGGCAAGACGGAAGAAGATGGGA CAAAAATACAGGGCATCAATGAATACATCAACCTTTATAACCAACATCACCCAGAT GTAAAGTTGCCGTTCTTGAAACCTCTATACAAGATGATTCTTAGCGACAAGGTTG CGCTTTCATGGTTGCCGGAGGAGTTTGAGAATGATGAAGAGATGTTGACGGCCA TAAATGATTTTTACAAGTCAGTTCAGCCTGTCGTTTTCGGGGATGACGAGAATTGT ATCCGTCATCTTCTGACGAATATTGCCGAATACAATACGGATCACATATACATTTC AAACGATTTAGGATTGACTGGAATATCCCAGCAATTGTTCGACCAATACAGCATCT TTGAAGACGTCATTAAAGATGAGTTGAGGCGTAATGTCAAACAGACGCCCAAAGA GAAACGCAATCCTGAATTGTTGGAAGAAAGAATAAAGAACTTGTTCAAGAAAGAG AAGAGTTTCTCCATCTCTTACCTGGACTCTCTCATTAAGGATAAGGGTGAGGATA CGATCGAGTCTTATTATGCCAAACTTGGTGCGTTTGACAGAGACGGTAAGCAAAC AGTGAATTTGCTCACGCAAATTGAAATGGCATACATAGCGGCAAAGGAGGTGCTT GATGGTAAGTATGACAACATTAACCAGTCTGAAGAAGCAACGAAATATATTAAAGA TCTTCTTGATGCGTTCAAGTCTTTGCAACACTACATCAAACCGCTGTTAGGTAGTG GCGAAGAAGCAGAAAAGGATAATGTGTTTAGTTCGCAACTGCTCAATGTTTGGGA GGCGTTAGACGTTGTGACTCCTCTTTATAACAAAGTTCGCAACTGGCTCACACGC AAGCCTTACTCAACAAAAAAGATAAAGCTGAACTTTGAGAATGTCCAACTGCTTG GCGGCTGGCCAAATATAGAAGCGTATTCATGTGCTATTTTTATGAAGGATGATAAT ACTTACTATCTTGGAATACTGGACAATGCATATAAAACTTTATTAAGAGATTTTCCA GAGCCTGCCGAAGAGAAGGATACTATTGGGCTAATGCATTACCTCCAAGGAGGC GATATGGGAAAAAATATTCAGAATTTGATGGTGGTAGATGGAAAGGTTCGGAAAG TTAATGGGCGCAAAGAGAAGTCAGGAATTAATGTTGGGCAGAATATTCGATTAGA AGAAGCAAAAAAGAGATACCTGCCAACAGAAATCAATAGAATAAGGAAGTTGGGA ACGTATTCTGTTTCAAATCCAAATTATAACAAACAAGATTTGATAACCATAATCGAT TATTACAAGCCACTGGCTTGTGAATACTATGCTTCCTATACATTCCATTTCAAGGA TTCTTCCGAGTATAATTCGTTCGCGGAGTTTACAGACGATATCAATCAGCAAGCG TATCAACTTGGGTTTGTACCTTTTTCTCAACAATACTTAAACAAACTTGTAGACGAA GGCAAACTCTACCTTTTCCAAATATGGAATAAAGATTTCTCTGATTATAGTAAAGG CACTCCCAATATGCATACCCTTTATTGGAAGGCGCTCTTTGATAAAGCAAATCTTG CCGATGTTGTCTACAAACTTAATGGTCGTCAGGCAGAGGTGTTCTATCGGAAAAG AAGCCTCCAAAAAGAGAATACGACTGTGCACAAAGCATTGCAGCCTATAAAGAAT AAAAACACGCAGAATGAGAAAAGCACCAGTACGTTTGACTATGACATCGTAAAAG ATCGTCGTTATACAGTTGATAAATTCCATTTCCATGTGCCCATTACTATTAACTTTA AGTCATCTGGAAAACCTAATATCAATGAACACGTTTTAGATATTATCCGTCACCAT GGCATTGAGCATGTCATCGGAATCGACCGTGGCGAGCGCCATCTATTATATCTTT CTCTTATAGATCTCAAGGGAAGAATAATCAAGCAAATGACGCTTAATGAGATAAAG CAGCAAACAGGCGGTAACTATGGCACAAATTATAAAGAACTCTTGGCCGCAAGAG AAGGCGATCGTGCGGAAGCGCGTCGTAACTGGAAAAAGATAGAGAATATTAAAG ACCTTAAAGCTGGCTATCTCAGTCAGGTTGTACATGTGATAGCCCAAATGATGGT GGAATACAATGCCATCGTTGTGCTCGAAGACCTCAATATGGGCTTTATGCGTGGG CGGCAGAAAATCGAGCGGAGCGTATACGAGCAGTTCGAACACATGCTGATAGAT AAGTTGAACTTCTATGTTGATAAGAAAAAGGAAGCATGTGCCCCCGGAGGTCTGC TTCATGGTCTCCAATTAGCCAATAAATTTGAGAGCTTCAATAAGCTTGGGAAACAG AGCGGTTGCCTTTTTTATGTACCGGCATGGAATACCAGCAAAATAGATCCTGTCA CAGGGTTTGTCAATATGCTTGATGCACGCTATGAAAGTGTAGAAAGTTCGCGCCG CTTCTTCTCTCGTTTCGATGTTATTCGTTACAATGAGGAAAAGAATTGGTTTGAAT TTACTTTTGATTATAATAACTTCCATGCAAAGTTGGACGGGACAAAAACCCAATGG ACGCTTTGCACATACGGCAGTCGCATCAAAACATTCCGCAACCCCGCAAAACTCA ATCAATGGGATAATGAAGAGGTGGTTCTTACCGATGAATTTAAGAAGGTATTTGC CAATGCTGGTATCAATATTCATGGGAATTTGAAAGAGGCCATTTGCTCTCTTGCTA AACGGGAGCATTTAGAACCGTTGATGCATTTGATGAAACTGCTTTTACAGTTGCG CAACAGCAAGACCAACTCAGAGGTCGACTATATGCTTTCTCCTGTGGCAGATAAT GGCGTGTTTTACGACAGCCGTTCTTGCAATGGCAATTTGCCTATAGATGCCGATG CCAATGGGGCATACAACATTGCCCGGAAAGGATTATGGGTTTTGCGCCAAATTCA GGACTCTAAGCCTGGCGACAAACTGAATTTGGCTTTGTCGAACAAGGAATGGTTG CGATTTGTTCAAGAAAAGAGCAACTTTGAATAA Codon AAGGATCTGACAGGCCAGTACAGCCTCTCTAAGACCCTCAGATTTGAACTGAAGC 59 optimized CTATCGGCAAGACCCTGGAGCACATCGAGCAAAAGGGCCTGCTGACCCAGGAC coding GAGCAGAGAGCCGAGGAATACGAGCAGATGAAGGGAATTATTGACAGATACCAC sequence(no AAGGCCTTCATCACTATGTGCCTGAGAAATTGCAAGATCAAGGTGAACAACACCG N-terminal ACGATGAGCTGGACAGCCTGGAAGAGTACAGCAGCCTGCTGTCAAAGTCTAAGC methionine,no GGGACGCCGACGACGAGAACAAACTGGAGAAGATCAAGGAAAACCTGAGAAAG stopcodon) CAGATCGTCAATGCCTTCAAGAGCGGAAACACCTACGGCGATCTGTTCACCAAG GAGCTGATCAAGAACCACCTCCCCGATTTTGTGACCGACGAGGAAGAAAAGCAG GTGGTGGAACACTTCTGCAACTTCACCACCTACTTCACCGGCTTTCACGACAACC GCAAGAACATGTACAGCGACAAGGCCAAGAGCACAGCCATCGCCTACAGACTGA TCCACGAGAACTTTCCAAGATTTTTCGATAATCTGCGGAGCTTTGCCAAGATCTC CGAATCTGAAGTGGCCAACAGATTCCCAGAAATCGAGAGCGCCTTTAGCCTGTA CCTGAATGTGGAACATATCGCCGATATGTTCCACGTGGACTACTTCCCAGTGGTG CTGACCCAGGAGCAGATTGACGTGTACAACAACATCATCGGAGGCAAGACCGAG GAAGATGGCACAAAGATTCAGGGCATCAACGAGTATATCAACCTGTACAACCAAC ACCATCCTGACGTCAAACTGCCCTTCCTGAAGCCTCTGTATAAGATGATCCTGAG CGACAAGGTGGCCCTGAGCTGGCTGCCTGAAGAGTTCGAGAACGACGAGGAAA TGCTGACCGCCATCAATGATTTCTACAAGTCTGTGCAGCCTGTGGTGTTCGGCGA TGACGAGAACTGTATCAGACACCTGCTGACAAACATCGCCGAGTACAACACCGAT CACATTTACATCAGCAATGACCTGGGACTGACTGGCATCTCTCAGCAGCTGTTCG ACCAGTACTCTATCTTCGAAGATGTGATCAAGGACGAGCTACGGCGGAACGTGA AGCAAACACCTAAGGAGAAGCGGAACCCCGAACTGCTGGAAGAGAGAATCAAGA ACCTGTTCAAGAAAGAAAAGAGCTTCTCCATCAGCTACCTGGATAGCCTGATCAA GGACAAAGGAGAAGATACCATCGAGAGCTACTACGCCAAGCTGGGCGCCTTCGA CAGAGATGGCAAGCAGACAGTGAACCTGCTCACCCAGATCGAGATGGCCTACAT CGCCGCTAAGGAAGTGCTGGATGGCAAGTACGACAACATCAACCAGAGCGAGGA AGCTACAAAGTACATCAAGGATCTGCTTGACGCCTTCAAGAGCCTGCAGCACTAC ATCAAGCCCCTGCTGGGCAGCGGCGAGGAGGCCGAAAAAGACAACGTGTTCAG CAGCCAGCTCCTGAACGTGTGGGAGGCTCTGGACGTGGTGACGCCTCTGTACAA CAAGGTCAGAAATTGGCTGACAAGAAAGCCCTACAGTACCAAGAAAATCAAACTG AACTTCGAGAATGTTCAACTGCTGGGCGGATGGCCTAACATCGAGGCCTATAGC TGCGCCATTTTTATGAAAGACGACAACACCTACTACTTAGGCATCCTGGACAACG CCTATAAAACACTACTTCGGGACTTTCCTGAACCTGCTGAAGAAAAGGACACAAT CGGCCTGATGCACTACCTGCAAGGAGGCGACATGGGCAAGAACATCCAGAACCT GATGGTCGTCGACGGGAAGGTGCGGAAGGTGAACGGCCGTAAGGAAAAGTCCG GCATCAACGTGGGCCAGAATATCCGGCTGGAGGAGGCCAAGAAGAGATACCTG CCTACAGAGATCAACAGAATCAGAAAGCTGGGCACCTACTCTGTGAGCAACCCTA ATTATAACAAGCAGGATCTGATTACAATCATCGACTACTACAAGCCACTGGCCTG CGAGTACTACGCCTCTTATACATTCCACTTCAAGGACAGCAGCGAGTACAACAGC TTCGCCGAGTTCACCGATGATATCAACCAGCAGGCCTACCAGTTGGGCTTCGTG CCTTTCTCCCAGCAATACCTCAACAAACTGGTGGACGAGGGCAAGCTGTACCTGT TCCAGATCTGGAATAAGGACTTCTCTGACTACTCTAAGGGCACCCCCAACATGCA CACCCTGTACTGGAAGGCCCTGTTTGACAAGGCCAATCTGGCTGATGTGGTTTAC AAGCTGAACGGCAGACAGGCCGAGGTGTTTTACAGAAAGAGAAGCCTGCAGAAA GAGAACACAACCGTGCACAAGGCTCTGCAGCCCATCAAGAATAAGAACACACAG AACGAGAAATCTACCAGCACATTCGATTACGATATCGTGAAGGACAGAAGATACA CCGTGGACAAGTTCCATTTCCACGTTCCTATCACCATCAACTTCAAGTCCAGCGG CAAGCCTAACATCAACGAGCATGTGCTGGATATCATCAGACACCACGGCATCGA GCACGTGATCGGCATCGACCGCGGCGAAAGGCACCTGCTGTACCTGTCCCTGAT CGACCTGAAAGGACGGATCATAAAGCAGATGACCCTTAACGAGATCAAACAACA GACCGGCGGCAACTACGGCACAAACTACAAAGAGCTGCTGGCCGCCAGAGAAG GCGACAGAGCCGAGGCTAGAAGAAACTGGAAGAAAATCGAGAACATCAAGGACC TGAAGGCCGGCTACCTGAGCCAGGTGGTGCACGTGATTGCTCAGATGATGGTGG AATACAACGCCATTGTAGTGCTGGAGGACCTGAACATGGGCTTCATGAGAGGCA GACAGAAGATCGAGAGAAGCGTGTACGAGCAGTTCGAGCACATGCTGATTGACA AGCTGAACTTCTACGTGGACAAAAAGAAGGAAGCATGCGCCCCTGGCGGACTTC TGCACGGCCTGCAGCTGGCCAACAAATTCGAGTCTTTCAACAAACTGGGCAAGC AATCCGGCTGTCTGTTCTACGTGCCCGCCTGGAACACCAGCAAGATCGATCCTG TGACCGGATTCGTGAACATGCTGGACGCCCGGTACGAGAGCGTGGAGAGCTCC CGGCGGTTCTTCTCCAGATTTGACGTGATCAGATACAACGAGGAGAAGAACTGG TTCGAGTTCACCTTTGATTATAACAACTTCCACGCCAAACTGGATGGCACCAAGA CCCAGTGGACACTGTGCACCTACGGCAGCAGAATCAAGACCTTTAGAAATCCTG CTAAGCTGAATCAGTGGGACAATGAAGAGGTGGTTCTGACCGACGAATTTAAGAA GGTGTTCGCCAACGCCGGAATCAATATCCACGGCAACCTGAAGGAAGCTATCTG CAGCCTGGCCAAAAGAGAGCACCTGGAACCTCTGATGCACCTGATGAAACTGCT GCTGCAACTTCGGAATAGCAAAACCAACAGCGAGGTCGACTACATGCTGTCTCC AGTGGCCGATAATGGAGTGTTCTACGACAGCAGAAGCTGTAACGGTAACCTGCC TATCGACGCCGACGCCAACGGAGCCTACAATATCGCTAGAAAAGGTCTGTGGGT CCTCAGGCAAATCCAGGATAGCAAGCCCGGCGACAAGCTGAACCTGGCTCTGAG CAACAAGGAATGGCTGCGATTTGTACAGGAGAAAAGCAATTTCGAG Expression ATGggcAAGGATCTGACAGGCCAGTACAGCCTCTCTAAGACCCTCAGATTTGAACT 60 construct(with GAAGCCTATCGGCAAGACCCTGGAGCACATCGAGCAAAAGGGCCTGCTGACCCA N-terminal GGACGAGCAGAGAGCCGAGGAATACGAGCAGATGAAGGGAATTATTGACAGATA methionine CCACAAGGCCTTCATCACTATGTGCCTGAGAAATTGCAAGATCAAGGTGAACAAC andstop ACCGACGATGAGCTGGACAGCCTGGAAGAGTACAGCAGCCTGCTGTCAAAGTCT codon, AAGCGGGACGCCGACGACGAGAACAAACTGGAGAAGATCAAGGAAAACCTGAG includesV5- AAAGCAGATCGTCAATGCCTTCAAGAGCGGAAACACCTACGGCGATCTGTTCAC tagandC- CAAGGAGCTGATCAAGAACCACCTCCCCGATTTTGTGACCGACGAGGAAGAAAA terminalNLS) GCAGGTGGTGGAACACTTCTGCAACTTCACCACCTACTTCACCGGCTTTCACGAC AACCGCAAGAACATGTACAGCGACAAGGCCAAGAGCACAGCCATCGCCTACAGA CTGATCCACGAGAACTTTCCAAGATTTTTCGATAATCTGCGGAGCTTTGCCAAGA TCTCCGAATCTGAAGTGGCCAACAGATTCCCAGAAATCGAGAGCGCCTTTAGCCT GTACCTGAATGTGGAACATATCGCCGATATGTTCCACGTGGACTACTTCCCAGTG GTGCTGACCCAGGAGCAGATTGACGTGTACAACAACATCATCGGAGGCAAGACC GAGGAAGATGGCACAAAGATTCAGGGCATCAACGAGTATATCAACCTGTACAACC AACACCATCCTGACGTCAAACTGCCCTTCCTGAAGCCTCTGTATAAGATGATCCT GAGCGACAAGGTGGCCCTGAGCTGGCTGCCTGAAGAGTTCGAGAACGACGAGG AAATGCTGACCGCCATCAATGATTTCTACAAGTCTGTGCAGCCTGTGGTGTTCGG CGATGACGAGAACTGTATCAGACACCTGCTGACAAACATCGCCGAGTACAACAC CGATCACATTTACATCAGCAATGACCTGGGACTGACTGGCATCTCTCAGCAGCTG TTCGACCAGTACTCTATCTTCGAAGATGTGATCAAGGACGAGCTACGGCGGAAC GTGAAGCAAACACCTAAGGAGAAGCGGAACCCCGAACTGCTGGAAGAGAGAATC AAGAACCTGTTCAAGAAAGAAAAGAGCTTCTCCATCAGCTACCTGGATAGCCTGA TCAAGGACAAAGGAGAAGATACCATCGAGAGCTACTACGCCAAGCTGGGCGCCT TCGACAGAGATGGCAAGCAGACAGTGAACCTGCTCACCCAGATCGAGATGGCCT ACATCGCCGCTAAGGAAGTGCTGGATGGCAAGTACGACAACATCAACCAGAGCG AGGAAGCTACAAAGTACATCAAGGATCTGCTTGACGCCTTCAAGAGCCTGCAGC ACTACATCAAGCCCCTGCTGGGCAGCGGCGAGGAGGCCGAAAAAGACAACGTG TTCAGCAGCCAGCTCCTGAACGTGTGGGAGGCTCTGGACGTGGTGACGCCTCTG TACAACAAGGTCAGAAATTGGCTGACAAGAAAGCCCTACAGTACCAAGAAAATCA AACTGAACTTCGAGAATGTTCAACTGCTGGGCGGATGGCCTAACATCGAGGCCT ATAGCTGCGCCATTTTTATGAAAGACGACAACACCTACTACTTAGGCATCCTGGA CAACGCCTATAAAACACTACTTCGGGACTTTCCTGAACCTGCTGAAGAAAAGGAC ACAATCGGCCTGATGCACTACCTGCAAGGAGGCGACATGGGCAAGAACATCCAG AACCTGATGGTCGTCGACGGGAAGGTGCGGAAGGTGAACGGCCGTAAGGAAAA GTCCGGCATCAACGTGGGCCAGAATATCCGGCTGGAGGAGGCCAAGAAGAGAT ACCTGCCTACAGAGATCAACAGAATCAGAAAGCTGGGCACCTACTCTGTGAGCA ACCCTAATTATAACAAGCAGGATCTGATTACAATCATCGACTACTACAAGCCACTG GCCTGCGAGTACTACGCCTCTTATACATTCCACTTCAAGGACAGCAGCGAGTACA ACAGCTTCGCCGAGTTCACCGATGATATCAACCAGCAGGCCTACCAGTTGGGCT TCGTGCCTTTCTCCCAGCAATACCTCAACAAACTGGTGGACGAGGGCAAGCTGT ACCTGTTCCAGATCTGGAATAAGGACTTCTCTGACTACTCTAAGGGCACCCCCAA CATGCACACCCTGTACTGGAAGGCCCTGTTTGACAAGGCCAATCTGGCTGATGT GGTTTACAAGCTGAACGGCAGACAGGCCGAGGTGTTTTACAGAAAGAGAAGCCT GCAGAAAGAGAACACAACCGTGCACAAGGCTCTGCAGCCCATCAAGAATAAGAA CACACAGAACGAGAAATCTACCAGCACATTCGATTACGATATCGTGAAGGACAGA AGATACACCGTGGACAAGTTCCATTTCCACGTTCCTATCACCATCAACTTCAAGTC CAGCGGCAAGCCTAACATCAACGAGCATGTGCTGGATATCATCAGACACCACGG CATCGAGCACGTGATCGGCATCGACCGCGGCGAAAGGCACCTGCTGTACCTGTC CCTGATCGACCTGAAAGGACGGATCATAAAGCAGATGACCCTTAACGAGATCAAA CAACAGACCGGCGGCAACTACGGCACAAACTACAAAGAGCTGCTGGCCGCCAG AGAAGGCGACAGAGCCGAGGCTAGAAGAAACTGGAAGAAAATCGAGAACATCAA GGACCTGAAGGCCGGCTACCTGAGCCAGGTGGTGCACGTGATTGCTCAGATGAT GGTGGAATACAACGCCATTGTAGTGCTGGAGGACCTGAACATGGGCTTCATGAG AGGCAGACAGAAGATCGAGAGAAGCGTGTACGAGCAGTTCGAGCACATGCTGAT TGACAAGCTGAACTTCTACGTGGACAAAAAGAAGGAAGCATGCGCCCCTGGCGG ACTTCTGCACGGCCTGCAGCTGGCCAACAAATTCGAGTCTTTCAACAAACTGGGC AAGCAATCCGGCTGTCTGTTCTACGTGCCCGCCTGGAACACCAGCAAGATCGAT CCTGTGACCGGATTCGTGAACATGCTGGACGCCCGGTACGAGAGCGTGGAGAG CTCCCGGCGGTTCTTCTCCAGATTTGACGTGATCAGATACAACGAGGAGAAGAA CTGGTTCGAGTTCACCTTTGATTATAACAACTTCCACGCCAAACTGGATGGCACC AAGACCCAGTGGACACTGTGCACCTACGGCAGCAGAATCAAGACCTTTAGAAAT CCTGCTAAGCTGAATCAGTGGGACAATGAAGAGGTGGTTCTGACCGACGAATTTA AGAAGGTGTTCGCCAACGCCGGAATCAATATCCACGGCAACCTGAAGGAAGCTA TCTGCAGCCTGGCCAAAAGAGAGCACCTGGAACCTCTGATGCACCTGATGAAAC TGCTGCTGCAACTTCGGAATAGCAAAACCAACAGCGAGGTCGACTACATGCTGT CTCCAGTGGCCGATAATGGAGTGTTCTACGACAGCAGAAGCTGTAACGGTAACC TGCCTATCGACGCCGACGCCAACGGAGCCTACAATATCGCTAGAAAAGGTCTGT GGGTCCTCAGGCAAATCCAGGATAGCAAGCCCGGCGACAAGCTGAACCTGGCT CTGAGCAACAAGGAATGGCTGCGATTTGTACAGGAGAAAAGCAATTTCGAGtctaga AAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTG ggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0115] In some embodiments a ZPPX Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 55, SEQ ID NO:56, or SEQ ID NO:57. In some embodiments, a ZPPX Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D877 substitution, wherein the position of the D877 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E969 substitution, wherein the position of the E969 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1181 substitution, wherein the position of the R1181 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1217 substitution, wherein the position of the D1217 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZPPX Type V Cas protein is catalytically inactive, for example due to a R1181 substitution in combination with a D877 substitution, a E969 substitution, and/or D1217 substitution.

6.2.11. ZXHQ Type V Cas Proteins

[0116] In one aspect, the disclosure provides ZXHQ Type V Cas proteins. ZXHQ Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZXHQ Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:61. In some embodiments, the ZXHQ Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61. In some embodiments, a ZXHQ Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:61.

[0117] Exemplary ZXHQ Type V Cas protein sequences and nucleotide sequences encoding exemplary ZXHQ Type V Cas proteins are set forth in Table 1K.

TABLE-US-00012 TABLE1K ZXHQTypeVCasSequences SEQID Name Sequence NO. Wildtype TNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPLFDEL 61 aminoacid HFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITDTFES sequence TGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFFTYFS (withoutN- GFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFEDILSL terminal TTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGSPKED methionine) KRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETILSEV YFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELKQVLD SLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQECNAVKE DTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSLRSYYD AFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYFLAVMN KKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEKYFIIPE EIVQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEEYEDVS KFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGNKNLHTL LFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPILEAKRY YEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLYYYVIKP DGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFESTGKKVD YIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNAVVVLE DLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQLTPAITA DTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYFCKQGE KEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPVEIFDRL RELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDGDFIQSP VAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTFEKMREK DGLNLYISDAEWDTEIS Wildtype MTNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPLFDE 62 aminoacid LHFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITDTFE sequence(with STGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFFTYF N-terminal SGFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFEDIL methionine) SLTTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGSPKE DKRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETILSE VYFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELKQVL DSLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQECNAV KEDTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSLRSY YDAFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYFLAV MNKKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEKYFII PEEIVQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEEYED VSKFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGNKNLH TLLFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPILEAK RYYEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLYYYVI KPDGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFESTGKK VDYIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNAVVV LEDLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQLTPAI TADTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYFCKQ GEKEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPVEIF DRLRELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDGDFI QSPVAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTFEKM REKDGLNLYISDAEWDTEIS Expression MGSGTNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPL 63 construct(with FDELHFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITD N-terminal TFESTGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFF methionine, TYFSGFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFE V5-tagandC- DILSLTTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGS terminalNLS) PKEDKRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETI aasequence LSEVYFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELK QVLDSLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQEC NAVKEDTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSL RSYYDAFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYF LAVMNKKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEK YFIIPEEIVQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEE YEDVSKFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGN KNLHTLLFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPIL EAKRYYEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLY YYVIKPDGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFEST GKKVDYIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNA WVVLEDLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQL TPAITADTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYF CKQGEKEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPV EIFDRLRELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDG DFIQSPVAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTF EKMREKDGLNLYISDAEWDTEISSRKRTADGSEFESPKKKRKVGSGKPIPNPLLG LDST Wildtype ATGACTAACACATCTATTTTCAAAACCTTCACTAATCAATATTCACTTTCAAAAA 64 coding CGTTGCGGTTTGAGTTGAGACCTCATCCGATGACTAGTGGTCTAGATGATATC sequence(with ATTTCATTAGATACTGGCATAAAAAAATTGTATGAAAACGAGATGAAGCCGCTA N-terminal TTTGATGAACTTCATTTTGAATTTATCTCTCAGTCGCTAGTTCAAGTATCATTCC methionine CTTCAGAAAAACTGGAAGTTTTGCTAAACAAGTATAGGTCTCTTAAGGATCAG andstop AAAGCTAAAAATATAGAAAAAGAACTGGAAGGCCCATTACAGGAACTAAGAAC codon) AATTATTACTGACACCTTTGAATCCACTGGTAACAACTGGAAAAAAGAATGGCT ACAACAAGGGTTTAAAATCAAAAGCTCGGGATACAAAGTACTAACAGAAGAGG GAATATTAGAAGTATTGTCTGTTCGTAAAAAAGATAAAGCGGATGCAATCAATA AATTTAAAGGATTCTTCACGTACTTTTCAGGGTTTAACATGAACCGTGAAAATT ATTATTCATCGGAAGATAAAAAAACAGCTGTAGCGTATAGGGTAATTAATGAAA ACCTTATCCGGTATATGGATAACATTCTCCTCCTTCAGAATGTTTTAGCAAAAG CTCCTGAGTTTAAAAAGTTTGAAGATATTTTAAGTCTTACTACATTTGGAAAATA CATAAATCAGGAAGGAATAACTACATATAATAATAACGTAGTTGCAACAATTAA TCTTGAACTTAATACGTACCATCAGCATAATCCAAAAATCTTTTCTCGCCTGCC AAAGTTAAAATTGCTTTATAAACAAATTGGTTCACCAAAAGAGGACAAACGCAT TTTTACTATTGAAAAAAGAACGGAATGGCAGAGTTTGGAAGACTTAATACAAAA ACAGAATAAAGTTGTTGAACACGAAAAAAAGAATGTTGAAATCCTGTCAAATTT GAAAGCAGCATACATTTCTTTTTTCACGAACACAGATGAAACAATCTTAAGCGA GGTATATTTCAATAAGCGTTCTCTTAATACAATTTCTTCTTTCTGGTTTACGGGT GGCTGGCAAACACTGCTTCTTAAACTAAAAGAGTTTAAATTGGCCAATCAAAA CAAAGATGGTGATATAGTAGTCCCTAAAGCATTATCCCTTGCTGAACTAAAAC AGGTGCTTGATTCGTTAGAAGAGCAAGACCCTGCTGTTAATCATTTATTTAAG GAAATGTACTCAGATTGTTACAAAGAAAACCTATGGCAGACCTTTATAGCTATC TGGCAATGTGAAATTACATCAAAATTTAACCTGCTCGAAGGGTATATTCAAGAA TGTAATGCTGTTAAAGAAGACACCTTTGATAAAAAAAAGCATAAAAATATTATC AAAAACATCTGCGATACATACCTGGATATTGAGCAGATATCAAAATACATAATA GTACATGAAAGTCTTCCTAAATATGATGCGCTATATGATGCGGTAATACTTTAT TTGCAGGAATCTTCTTTACGCAGTTATTACGATGCCTTCCGCAACCTTATTAGC AAGCGACCTGTTAACGAAGAAAAAGTTAAGCTCAACTTTCAGAACTCTACCCT GCTTGATGGCTGGGATATGAATAAAGAAAGCGCTAACTTATGCGTATTACTGA AAAACAATATAGGTGAATACTTCCTTGCTGTAATGAATAAAAAGAGCAACATGG TTTTTGATCAGAAGAAAAACTCTGCCCTTTACTCTGCTGGGAATGAAAGTAGTT TTCAGAAGCTGGAGTATAAACTGTTGCCTGGGCCTAACAAAATGCTGCCAAAA GTAATTTTTGCAAAATCGAACGAAAAATATTTCATCATACCGGAAGAAATTGTG CAGATTAGAGAAGAAGAATCGTTTAAAAAAGGAAAAAAATTTGATAAGCATGC ATTGAAAACGTGGATCAGGTTTATGCAGGAATCAATTGAAAAATACCCAGGTT GGAAGACATTCGACTTTACCTTTAAAAAACCGGAAGAGTACGAAGATGTCAGC AAGTTCTATAAAGATGTAGAAGAACAGGGGTATAAACTAAACTGGAAAGATAT TAACGAGGAAGAGCTCCTGTCACTTGTAGAACAAAAAAAAGTATATCTGTTTC AGATAAAAAGCAAAGATATCGGAGAAACAAAGGAGCACGGCAACAAGAACCT TCACACATTGTTATTTTTAGAACTCCTCAAACCGGAAAATACCAGCAGGTTAAA GCTACTGGGCGGTGGCGAAATGTTTTATCGTGCGCCAAGTATGGAAAAGGTA TACAAAACCGTAAATGAAAAACAGGTTCTGGATTCAAAAGGTAACCCCATTTTA GAAGCAAAACGGTACTATGAACCAAAGTTTTTCCTTCACTTCCCTATTCAGGTC AAAGGGAGCGAAAATGGTTATAAAACAGAAATGAATCCGAAAATATTGCGGGC AATTAGCACTTCAAAAGAAGTAAATATAATAGGAATAGACCGTGGAGAAAAGC ATTTACTCTATTATTACGTTATAAAGCCAGACGGAACTCCAATTACTCAAGGAA GCCTGAATACAATTAGTTTAGGTTTAGATAAAAATCAAAATCCCAGACTTGTTG ACGAGCGTACCTTCAAGATTTTGGAGAGAGATTCCAAGGGAAAACCATCAAAA ATATCAGATTTTGAATCTACAGGGAAAAAAGTTGATTACATAGATTATCACAAT ATACTTACCTATTACGAAACAAAACGCAATATAGCACGCCGTTCGTGGGATAC TATTGGGGCAATAAAAAACTTTAAAGAGGGGTACTTGTCTCAGGCGATTCACC AGATTTATCAGCTTATGTTGAAGTATAACGCTGTGGTAGTTTTGGAAGATCTTA ATACGGAGTTTAAGGCAAAACGAACCGCAAAAGTTGAAAAATCCGTGTACGAA AAGTTTGAAATTGCCCTTGCTAAAAAACTGAACCACTTAATTATTAAAGGAACT GACCCTGCAGAAGCAGGAAGCGTAATAAATCCGTATCAGCTTACTCCAGCAAT TACAGCTGATACATTAAGCGACTTTAAGAAATCAAAACAATGGGGTCCGCTTT TCTATATTAGAGCAAACTATACCTCTACGACTGACCCTATAACCGGCTGGCGT AAACACATATATATCCCGTCCGGAGCTTCAGATAAAGAAATTAAAACATATTTC TGTAAACAGGGCGAAAAAGAACCTTTGATTCAGATTTCATATGATACAGCGCT TACCGCGTTTGCATTTACCTATACCCATGAAGGCAAAGAATGGACATTACACG CAACGAAAGATACTCAGCGTATGCGTTATGACAGTAAGAAGCGGAAGATGGA ACCCGTAGAAATATTTGATAGACTACGAGAGCTTTTTATAGATTTTAGTTTCGA AGAATCGTTAACAGATCAACTAGAAGCAACACTTTCCTTTGACTGGAAAACAC TGGCCTTTTTGTGGACAATGTTAAACCAGATACGTAATACCGACAGAGAAGCA GAAGGGAATGACGGTGACTTTATTCAGTCTCCGGTTGCTCCGTTTTATGATAG TCGAGATCCGGAAAATAAAACAAATGGACTTCCTGTTAACGGAGATGCTAATG GGGCTTTCAATATAGCCAGAAAAGGTGCAATCCTGATAAAACGTATTCAAGAA TATGCAAAAAAAGACCCCACCTTTGAAAAGATGAGAGAAAAAGATGGTCTCAA TTTGTATATATCTGATGCAGAGTGGGATACAGAAATAAGCTAA Codon ACAAACACTAGCATCTTCAAGACATTCACCAACCAATACAGCCTCTCCAAGAC 65 optimized CCTGCGGTTTGAGCTCAGACCCCACCCTATGACCTCCGGCCTGGACGACATC coding ATCAGCCTGGACACCGGAATCAAAAAGCTGTACGAGAACGAAATGAAGCCTC sequence(no TGTTCGACGAGCTGCACTTCGAGTTCATCAGCCAGAGCCTGGTCCAGGTCAG N-terminal CTTCCCTAGCGAGAAGCTCGAAGTGCTGCTGAACAAGTACCGGAGCCTGAAG methionine,no GACCAGAAAGCTAAGAACATCGAGAAGGAACTGGAGGGCCCCCTGCAGGAG stopcodon) CTGAGAACCATCATCACCGACACCTTCGAGAGCACCGGCAACAACTGGAAGA AAGAGTGGCTGCAGCAGGGGTTCAAGATCAAAAGCAGTGGATACAAGGTGCT GACAGAGGAGGGCATCCTGGAAGTGCTTTCCGTGCGGAAGAAGGATAAGGC CGATGCTATAAACAAGTTCAAAGGATTCTTCACCTACTTCAGCGGCTTCAACA TGAACAGAGAGAACTACTACAGCAGCGAAGATAAAAAAACAGCCGTGGCCTA CAGAGTGATCAACGAGAACCTGATCCGGTACATGGATAACATCCTGCTCCTG CAGAACGTGCTGGCCAAAGCCCCTGAGTTCAAGAAATTTGAAGATATCCTGA GTCTGACCACCTTCGGCAAGTACATCAACCAGGAGGGCATCACAACCTACAA CAACAACGTTGTGGCCACCATCAACCTGGAGCTGAACACCTACCACCAGCAC AACCCAAAAATCTTCAGCAGACTGCCCAAACTGAAGCTGCTGTACAAGCAGAT CGGTTCTCCAAAGGAGGACAAGCGCATCTTCACCATCGAGAAGAGAACAGAA TGGCAGAGCCTGGAGGACCTGATCCAGAAGCAGAACAAGGTCGTGGAACAC GAAAAGAAGAACGTGGAGATCCTGTCTAATCTGAAGGCCGCCTATATCAGCTT CTTCACAAACACCGACGAAACCATCCTGTCTGAGGTGTACTTCAACAAGAGAA GCCTGAATACGATCAGCAGCTTCTGGTTCACCGGCGGATGGCAAACCCTGCT GCTGAAACTGAAGGAATTTAAGCTGGCTAATCAGAACAAAGACGGCGATATC GTGGTTCCCAAGGCCCTGAGCCTGGCCGAGCTGAAGCAGGTGCTGGACTCC CTGGAAGAGCAGGACCCCGCCGTGAATCACCTGTTCAAGGAAATGTACAGCG ACTGCTACAAGGAAAACCTGTGGCAAACATTTATCGCCATCTGGCAATGTGAA ATCACAAGCAAGTTCAACCTGCTGGAGGGCTATATCCAAGAGTGCAACGCCG TGAAAGAGGACACCTTTGACAAGAAAAAGCACAAAAACATCATCAAGAACATC TGCGACACGTACCTGGACATTGAGCAGATCAGTAAGTACATCATCGTGCACG AAAGCCTGCCTAAATACGACGCCCTCTATGATGCCGTCATCCTGTACCTGCAG GAGTCTAGTCTGCGGTCCTACTACGACGCCTTTAGAAACCTGATTTCTAAGCG GCCAGTGAACGAGGAAAAGGTGAAGCTGAATTTCCAGAATAGCACCCTGCTG GATGGCTGGGACATGAATAAAGAAAGCGCCAATCTTTGTGTGCTGCTGAAGA ACAACATCGGAGAGTACTTTCTGGCCGTGATGAACAAAAAAAGCAACATGGTT TTTGACCAGAAAAAAAACAGCGCCCTGTATAGCGCTGGCAATGAATCTAGCTT CCAGAAGCTGGAGTACAAGCTGTTGCCCGGCCCTAACAAGATGCTGCCTAAG GTGATCTTTGCCAAGTCCAATGAGAAGTACTTCATCATCCCTGAGGAGATCGT GCAGATCAGGGAGGAAGAGAGCTTCAAGAAAGGCAAAAAATTCGATAAGCAC GCGCTGAAAACCTGGATCAGATTCATGCAGGAGTCTATCGAGAAGTATCCTG GCTGGAAAACCTTTGACTTCACATTCAAAAAGCCTGAGGAATACGAGGATGTG TCCAAGTTCTACAAAGACGTGGAAGAGCAGGGCTACAAACTGAACTGGAAGG ATATCAACGAGGAAGAACTGCTGAGCCTGGTGGAACAGAAGAAGGTGTACCT TTTTCAGATCAAGTCCAAAGACATAGGCGAGACAAAGGAACACGGAAATAAGA ACCTGCACACCCTGCTCTTCCTAGAATTGCTGAAGCCTGAGAACACAAGTCG GCTGAAGCTGTTGGGCGGCGGAGAAATGTTCTACCGGGCCCCTTCTATGGAA AAAGTCTACAAAACAGTGAACGAGAAGCAGGTGCTGGATTCTAAAGGCAACC CTATCCTGGAGGCCAAGCGCTACTACGAGCCTAAGTTTTTTCTGCATTTCCCC ATCCAGGTGAAGGGCTCTGAGAACGGCTATAAGACCGAGATGAACCCCAAAA TCCTCAGAGCCATCAGCACCAGCAAGGAAGTGAACATCATTGGCATCGACAG AGGCGAGAAGCACCTGCTGTACTATTACGTGATCAAGCCCGACGGAACACCT ATCACCCAGGGCAGCCTGAACACCATCTCCCTGGGCCTTGATAAGAATCAAA ATCCTAGACTGGTGGACGAGAGAACCTTCAAGATCCTGGAAAGAGATAGCAA GGGCAAGCCAAGCAAGATCTCAGATTTTGAAAGCACAGGCAAGAAGGTCGAC TACATCGACTACCACAACATCCTGACATACTATGAAACCAAGAGAAATATCGC CAGAAGAAGCTGGGACACAATTGGCGCCATCAAGAATTTCAAGGAGGGATAC CTCTCTCAGGCCATCCACCAGATCTACCAGCTGATGCTGAAATATAACGCCGT GGTGGTGCTAGAGGACCTGAACACCGAGTTCAAGGCAAAGAGAACCGCCAA GGTGGAAAAAAGCGTGTACGAAAAGTTTGAGATAGCTCTGGCCAAGAAGCTG AATCACCTGATCATCAAGGGCACCGACCCAGCCGAGGCCGGATCTGTGATCA ACCCTTACCAGCTGACCCCTGCTATTACAGCCGACACACTGAGCGATTTCAAG AAGAGCAAACAATGGGGCCCTCTGTTCTACATCCGGGCCAACTACACCAGCA CAACCGACCCTATCACAGGCTGGAGAAAGCACATCTACATCCCCAGCGGAGC CAGTGACAAGGAAATCAAGACCTACTTCTGCAAGCAGGGCGAGAAGGAGCCT CTGATCCAGATTAGCTACGACACCGCCCTGACCGCCTTCGCCTTCACATACA CCCACGAAGGCAAGGAGTGGACCCTACATGCCACAAAGGATACCCAAAGAAT GCGGTACGACAGCAAGAAGAGAAAGATGGAACCCGTGGAAATCTTCGACAGA CTGAGAGAGCTGTTCATCGACTTCTCTTTCGAGGAAAGCCTGACCGACCAGC TGGAGGCAACCCTGTCCTTCGACTGGAAAACCCTGGCTTTTCTGTGGACAAT GCTGAATCAGATCAGAAACACCGATAGAGAGGCTGAAGGCAACGACGGCGA CTTCATCCAGTCTCCTGTGGCCCCTTTCTATGATAGCCGGGACCCAGAGAAC AAGACCAATGGCCTGCCCGTTAACGGCGACGCCAACGGCGCCTTCAACATCG CTAGAAAGGGGGCTATCCTGATCAAGAGAATCCAGGAATACGCCAAGAAGGA CCCTACATTCGAGAAGATGCGGGAAAAGGACGGTTTAAACCTGTACATCAGC GATGCTGAGTGGGATACCGAGATCAGC Expression ATGggctccggaACAAACACTAGCATCTTCAAGACATTCACCAACCAATACAGCCT 66 construct(with CTCCAAGACCCTGCGGTTTGAGCTCAGACCCCACCCTATGACCTCCGGCCTG N-terminal GACGACATCATCAGCCTGGACACCGGAATCAAAAAGCTGTACGAGAACGAAA methionine TGAAGCCTCTGTTCGACGAGCTGCACTTCGAGTTCATCAGCCAGAGCCTGGT andstop CCAGGTCAGCTTCCCTAGCGAGAAGCTCGAAGTGCTGCTGAACAAGTACCGG codon, AGCCTGAAGGACCAGAAAGCTAAGAACATCGAGAAGGAACTGGAGGGCCCC includesV5- CTGCAGGAGCTGAGAACCATCATCACCGACACCTTCGAGAGCACCGGCAACA tagandC- ACTGGAAGAAAGAGTGGCTGCAGCAGGGGTTCAAGATCAAAAGCAGTGGATA terminalNLS) CAAGGTGCTGACAGAGGAGGGCATCCTGGAAGTGCTTTCCGTGCGGAAGAA GGATAAGGCCGATGCTATAAACAAGTTCAAAGGATTCTTCACCTACTTCAGCG GCTTCAACATGAACAGAGAGAACTACTACAGCAGCGAAGATAAAAAAACAGCC GTGGCCTACAGAGTGATCAACGAGAACCTGATCCGGTACATGGATAACATCC TGCTCCTGCAGAACGTGCTGGCCAAAGCCCCTGAGTTCAAGAAATTTGAAGA TATCCTGAGTCTGACCACCTTCGGCAAGTACATCAACCAGGAGGGCATCACA ACCTACAACAACAACGTTGTGGCCACCATCAACCTGGAGCTGAACACCTACC ACCAGCACAACCCAAAAATCTTCAGCAGACTGCCCAAACTGAAGCTGCTGTAC AAGCAGATCGGTTCTCCAAAGGAGGACAAGCGCATCTTCACCATCGAGAAGA GAACAGAATGGCAGAGCCTGGAGGACCTGATCCAGAAGCAGAACAAGGTCG TGGAACACGAAAAGAAGAACGTGGAGATCCTGTCTAATCTGAAGGCCGCCTA TATCAGCTTCTTCACAAACACCGACGAAACCATCCTGTCTGAGGTGTACTTCA ACAAGAGAAGCCTGAATACGATCAGCAGCTTCTGGTTCACCGGCGGATGGCA AACCCTGCTGCTGAAACTGAAGGAATTTAAGCTGGCTAATCAGAACAAAGACG GCGATATCGTGGTTCCCAAGGCCCTGAGCCTGGCCGAGCTGAAGCAGGTGC TGGACTCCCTGGAAGAGCAGGACCCCGCCGTGAATCACCTGTTCAAGGAAAT GTACAGCGACTGCTACAAGGAAAACCTGTGGCAAACATTTATCGCCATCTGG CAATGTGAAATCACAAGCAAGTTCAACCTGCTGGAGGGCTATATCCAAGAGTG CAACGCCGTGAAAGAGGACACCTTTGACAAGAAAAAGCACAAAAACATCATCA AGAACATCTGCGACACGTACCTGGACATTGAGCAGATCAGTAAGTACATCATC GTGCACGAAAGCCTGCCTAAATACGACGCCCTCTATGATGCCGTCATCCTGT ACCTGCAGGAGTCTAGTCTGCGGTCCTACTACGACGCCTTTAGAAACCTGATT TCTAAGCGGCCAGTGAACGAGGAAAAGGTGAAGCTGAATTTCCAGAATAGCA CCCTGCTGGATGGCTGGGACATGAATAAAGAAAGCGCCAATCTTTGTGTGCT GCTGAAGAACAACATCGGAGAGTACTTTCTGGCCGTGATGAACAAAAAAAGC AACATGGTTTTTGACCAGAAAAAAAACAGCGCCCTGTATAGCGCTGGCAATGA ATCTAGCTTCCAGAAGCTGGAGTACAAGCTGTTGCCCGGCCCTAACAAGATG CTGCCTAAGGTGATCTTTGCCAAGTCCAATGAGAAGTACTTCATCATCCCTGA GGAGATCGTGCAGATCAGGGAGGAAGAGAGCTTCAAGAAAGGCAAAAAATTC GATAAGCACGCGCTGAAAACCTGGATCAGATTCATGCAGGAGTCTATCGAGA AGTATCCTGGCTGGAAAACCTTTGACTTCACATTCAAAAAGCCTGAGGAATAC GAGGATGTGTCCAAGTTCTACAAAGACGTGGAAGAGCAGGGCTACAAACTGA ACTGGAAGGATATCAACGAGGAAGAACTGCTGAGCCTGGTGGAACAGAAGAA GGTGTACCTTTTTCAGATCAAGTCCAAAGACATAGGCGAGACAAAGGAACAC GGAAATAAGAACCTGCACACCCTGCTCTTCCTAGAATTGCTGAAGCCTGAGAA CACAAGTCGGCTGAAGCTGTTGGGCGGCGGAGAAATGTTCTACCGGGCCCC TTCTATGGAAAAAGTCTACAAAACAGTGAACGAGAAGCAGGTGCTGGATTCTA AAGGCAACCCTATCCTGGAGGCCAAGCGCTACTACGAGCCTAAGTTTTTTCTG CATTTCCCCATCCAGGTGAAGGGCTCTGAGAACGGCTATAAGACCGAGATGA ACCCCAAAATCCTCAGAGCCATCAGCACCAGCAAGGAAGTGAACATCATTGG CATCGACAGAGGCGAGAAGCACCTGCTGTACTATTACGTGATCAAGCCCGAC GGAACACCTATCACCCAGGGCAGCCTGAACACCATCTCCCTGGGCCTTGATA AGAATCAAAATCCTAGACTGGTGGACGAGAGAACCTTCAAGATCCTGGAAAG AGATAGCAAGGGCAAGCCAAGCAAGATCTCAGATTTTGAAAGCACAGGCAAG AAGGTCGACTACATCGACTACCACAACATCCTGACATACTATGAAACCAAGAG AAATATCGCCAGAAGAAGCTGGGACACAATTGGCGCCATCAAGAATTTCAAG GAGGGATACCTCTCTCAGGCCATCCACCAGATCTACCAGCTGATGCTGAAAT ATAACGCCGTGGTGGTGCTAGAGGACCTGAACACCGAGTTCAAGGCAAAGAG AACCGCCAAGGTGGAAAAAAGCGTGTACGAAAAGTTTGAGATAGCTCTGGCC AAGAAGCTGAATCACCTGATCATCAAGGGCACCGACCCAGCCGAGGCCGGAT CTGTGATCAACCCTTACCAGCTGACCCCTGCTATTACAGCCGACACACTGAG CGATTTCAAGAAGAGCAAACAATGGGGCCCTCTGTTCTACATCCGGGCCAAC TACACCAGCACAACCGACCCTATCACAGGCTGGAGAAAGCACATCTACATCC CCAGCGGAGCCAGTGACAAGGAAATCAAGACCTACTTCTGCAAGCAGGGCGA GAAGGAGCCTCTGATCCAGATTAGCTACGACACCGCCCTGACCGCCTTCGCC TTCACATACACCCACGAAGGCAAGGAGTGGACCCTACATGCCACAAAGGATA CCCAAAGAATGCGGTACGACAGCAAGAAGAGAAAGATGGAACCCGTGGAAAT CTTCGACAGACTGAGAGAGCTGTTCATCGACTTCTCTTTCGAGGAAAGCCTGA CCGACCAGCTGGAGGCAACCCTGTCCTTCGACTGGAAAACCCTGGCTTTTCT GTGGACAATGCTGAATCAGATCAGAAACACCGATAGAGAGGCTGAAGGCAAC GACGGCGACTTCATCCAGTCTCCTGTGGCCCCTTTCTATGATAGCCGGGACC CAGAGAACAAGACCAATGGCCTGCCCGTTAACGGCGACGCCAACGGCGCCT TCAACATCGCTAGAAAGGGGGCTATCCTGATCAAGAGAATCCAGGAATACGC CAAGAAGGACCCTACATTCGAGAAGATGCGGGAAAAGGACGGTTTAAACCTG T ACATCAGCGATGCTGAGTGGGATACCGAGATCAGCtctagaAAGCGGACAGCAG ACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAAC CTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0118] In some embodiments a ZXHQ Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 61, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, a ZXHQ Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D836 substitution, wherein the position of the D836 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E963 substitution, wherein the position of the E963 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1172 substitution, wherein the position of the R1172 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1211 substitution, wherein the position of the D1211 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZXHQ Type V Cas protein is catalytically inactive, for example due to a R1172 substitution in combination with a D836 substitution, a E963 substitution, and/or D1211 substitution.

6.2.12. ZQKH Type V Cas Proteins

[0119] In one aspect, the disclosure provides ZQKH Type V Cas proteins. ZQKH Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZQKH Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:67. In some embodiments, the ZQKH Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67. In some embodiments, a ZQKH Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:67.

[0120] Exemplary ZQKH Type V Cas protein sequences and nucleotide sequences encoding exemplary ZQKH Type V Cas proteins are set forth in Table 1L.

TABLE-US-00013 TABLE1L ZQKHTypeVCasSequences SEQ ID Name Sequence NO. Wildtype AYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDLIGIINK 67 aminoacid KLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNGLEDFC sequence NLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYESLILINE (withoutN- TFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHRRFKKLN terminal KQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYDEHIYYF methionine) RKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDSSYTDIE KCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGEEEDRNE DFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWKEKAAYI KRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKANAGKG RPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSHPDYNR FPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLYNKDYA KNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELNMRLIPH RYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYYSVLRL SDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLSQAIHQL TSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEGQAGNIY SALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNICELLYR FENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGSERNKN RRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLMLSLRNS IVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGILTIRKL ENGNEKALTLDEWVISTQKGNIRM Wildtype MAYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDLIGII 68 aminoacid NKKLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNGLED sequence(with FCNLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYESLILI N-terminal NETFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHRRFKK methionine) LNKQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYDEHIY YFRKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDSSYT DIEKCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGEEED RNEDFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWKEKA AYIKRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKANA GKGRPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSHPD YNRFPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLYNK DYAKNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELNMR LIPHRYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYYSV LRLSDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLSQAI HQLTSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEGQA GNIYSALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNICE LLYRFENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGSER NKNRRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLMLSL RNSIVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGILT IRKLENGNEKALTLDEWVISTQKGNIRM Expression MGSGAYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDL 69 construct(with IGIINKKLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNG N-terminal LEDFCNLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYE methionine, SLILINETFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHR V5-tagandC- RFKKLNKQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYD terminalNLS) EHIYYFRKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDS aasequence SYTDIEKCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGE EEDRNEDFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWK EKAAYIKRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKA NAGKGRPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSH PDYNRFPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLY NKDYAKNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELN MRLIPHRYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYY SVLRLSDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLS QAIHQLTSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEG QAGNIYSALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNI CELLYRFENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGS ERNKNRRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLML SLRNSIVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGI LTIRKLENGNEKALTLDEWVISTQKGNIRMSRKRTADGSEFESPKKKRKVGSGKPIPN PLLGLDST Wildtype ATGGCATACCAAGTGGTTAAATGCCTAATCAACGACTATTGCCAGAATGAAATCAT 70 coding TGCACCTCAATTGCAGAAAGTTTCCTGTGATAACACTTGGATTGTAAAACTTCGCG sequence(with AGTTTCAAGAGGCTGCCAATTGGGAAGCCCAAAAAATTATCCAGCAAGATCTTAT N-terminal TGGTATCATAAACAAGAAACTTCCTAAAAAGTTCAATAGCAAGGCATTGATAGAAG methionine CCATTCCTGACTATTTACAAGGCAAGTCTAAAGAAGATCTGCAACGTATGTTGAGT andstop GGTATACATGACTATGAGATTAAGGTAAAAAATCAGAACCTTCAGGTGGCTTGGA codon) ATAATGGGTTAGAAGATTTTTGTAACCTCTGCTATCAACAATTTAGAGGATTTTCT GGCTATCTTGACGCTTTATCTGAGAACCTGAAATTTCTATTCTCGGGCAGAAAAAA TGGTATAGCCTATAGAATAGTGTATCAGAACCTTGTTACATTTGAGAGGAATAGGA GAGCTTATGAATCCCTAATATTAATAAATGAGACTTTTAGGGTACAAGATGAGGCT CTACTTCTTAATTACTCCAGTAGTCTGACCCAAGAAGGTATCAACACCTATAATGA ACGAATAGGGCAACTTGTCAAAAATCTGAAAGAATTTGGCGATACAGACAGATCT TTCAGAAACTGGCATCGCCGATTCAAGAAACTGAACAAGCAAATCCTAAGCCCTC GTGTTGCTCCACCTTGGTTGGCACGCGCCTACAGAAGCGATGAAGAGATGGTGA TGTCGCTACAGTCTTTTCTCGACGAGTTCAATCCATTAAAACCTCGTTTGAAGCAA CTTATTGCTAATCTGGAATCTTACGATGAGCATATCTATTACTTCCGCAAGTCTCT TTCTCTATTATCGGTGACCTTGAGGAATGATTATAAGGCACTTGATGAAGAACTCT CAATACCACAAGAACAGGCCAATTGCAGAAGTTTAAGCCTTTCGTGGATTCCGTT TCGCCAAGAATTGATAAACGAAATAGAACGAATTATTGACAGTTCATATACAGACA TAGAGAAGTGTCTTGCCTCTGCCTCGGAATATCTGAACACGGAGAGAGCAAAAC GGAACGACTATCGTCTAGATAATACTGTGTCTTTCACAATCAAGAAACTGATGGA CGTATTCCTGTCATTGTATCGTGCGGTGAAGCCTCTGACTGGAACAGGAGAGGA GGAGGATCGAAACGAGGACTTCTATGATGAGTTTACAACAATCTGGGATGTGCTT CAATATGTACAAAAACTTTATAATGCAGTTTTTGCATGGCTGAACAAGAAGCCTTA TGAGAACAACAGCTATCCTGCCTATTTGGACGAGTTTACACTTCTTAAAAACTGGA AGGAGAAAGCCGCGTATATAAAACGGAATGGGAAGTTCTATTTTATCATGTTCAAT GGTATTGATGAACAAGACATTATCGAGCATCGAGGTGATTCTGCAATCTTGTATC ATGTGGAAAGTCAATCCCCCGATAGGATTAAGGCAAATCTCACCAAACAATTTGT TTTTTCCAAAAAAGCAAATGCAGGAAAGGGGCGACCAAATCCTTCTAAAGCCAAA TTCGTGCGTGACAATCCAGAATTCCAAGCTGACTGGGAACGTGTGAAAACTGAAG CATATAAAGTAGCTGGAAACACAGAAGCGCTTGCTCATGCCATTCGATATTTTCAA CGCTGCCTTCAATCACATCCTGACTATAATAGGTTTCCGTTCAATTTTAGACCAGC GAATGACTACACTAGTTTAGATGATTTTGTTGACTCCATTAAAGACAAATTGTTTAT GATGGAAGAAACTGCTATTAACTGGTCGTATGTGAGGCAATTAGCAGAAGAAGGA ACAATTTACTTGTTTAAACTCTACAATAAAGATTATGCCAAGAATAGAGTTGGCGG GTCTAAACCCAACTTGCATACGCTCTATTGGGAGGCGATGTTCAGCTCTGAGAAC CTTCGTGAAAATAATATAAAGTTGGAGGAACCCAAACTCTTCTATCGTGAAGTTGC AACTAACCGTGATGGTGAATTGAATATGCGCTTGATACCTCACAGATATGCAACA GACCAACTTGAGCTGCATGTTCCAATTCACTTAAATGTGAATGCAACCGCTTCAA GCGATATAAATATGATGGTGTTGGATGCAATACGAGAAGGGAGTATTGAAAATGT CATTGGTATTGACCGTGGAGAGAGGAACCTTCTCTACTATTCAGTCTTGCGGTTG TCAGATGGTGAAATTGTTGACCAAAAAAGTTTGAATATTACTTTCAATGATGTTGA CTACCACGCCAAACTGTCGACTAAAGAGGAGGAAATCCATGACGAACAAAGAGA ATGGAAAGCAAAAACAAGTATTCGGAAACTGAAAGAAGGATACCTTAGTCAAGCT ATCCACCAACTAACATCGCTGATTGTCAAGTACCATGCTGTGGTAGTGCTAGAAG ACTTATCAGAGGACTTCTATTCGAAGCGCCAGAAGATAAACAAGCAAATCTATCA GATATTTGAAAAAAGGCTGATAGAAAAACTGAGTTATTTTGTCGATAAGGATGCTG CAGAAGGTCAGGCAGGCAATATATATTCAGCATTGCAGTTGTCAAGCCCCAACTT GGTGAGGAAAGATAATAAAAAAATCTTTCAGAACGGCATCGTCTTTTTTGTGCCAC CTGAATATACAAGTGCCATTGACCCTGTAACAGGGTTCTGCAATCTCTTTGACAA GAATCGGGTAAGAAATATTTGCGAACTTCTCTACAGATTTGAAAACATCTGCTATA ATAGGAAAAATGACCGATTTGAGTTCACATGGGACTATCGTAATGTTATGACTTAT ACGCGTCTGGAGCAGGACAATATTTCACATCTTTGGACAGCATGCTCTTTAGGAA ACAGGATTGAATGGTCTGGTAGCGAACGTAATAAAAACAGAAGGTGCGAAATTGT AAACCTTACGCAATCTATGAAAGTTTTGTTTGAAAAACATGGTATCCAATACCAAA CAGGAAAAGATGTAAGGGAGGCTGTATGCAGCATAAGAAACAACGATTTTAAAAA AGAATTGAAGCGCCTGTTCTTCTTGATGTTATCTTTAAGGAATAGCATTGTTGATG GAAAAGTGAAAAAAGACTATATATTATCCCCCGTTCAGAACCAACGAGGCAGTTT TTTCGATAGTAGAGAATATGAAGAGTTGGACAATCCAAAACTCCCTAAATGTGGA GATGCAAATGGCGCATATAATATTGCAAGGAAAGGGATACTGACAATTAGAAAGT TGGAAAATGGCAATGAAAAGGCATTAACCCTTGATGAGTGGGTTATTTCTACGCA AAAAGGGAATATACGCATGTAA Codon GCCTACCAGGTGGTGAAATGCCTGATTAACGACTACTGCCAGAACGAGATCATC 71 optimized GCCCCTCAGCTGCAAAAGGTGAGCTGCGACAATACCTGGATCGTGAAGCTCAGA coding GAGTTCCAGGAGGCCGCAAACTGGGAAGCCCAGAAGATCATCCAGCAGGACCT sequence(no GATCGGCATTATCAATAAGAAACTGCCTAAGAAATTCAACTCTAAGGCCCTGATC N-terminal GAGGCTATACCTGATTACCTCCAGGGCAAGAGCAAGGAAGATCTGCAGAGAATG methionine,no CTGTCCGGCATCCACGACTATGAGATCAAGGTGAAGAACCAGAACCTGCAGGTA stopcodon) GCTTGGAACAATGGCCTGGAAGATTTCTGTAACTTGTGCTACCAACAATTTAGAG GCTTTTCCGGCTACCTTGATGCTCTGTCAGAAAATCTGAAGTTCCTGTTCAGCGG CAGAAAAAACGGCATCGCCTACAGGATCGTCTACCAGAACCTGGTGACCTTCGA GCGGAACCGGAGAGCTTACGAGAGCCTGATCCTGATCAACGAGACATTTAGAGT GCAGGACGAGGCCCTGCTGCTCAACTACTCTAGCTCTCTGACACAGGAGGGAAT CAACACGTACAACGAGCGGATCGGCCAGCTGGTGAAGAACCTGAAGGAGTTCG GCGACACCGACCGGAGCTTTCGGAACTGGCACAGACGGTTCAAGAAACTGAACA AGCAGATCCTGAGCCCTAGAGTGGCCCCTCCTTGGCTGGCTCGTGCCTACAGAA GCGATGAGGAAATGGTGATGAGCCTGCAGAGCTTCCTGGATGAGTTCAACCCTC TGAAACCTAGACTCAAACAGCTGATCGCCAATCTGGAGTCCTACGACGAGCACAT CTACTACTTCAGAAAGTCCCTGTCTCTGCTGTCAGTGACACTGAGGAACGACTAT AAGGCACTGGATGAAGAGCTGAGCATCCCTCAGGAGCAGGCCAACTGCAGATCT CTTAGCCTGAGCTGGATTCCTTTCAGACAGGAACTGATCAACGAGATCGAGAGAA TCATCGATAGCAGCTACACAGACATTGAGAAGTGCCTGGCCAGCGCCTCCGAGT ACCTGAACACCGAGAGAGCCAAGAGAAACGACTACCGGCTAGATAATACCGTGT CCTTCACCATCAAGAAGCTGATGGACGTGTTCCTGAGCCTGTACCGCGCCGTGA AGCCTCTGACCGGAACAGGCGAAGAGGAGGACAGAAATGAAGATTTCTACGACG AGTTCACCACCATCTGGGATGTGCTGCAATACGTGCAGAAGCTGTACAACGCTGT TTTCGCCTGGCTGAACAAGAAGCCCTACGAGAACAATAGCTACCCTGCCTACCTG GATGAATTTACCCTGCTGAAGAACTGGAAGGAAAAGGCCGCCTACATCAAGAGG AATGGAAAATTCTACTTCATCATGTTCAACGGCATCGACGAGCAGGATATCATCG AACACAGAGGAGATTCTGCCATCCTGTACCATGTGGAAAGCCAGAGCCCTGATA GAATCAAGGCCAATCTGACCAAGCAGTTCGTGTTCAGCAAGAAAGCCAATGCCG GCAAGGGCCGGCCCAATCCCAGCAAGGCCAAGTTCGTGAGAGATAACCCCGAG TTTCAGGCCGACTGGGAGCGGGTGAAAACCGAGGCCTACAAGGTGGCCGGAAA CACCGAGGCCCTGGCCCACGCCATCAGATACTTCCAAAGATGCCTGCAAAGCCA CCCCGATTATAATCGGTTCCCCTTCAACTTCAGACCTGCCAACGACTACACATCT CTGGATGACTTCGTGGACAGCATCAAGGACAAGCTGTTCATGATGGAAGAAACC GCCATCAACTGGAGTTATGTGAGACAGCTGGCCGAAGAAGGCACAATCTACCTG TTCAAGCTGTATAACAAAGACTACGCCAAGAACCGGGTGGGCGGCAGCAAGCCT AACCTGCACACCCTGTACTGGGAGGCCATGTTCAGCTCTGAGAATCTGAGAGAA AACAACATCAAACTGGAAGAACCCAAACTGTTCTACAGAGAGGTGGCCACAAACC GGGACGGCGAGCTGAACATGAGACTGATCCCCCACAGATACGCCACCGACCAG CTGGAACTGCACGTGCCTATCCACCTGAATGTGAACGCCACAGCCAGCAGCGAC ATCAACATGATGGTCCTTGATGCCATCCGGGAAGGATCTATTGAGAACGTGATCG GCATCGACCGGGGAGAACGGAACCTGCTGTACTACAGCGTCCTGCGACTGTCC GACGGCGAGATCGTGGACCAGAAGAGCCTGAATATCACCTTTAACGATGTGGAC TACCACGCAAAGTTGTCTACCAAGGAGGAAGAAATCCATGATGAGCAGAGAGAG TGGAAAGCCAAGACCTCCATCAGAAAGCTGAAGGAAGGTTACCTGTCTCAGGCT ATCCACCAGCTGACCAGCCTGATCGTGAAGTACCACGCTGTGGTAGTGCTGGAA GATCTGAGCGAAGATTTCTACAGCAAGCGGCAGAAAATCAACAAGCAGATCTACC AGATTTTCGAGAAAAGACTTATCGAGAAGCTGAGCTACTTTGTGGACAAAGACGC CGCCGAGGGCCAGGCAGGCAACATCTACAGCGCCCTGCAGCTGAGCTCCCCAA ATCTGGTGAGAAAGGACAACAAGAAGATCTTCCAGAACGGCATCGTGTTCTTCGT GCCACCTGAGTACACGAGTGCGATTGACCCCGTGACCGGCTTCTGCAACCTGTT TGACAAGAACAGAGTGCGCAATATCTGTGAGCTGCTCTACAGATTCGAAAACATT TGCTACAACAGAAAGAATGACCGGTTTGAGTTCACATGGGACTATAGAAACGTGA TGACCTACACCAGACTTGAGCAGGACAACATCTCTCACCTGTGGACCGCTTGTAG CCTCGGCAACCGGATCGAGTGGAGCGGCTCTGAAAGAAATAAGAACAGAAGATG CGAGATCGTGAACCTGACACAAAGCATGAAGGTCCTGTTTGAGAAGCACGGCAT CCAGTACCAGACCGGCAAGGACGTGCGGGAAGCTGTGTGTAGTATCAGAAACAA CGACTTTAAGAAAGAACTGAAGAGACTGTTTTTCCTGATGCTGAGCCTGCGTAAC AGCATCGTGGATGGAAAGGTGAAAAAGGACTACATCCTGAGCCCAGTGCAAAAC CAGCGGGGTAGCTTTTTCGACTCCAGAGAATATGAAGAACTGGACAACCCGAAG TTGCCTAAGTGCGGGGACGCCAACGGCGCCTACAACATCGCCAGAAAAGGAATC CTGACAATCAGAAAGCTGGAGAACGGCAACGAGAAAGCCCTGACCCTGGACGAA TGGGTGATCAGCACCCAGAAGGGCAACATCAGAATG Expression ATGggctccggaGCCTACCAGGTGGTGAAATGCCTGATTAACGACTACTGCCAGAAC 72 construct(with GAGATCATCGCCCCTCAGCTGCAAAAGGTGAGCTGCGACAATACCTGGATCGTG N-terminal AAGCTCAGAGAGTTCCAGGAGGCCGCAAACTGGGAAGCCCAGAAGATCATCCAG methionine CAGGACCTGATCGGCATTATCAATAAGAAACTGCCTAAGAAATTCAACTCTAAGG andstop CCCTGATCGAGGCTATACCTGATTACCTCCAGGGCAAGAGCAAGGAAGATCTGC codon, AGAGAATGCTGTCCGGCATCCACGACTATGAGATCAAGGTGAAGAACCAGAACC includesV5- TGCAGGTAGCTTGGAACAATGGCCTGGAAGATTTCTGTAACTTGTGCTACCAACA tagandC- ATTTAGAGGCTTTTCCGGCTACCTTGATGCTCTGTCAGAAAATCTGAAGTTCCTGT terminalNLS) TCAGCGGCAGAAAAAACGGCATCGCCTACAGGATCGTCTACCAGAACCTGGTGA CCTTCGAGCGGAACCGGAGAGCTTACGAGAGCCTGATCCTGATCAACGAGACAT TTAGAGTGCAGGACGAGGCCCTGCTGCTCAACTACTCTAGCTCTCTGACACAGG AGGGAATCAACACGTACAACGAGCGGATCGGCCAGCTGGTGAAGAACCTGAAG GAGTTCGGCGACACCGACCGGAGCTTTCGGAACTGGCACAGACGGTTCAAGAAA CTGAACAAGCAGATCCTGAGCCCTAGAGTGGCCCCTCCTTGGCTGGCTCGTGCC TACAGAAGCGATGAGGAAATGGTGATGAGCCTGCAGAGCTTCCTGGATGAGTTC AACCCTCTGAAACCTAGACTCAAACAGCTGATCGCCAATCTGGAGTCCTACGACG AGCACATCTACTACTTCAGAAAGTCCCTGTCTCTGCTGTCAGTGACACTGAGGAA CGACTATAAGGCACTGGATGAAGAGCTGAGCATCCCTCAGGAGCAGGCCAACTG CAGATCTCTTAGCCTGAGCTGGATTCCTTTCAGACAGGAACTGATCAACGAGATC GAGAGAATCATCGATAGCAGCTACACAGACATTGAGAAGTGCCTGGCCAGCGCC TCCGAGTACCTGAACACCGAGAGAGCCAAGAGAAACGACTACCGGCTAGATAAT ACCGTGTCCTTCACCATCAAGAAGCTGATGGACGTGTTCCTGAGCCTGTACCGC GCCGTGAAGCCTCTGACCGGAACAGGCGAAGAGGAGGACAGAAATGAAGATTTC TACGACGAGTTCACCACCATCTGGGATGTGCTGCAATACGTGCAGAAGCTGTAC AACGCTGTTTTCGCCTGGCTGAACAAGAAGCCCTACGAGAACAATAGCTACCCTG CCTACCTGGATGAATTTACCCTGCTGAAGAACTGGAAGGAAAAGGCCGCCTACAT CAAGAGGAATGGAAAATTCTACTTCATCATGTTCAACGGCATCGACGAGCAGGAT ATCATCGAACACAGAGGAGATTCTGCCATCCTGTACCATGTGGAAAGCCAGAGC CCTGATAGAATCAAGGCCAATCTGACCAAGCAGTTCGTGTTCAGCAAGAAAGCCA ATGCCGGCAAGGGCCGGCCCAATCCCAGCAAGGCCAAGTTCGTGAGAGATAAC CCCGAGTTTCAGGCCGACTGGGAGCGGGTGAAAACCGAGGCCTACAAGGTGGC CGGAAACACCGAGGCCCTGGCCCACGCCATCAGATACTTCCAAAGATGCCTGCA AAGCCACCCCGATTATAATCGGTTCCCCTTCAACTTCAGACCTGCCAACGACTAC ACATCTCTGGATGACTTCGTGGACAGCATCAAGGACAAGCTGTTCATGATGGAAG AAACCGCCATCAACTGGAGTTATGTGAGACAGCTGGCCGAAGAAGGCACAATCT ACCTGTTCAAGCTGTATAACAAAGACTACGCCAAGAACCGGGTGGGCGGCAGCA AGCCTAACCTGCACACCCTGTACTGGGAGGCCATGTTCAGCTCTGAGAATCTGA GAGAAAACAACATCAAACTGGAAGAACCCAAACTGTTCTACAGAGAGGTGGCCA CAAACCGGGACGGCGAGCTGAACATGAGACTGATCCCCCACAGATACGCCACC GACCAGCTGGAACTGCACGTGCCTATCCACCTGAATGTGAACGCCACAGCCAGC AGCGACATCAACATGATGGTCCTTGATGCCATCCGGGAAGGATCTATTGAGAAC GTGATCGGCATCGACCGGGGAGAACGGAACCTGCTGTACTACAGCGTCCTGCG ACTGTCCGACGGCGAGATCGTGGACCAGAAGAGCCTGAATATCACCTTTAACGA TGTGGACTACCACGCAAAGTTGTCTACCAAGGAGGAAGAAATCCATGATGAGCA GAGAGAGTGGAAAGCCAAGACCTCCATCAGAAAGCTGAAGGAAGGTTACCTGTC TCAGGCTATCCACCAGCTGACCAGCCTGATCGTGAAGTACCACGCTGTGGTAGT GCTGGAAGATCTGAGCGAAGATTTCTACAGCAAGCGGCAGAAAATCAACAAGCA GATCTACCAGATTTTCGAGAAAAGACTTATCGAGAAGCTGAGCTACTTTGTGGAC AAAGACGCCGCCGAGGGCCAGGCAGGCAACATCTACAGCGCCCTGCAGCTGAG CTCCCCAAATCTGGTGAGAAAGGACAACAAGAAGATCTTCCAGAACGGCATCGT GTTCTTCGTGCCACCTGAGTACACGAGTGCGATTGACCCCGTGACCGGCTTCTG CAACCTGTTTGACAAGAACAGAGTGCGCAATATCTGTGAGCTGCTCTACAGATTC GAAAACATTTGCTACAACAGAAAGAATGACCGGTTTGAGTTCACATGGGACTATA GAAACGTGATGACCTACACCAGACTTGAGCAGGACAACATCTCTCACCTGTGGA CCGCTTGTAGCCTCGGCAACCGGATCGAGTGGAGCGGCTCTGAAAGAAATAAGA ACAGAAGATGCGAGATCGTGAACCTGACACAAAGCATGAAGGTCCTGTTTGAGA AGCACGGCATCCAGTACCAGACCGGCAAGGACGTGCGGGAAGCTGTGTGTAGT ATCAGAAACAACGACTTTAAGAAAGAACTGAAGAGACTGTTTTTCCTGATGCTGA GCCTGCGTAACAGCATCGTGGATGGAAAGGTGAAAAAGGACTACATCCTGAGCC CAGTGCAAAACCAGCGGGGTAGCTTTTTCGACTCCAGAGAATATGAAGAACTGG ACAACCCGAAGTTGCCTAAGTGCGGGGACGCCAACGGCGCCTACAACATCGCCA GAAAAGGAATCCTGACAATCAGAAAGCTGGAGAACGGCAACGAGAAAGCCCTGA CCCTGGACGAATGGGTGATCAGCACCCAGAAGGGCAACATCAGAATGtctagaAAG CGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggat ccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0121] In some embodiments a ZQKH Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, a ZQKH Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D744 substitution, wherein the position of the D744 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E831 substitution, wherein the position of the E831 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1048 substitution, wherein the position of the R1048 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1091 substitution, wherein the position of the D1091 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZQKH Type V Cas protein is catalytically inactive, for example due to a R1048 substitution in combination with a D744 substitution, a E831 substitution, and/or D1091 substitution.

6.2.13. ZRGM Type V Cas Protein

[0122] In one aspect, the disclosure provides ZRGM Type V Cas proteins. ZRGM Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZRGM Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:73. In some embodiments, the ZRGM Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73. In some embodiments, a ZRGM Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:73.

[0123] Exemplary ZRGM Type V Cas protein sequences and nucleotide sequences encoding exemplary ZRGM Type V Cas proteins are set forth in Table 1M.

TABLE-US-00014 TABLE1M ZRGMTypeVCasSequences SEQID Name Sequence NO. Wildtype ERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMKMVLDQ 73 aminoacid LHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSLQASLR sequence KEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFKKYNET (withoutN- RANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNLTNYFSI terminal KNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQILSDREE methionine) QSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAEVSIKMK MGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEHEEDWK FVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDSCLQLVR WCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQARKMHLMF NYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYERLSYML FPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYFVNLYDD KKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPISNFRSLD EFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNLFTLYW EQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGVRKTLP DTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFHVPITL NYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQGTFNKI VGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQMMVK YDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIEGSVSN AYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKARKAFFE NFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKERKEKQID VTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLRSSQIDN GEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLLAENVKK TGRGANGKWNSSVKNISNKDWFAFVQK Wildtype MERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMKMVLD 74 aminoacid QLHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSLQASL sequence(with RKEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFKKYNE N-terminal TRANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNLTNYF methionine) SIKNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQILSDRE EQSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAEVSIKM KMGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEHEEDW KFVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDSCLQLV RWCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQARKMHLM FNYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYERLSYM LFPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYFVNLYD DKKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPISNFRSL DEFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNLFTLY WEQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGVRKTL PDTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFHVPIT LNYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQGTEN KIVGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQMMV KYDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIEGSVS NAYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKARKAFF ENFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKERKEKQI DVTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLRSSQID NGEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLLAENVK KTGRGANGKWNSSVKNISNKDWFAFVQK Expression MGSGERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMK 75 construct(with MVLDQLHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSL N-terminal QASLRKEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFK methionine, KYNETRANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNL V5-tagandC- TNYFSIKNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQIL terminalNLS) SDREEQSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAE aasequence VSIKMKMGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEH EEDWKFVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDS CLQLVRWCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQAR KMHLMFNYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYE RLSYMLFPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYF VNLYDDKKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPIS NFRSLDEFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNL FTLYWEQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGV RKTLPDTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFH VPITLNYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQG TFNKIVGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQ MMVKYDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIE GSVSNAYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKA RKAFFENFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKER KEKQIDVTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLR SSQIDNGEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLL AENVKKTGRGANGKWNSSVKNISNKDWFAFVQKSRKRTADGSEFESPKKKRKVG SGKPIPNPLLGLDST Wildtype ATGGAGAGAATGTACGAAGAATTTAGAAATTGTTATTCAGTACGAAAAACATTGT 76 coding CATTTAAGGCAATCCCAACAGAGGAAACAAAAAAACATTTACAATTACAATGGGA sequence(with AGTGTTGGGGGATGAGATACGTTTTGAAAACTATGATAAAATGAAAATGGTTTTG N-terminal GATCAACTTCATCAATCATATATTTCGAGAAAATTAGATAATATAGGAGAAGAAAA methionine TCAAAAAAAGATAGTTGAAATCTTAGAGAAACTCGTATTAGTTATGAAAAAGATA andstop GATACTACGCATCAAAAGGATAAAGAGAAAGCGCAAAATCAGCTTCAATCGTTA codon) CAAGCTTCATTAAGGAAAGAAATAGGAATGTTTTTTCCTAAAAACGAATGGCAAC AATTACAGGGAAAAAATGTATTTAAGAAGGATGGGGTACTAAGCGAGTATAACAT TTCGGAAGAGAATAAGAAAAATATTCAATGTTATGATGGTTTTATGACATTCTTTA AAAAATATAATGAAACTAGAGCAAATATATATAGTACAGAGGAAAAAAGCACGGC AATCACTTTTCGAATTGTGAATGATAATCTTCCAAAATATGTGAGAAATGCGGAT AATTACGAACAGATTAAAAAATTAATTCCTGAAGCTCTTGAAGAAGTAGAAAAAA CATACCCAAATTTGACGAATTATTTCTCGATTAAAAACTATTTGAAGTATTGGAGT CAGAAGGGGATTGAAACATACAATACTGTTATTGGAGAAATAAATAAGCAGGTTA ATCTTGTAGTACAACAAAGAAAAGATTCGAAATTTAGAAAATACAAGATGCAGGT GTTGTATCAACAAATTCTAAGTGATAGAGAGGAACAGTCTTTTGTGTATCAACAG GATCAGGAAGTTTTTGCTGCTGTTAATGAACTTGCAGAACTTGTGAACGGTAGT GCTTTTAACGAGGCAATTGAATTGTTGAAATCACCTAATATTAACGAAAATGAGA TATTTATTCCCTATGCAAAATTAGCAGAAGTATCCATAAAAATGAAAATGGGATG GAATGGATTAGAGGAGGCTTTTATAAACGATTTGCAACAGCAGTATCCAAAGAA GGATCATGAAAAATTGGTGCAAAAATTAAAAAAAGAGAAAAAAGTTTTTTCTTTGA ATGAAATTAAAGATGTTGTTATGAAAATTGAACATGAAGAAGATTGGAAATTTGTT AGTTTGCTGGATTGTGTTGAGGATTATCAAAAACAGTTGACAGAGACAAGAGAT GCATATGTGGAATATGCAAAAACTTATGCAGGTTCAACCGGTACATCATTACAAG GAAATGATGTAGCACCGATAAAAGCATTTTTAGATAGTTGTTTGCAATTGGTACG ATGGTGTAAGTTGTTTGAATATTCTGATTTGTATGGAAATCGAGATAAAATATTTT ATGGAGGAGCAGAGTCGATTATACTTGCATTAGATTCCTTAATATCTGTGTATAA TAAAACAAGAAATTATGTGACTATGCGACCGGGGCAGGCTAGAAAAATGCATTT AATGTTTAATTATCCGGAATTCGGTGATGGCTTTAGTAATAGTAAAGTGGATTCT TATGGTACGATTTTGCTTCGTGAAGGAAAGAAATATTATTTAGCTGTTATTAAAAA AGGCATAAAAGTCTTGCTGGAAGATACCATAAATGAAAATGACAGTTATGAACGT TTGAGTTATATGTTGTTTCCTGATGTAAAAAAAATGATACCGAAATGTTCTATTAG TACGAAGAAAGTTAAAGAACATTTTGAAAATTCGGATGATGATTATACGATTCGT AAAGGTGAATCTTATGCAAAAGAATTACTTGTGAAAAAAGAAGATTATGACCTTT ACTTTGTAAATCTTTATGATGATAAGAAGATGTTTCAAAAGGACTATTTGAGTAAA ACTGGAGATAAAAAAGGATATAGACAGGCGTTAGAACGCTGGATACGTTTTTGC ATTCGATTTTTACAAGCTTATAAGAGTACAAAGGATTATGATCTCAGTGAATTAGA GCCAATTTCGAATTTTCGTTCCTTAGATGAGTTTTATGATAAATTGGATACTTTGT TATACAAGATAGAGTGGAAAACAATTTCAAGAGAACAAATTAAGCAAATGGAGTC ATCTGGTCAGTTGTTTTTATTTGAATTATATAACAAAGATTTCTCTGAACATGCAA AAGGAAAGAAAAATTTATTTACATTGTATTGGGAACAGATTTTCTGTGAAGAGAA TTTAAAACAGCCAGTGATTAAACTTTGTGGCGGGGCAGAGATGTTTTATCGTAAG GTTGCCATTCAAAAAAAATATGTACATAAAAAAGACTCCATTTTGGTGGATAAAA CGTATGTGGATCAGAATGGAGTCAGAAAAACACTTCCGGATACTATATATAAAGA GTGGTCGGATTTTATGAATAAAAAGATAACATCTGTCAGCCAGGAGGCAAGTAA ATATAAAGGTTTGGTTAATTGTCATGAGGCAAAATATGATATTACAAAAGATAAAA GATATACGGAAGATCAATTTGAGTTTCATGTGCCAATTACTTTAAATTATTCAGCA TTAGGAAAAGGGCAATTAAATGATAGTGTTCTGGATTGTCTATGTCAGAAAGAAA AATATAATGTGATAGGAATTGACCGTGGAGAAAGAAACTTGTTGGCTTACTGTGT CGTAAATCAAGATGGACAGATTTTAGAACAAGGGACATTTAATAAGATTGTAGGT GGAAATAAACAGGAAGTAGATTACAAACAGAAGTTACAGGAGAAAGAAGTAAAT CGACAACAAGCAAGAAAAGAGTGGAAAAATATTGGAAAAATTAAAGAATTAAAGA ACGGTTATTTGTCTCAGGTTATTTATCAACTGACGCAAATGATGGTAAAATATGA TGCTATTGTTGTTATGGAAGATTTGAATGTTGGCTTTAAACGTGGTCGATTTAAG GTGGAACGACAGGTTTACCAGAAATTTGAAAAAGCGCTGATTGACAAATTAAATT ATTTAGTAACTAAAAAAGATGAAAATCAATATGGAATAGAGGGTAGCGTAAGCAA TGCATATCAACTGACAGAAAAAATCAAATCATTTAAAGATATTGGCAAACAAAAC GGGATGATATTTTATGTGCCAGCGGGATATACCTCTAAAATAGATCCTACAACAG GATTTGTGGATGTGCTAAATCGAACAGGATTAACAAATGCCAAAGCCAGAAAAG CGTTCTTTGAAAATTTTGATGATATTAACTATTCAAAAGAAGATAATATGTTTGCC TTTTCTTTTGATTATAGCAAGTTTAAGACATTTCAAGAAATGCATAGAAAAAAATG GACAGTTTACACAAATGGTAAAAAGTACATTTATTCAAAAAAAGAACGAAAAGAA AAACAAATTGATGTTACTGAGTTGATGAAAGAAGAATTGAGAAAAGTAGGAATTA CAGAGTATGATAATCTTTATTCGCAAATTACTAATGTGGAAGATGATAAAGAACA TGCAGATTTTTGGAAATCTTTACAGTTTGTATTTGATAGAACGATGCAGTTGAGA AGTAGTCAAATTGACAATGGAGAGGATAATCTTGAGGATAAGATTATATCTCCGG TGAAAAATGCAGAGGGTGTATTTTATGAATCAAATGGAAATTATGGTGACACTTC ACAACCTGCAGATGCAGATACAAATGGTGCTTTTCATATTGCAAGGAAGGGATT ACTACTTGCAGAAAATGTGAAAAAAACAGGTAGAGGAGCAAATGGAAAATGGAA TTCTTCTGTAAAAAATATTTCTAATAAGGATTGGTTTGCATTTGTTCAAAAATAA Codon GAACGGATGTACGAGGAGTTCAGAAACTGCTACTCCGTGCGGAAAACACTGTC 77 optimized CTTCAAAGCCATCCCTACCGAGGAGACAAAGAAGCACCTGCAGCTGCAGTGGG coding AAGTGCTCGGCGACGAGATTAGATTTGAGAATTATGATAAGATGAAAATGGTGC sequence(no TGGACCAGCTGCACCAGTCTTACATCAGCCGGAAGCTGGACAACATCGGCGAG N-terminal GAGAACCAGAAAAAGATTGTAGAAATCCTGGAGAAGCTGGTGCTGGTGATGAAG methionine,no AAGATCGATACAACCCACCAGAAGGACAAGGAGAAGGCCCAGAATCAACTGCA stopcodon) GAGCCTGCAGGCTTCCCTGCGGAAGGAAATTGGTATGTTTTTCCCAAAGAACGA GTGGCAGCAGCTGCAGGGCAAAAACGTGTTCAAGAAGGACGGCGTTCTCAGCG AATACAACATCAGCGAGGAAAACAAGAAGAACATCCAGTGTTACGACGGCTTTA TGACCTTCTTCAAGAAGTACAACGAGACACGGGCCAATATCTATTCTACGGAGG AAAAGAGCACCGCCATCACCTTCAGGATCGTGAATGATAATCTGCCTAAGTATG TGCGAAACGCTGACAACTACGAGCAGATAAAGAAGCTGATCCCCGAAGCTCTG GAAGAAGTCGAAAAGACCTATCCTAATCTGACCAACTACTTCAGCATCAAGAACT ATCTGAAGTACTGGAGCCAGAAGGGGATCGAAACATACAACACCGTGATCGGC GAGATCAACAAGCAGGTGAACCTGGTGGTCCAACAGAGAAAGGACAGCAAGTT CAGGAAGTACAAAATGCAGGTGCTGTACCAGCAGATCCTATCCGACAGAGAGG AGCAGAGCTTCGTGTACCAGCAGGACCAGGAGGTGTTCGCCGCCGTGAACGAG CTGGCCGAGCTGGTGAATGGCAGCGCCTTCAATGAAGCTATCGAATTGCTGAAA AGCCCAAACATCAACGAGAATGAGATTTTCATCCCCTACGCCAAGCTCGCCGAG GTGTCTATCAAGATGAAAATGGGATGGAACGGCCTGGAGGAGGCCTTCATCAA CGATCTGCAGCAACAATACCCCAAGAAAGACCACGAAAAATTGGTTCAGAAGCT GAAGAAAGAGAAGAAGGTGTTTAGCCTGAATGAAATCAAGGATGTGGTCATGAA GATCGAACACGAGGAAGATTGGAAATTCGTGAGCCTGCTGGACTGCGTGGAGG ATTACCAGAAGCAGCTTACAGAGACAAGAGATGCCTACGTGGAGTACGCTAAGA CATACGCCGGCAGCACAGGCACCAGCCTGCAGGGCAACGACGTGGCCCCTAT CAAGGCCTTCCTGGACTCCTGCCTGCAACTGGTGCGGTGGTGCAAGCTGTTCG AGTACAGCGACCTGTACGGCAACAGAGACAAGATCTTCTACGGAGGCGCCGAG AGCATCATCCTGGCCCTGGATAGCCTGATTTCCGTGTACAACAAAACCAGAAAC TACGTGACCATGCGGCCTGGCCAGGCCAGAAAAATGCACCTGATGTTCAACTAC CCCGAGTTTGGCGACGGCTTCAGCAACAGCAAAGTGGATTCTTACGGCACCAT CCTGCTGAGAGAAGGCAAGAAGTACTACCTGGCTGTGATCAAGAAGGGCATCA AAGTGCTGCTGGAGGACACCATTAACGAGAATGACTCTTACGAGCGGCTGTCCT ACATGCTGTTCCCCGACGTGAAAAAGATGATCCCTAAGTGCAGCATCAGTACCA AGAAGGTGAAAGAGCATTTCGAGAACAGCGACGACGACTACACCATCAGAAAG GGCGAGAGCTATGCCAAGGAGCTGCTGGTGAAGAAGGAAGATTACGACCTGTA TTTCGTGAACCTGTACGACGACAAAAAGATGTTCCAGAAAGACTACCTGAGCAA AACCGGCGACAAGAAGGGATACAGACAGGCCCTGGAGAGGTGGATCAGATTCT GCATCAGATTCCTGCAGGCTTACAAGTCTACAAAGGATTATGACCTGTCTGAACT GGAACCTATCAGCAACTTCAGAAGCCTGGACGAGTTCTACGATAAGCTGGACAC CCTACTGTACAAGATCGAGTGGAAAACCATCTCCAGAGAGCAGATCAAGCAAAT GGAATCCTCTGGCCAGCTCTTCCTGTTCGAGTTGTACAACAAGGACTTCTCTGA ACACGCCAAGGGAAAGAAGAACCTGTTCACCCTGTACTGGGAGCAAATTTTTTG TGAAGAGAACCTGAAGCAGCCTGTGATCAAGCTGTGCGGCGGAGCCGAGATGT TCTACAGAAAGGTTGCCATCCAGAAAAAGTACGTGCACAAGAAGGACAGCATCC TGGTAGACAAGACCTACGTGGATCAGAACGGCGTTCGCAAGACCCTGCCTGAT ACCATCTACAAGGAATGGTCCGACTTCATGAACAAAAAGATCACCAGCGTGTCC CAAGAAGCCTCTAAATACAAGGGCCTGGTGAACTGTCACGAGGCCAAGTACGA CATCACCAAGGACAAGAGATACACCGAAGATCAATTCGAATTTCACGTGCCAAT CACACTGAACTACAGCGCCCTCGGAAAAGGTCAGCTGAACGACAGCGTGCTGG ACTGCCTGTGTCAGAAAGAGAAGTACAACGTGATTGGAATCGACCGGGGAGAA AGAAACCTGCTGGCCTACTGCGTGGTGAACCAGGATGGCCAGATCCTGGAACA GGGCACCTTCAACAAGATCGTGGGCGGCAATAAGCAGGAGGTGGACTATAAGC AGAAACTGCAGGAGAAGGAGGTGAATAGACAGCAGGCCAGGAAGGAGTGGAA GAACATCGGCAAGATCAAGGAGTTGAAAAACGGCTACCTGAGCCAAGTAATCTA CCAGCTGACACAGATGATGGTGAAGTACGATGCCATCGTGGTGATGGAAGATCT GAACGTGGGCTTTAAGAGAGGCAGATTCAAGGTTGAGCGGCAGGTGTACCAGA AGTTCGAAAAGGCTCTGATCGATAAGCTGAATTATCTGGTCACCAAGAAGGACG AGAACCAATACGGGATCGAGGGCAGCGTTTCGAATGCCTACCAGCTGACCGAG AAAATCAAGAGCTTCAAAGACATCGGAAAACAGAACGGCATGATCTTCTACGTG CCTGCTGGCTATACAAGCAAAATCGACCCTACGACCGGATTCGTCGATGTGCTG AACAGAACCGGCCTGACAAACGCCAAGGCTAGAAAAGCCTTCTTCGAGAATTTT GACGACATCAACTACTCTAAGGAGGACAACATGTTCGCCTTCAGCTTCGATTAC AGCAAGTTCAAGACCTTTCAGGAAATGCATAGAAAAAAGTGGACAGTGTACACA AACGGAAAAAAATACATCTACAGCAAGAAGGAACGGAAGGAAAAGCAGATAGAC GTGACCGAACTGATGAAAGAAGAGCTGAGAAAGGTGGGCATAACCGAGTACGA CAACCTCTACAGCCAGATCACCAACGTGGAAGATGATAAGGAGCACGCCGACTT TTGGAAGTCTCTGCAGTTCGTGTTCGACAGAACAATGCAGCTGAGAAGCAGCCA GATCGACAACGGCGAGGACAATCTGGAAGATAAGATCATTTCACCTGTGAAAAA CGCCGAGGGCGTGTTCTATGAAAGCAACGGCAACTACGGCGATACGAGCCAGC CCGCCGACGCGGACACCAACGGCGCCTTCCACATCGCGCGGAAGGGCCTGCT GCTCGCCGAGAATGTGAAGAAAACCGGAAGAGGCGCCAATGGCAAATGGAATA GCAGCGTGAAGAACATCTCTAACAAGGATTGGTTCGCCTTTGTGCAGAAA Expression ATGggctccggaGAACGGATGTACGAGGAGTTCAGAAACTGCTACTCCGTGCGGAA 78 construct(with AACACTGTCCTTCAAAGCCATCCCTACCGAGGAGACAAAGAAGCACCTGCAGCT N-terminal GCAGTGGGAAGTGCTCGGCGACGAGATTAGATTTGAGAATTATGATAAGATGAA methionine AATGGTGCTGGACCAGCTGCACCAGTCTTACATCAGCCGGAAGCTGGACAACA andstop TCGGCGAGGAGAACCAGAAAAAGATTGTAGAAATCCTGGAGAAGCTGGTGCTG codon, GTGATGAAGAAGATCGATACAACCCACCAGAAGGACAAGGAGAAGGCCCAGAA includesV5- TCAACTGCAGAGCCTGCAGGCTTCCCTGCGGAAGGAAATTGGTATGTTTTTCCC tagandC- AAAGAACGAGTGGCAGCAGCTGCAGGGCAAAAACGTGTTCAAGAAGGACGGCG terminalNLS) TTCTCAGCGAATACAACATCAGCGAGGAAAACAAGAAGAACATCCAGTGTTACG ACGGCTTTATGACCTTCTTCAAGAAGTACAACGAGACACGGGCCAATATCTATTC TACGGAGGAAAAGAGCACCGCCATCACCTTCAGGATCGTGAATGATAATCTGCC TAAGTATGTGCGAAACGCTGACAACTACGAGCAGATAAAGAAGCTGATCCCCGA AGCTCTGGAAGAAGTCGAAAAGACCTATCCTAATCTGACCAACTACTTCAGCAT CAAGAACTATCTGAAGTACTGGAGCCAGAAGGGGATCGAAACATACAACACCGT GATCGGCGAGATCAACAAGCAGGTGAACCTGGTGGTCCAACAGAGAAAGGACA GCAAGTTCAGGAAGTACAAAATGCAGGTGCTGTACCAGCAGATCCTATCCGACA GAGAGGAGCAGAGCTTCGTGTACCAGCAGGACCAGGAGGTGTTCGCCGCCGT GAACGAGCTGGCCGAGCTGGTGAATGGCAGCGCCTTCAATGAAGCTATCGAAT TGCTGAAAAGCCCAAACATCAACGAGAATGAGATTTTCATCCCCTACGCCAAGC TCGCCGAGGTGTCTATCAAGATGAAAATGGGATGGAACGGCCTGGAGGAGGCC TTCATCAACGATCTGCAGCAACAATACCCCAAGAAAGACCACGAAAAATTGGTT CAGAAGCTGAAGAAAGAGAAGAAGGTGTTTAGCCTGAATGAAATCAAGGATGTG GTCATGAAGATCGAACACGAGGAAGATTGGAAATTCGTGAGCCTGCTGGACTG CGTGGAGGATTACCAGAAGCAGCTTACAGAGACAAGAGATGCCTACGTGGAGT ACGCTAAGACATACGCCGGCAGCACAGGCACCAGCCTGCAGGGCAACGACGT GGCCCCTATCAAGGCCTTCCTGGACTCCTGCCTGCAACTGGTGCGGTGGTGCA AGCTGTTCGAGTACAGCGACCTGTACGGCAACAGAGACAAGATCTTCTACGGA GGCGCCGAGAGCATCATCCTGGCCCTGGATAGCCTGATTTCCGTGTACAACAA AACCAGAAACTACGTGACCATGCGGCCTGGCCAGGCCAGAAAAATGCACCTGA TGTTCAACTACCCCGAGTTTGGCGACGGCTTCAGCAACAGCAAAGTGGATTCTT ACGGCACCATCCTGCTGAGAGAAGGCAAGAAGTACTACCTGGCTGTGATCAAG AAGGGCATCAAAGTGCTGCTGGAGGACACCATTAACGAGAATGACTCTTACGAG CGGCTGTCCTACATGCTGTTCCCCGACGTGAAAAAGATGATCCCTAAGTGCAGC ATCAGTACCAAGAAGGTGAAAGAGCATTTCGAGAACAGCGACGACGACTACACC ATCAGAAAGGGCGAGAGCTATGCCAAGGAGCTGCTGGTGAAGAAGGAAGATTA CGACCTGTATTTCGTGAACCTGTACGACGACAAAAAGATGTTCCAGAAAGACTA CCTGAGCAAAACCGGCGACAAGAAGGGATACAGACAGGCCCTGGAGAGGTGG ATCAGATTCTGCATCAGATTCCTGCAGGCTTACAAGTCTACAAAGGATTATGACC TGTCTGAACTGGAACCTATCAGCAACTTCAGAAGCCTGGACGAGTTCTACGATA AGCTGGACACCCTACTGTACAAGATCGAGTGGAAAACCATCTCCAGAGAGCAGA TCAAGCAAATGGAATCCTCTGGCCAGCTCTTCCTGTTCGAGTTGTACAACAAGG ACTTCTCTGAACACGCCAAGGGAAAGAAGAACCTGTTCACCCTGTACTGGGAGC AAATTTTTTGTGAAGAGAACCTGAAGCAGCCTGTGATCAAGCTGTGCGGCGGAG CCGAGATGTTCTACAGAAAGGTTGCCATCCAGAAAAAGTACGTGCACAAGAAGG ACAGCATCCTGGTAGACAAGACCTACGTGGATCAGAACGGCGTTCGCAAGACC CTGCCTGATACCATCTACAAGGAATGGTCCGACTTCATGAACAAAAAGATCACC AGCGTGTCCCAAGAAGCCTCTAAATACAAGGGCCTGGTGAACTGTCACGAGGC CAAGTACGACATCACCAAGGACAAGAGATACACCGAAGATCAATTCGAATTTCA CGTGCCAATCACACTGAACTACAGCGCCCTCGGAAAAGGTCAGCTGAACGACA GCGTGCTGGACTGCCTGTGTCAGAAAGAGAAGTACAACGTGATTGGAATCGAC CGGGGAGAAAGAAACCTGCTGGCCTACTGCGTGGTGAACCAGGATGGCCAGAT CCTGGAACAGGGCACCTTCAACAAGATCGTGGGCGGCAATAAGCAGGAGGTGG ACTATAAGCAGAAACTGCAGGAGAAGGAGGTGAATAGACAGCAGGCCAGGAAG GAGTGGAAGAACATCGGCAAGATCAAGGAGTTGAAAAACGGCTACCTGAGCCA AGTAATCTACCAGCTGACACAGATGATGGTGAAGTACGATGCCATCGTGGTGAT GGAAGATCTGAACGTGGGCTTTAAGAGAGGCAGATTCAAGGTTGAGCGGCAGG TGTACCAGAAGTTCGAAAAGGCTCTGATCGATAAGCTGAATTATCTGGTCACCA AGAAGGACGAGAACCAATACGGGATCGAGGGCAGCGTTTCGAATGCCTACCAG CTGACCGAGAAAATCAAGAGCTTCAAAGACATCGGAAAACAGAACGGCATGATC TTCTACGTGCCTGCTGGCTATACAAGCAAAATCGACCCTACGACCGGATTCGTC GATGTGCTGAACAGAACCGGCCTGACAAACGCCAAGGCTAGAAAAGCCTTCTTC GAGAATTTTGACGACATCAACTACTCTAAGGAGGACAACATGTTCGCCTTCAGC TTCGATTACAGCAAGTTCAAGACCTTTCAGGAAATGCATAGAAAAAAGTGGACA GTGTACACAAACGGAAAAAAATACATCTACAGCAAGAAGGAACGGAAGGAAAAG CAGATAGACGTGACCGAACTGATGAAAGAAGAGCTGAGAAAGGTGGGCATAAC CGAGTACGACAACCTCTACAGCCAGATCACCAACGTGGAAGATGATAAGGAGC ACGCCGACTTTTGGAAGTCTCTGCAGTTCGTGTTCGACAGAACAATGCAGCTGA GAAGCAGCCAGATCGACAACGGCGAGGACAATCTGGAAGATAAGATCATTTCAC CTGTGAAAAACGCCGAGGGCGTGTTCTATGAAAGCAACGGCAACTACGGCGAT ACGAGCCAGCCCGCCGACGCGGACACCAACGGCGCCTTCCACATCGCGCGGA AGGGCCTGCTGCTCGCCGAGAATGTGAAGAAAACCGGAAGAGGCGCCAATGG CAAATGGAATAGCAGCGTGAAGAACATCTCTAACAAGGATTGGTTCGCCTTTGT GCAGAAAtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAA AAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACA GCACCTGA

[0124] In some embodiments a ZRGM Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 73, SEQ ID NO:74, or SEQ ID NO:75. In some embodiments, a ZRGM Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D890 substitution, wherein the position of the D890 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E980 substitution, wherein the position of the E980 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1194 substitution, wherein the position of the R1194 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1237 substitution, wherein the position of the D1237 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZRGM Type V Cas protein is catalytically inactive, for example due to a R1194 substitution in combination with a D890 substitution, a E980 substitution, and/or D1237 substitution.

6.2.14. ZTAE Type V Cas Protein

[0125] In one aspect, the disclosure provides ZTAE Type V Cas proteins. ZTAE Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZTAE Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:79. In some embodiments, the ZTAE Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79. In some embodiments, a ZTAE Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:79.

[0126] Exemplary ZTAE Type V Cas protein sequences and nucleotide sequences encoding exemplary ZTAE Type V Cas proteins are set forth in Table 1N.

TABLE-US-00015 TABLE1N ZTAETypeVCasSequences SEQ ID Name Sequence NO. Wildtype SFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDEQHKE 79 aminoacid LLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRAGEVE sequence KNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKFACYF (withoutN- TGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEMELM terminal DELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQQHE methionine) EANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELNAQK FNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFTRAK DRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDAELA MKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADFYS DFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNKAV LFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVFFA DSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQFS ATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASSGK PNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGEHG KGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKFFF HVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIEQR SFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVTNN AIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAYQL TDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAMN SIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPTQI LHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTDE DYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDLS IKHEDWFAFAQKRCQR Wildtype MSFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDEQHK 80 aminoacid ELLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRAGEV sequence(with EKNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKFACY N-terminal FTGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEMELM methionine) DELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQQHE EANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELNAQK FNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFTRAK DRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDAELA MKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADFYS DFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNKAV LFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVFFA DSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQFS ATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASSGK PNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGEHG KGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKFFF HVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIEQR SFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVTNN AIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAYQL TDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAMN SIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPTQI LHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTDE DYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDLS IKHEDWFAFAQKRCQR Expression MGSGSFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDE 81 construct(with QHKELLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRA N-terminal GEVEKNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKF methionine, ACYFTGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEM V5-tagandC- ELMDELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQ terminalNLS) QHEEANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELN aasequence AQKFNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFT RAKDRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDA ELAMKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADF YSDFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNK AVLFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVF FADSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQ FSATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASS GKPNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGE HGKGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKF FFHVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIE QRSFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVT NNAIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAY QLTDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAM NSIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPT QILHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTD EDYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDL SIKHEDWFAFAQKRCQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAGTTTTGAATCATTCACTAACGTTTATCCCGTTTCCAAGACTTTGCGCTTTGA 82 coding GCTGAGGCCCGTTGGTGCAACTGCAGAGAAGCTTAAGGAAAGTGGTATCCTTGA sequence(with GCATGATACGAAACGAGGTAAGGAATATGCGACTCTCAAGGATCTGCTTGATGAG N-terminal CAACATAAGGAGTTACTTGCTGACGCCCTAAAACCTGAACGTGTGAAGAATGCGC methionine TTAAGCCCAATAGTGGTAAGAGTAAAAAAGATAAATTGGTTGAAGAGAATTACATT andstop ACGGAAGACGGGGAGATTCGATGGGAAACTCTTGCGGCTGCGATGGAGGCATTT codon) CGCGCCGGTGAGGTAGAGAAAAATGTGCTTGAAGCAATACAGACGCAATTTAGA AAGCTGATTGTAACGATACTGAAGGCGGATGAGCGGTATCCGGGACTGACAGCT TCAACGCCTTCGGCTGTCATTAAGACTCTTCTTAAGCAGGATGTTCATCCAGAAG CAGTAGAGACATTTGCAAAATTTGCCTGTTATTTTACCGGTTTTCAGGAAAATCGG AAGAATATCTATGCGGAAGAAAAGCAAGCAACTGCAGTTGCAACGCGAGTTGTTC ATGATAATTTCGCAAAGTTCCATACACAATCGAAAATAATAGGTGTCATAAAGAAT AAATATCCAGAAATCCTTCAGTCGGTAGAAATGGAATTGATGGACGAATTAGGTG GGATGAAAATCACTGATATCTTTTCTATCAACAGCTATTCCAAATGGATGACGCAA GAAGGGATAGACTTTATTAATAAGATTATAGGTGGCTATAGCCCATCTGTTGGTGT GAAGGTGCGTGGTCTGAACGAGTTCATTAATCTTTATCGGCAGCAGCATGAAGAG GCAAATGCAGATCGGCGGAATCTCGCAAAAATGCCGATGCTGTTTAAACAAATTT TAAGTGATATTTCGACACGATCATTCATTCCGGTGATGTTTGAAAATGATGCGGAA CTAAAGGATTCAATAGAAGCATTCTTGACAGGTCTGAATGATTTTGAGTTGAATGC TCAGAAGTTTAACGTTGTCGTTGCATTAGGTAATCTTTTCCAAAAAATTGTGCCTT GCGAAGGTATTTTCTTGGATGCAGCATTGATGGAAAAAGTTTCGAAGACGGCTAC AGGAGATTGGAGTCTTCTTGCTCAGTCGATGGAGGCGTATGCAGAGACAGCATT CACAAGAGCAAAAGACCGAGACGCATGGCTAAGGAAAAATTATTATTCGCTGTCC GAGCTGAGCCAAGTTCCGATTTTGAAGAACACTGATGAAGGAATGTTGAAGTTTG AACTATCTGCCTATTGGTCAGGCGAAAAGATGGAAAGTTTTGTTAAAGGAATCAT GGATGCTGAATTGGCAATGAAACCAGTTCTTGCCAGCATTGGTCAGAAAACCGAA GAGGTGCGTCTTCGTGATCGGATTGACGATGTCGTAAAAATCAAGGGATATCTTG ATTCAATTCAGAATTTTTTACATCACCTAAAACCGTTTTGTGCTCCAACTGAATTGA ATCGTGATGCGGATTTTTATTCTGACTTTGACGCATTGTATAATCAGCTTGTACTG GTTATACCGCTTTATAACTGTGTCCGCAATTACGTGACACAGAAAGTGACAGAGG TTCAGAAACTGAGGCTAAAGTTTGATGCCCCTACATTGGCGGACGGATGGGACG CGAATAAAGAAAATGATAATAAGGCAGTTCTGTTTGAAAAGGACGGGCTATATTTT CTTGGAATCCTGAATCCTAACCTGAAGGCGAAAGATCGTCCAGTCTTTGAGCATG AAAGTAATGTTACAAAGAAATCTTGTTATCGCAAGATTGTCTATAAACTTTTGCCA GGACCAAATAAAATGCTTCCCAAGGTCTTTTTTGCTGATTCCAATAGGACACTGTA CCATCCTTCCAAGTCGTTGCTGGATCGTTATCACAACGGTGAATACAAGAAAGGC GATTCATTCGACATCAAATTCTGTCATGAATTGATTGATTATTTTAAAGCCTCGATT AGTATTCACCCCGATTGGAAGGAATTCGGTTTCCAATTCAGTGCGACAAAAACAT ATGAGAGCATTGATGGTTTTTATCGTGAGGTTGAGGAGCAAGGATATAAAGTTAA TTTTGCTTTTGTAAGGGCGGATTTAATTGACAAATATGTGGAAAGTGGAAGTTTGT TCCTTTTCCAATTGTATAACAAGGATTTCTCTTGTGCGTCATCTGGGAAGCCAAAC CTCCACACGCTTTACTGGAAGAGCCTCTTTGCAAAAGAAAACCTTGATGAGCCGA TTCTGAAGTTGTGTGGGGGTGCAGAGCTATTCTTCCGCCCAGTTGCAATCCAGAA GCCGTATGTACATACCTTGGGAGAAAAGTTGGTCAATCGCAGGCTTGGCGAGCA CGGTAAGGGAGAGGCAATCCCGGAGAGAGTTCACAAGGAACTCGTGGACTACTA CAACCATCGTGTGTCGGTGCTGAGTCATGATGGGAAGGCATTTAAAGACAAGGTT GTTGTTCGGGATGTCGCACATTCGATTACAAAAGATCGTCGATTCTCAGAGGCAA AGTTTTTTTTCCATGTTCCGATCATGTTTAACCGTACAGCATCGAAGAGTGCAAAG TTTAACGACAAAGTTGTGGACTATCTCAAGACCACTCAGAATGTAAACGTTATCG GGTTGGATCGAGGAGAAAGAAATCTGATTTATCTGACAATGGTAAATTTGCACGG AAAGCTGATAGAGCAGCGTAGTTTCAACCTAGTTAATGGTGTGGATTATCATTCAA AGCTAGATTTGCGAGAAAAGGAGCGCATGGACGCACGCGTTAATTGGGAGAACA TTGGGGGAATTAAAGATCTTAAGACCGGATATCTTTCCGCGGTTGTTCATGAGAT TGCGAAGATGATGGTGACGAATAATGCCATTGTTGTCTTGGAGGACTTGAACTTC GGTTTCAAACGTGGGCGGTTCAAGGTTGAGAAACAGGTCTATCAGAAGTTTGAGA AGATGCTGATTGATAAACTGAATTTCCTGATGTTCAAGGAATGCAATCAAGCGGC TCTCGGTGGTGTTCGCCGTGCATATCAATTGACGGATAAATTCGTGAGTTTTGAA AAACTTGGTAAACAAACGGGTTTCCTGTTTTATGTTCCGGCGGGCTACACATCGA AGATTGATCCAACAACTGGATTCACCAACCTCTTCAACACGAAAAAATGCACTAAT GCCGAAGGTCGGAAGGTCTTCTTTGAGGCGATGAACTCTATCATATATGACGGAT CAAGGAAGTCGTTTGCGTTCTCATTTGATTACGGCAACCCAGTTTTTAGAGCAAG TCAAACGAGTTTTAAAAAAGAATGGACCGTCTATTCCGCTGATACGCGCATTGTC TACAATCGTGGCGAGAAAACTGTTAATACGATCCATCCGACACAAATTCTTCATGA TGCTTTGTGTGCACTCGGCATTGACGTTCATGACGGATTGAACGTCTTGAACGTA GTTCGTGAGACGCCAGCGGACAAGATTCATGCTAAGTTTTTCTCAGACTTGTTCT ATGCGTTTGATCGTACACTTCAGATGCGTAACAGTGTTTCAGGAACAGATGAAGA CTATATCCAATCGCCTGTTTTGAATGCGACAGGTGAGTTTTTTGATTCGCGGAAA GCAGACAGTACTCTTCCGCAGGATGCCGATGCCAATGGTGCCTACCACATCGCA TTAAAGGGACTTTTGCTGCTACAACGCATGAAAGATATTGGCAGTGATATCAAGC TTGATCTATCCATTAAGCATGAGGACTGGTTTGCGTTTGCACAAAAGCGTTGCCA GAGATAA Codon AGCTTCGAGTCTTTCACTAACGTATACCCTGTGTCTAAAACCCTGCGTTTTGAACT 83 optimized GCGGCCTGTGGGCGCCACTGCCGAGAAGCTGAAGGAGAGCGGCATCCTGGAGC coding ACGATACCAAGCGGGGCAAGGAATACGCTACACTGAAGGACCTGCTGGACGAG sequence(no CAGCACAAAGAGCTACTGGCCGACGCCCTGAAGCCAGAGAGAGTGAAGAACGC N-terminal CCTGAAGCCCAACAGCGGCAAGTCCAAAAAGGACAAGCTGGTCGAAGAGAACTA methionine,no CATTACAGAAGATGGAGAGATCAGATGGGAGACACTGGCCGCTGCTATGGAGGC stopcodon) CTTCAGAGCTGGCGAAGTGGAGAAGAACGTGCTGGAAGCGATCCAGACACAGTT TCGGAAGCTGATCGTGACCATCCTGAAAGCCGACGAGAGATACCCTGGACTGAC CGCCTCTACACCTAGCGCCGTCATCAAGACCTTGCTGAAGCAGGACGTGCACCC CGAGGCCGTAGAGACATTCGCTAAATTTGCCTGTTACTTCACCGGCTTTCAGGAA AACAGAAAGAATATCTACGCCGAAGAAAAACAGGCCACCGCCGTGGCCACACGG GTTGTCCACGACAACTTCGCCAAATTTCACACCCAGTCTAAGATTATCGGCGTGA TCAAAAACAAGTACCCCGAGATCCTGCAGAGCGTCGAGATGGAACTGATGGACG AACTTGGGGGAATGAAGATCACCGATATCTTCAGTATCAACAGCTACAGCAAGTG GATGACCCAGGAGGGAATCGACTTCATCAACAAAATCATCGGCGGCTACAGCCC TAGCGTGGGCGTCAAAGTGAGAGGCCTGAACGAGTTCATCAACCTGTACAGACA GCAGCACGAGGAAGCCAACGCCGACCGGCGGAACCTGGCTAAGATGCCTATGC TGTTTAAACAAATTCTGAGCGACATCAGCACCCGGAGCTTCATCCCTGTGATGTT CGAGAATGACGCCGAGCTCAAGGACAGCATCGAGGCCTTCCTGACAGGCCTGAA TGATTTCGAGCTGAACGCTCAGAAGTTCAACGTTGTGGTGGCCCTGGGGAACCT GTTTCAGAAGATTGTGCCTTGTGAAGGCATCTTCCTGGACGCTGCCCTGATGGAA AAGGTTTCCAAGACAGCTACAGGCGACTGGAGCCTGCTCGCACAGTCTATGGAA GCCTACGCCGAAACAGCCTTTACAAGAGCCAAGGACCGGGACGCCTGGCTGAG AAAGAATTACTACAGCCTGTCCGAGCTGAGCCAGGTGCCAATCCTGAAGAACACT GATGAGGGCATGCTGAAGTTCGAGCTGAGCGCCTACTGGTCCGGCGAGAAAATG GAATCTTTCGTGAAGGGCATCATGGACGCCGAGCTGGCCATGAAGCCAGTGCTG GCCAGCATCGGCCAGAAAACCGAAGAGGTGCGGCTGAGAGATAGAATCGACGA CGTGGTGAAGATCAAGGGCTACCTGGACAGCATCCAGAATTTCCTGCACCACCT GAAGCCTTTCTGTGCCCCTACCGAGCTGAACCGGGACGCCGACTTCTACTCTGA CTTCGATGCTCTGTACAATCAACTGGTGCTGGTGATTCCCCTGTACAACTGCGTG AGAAACTACGTCACCCAAAAGGTTACCGAGGTGCAGAAGCTGCGCCTCAAGTTC GATGCACCTACCCTGGCCGATGGATGGGACGCCAATAAAGAGAATGACAACAAA GCCGTCCTGTTCGAGAAAGACGGCCTGTATTTCCTCGGCATCCTCAACCCTAACC TGAAAGCCAAGGACCGGCCTGTGTTCGAACATGAAAGCAACGTGACCAAGAAGT CATGCTACCGGAAGATTGTGTACAAACTGCTGCCAGGCCCTAACAAGATGCTGC CTAAGGTGTTCTTTGCCGATAGCAACAGGACACTGTACCACCCTAGCAAGAGCCT GCTGGACCGGTATCACAACGGCGAGTACAAGAAGGGCGATAGCTTTGATATCAA GTTTTGCCACGAGCTGATCGACTACTTCAAGGCCTCTATCTCTATTCACCCTGAC TGGAAGGAGTTCGGCTTTCAATTTTCTGCCACAAAGACCTACGAGTCTATCGACG GCTTCTATAGAGAGGTGGAAGAGCAGGGCTACAAGGTGAACTTCGCCTTTGTGC GTGCTGACCTGATCGATAAGTACGTGGAAAGCGGCTCCCTGTTCCTGTTCCAGC TCTATAACAAGGACTTCAGCTGTGCCTCTAGCGGCAAGCCGAATCTTCATACACT GTACTGGAAAAGCCTGTTCGCCAAGGAGAACCTGGACGAGCCTATACTGAAGCT GTGCGGCGGCGCCGAGCTGTTCTTCAGACCCGTGGCGATCCAGAAACCCTACG TGCACACATTGGGCGAAAAGCTGGTGAATAGACGGCTCGGCGAGCACGGCAAG GGCGAGGCTATCCCTGAGCGGGTGCACAAGGAACTGGTGGACTACTACAACCAC AGAGTGAGCGTGCTCAGTCACGATGGAAAGGCCTTCAAGGACAAGGTGGTGGTT CGGGACGTGGCCCACAGCATCACCAAGGACCGACGGTTTAGCGAGGCCAAGTT CTTCTTCCACGTGCCCATCATGTTTAACCGGACCGCCAGCAAGAGCGCCAAGTT CAACGACAAGGTGGTGGACTACCTGAAAACCACCCAAAACGTGAACGTGATCGG ACTGGACAGAGGTGAAAGAAACCTGATCTACCTCACAATGGTGAACCTGCATGG CAAGCTCATCGAGCAGCGGAGCTTCAACCTGGTGAATGGCGTGGACTACCATTC TAAGCTGGATCTGCGCGAGAAGGAACGTATGGATGCTAGAGTGAACTGGGAGAA TATCGGCGGCATAAAGGATCTGAAAACCGGCTACCTGAGCGCCGTGGTGCACGA GATCGCCAAAATGATGGTGACAAACAACGCCATCGTGGTGCTGGAAGATCTGAA CTTTGGATTCAAGAGAGGCAGATTCAAAGTGGAAAAGCAGGTGTACCAGAAATTC GAGAAGATGCTGATCGACAAACTGAACTTCCTGATGTTCAAAGAGTGCAACCAGG CCGCCCTGGGCGGCGTGCGGCGGGCCTATCAGCTGACCGACAAGTTCGTGAGC TTCGAGAAGCTGGGAAAGCAGACCGGCTTCCTGTTCTATGTGCCCGCCGGCTAT ACAAGCAAAATCGATCCTACAACCGGTTTCACCAACCTGTTCAATACCAAGAAAT GCACCAACGCCGAGGGAAGAAAGGTGTTCTTCGAGGCTATGAACAGCATCATCT ACGACGGCTCCAGAAAATCTTTCGCCTTTAGCTTCGACTACGGCAACCCCGTGTT TCGAGCCTCCCAGACCAGCTTCAAGAAGGAATGGACCGTGTACAGCGCCGATAC AAGAATCGTGTATAATCGGGGCGAAAAGACCGTAAACACCATCCACCCTACCCA GATCCTGCACGACGCCCTGTGCGCCTTGGGAATCGACGTGCACGATGGGTTAAA TGTCTTGAACGTCGTGAGAGAGACACCCGCTGATAAGATCCACGCCAAGTTCTTC AGCGATCTCTTCTACGCCTTCGACAGAACCCTGCAGATGAGGAACTCTGTGAGC GGGACCGACGAAGATTACATCCAGAGCCCTGTGCTGAATGCTACCGGCGAGTTC TTTGACAGCAGAAAAGCCGACAGCACCCTGCCCCAGGACGCAGACGCTAATGGA GCCTACCACATCGCCCTGAAGGGCCTGCTGCTCCTGCAGAGAATGAAGGATATC GGCTCAGATATCAAGCTGGATCTGTCTATTAAGCACGAGGATTGGTTCGCCTTCG CTCAGAAGCGGTGCCAGAGA Expression ATGggctccggaAGCTTCGAGTCTTTCACTAACGTATACCCTGTGTCTAAAACCCTGC 84 construct(with GTTTTGAACTGCGGCCTGTGGGCGCCACTGCCGAGAAGCTGAAGGAGAGCGGC N-terminal ATCCTGGAGCACGATACCAAGCGGGGCAAGGAATACGCTACACTGAAGGACCTG methionine CTGGACGAGCAGCACAAAGAGCTACTGGCCGACGCCCTGAAGCCAGAGAGAGT andstop GAAGAACGCCCTGAAGCCCAACAGCGGCAAGTCCAAAAAGGACAAGCTGGTCGA codon, AGAGAACTACATTACAGAAGATGGAGAGATCAGATGGGAGACACTGGCCGCTGC includesV5- TATGGAGGCCTTCAGAGCTGGCGAAGTGGAGAAGAACGTGCTGGAAGCGATCCA tagandC- GACACAGTTTCGGAAGCTGATCGTGACCATCCTGAAAGCCGACGAGAGATACCC terminalNLS) TGGACTGACCGCCTCTACACCTAGCGCCGTCATCAAGACCTTGCTGAAGCAGGA CGTGCACCCCGAGGCCGTAGAGACATTCGCTAAATTTGCCTGTTACTTCACCGG CTTTCAGGAAAACAGAAAGAATATCTACGCCGAAGAAAAACAGGCCACCGCCGT GGCCACACGGGTTGTCCACGACAACTTCGCCAAATTTCACACCCAGTCTAAGATT ATCGGCGTGATCAAAAACAAGTACCCCGAGATCCTGCAGAGCGTCGAGATGGAA CTGATGGACGAACTTGGGGGAATGAAGATCACCGATATCTTCAGTATCAACAGCT ACAGCAAGTGGATGACCCAGGAGGGAATCGACTTCATCAACAAAATCATCGGCG GCTACAGCCCTAGCGTGGGCGTCAAAGTGAGAGGCCTGAACGAGTTCATCAACC TGTACAGACAGCAGCACGAGGAAGCCAACGCCGACCGGCGGAACCTGGCTAAG ATGCCTATGCTGTTTAAACAAATTCTGAGCGACATCAGCACCCGGAGCTTCATCC CTGTGATGTTCGAGAATGACGCCGAGCTCAAGGACAGCATCGAGGCCTTCCTGA CAGGCCTGAATGATTTCGAGCTGAACGCTCAGAAGTTCAACGTTGTGGTGGCCC TGGGGAACCTGTTTCAGAAGATTGTGCCTTGTGAAGGCATCTTCCTGGACGCTG CCCTGATGGAAAAGGTTTCCAAGACAGCTACAGGCGACTGGAGCCTGCTCGCAC AGTCTATGGAAGCCTACGCCGAAACAGCCTTTACAAGAGCCAAGGACCGGGACG CCTGGCTGAGAAAGAATTACTACAGCCTGTCCGAGCTGAGCCAGGTGCCAATCC TGAAGAACACTGATGAGGGCATGCTGAAGTTCGAGCTGAGCGCCTACTGGTCCG GCGAGAAAATGGAATCTTTCGTGAAGGGCATCATGGACGCCGAGCTGGCCATGA AGCCAGTGCTGGCCAGCATCGGCCAGAAAACCGAAGAGGTGCGGCTGAGAGAT AGAATCGACGACGTGGTGAAGATCAAGGGCTACCTGGACAGCATCCAGAATTTC CTGCACCACCTGAAGCCTTTCTGTGCCCCTACCGAGCTGAACCGGGACGCCGAC TTCTACTCTGACTTCGATGCTCTGTACAATCAACTGGTGCTGGTGATTCCCCTGTA CAACTGCGTGAGAAACTACGTCACCCAAAAGGTTACCGAGGTGCAGAAGCTGCG CCTCAAGTTCGATGCACCTACCCTGGCCGATGGATGGGACGCCAATAAAGAGAA TGACAACAAAGCCGTCCTGTTCGAGAAAGACGGCCTGTATTTCCTCGGCATCCTC AACCCTAACCTGAAAGCCAAGGACCGGCCTGTGTTCGAACATGAAAGCAACGTG ACCAAGAAGTCATGCTACCGGAAGATTGTGTACAAACTGCTGCCAGGCCCTAACA AGATGCTGCCTAAGGTGTTCTTTGCCGATAGCAACAGGACACTGTACCACCCTAG CAAGAGCCTGCTGGACCGGTATCACAACGGCGAGTACAAGAAGGGCGATAGCTT TGATATCAAGTTTTGCCACGAGCTGATCGACTACTTCAAGGCCTCTATCTCTATTC ACCCTGACTGGAAGGAGTTCGGCTTTCAATTTTCTGCCACAAAGACCTACGAGTC TATCGACGGCTTCTATAGAGAGGTGGAAGAGCAGGGCTACAAGGTGAACTTCGC CTTTGTGCGTGCTGACCTGATCGATAAGTACGTGGAAAGCGGCTCCCTGTTCCT GTTCCAGCTCTATAACAAGGACTTCAGCTGTGCCTCTAGCGGCAAGCCGAATCTT CATACACTGTACTGGAAAAGCCTGTTCGCCAAGGAGAACCTGGACGAGCCTATA CTGAAGCTGTGCGGCGGCGCCGAGCTGTTCTTCAGACCCGTGGCGATCCAGAA ACCCTACGTGCACACATTGGGCGAAAAGCTGGTGAATAGACGGCTCGGCGAGCA CGGCAAGGGCGAGGCTATCCCTGAGCGGGTGCACAAGGAACTGGTGGACTACT ACAACCACAGAGTGAGCGTGCTCAGTCACGATGGAAAGGCCTTCAAGGACAAGG TGGTGGTTCGGGACGTGGCCCACAGCATCACCAAGGACCGACGGTTTAGCGAG GCCAAGTTCTTCTTCCACGTGCCCATCATGTTTAACCGGACCGCCAGCAAGAGC GCCAAGTTCAACGACAAGGTGGTGGACTACCTGAAAACCACCCAAAACGTGAAC GTGATCGGACTGGACAGAGGTGAAAGAAACCTGATCTACCTCACAATGGTGAAC CTGCATGGCAAGCTCATCGAGCAGCGGAGCTTCAACCTGGTGAATGGCGTGGAC TACCATTCTAAGCTGGATCTGCGCGAGAAGGAACGTATGGATGCTAGAGTGAACT GGGAGAATATCGGCGGCATAAAGGATCTGAAAACCGGCTACCTGAGCGCCGTG GTGCACGAGATCGCCAAAATGATGGTGACAAACAACGCCATCGTGGTGCTGGAA GATCTGAACTTTGGATTCAAGAGAGGCAGATTCAAAGTGGAAAAGCAGGTGTACC AGAAATTCGAGAAGATGCTGATCGACAAACTGAACTTCCTGATGTTCAAAGAGTG CAACCAGGCCGCCCTGGGCGGCGTGCGGCGGGCCTATCAGCTGACCGACAAGT TCGTGAGCTTCGAGAAGCTGGGAAAGCAGACCGGCTTCCTGTTCTATGTGCCCG CCGGCTATACAAGCAAAATCGATCCTACAACCGGTTTCACCAACCTGTTCAATAC CAAGAAATGCACCAACGCCGAGGGAAGAAAGGTGTTCTTCGAGGCTATGAACAG CATCATCTACGACGGCTCCAGAAAATCTTTCGCCTTTAGCTTCGACTACGGCAAC CCCGTGTTTCGAGCCTCCCAGACCAGCTTCAAGAAGGAATGGACCGTGTACAGC GCCGATACAAGAATCGTGTATAATCGGGGCGAAAAGACCGTAAACACCATCCAC CCTACCCAGATCCTGCACGACGCCCTGTGCGCCTTGGGAATCGACGTGCACGAT GGGTTAAATGTCTTGAACGTCGTGAGAGAGACACCCGCTGATAAGATCCACGCC AAGTTCTTCAGCGATCTCTTCTACGCCTTCGACAGAACCCTGCAGATGAGGAACT CTGTGAGCGGGACCGACGAAGATTACATCCAGAGCCCTGTGCTGAATGCTACCG GCGAGTTCTTTGACAGCAGAAAAGCCGACAGCACCCTGCCCCAGGACGCAGAC GCTAATGGAGCCTACCACATCGCCCTGAAGGGCCTGCTGCTCCTGCAGAGAATG AAGGATATCGGCTCAGATATCAAGCTGGATCTGTCTATTAAGCACGAGGATTGGT TCGCCTTCGCTCAGAAGCGGTGCCAGAGAtctagaAAGCGGACAGCAGACGGCTC CGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCA ATCCCCTGCTGGGCCTGGACAGCACCTGA

[0127] In some embodiments a ZTAE Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 79, SEQ ID NO:80, or SEQ ID NO:81. In some embodiments, a ZTAE Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:79, SEQ ID NO: 80, or SEQ ID NO:81. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D905 substitution, wherein the position of the D905 substitution is defined with respect to the amino acid numbering of SEQ ID NO:80 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E990 substitution, wherein the position of the E990 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 80 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1206 substitution, wherein the position of the R1206 substitution is defined with respect to the amino acid numbering of SEQ ID NO:80 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1243 substitution, wherein the position of the D1243 substitution is defined with respect to the amino acid numbering of SEQ ID NO:80 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZTAE Type V Cas protein is catalytically inactive, for example due to a R1206 substitution in combination with a D905 substitution, a E990 substitution, and/or D1243 substitution.

6.2.15. ZSQQ Type V Cas Protein

[0128] In one aspect, the disclosure provides ZSQQ Type V Cas proteins. ZSQQ Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZSQQ Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:85. In some embodiments, the ZSQQ Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:85. In some embodiments, a ZSQQ Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:85.

[0129] Exemplary ZSQQ Type V Cas protein sequences and nucleotide sequences encoding exemplary ZSQQ Type V Cas proteins are set forth in Table 10.

TABLE-US-00016 TABLE10 ZSQQTypeVCasSequences SEQID Name Sequence NO. Wildtype STINKFCGQGNGYSRSITLRNKLIPIGKTEENLKWFLEKDLERAIAYPEIKNLIDNIHRS 85 aminoacid VIEDTLSKVALNWNEIFNTLAAYQNEKDKKKKAAIKKDLEKLQGCARKKIVDTFKKNP sequence DYEKLFKEGLFKELLPELIKTAPVSEIEDKTKALECFNRFSTYFTGFHENRKNMYSED (withoutN- AKSTAISYRIVNENFPKFFANIKLYNYLKEKFPQIIINTEESLKDYLKGKKLDSVFSIDGF terminal NDVLAQSGIDFYNTVIGGISGEAGTEKTQGLNEKINLARQQLPKDEKDKLRGKMVDL methionine) FKQILSDRETSSFIPTGFENKKEVYSTVKKFSEIVVEKSVSKVKEIFTQNEEYNLNEIF VPAKSLTNFSQNIFGNWSILSEGLFLLEKDNVKKQLSEKQIETLHKEIAKKDCSFTEL QNAYERWCAENSVDATKNINRYFSIVDLRTKNDSFEKEEINILDEITNAFSKIDFDDIH DLQQEKEAATPIKNYLDEVQNLYHHLKLVDYRGEERKDANFYSKLDYILRKDRKDYL NLAEVVPLYNKVRNFVTKKPGEVKKIKMMFDCSSLLGGWGTDYETKEAHIFIDSGKY YLGIINEKLSKDDVELLKKSSERMITKVIYDFQKPDNKNTPRLFIRSKGTNYAPAVFQY NLPIESVIDIYDRGLFKTEYRKINSKVYKESLIKMIDYFKMGFERHESYKHYKFCWKE SSKYNDIGEFYKDVINSCYQLNFEKVNYENLLKLVENNKLFLFQIYNKDFAEKKSGKK NLHTLYWENLFSEENLKDVCLKLNGEAELFWRKASLDKGKVIVHRMGSILVNRTTSE GKSIPEDIYQEIYQYKNKMKDKISDEAKSLLDSGTVICKEATHDITKDKRFTEDTYLFH CPITMNFKATDKKNKEFNNHVLEVLKENPDVKIIGLDRGERHLIYLSLINQKGEIELQK TLNLVEQVRNDKTVKVDYQEKLVHKEGDRDKARKNWQTIGNIKELKEGYLSAVVHEI AMLMVENNAIVVMEDLNFGFKRGRFAVERQIYQKFENMLIEKLNYLVFKDKNATEPG GVLNAYQLTNKSANVTDVYKQCGWLFYIPAAYTSKIDPKTGFANLFITKGLTNVEKK KEFFDKFDSIRYDSKEDCFVFGFDYAKLCDNASFRKKWEVYTRGERLVYNKDKHKN EPINPTEELKGIFDAFDINWNTDDNFIDSVQTIQAEKANAKFFDILLRMFNATLQMRN SKTNSSASEDDYLISPVKAEDGTFFDTREELKKGKDAKLPIDSDANGAYHIALKGLFL LENDFNRDEKGVIQNISNADWFKFVQEKKYKD Wildtype MSTINKFCGQGNGYSRSITLRNKLIPIGKTEENLKWFLEKDLERAIAYPEIKNLIDNIHR 86 aminoacid SVIEDTLSKVALNWNEIFNTLAAYQNEKDKKKKAAIKKDLEKLQGCARKKIVDTFKKN sequence(with PDYEKLFKEGLFKELLPELIKTAPVSEIEDKTKALECFNRFSTYFTGFHENRKNMYSE N-terminal DAKSTAISYRIVNENFPKFFANIKLYNYLKEKFPQIIINTEESLKDYLKGKKLDSVFSID methionine) GFNDVLAQSGIDFYNTVIGGISGEAGTEKTQGLNEKINLARQQLPKDEKDKLRGKMV DLFKQILSDRETSSFIPTGFENKKEVYSTVKKFSEIVVEKSVSKVKEIFTQNEEYNLNE IFVPAKSLTNFSQNIFGNWSILSEGLFLLEKDNVKKQLSEKQIETLHKEIAKKDCSFTE LQNAYERWCAENSVDATKNINRYFSIVDLRTKNDSFEKEEINILDEITNAFSKIDFDDI HDLQQEKEAATPIKNYLDEVQNLYHHLKLVDYRGEERKDANFYSKLDYILRKDRKDY LNLAEVVPLYNKVRNFVTKKPGEVKKIKMMFDCSSLLGGWGTDYETKEAHIFIDSGK YYLGIINEKLSKDDVELLKKSSERMITKVIYDFQKPDNKNTPRLFIRSKGTNYAPAVFQ YNLPIESVIDIYDRGLFKTEYRKINSKVYKESLIKMIDYFKMGFERHESYKHYKFCWK ESSKYNDIGEFYKDVINSCYQLNFEKVNYENLLKLVENNKLFLFQIYNKDFAEKKSGK KNLHTLYWENLFSEENLKDVCLKLNGEAELFWRKASLDKGKVIVHRMGSILVNRTTS EGKSIPEDIYQEIYQYKNKMKDKISDEAKSLLDSGTVICKEATHDITKDKRFTEDTYLF HCPITMNFKATDKKNKEFNNHVLEVLKENPDVKIIGLDRGERHLIYLSLINQKGEIELQ KTLNLVEQVRNDKTVKVDYQEKLVHKEGDRDKARKNWQTIGNIKELKEGYLSAVVH EIAMLMVENNAIVVMEDLNFGFKRGRFAVERQIYQKFENMLIEKLNYLVFKDKNATE PGGVLNAYQLTNKSANVTDVYKQCGWLFYIPAAYTSKIDPKTGFANLFITKGLTNVE KKKEFFDKFDSIRYDSKEDCFVFGFDYAKLCDNASFRKKWEVYTRGERLVYNKDKH KNEPINPTEELKGIFDAFDINWNTDDNFIDSVQTIQAEKANAKFFDILLRMFNATLQM RNSKTNSSASEDDYLISPVKAEDGTFFDTREELKKGKDAKLPIDSDANGAYHIALKG LFLLENDFNRDEKGVIQNISNADWFKFVQEKKYKD Expression MGSGSTINKFCGQGNGYSRSITLRNKLIPIGKTEENLKWFLEKDLERAIAYPEIKNLID 87 construct(with NIHRSVIEDTLSKVALNWNEIFNTLAAYQNEKDKKKKAAIKKDLEKLQGCARKKIVDT N-terminal FKKNPDYEKLFKEGLFKELLPELIKTAPVSEIEDKTKALECFNRFSTYFTGFHENRKN methionine, MYSEDAKSTAISYRIVNENFPKFFANIKLYNYLKEKFPQIIINTEESLKDYLKGKKLDSV V5-tagandC- FSIDGFNDVLAQSGIDFYNTVIGGISGEAGTEKTQGLNEKINLARQQLPKDEKDKLRG terminalNLS) KMVDLFKQILSDRETSSFIPTGFENKKEVYSTVKKFSEIVVEKSVSKVKEIFTQNEEY aasequence NLNEIFVPAKSLTNFSQNIFGNWSILSEGLFLLEKDNVKKQLSEKQIETLHKEIAKKDC SFTELQNAYERWCAENSVDATKNINRYFSIVDLRTKNDSFEKEEINILDEITNAFSKID FDDIHDLQQEKEAATPIKNYLDEVQNLYHHLKLVDYRGEERKDANFYSKLDYILRKD RKDYLNLAEVVPLYNKVRNFVTKKPGEVKKIKMMFDCSSLLGGWGTDYETKEAHIFI DSGKYYLGIINEKLSKDDVELLKKSSERMITKVIYDFQKPDNKNTPRLFIRSKGTNYA PAVFQYNLPIESVIDIYDRGLFKTEYRKINSKVYKESLIKMIDYFKMGFERHESYKHYK FCWKESSKYNDIGEFYKDVINSCYQLNFEKVNYENLLKLVENNKLFLFQIYNKDFAE KKSGKKNLHTLYWENLFSEENLKDVCLKLNGEAELFWRKASLDKGKVIVHRMGSIL VNRTTSEGKSIPEDIYQEIYQYKNKMKDKISDEAKSLLDSGTVICKEATHDITKDKRFT EDTYLFHCPITMNFKATDKKNKEFNNHVLEVLKENPDVKIIGLDRGERHLIYLSLINQK GEIELQKTLNLVEQVRNDKTVKVDYQEKLVHKEGDRDKARKNWQTIGNIKELKEGY LSAVVHEIAMLMVENNAIVVMEDLNFGFKRGRFAVERQIYQKFENMLIEKLNYLVFK DKNATEPGGVLNAYQLTNKSANVTDVYKQCGWLFYIPAAYTSKIDPKTGFANLFITK GLTNVEKKKEFFDKFDSIRYDSKEDCFVFGFDYAKLCDNASFRKKWEVYTRGERLV YNKDKHKNEPINPTEELKGIFDAFDINWNTDDNFIDSVQTIQAEKANAKFFDILLRMF NATLQMRNSKTNSSASEDDYLISPVKAEDGTFFDTREELKKGKDAKLPIDSDANGAY HIALKGLFLLENDFNRDEKGVIQNISNADWFKFVQEKKYKDSRKRTADGSEFESPKK KRKVGSGKPIPNPLLGLDST Wildtype ATGTCAACTATTAACAAATTTTGTGGACAGGGGAATGGGTATTCTCGTTCAATTA 88 coding CTTTGAGGAATAAGTTAATTCCTATTGGAAAAACTGAAGAAAATTTGAAATGGTTT sequence(with TTAGAAAAAGATTTGGAAAGGGCAATTGCTTATCCGGAGATAAAGAATCTTATAG N-terminal ATAATATTCATCGTAGTGTAATTGAGGATACTTTATCCAAAGTTGCTTTGAATTGG methionine AATGAAATATTCAATACACTTGCTGCTTATCAAAATGAAAAAGATAAAAAAAAGAA andstop AGCAGCAATAAAAAAGGATTTGGAGAAATTACAAGGTTGTGCAAGAAAGAAAATA codon) GTTGATACTTTTAAAAAGAATCCTGATTATGAAAAATTGTTTAAGGAAGGATTATT CAAAGAACTATTACCTGAGTTAATAAAAACTGCTCCTGTTAGTGAAATAGAAGAT AAAACAAAAGCTTTGGAATGTTTTAATAGATTTAGTACATATTTTACAGGATTTCA TGAAAATAGAAAAAATATGTATAGCGAAGATGCAAAATCAACTGCAATAAGTTAC CGTATTGTAAATGAGAATTTCCCCAAATTTTTTGCAAATATAAAGTTATATAATTAT TTAAAAGAAAAGTTTCCACAAATTATTATTAATACAGAAGAATCTTTAAAAGATTAT CTAAAAGGTAAAAAACTTGATTCTGTATTTAGTATTGATGGATTTAATGATGTTTT AGCTCAAAGTGGAATCGATTTTTATAATACAGTAATTGGTGGAATTTCTGGTGAA GCCGGAACAGAAAAGACTCAAGGATTAAATGAAAAAATCAATCTTGCAAGACAA CAATTACCAAAAGATGAAAAAGATAAACTTCGTGGAAAAATGGTTGATTTATTTAA GCAGATTTTAAGTGATAGAGAAACATCTTCGTTTATTCCAACTGGTTTTGAAAATA AAAAAGAAGTTTATTCTACTGTAAAGAAATTTAGTGAAATTGTTGTTGAAAAGTCT GTTTCAAAAGTAAAAGAAATTTTTACACAAAATGAAGAATATAATCTTAATGAAAT CTTTGTTCCAGCAAAATCATTAACAAATTTTTCTCAAAATATTTTTGGAAATTGGT CTATTTTATCAGAAGGGTTATTTTTGCTTGAAAAAGATAATGTTAAAAAACAATTA TCTGAAAAACAAATTGAAACATTACACAAAGAAATTGCAAAAAAAGATTGTTCTTT TACTGAACTACAAAATGCTTATGAAAGATGGTGTGCTGAAAATAGTGTTGATGCA ACAAAAAATATCAATAGGTATTTTTCAATAGTTGATTTAAGAACAAAAAATGATTC GTTTGAAAAAGAAGAAATTAATATTTTGGATGAAATTACAAATGCTTTTTCAAAAA TTGATTTTGATGATATTCATGATTTACAACAAGAAAAAGAAGCTGCAACACCAATA AAAAATTATTTGGATGAAGTTCAAAATCTTTATCATCACTTAAAACTTGTTGATTAT CGTGGTGAAGAACGAAAGGATGCAAACTTTTATTCAAAGCTAGATTATATATTAA GGAAAGATAGGAAAGATTACCTTAATCTTGCTGAAGTTGTACCTTTGTATAACAA AGTTCGTAATTTTGTAACAAAGAAACCTGGTGAAGTAAAAAAGATTAAAATGATG TTTGATTGTAGTTCTTTATTAGGGGGGTGGGGAACTGATTACGAAACAAAAGAA GCTCATATTTTTATTGATTCTGGAAAATATTATTTGGGAATTATAAACGAAAAATT ATCAAAAGATGATGTTGAGTTATTAAAAAAATCAAGTGAAAGAATGATAACAAAA GTAATTTATGATTTTCAGAAACCTGATAATAAAAATACACCTCGTTTATTTATTCG TTCAAAAGGAACAAATTATGCACCTGCTGTTTTTCAATATAATTTACCAATAGAAT CTGTTATTGATATTTATGATAGAGGATTGTTTAAAACCGAATATAGAAAAATCAAT TCAAAAGTTTACAAAGAATCATTAATAAAAATGATTGATTATTTCAAGATGGGCTT TGAAAGACATGAATCATATAAGCATTATAAATTCTGTTGGAAGGAATCTTCAAAAT ATAATGATATTGGTGAATTTTACAAGGATGTGATAAATTCATGCTATCAATTAAAT TTCGAAAAAGTGAATTATGAAAATTTATTAAAATTGGTTGAAAACAATAAATTATT CCTTTTCCAAATATATAACAAAGATTTTGCAGAAAAAAAATCTGGAAAGAAAAATC TTCATACTTTGTATTGGGAAAATCTTTTTAGTGAAGAAAACTTGAAAGATGTTTGC TTAAAATTGAATGGTGAAGCTGAACTTTTCTGGCGCAAAGCAAGTTTAGACAAAG GAAAAGTTATAGTTCATAGAATGGGTTCTATTCTTGTAAATAGAACTACATCTGAA GGTAAATCAATTCCAGAAGATATTTATCAGGAAATTTATCAATATAAAAATAAAAT GAAAGATAAAATTTCTGATGAAGCAAAAAGTCTTTTAGATTCAGGAACAGTTATTT GTAAAGAAGCAACTCACGATATTACAAAAGACAAGCGCTTTACAGAAGATACATA TCTTTTCCATTGTCCAATTACAATGAACTTTAAAGCAACTGATAAAAAAAATAAAG AATTTAATAATCATGTTCTTGAAGTTTTAAAAGAAAATCCAGATGTTAAAATTATTG GTCTTGACCGTGGTGAAAGACATTTGATTTATCTTTCTTTGATTAATCAAAAAGGT GAAATTGAACTTCAAAAAACATTGAATCTTGTAGAACAAGTTAGAAATGATAAAAC TGTAAAAGTAGATTATCAAGAAAAACTTGTACATAAAGAAGGCGACAGAGACAAA GCTCGTAAAAACTGGCAAACAATTGGAAATATCAAAGAACTAAAAGAAGGTTATT TATCTGCTGTTGTTCATGAAATTGCAATGTTGATGGTAGAAAATAATGCAATTGTT GTAATGGAAGATTTGAATTTTGGATTTAAACGTGGTCGATTTGCTGTAGAAAGAC AAATTTATCAAAAGTTTGAAAATATGCTCATTGAAAAACTTAATTATCTTGTGTTTA AGGATAAAAATGCTACAGAACCAGGTGGTGTCCTTAATGCATATCAATTAACAAA TAAATCTGCAAATGTAACTGACGTTTATAAACAATGTGGATGGCTTTTCTATATTC CAGCAGCGTATACTTCAAAAATTGATCCAAAAACAGGTTTTGCAAATTTATTCATA ACAAAAGGATTAACAAATGTAGAAAAGAAAAAAGAATTCTTTGATAAATTCGATTC CATTCGTTATGACTCAAAAGAAGACTGTTTTGTATTTGGTTTTGATTATGCAAAAC TTTGTGATAATGCAAGTTTTAGAAAAAAATGGGAAGTATACACAAGAGGGGAAAG ATTAGTTTACAATAAAGATAAACATAAAAATGAACCTATTAATCCAACAGAAGAAT TAAAAGGAATTTTTGATGCATTCGATATAAATTGGAATACGGATGATAATTTTATT GATTCCGTACAGACAATACAAGCAGAAAAAGCAAATGCCAAATTCTTTGATATTC TTTTGCGAATGTTTAATGCAACTCTTCAAATGCGAAATTCAAAAACAAATTCTTCA GCATCAGAAGATGATTATTTGATATCTCCGGTAAAAGCAGAGGATGGAACATTCT TTGATACTCGTGAAGAATTAAAGAAAGGCAAAGATGCAAAACTTCCTATAGATTC AGATGCAAACGGAGCTTATCATATTGCACTAAAAGGACTTTTCTTACTTGAAAAT GACTTCAATAGAGATGAAAAAGGTGTGATTCAGAATATCTCCAACGCCGATTGG TTTAAGTTTGTGCAGGAGAAAAAATACAAAGATTAA Codon AGCACCATCAACAAATTCTGCGGCCAGGGCAACGGCTACAGCAGAAGCATCAC 89 optimized CCTGCGGAACAAACTGATCCCTATCGGCAAGACTGAGGAGAACCTGAAGTGGT coding TCCTGGAGAAGGACCTGGAGCGGGCTATCGCCTACCCCGAGATTAAAAACCTT sequence(no ATCGACAATATCCACAGAAGCGTGATAGAGGATACCCTGAGCAAGGTCGCCCT N-terminal GAACTGGAATGAGATCTTCAACACCCTGGCCGCCTACCAGAACGAGAAAGATAA methionine,no GAAAAAGAAGGCCGCTATCAAGAAGGACCTGGAGAAGTTGCAAGGATGTGCGA stopcodon) GAAAGAAAATCGTGGATACCTTCAAGAAGAACCCTGATTATGAGAAACTGTTTAA AGAGGGACTGTTCAAGGAGCTGCTGCCTGAACTGATCAAGACCGCCCCTGTGA GCGAAATTGAAGATAAAACCAAAGCCCTGGAGTGCTTCAACCGGTTCTCCACAT ACTTCACCGGCTTCCACGAAAATCGCAAAAATATGTACAGCGAGGACGCGAAGA GCACCGCCATCTCCTACCGGATCGTGAACGAGAACTTCCCCAAGTTCTTCGCTA ATATCAAGCTGTACAACTACCTCAAGGAAAAATTTCCACAGATTATCATCAACAC AGAAGAGTCTCTGAAGGATTACCTGAAGGGCAAGAAGCTGGATTCCGTGTTCTC CATCGACGGGTTCAATGACGTGCTGGCCCAGAGCGGCATAGACTTCTACAACA CCGTGATCGGTGGCATCTCAGGAGAGGCCGGCACAGAAAAGACCCAGGGCCT GAATGAGAAGATCAACCTAGCCAGACAGCAGCTGCCTAAGGATGAGAAGGACA AGCTAAGAGGCAAGATGGTCGACCTGTTCAAGCAGATTCTGAGCGATAGAGAAA CCAGCAGCTTCATCCCTACTGGCTTCGAGAATAAGAAGGAAGTGTACTCTACCG TGAAGAAGTTCAGCGAAATCGTGGTCGAAAAAAGCGTGTCCAAGGTGAAGGAG ATCTTCACTCAGAACGAAGAGTACAATCTGAACGAGATCTTCGTGCCTGCGAAG AGCCTGACCAATTTTAGCCAGAACATCTTTGGCAACTGGAGCATCCTTTCTGAA GGCCTGTTCCTGCTGGAAAAGGACAACGTGAAGAAACAGCTGAGTGAGAAACA AATCGAGACACTCCATAAGGAGATCGCCAAGAAGGACTGCAGCTTTACCGAACT GCAGAACGCCTACGAGCGGTGGTGCGCCGAGAACTCCGTGGACGCCACCAAG AACATTAACAGATACTTCAGCATCGTCGACCTGAGAACCAAGAATGACTCCTTC GAGAAGGAAGAGATCAATATCCTTGATGAGATAACCAACGCCTTCTCTAAGATT GACTTCGACGATATCCACGATCTGCAGCAAGAGAAGGAGGCCGCCACCCCTAT CAAGAACTACCTGGACGAGGTTCAAAACCTGTACCACCACCTGAAGCTGGTGGA CTACAGAGGTGAGGAACGAAAGGACGCTAACTTCTACTCTAAACTGGACTATAT CCTGAGAAAGGACAGAAAGGACTACCTGAACCTGGCCGAAGTGGTGCCATTGT ACAACAAGGTTAGAAACTTCGTGACCAAGAAGCCTGGCGAGGTGAAAAAGATCA AGATGATGTTCGACTGCAGCAGCCTGCTGGGCGGATGGGGCACAGATTACGAG ACAAAAGAGGCCCACATTTTCATCGACTCCGGCAAGTATTACCTTGGAATCATCA ACGAGAAGTTGTCAAAAGATGACGTGGAGCTGCTGAAGAAGAGCAGCGAACGG ATGATCACAAAGGTGATCTACGATTTCCAAAAGCCCGATAACAAGAATACACCTA GACTGTTCATCAGGAGCAAGGGCACAAATTATGCTCCTGCTGTTTTCCAATACAA TCTGCCAATAGAGTCTGTGATCGATATTTACGACCGTGGCCTGTTTAAGACCGA GTACAGAAAAATCAACAGCAAGGTGTACAAGGAGAGCCTGATTAAGATGATCGA TTACTTCAAGATGGGCTTTGAGAGACACGAGAGCTACAAGCACTACAAGTTTTG CTGGAAGGAATCTAGCAAGTACAACGACATCGGCGAATTTTACAAGGATGTGAT TAACTCTTGTTACCAGCTGAACTTCGAGAAGGTGAACTATGAGAACCTCCTGAA GTTAGTGGAAAACAACAAGCTGTTCCTGTTTCAGATCTACAACAAGGATTTTGCC GAAAAGAAAAGCGGTAAGAAGAACCTGCACACCCTGTACTGGGAGAACCTGTTT TCTGAGGAGAACCTGAAGGACGTTTGTCTGAAGCTGAATGGCGAGGCCGAGCT GTTCTGGCGGAAGGCTTCTCTGGACAAGGGCAAGGTGATCGTGCACAGAATGG GCTCTATCCTGGTGAACAGAACAACAAGCGAGGGCAAGTCAATCCCTGAGGAC ATCTACCAGGAGATCTATCAGTACAAGAACAAAATGAAGGATAAGATCAGCGAC GAAGCCAAAAGCCTGCTGGACAGCGGCACCGTGATCTGTAAAGAAGCCACCCA CGACATCACCAAGGACAAACGGTTCACAGAGGACACCTACCTGTTCCACTGCCC TATCACCATGAACTTCAAGGCCACCGACAAGAAAAACAAAGAGTTCAACAACCA CGTGCTGGAAGTGCTGAAAGAGAATCCCGACGTGAAGATCATCGGCCTGGACA GAGGCGAACGGCACCTGATCTACCTGAGCCTGATCAACCAGAAGGGCGAGATC GAGCTGCAGAAAACCCTGAATCTGGTGGAACAGGTGCGGAACGACAAAACCGT GAAGGTGGACTACCAGGAGAAGCTGGTGCATAAGGAAGGCGACCGCGACAAA GCCAGAAAGAACTGGCAGACAATCGGAAACATCAAGGAACTGAAGGAGGGCTA CCTGTCTGCCGTGGTGCACGAAATCGCCATGCTGATGGTGGAAAACAACGCCA TCGTGGTGATGGAGGACCTGAACTTCGGCTTCAAGAGAGGCAGATTCGCCGTG GAACGGCAGATCTACCAGAAGTTCGAGAACATGCTGATCGAAAAGCTGAACTAC CTAGTGTTCAAGGACAAGAACGCCACCGAACCTGGCGGCGTGCTGAATGCGTA TCAGCTCACCAACAAGAGCGCCAACGTCACCGACGTGTACAAACAGTGCGGCT GGCTGTTCTACATCCCCGCCGCTTATACAAGCAAGATCGACCCCAAGACCGGAT TCGCCAACCTGTTCATCACAAAGGGACTGACAAACGTGGAAAAGAAGAAGGAGT TCTTCGATAAGTTCGACAGCATCCGGTACGACAGCAAAGAGGACTGCTTTGTGT TCGGCTTCGACTACGCCAAGCTGTGCGACAACGCCTCCTTTAGAAAGAAGTGG GAAGTTTACACCAGAGGAGAGAGGCTGGTCTACAACAAAGACAAGCACAAAAAC GAACCTATCAACCCCACCGAGGAGCTGAAGGGCATCTTCGATGCTTTTGATATT AACTGGAACACCGACGACAACTTCATTGATTCAGTGCAGACCATCCAGGCCGAG AAGGCCAACGCCAAGTTCTTTGACATCCTGCTGAGAATGTTCAACGCCACACTG CAGATGAGAAACAGCAAGACTAACTCCTCTGCCAGCGAGGACGACTACCTGATC AGCCCTGTCAAAGCCGAGGATGGCACCTTCTTCGACACAAGAGAGGAATTAAAG AAGGGCAAAGATGCCAAGCTGCCGATCGACAGCGACGCTAATGGCGCCTACCA CATCGCCCTGAAAGGACTGTTCCTGCTGGAAAATGACTTTAACCGGGACGAGAA GGGAGTGATCCAAAATATCAGCAACGCTGATTGGTTCAAGTTTGTGCAGGAGAA GAAATACAAGGAT Expression ATGggctccggaAGCACCATCAACAAATTCTGCGGCCAGGGCAACGGCTACAGCAG 90 construct(with AAGCATCACCCTGCGGAACAAACTGATCCCTATCGGCAAGACTGAGGAGAACCT N-terminal GAAGTGGTTCCTGGAGAAGGACCTGGAGCGGGCTATCGCCTACCCCGAGATTA methionine AAAACCTTATCGACAATATCCACAGAAGCGTGATAGAGGATACCCTGAGCAAGG andstop TCGCCCTGAACTGGAATGAGATCTTCAACACCCTGGCCGCCTACCAGAACGAG codon, AAAGATAAGAAAAAGAAGGCCGCTATCAAGAAGGACCTGGAGAAGTTGCAAGG includesV5- ATGTGCGAGAAAGAAAATCGTGGATACCTTCAAGAAGAACCCTGATTATGAGAA tagandC- ACTGTTTAAAGAGGGACTGTTCAAGGAGCTGCTGCCTGAACTGATCAAGACCGC terminalNLS) CCCTGTGAGCGAAATTGAAGATAAAACCAAAGCCCTGGAGTGCTTCAACCGGTT CTCCACATACTTCACCGGCTTCCACGAAAATCGCAAAAATATGTACAGCGAGGA CGCGAAGAGCACCGCCATCTCCTACCGGATCGTGAACGAGAACTTCCCCAAGT TCTTCGCTAATATCAAGCTGTACAACTACCTCAAGGAAAAATTTCCACAGATTAT CATCAACACAGAAGAGTCTCTGAAGGATTACCTGAAGGGCAAGAAGCTGGATTC CGTGTTCTCCATCGACGGGTTCAATGACGTGCTGGCCCAGAGCGGCATAGACT TCTACAACACCGTGATCGGTGGCATCTCAGGAGAGGCCGGCACAGAAAAGACC CAGGGCCTGAATGAGAAGATCAACCTAGCCAGACAGCAGCTGCCTAAGGATGA GAAGGACAAGCTAAGAGGCAAGATGGTCGACCTGTTCAAGCAGATTCTGAGCG ATAGAGAAACCAGCAGCTTCATCCCTACTGGCTTCGAGAATAAGAAGGAAGTGT ACTCTACCGTGAAGAAGTTCAGCGAAATCGTGGTCGAAAAAAGCGTGTCCAAGG TGAAGGAGATCTTCACTCAGAACGAAGAGTACAATCTGAACGAGATCTTCGTGC CTGCGAAGAGCCTGACCAATTTTAGCCAGAACATCTTTGGCAACTGGAGCATCC TTTCTGAAGGCCTGTTCCTGCTGGAAAAGGACAACGTGAAGAAACAGCTGAGTG AGAAACAAATCGAGACACTCCATAAGGAGATCGCCAAGAAGGACTGCAGCTTTA CCGAACTGCAGAACGCCTACGAGCGGTGGTGCGCCGAGAACTCCGTGGACGC CACCAAGAACATTAACAGATACTTCAGCATCGTCGACCTGAGAACCAAGAATGA CTCCTTCGAGAAGGAAGAGATCAATATCCTTGATGAGATAACCAACGCCTTCTCT AAGATTGACTTCGACGATATCCACGATCTGCAGCAAGAGAAGGAGGCCGCCAC CCCTATCAAGAACTACCTGGACGAGGTTCAAAACCTGTACCACCACCTGAAGCT GGTGGACTACAGAGGTGAGGAACGAAAGGACGCTAACTTCTACTCTAAACTGGA CTATATCCTGAGAAAGGACAGAAAGGACTACCTGAACCTGGCCGAAGTGGTGC CATTGTACAACAAGGTTAGAAACTTCGTGACCAAGAAGCCTGGCGAGGTGAAAA AGATCAAGATGATGTTCGACTGCAGCAGCCTGCTGGGCGGATGGGGCACAGAT TACGAGACAAAAGAGGCCCACATTTTCATCGACTCCGGCAAGTATTACCTTGGA ATCATCAACGAGAAGTTGTCAAAAGATGACGTGGAGCTGCTGAAGAAGAGCAGC GAACGGATGATCACAAAGGTGATCTACGATTTCCAAAAGCCCGATAACAAGAAT ACACCTAGACTGTTCATCAGGAGCAAGGGCACAAATTATGCTCCTGCTGTTTTC CAATACAATCTGCCAATAGAGTCTGTGATCGATATTTACGACCGTGGCCTGTTTA AGACCGAGTACAGAAAAATCAACAGCAAGGTGTACAAGGAGAGCCTGATTAAGA TGATCGATTACTTCAAGATGGGCTTTGAGAGACACGAGAGCTACAAGCACTACA AGTTTTGCTGGAAGGAATCTAGCAAGTACAACGACATCGGCGAATTTTACAAGG ATGTGATTAACTCTTGTTACCAGCTGAACTTCGAGAAGGTGAACTATGAGAACCT CCTGAAGTTAGTGGAAAACAACAAGCTGTTCCTGTTTCAGATCTACAACAAGGAT TTTGCCGAAAAGAAAAGCGGTAAGAAGAACCTGCACACCCTGTACTGGGAGAAC CTGTTTTCTGAGGAGAACCTGAAGGACGTTTGTCTGAAGCTGAATGGCGAGGCC GAGCTGTTCTGGCGGAAGGCTTCTCTGGACAAGGGCAAGGTGATCGTGCACAG AATGGGCTCTATCCTGGTGAACAGAACAACAAGCGAGGGCAAGTCAATCCCTGA GGACATCTACCAGGAGATCTATCAGTACAAGAACAAAATGAAGGATAAGATCAG CGACGAAGCCAAAAGCCTGCTGGACAGCGGCACCGTGATCTGTAAAGAAGCCA CCCACGACATCACCAAGGACAAACGGTTCACAGAGGACACCTACCTGTTCCACT GCCCTATCACCATGAACTTCAAGGCCACCGACAAGAAAAACAAAGAGTTCAACA ACCACGTGCTGGAAGTGCTGAAAGAGAATCCCGACGTGAAGATCATCGGCCTG GACAGAGGCGAACGGCACCTGATCTACCTGAGCCTGATCAACCAGAAGGGCGA GATCGAGCTGCAGAAAACCCTGAATCTGGTGGAACAGGTGCGGAACGACAAAA CCGTGAAGGTGGACTACCAGGAGAAGCTGGTGCATAAGGAAGGCGACCGCGA CAAAGCCAGAAAGAACTGGCAGACAATCGGAAACATCAAGGAACTGAAGGAGG GCTACCTGTCTGCCGTGGTGCACGAAATCGCCATGCTGATGGTGGAAAACAAC GCCATCGTGGTGATGGAGGACCTGAACTTCGGCTTCAAGAGAGGCAGATTCGC CGTGGAACGGCAGATCTACCAGAAGTTCGAGAACATGCTGATCGAAAAGCTGAA CTACCTAGTGTTCAAGGACAAGAACGCCACCGAACCTGGCGGCGTGCTGAATG CGTATCAGCTCACCAACAAGAGCGCCAACGTCACCGACGTGTACAAACAGTGC GGCTGGCTGTTCTACATCCCCGCCGCTTATACAAGCAAGATCGACCCCAAGACC GGATTCGCCAACCTGTTCATCACAAAGGGACTGACAAACGTGGAAAAGAAGAAG GAGTTCTTCGATAAGTTCGACAGCATCCGGTACGACAGCAAAGAGGACTGCTTT GTGTTCGGCTTCGACTACGCCAAGCTGTGCGACAACGCCTCCTTTAGAAAGAAG TGGGAAGTTTACACCAGAGGAGAGAGGCTGGTCTACAACAAAGACAAGCACAA AAACGAACCTATCAACCCCACCGAGGAGCTGAAGGGCATCTTCGATGCTTTTGA TATTAACTGGAACACCGACGACAACTTCATTGATTCAGTGCAGACCATCCAGGC CGAGAAGGCCAACGCCAAGTTCTTTGACATCCTGCTGAGAATGTTCAACGCCAC ACTGCAGATGAGAAACAGCAAGACTAACTCCTCTGCCAGCGAGGACGACTACCT GATCAGCCCTGTCAAAGCCGAGGATGGCACCTTCTTCGACACAAGAGAGGAATT AAAGAAGGGCAAAGATGCCAAGCTGCCGATCGACAGCGACGCTAATGGCGCCT ACCACATCGCCCTGAAAGGACTGTTCCTGCTGGAAAATGACTTTAACCGGGACG AGAAGGGAGTGATCCAAAATATCAGCAACGCTGATTGGTTCAAGTTTGTGCAGG AGAAGAAATACAAGGATtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGC CCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGG GCCTGGACAGCACCTGA

[0130] In some embodiments a ZSQQ Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 85, SEQ ID NO:86, or SEQ ID NO:87. In some embodiments, a ZSQQ Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO:87. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D913 substitution, wherein the position of the D913 substitution is defined with respect to the amino acid numbering of SEQ ID NO:86 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E1006 substitution, wherein the position of the E1006 substitution is defined with respect to the amino acid numbering of SEQ ID NO:86 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1219 substitution, wherein the position of the R1219 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 86 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1264 substitution, wherein the position of the D1264 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 86 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZSQQ Type V Cas protein is catalytically inactive, for example due to a R1219 substitution in combination with a D913 substitution, a E1006 substitution, and/or D1264 substitution.

6.2.16. ZSYN Type V Cas Protein

[0131] In one aspect, the disclosure provides ZSYN Type V Cas proteins. ZSYN Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZSYN Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:91. In some embodiments, the ZSYN Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:91. In some embodiments, a ZSYN Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:91.

[0132] Exemplary ZSYN Type V Cas protein sequences and nucleotide sequences encoding exemplary ZSYN Type V Cas proteins are set forth in Table 1P.

TABLE-US-00017 TABLE1P ZSYNTypeVCasSequences SEQ ID Name Sequence NO. Wildtype GKFFETDEFIGQYSINKTLRFELIPQGKTKELLNNYMNDNSKIKQDILRADEKNNFKEVI 91 aminoacid DEYYRELIHDALTDEDIFSITPLVKDAYELYIASRKNTSDSSKKEYRDVKNKIRKEIANILN sequence KYKTIYGLDKFANIYKSESDKSVDDDESDNDDLDEKNTTNDDNAKSEDKRIYKWLNKK (withoutN- LRLKQISNEEYDRYYKSLNEYHGFTTGLQGLQNNKENMFSSENKSTAIAFRIIDDNMEK terminal YFSNILLLEFIKNKYKDLYEKIEEKANKMNVECFTKYFTQEGIDEYNQMIGRSIEEEYAK methionine) GINQEINLYKQSKGLNNKEIRTLSPLYKQILSKTSQNEIIVFKNDKETLEYIKNICDYIIEED IFGKMNHLIKTNLIDMCTGIYIKRNELSNISFKLYNDWGLLDRIICDYANEFKTKKEKNEF EKLNKEVISLNLLNDIFNKYKETRGNDTDLKEIVEYFKNVDEKMIEDEYSKIKSILNLERID IDRRVPSKDEEKGGEGFEQICMIKTFLDLLLESIHIYKPLSLIKNGEKVEIYNYNENFYNE YDILFSQLDNIINLYNKVRNYFSKKTYSKEKIKIYFSKPTLLNGWDVNKEISNYSIILRKDE EYFLAIMNSDNKIFTNERLEENCAITENNEECYEKMVYKQISDSNKMFSKVFFSEKNKKI YMPSEEIKNIRKNKTHLKVANNKDSQTKWIKFMIECYYKHPEWSKYFDINFKKPEEYES IVEFYNQVNEKIYNIKFVNIKCDYINSMVDSGELYLFKIYNKDFSKNKKKSGTDNLHTMY WKLLFSKENMNCGVYKLNGQAEVFFRKASLPDKITHERNKEIDNKNPIKDKKTSTFTY DLKKDKRFMEDKFFFHCPITINYKGLNAKDKEIRKYNEKINKFIAGNPDINIIGIDRGERH LLYYTIINQKGEILKQSTLNNVGIEGRDKDYQELLSNKEKERHLARKSWGTIGNIKELKE GYLSIWVHELAKLVKEYNAIIVLENLNAGFKRGRTKVEKQVYQKFELALIKKLNYLVFKN ENIQNKGGYLKGLQLTQPFDTFKDIGNQSGIIYYVIPSYTSKICPTTGFIDVIKPQYESVE KAKELFSKFKRIYFDNNKKCFIFEFMYKDFGRDYGLDKIWSICTLGEKRYYYDSKNKVS NVINVTESIISILQEKNINYINSDNIIDEILQYSDVKLYKELLFNLKVVLQMRYTKSGTNNE DDFILSPVLDENDKAFCSLNAKETEPQNADANGAYHIAMKGLNAIMSIKNGNVDRDINN LENWINFIQKFHIGK Wildtype MGKFFETDEFIGQYSINKTLRFELIPQGKTKELLNNYMNDNSKIKQDILRADEKNNFKEV 92 aminoacid IDEYYRELIHDALTDEDIFSITPLVKDAYELYIASRKNTSDSSKKEYRDVKNKIRKEIANIL sequence(with NKYKTIYGLDKFANIYKSESDKSVDDDESDNDDLDEKNTTNDDNAKSEDKRIYKWLNK N-terminal KLRLKQISNEEYDRYYKSLNEYHGFTTGLQGLQNNKENMFSSENKSTAIAFRIIDDNME methionine) KYFSNILLLEFIKNKYKDLYEKIEEKANKMNVECFTKYFTQEGIDEYNQMIGRSIEEEYA KGINQEINLYKQSKGLNNKEIRTLSPLYKQILSKTSQNEIIVFKNDKETLEYIKNICDYIIEE DIFGKMNHLIKTNLIDMCTGIYIKRNELSNISFKLYNDWGLLDRIICDYANEFKTKKEKNE FEKLNKEVISLNLLNDIFNKYKETRGNDTDLKEIVEYFKNVDEKMIEDEYSKIKSILNLERI DIDRRVPSKDEEKGGEGFEQICMIKTFLDLLLESIHIYKPLSLIKNGEKVEIYNYNENFYN EYDILFSQLDNIINLYNKVRNYFSKKTYSKEKIKIYFSKPTLLNGWDVNKEISNYSIILRKD EEYFLAIMNSDNKIFTNERLEENCAITENNEECYEKMVYKQISDSNKMFSKVFFSEKNK KIYMPSEEIKNIRKNKTHLKVANNKDSQTKWIKFMIECYYKHPEWSKYFDINFKKPEEY ESIVEFYNQVNEKIYNIKFVNIKCDYINSMVDSGELYLFKIYNKDFSKNKKKSGTDNLHT MYWKLLFSKENMNCGVYKLNGQAEVFFRKASLPDKITHERNKEIDNKNPIKDKKTSTF TYDLKKDKRFMEDKFFFHCPITINYKGLNAKDKEIRKYNEKINKFIAGNPDINIIGIDRGE RHLLYYTIINQKGEILKQSTLNNVGIEGRDKDYQELLSNKEKERHLARKSWGTIGNIKEL KEGYLSIWVHELAKLVKEYNAIIVLENLNAGFKRGRTKVEKQVYQKFELALIKKLNYLVF KNENIQNKGGYLKGLQLTQPFDTFKDIGNQSGIIYYVIPSYTSKICPTTGFIDVIKPQYES VEKAKELFSKFKRIYFDNNKKCFIFEFMYKDFGRDYGLDKIWSICTLGEKRYYYDSKNK VSNVINVTESIISILQEKNINYINSDNIIDEILQYSDVKLYKELLFNLKVVLQMRYTKSGTN NEDDFILSPVLDENDKAFCSLNAKETEPQNADANGAYHIAMKGLNAIMSIKNGNVDRDI NNLENWINFIQKFHIGK Expression MGSGGKFFETDEFIGQYSINKTLRFELIPQGKTKELLNNYMNDNSKIKQDILRADEKNN 93 construct(with FKEVIDEYYRELIHDALTDEDIFSITPLVKDAYELYIASRKNTSDSSKKEYRDVKNKIRKEI N-terminal ANILNKYKTIYGLDKFANIYKSESDKSVDDDESDNDDLDEKNTTNDDNAKSEDKRIYKW methionine, LNKKLRLKQISNEEYDRYYKSLNEYHGFTTGLQGLQNNKENMFSSENKSTAIAFRIIDD V5-tagandC- NMEKYFSNILLLEFIKNKYKDLYEKIEEKANKMNVECFTKYFTQEGIDEYNQMIGRSIEE terminalNLS) EYAKGINQEINLYKQSKGLNNKEIRTLSPLYKQILSKTSQNEIIVFKNDKETLEYIKNICDY aasequence IIEEDIFGKMNHLIKTNLIDMCTGIYIKRNELSNISFKLYNDWGLLDRIICDYANEFKTKKE KNEFEKLNKEVISLNLLNDIFNKYKETRGNDTDLKEIVEYFKNVDEKMIEDEYSKIKSILN LERIDIDRRVPSKDEEKGGEGFEQICMIKTFLDLLLESIHIYKPLSLIKNGEKVEIYNYNEN FYNEYDILFSQLDNIINLYNKVRNYFSKKTYSKEKIKIYFSKPTLLNGWDVNKEISNYSIIL RKDEEYFLAIMNSDNKIFTNERLEENCAITENNEECYEKMVYKQISDSNKMFSKVFFSE KNKKIYMPSEEIKNIRKNKTHLKVANNKDSQTKWIKFMIECYYKHPEWSKYFDINFKKP EEYESIVEFYNQVNEKIYNIKFVNIKCDYINSMVDSGELYLFKIYNKDFSKNKKKSGTDN LHTMYWKLLFSKENMNCGVYKLNGQAEVFFRKASLPDKITHERNKEIDNKNPIKDKKT STFTYDLKKDKRFMEDKFFFHCPITINYKGLNAKDKEIRKYNEKINKFIAGNPDINIIGIDR GERHLLYYTIINQKGEILKQSTLNNVGIEGRDKDYQELLSNKEKERHLARKSWGTIGNIK ELKEGYLSIWVHELAKLVKEYNAIIVLENLNAGFKRGRTKVEKQVYQKFELALIKKLNYLV FKNENIQNKGGYLKGLQLTQPFDTFKDIGNQSGIIYYVIPSYTSKICPTTGFIDVIKPQYE SVEKAKELFSKFKRIYFDNNKKCFIFEFMYKDFGRDYGLDKIWSICTLGEKRYYYDSKN KVSNVINVTESIISILQEKNINYINSDNIIDEILQYSDVKLYKELLFNLKVVLQMRYTKSGTN NEDDFILSPVLDENDKAFCSLNAKETEPQNADANGAYHIAMKGLNAIMSIKNGNVDRDI NNLENWINFIQKFHIGKSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGGGTAAATTTTTTGAAACAGATGAATTTATTGGACAGTATTCAATAAATAAAACAT 94 coding TACGATTCGAATTGATACCACAAGGTAAGACAAAGGAATTACTAAATAATTATATGA sequence(with ATGATAACAGCAAAATTAAACAGGATATTTTAAGAGCAGATGAAAAGAATAATTTTA N-terminal AAGAAGTAATTGATGAATATTATCGAGAGTTGATTCATGATGCTTTAACAGATGAAG methionine ATATTTTTTCCATTACACCATTAGTAAAGGATGCATATGAATTATATATTGCTTCTAG andstop AAAAAATACTTCTGATAGTTCTAAAAAAGAATATAGAGATGTTAAAAATAAAATTAG codon) GAAAGAAATAGCAAACATTCTTAATAAATATAAGACGATTTATGGACTAGATAAATT TGCAAATATATATAAATCCGAGAGTGATAAAAGTGTAGATGATGATGAATCTGATAA TGATGATTTAGATGAGAAAAATACTACTAATGATGATAATGCAAAATCAGAAGATAA AAGGATATACAAATGGCTAAATAAAAAATTAAGATTAAAACAAATTTCTAACGAGGA ATATGATAGATACTACAAATCTTTAAATGAATATCATGGTTTTACAACAGGTCTGCA AGGATTACAAAATAATAAAGAAAATATGTTCTCTTCAGAAAACAAAAGTACGGCAAT AGCATTTCGAATAATAGATGACAATATGGAAAAATATTTTTCAAATATACTGTTATTA GAATTTATTAAAAACAAATATAAAGATTTATATGAAAAAATTGAAGAAAAAGCAAATA AAATGAATGTGGAATGTTTTACTAAATATTTTACACAAGAGGGTATAGATGAATATA ATCAAATGATAGGTAGAAGTATAGAAGAAGAATATGCAAAAGGTATAAATCAAGAA ATAAATCTTTATAAACAATCAAAAGGATTAAATAATAAAGAAATTAGGACATTATCTC CATTGTATAAGCAAATATTATCAAAGACTTCACAAAATGAAATAATAGTATTCAAAAA TGATAAAGAAACTTTAGAATACATCAAGAATATATGTGATTATATAATAGAAGAAGA TATATTTGGAAAGATGAATCATTTAATTAAAACAAATTTGATTGATATGTGTACTGGT ATATATATAAAAAGAAATGAATTATCGAATATTTCATTTAAACTTTATAATGATTGGG GATTACTAGATAGAATAATATGTGATTATGCAAATGAATTTAAGACAAAAAAAGAAA AGAACGAATTTGAAAAATTAAATAAAGAAGTAATTTCACTTAATCTTTTAAATGATAT ATTTAATAAATATAAGGAAACAAGAGGGAATGATACAGATTTAAAAGAAATAGTAGA ATATTTTAAAAATGTAGATGAAAAAATGATAGAGGATGAATACTCTAAAATAAAAAG TATTTTAAATTTAGAAAGAATAGATATTGATAGAAGAGTACCAAGCAAAGATGAAGA AAAAGGTGGAGAAGGATTTGAACAAATTTGTATGATAAAAACATTTTTAGATTTATT GCTTGAGAGTATACATATTTACAAACCATTAAGTTTAATTAAAAATGGAGAGAAAGT GGAGATATATAATTATAATGAAAATTTTTACAATGAATATGATATATTGTTTTCACAA TTAGATAATATAATTAACTTATATAATAAAGTCAGAAATTATTTTTCTAAAAAAACATA TTCAAAAGAAAAAATCAAGATATATTTTTCTAAGCCAACGTTATTAAATGGATGGGA TGTAAATAAAGAAATATCAAATTATTCGATTATTTTGAGAAAAGATGAAGAATATTTC CTAGCCATAATGAATAGTGATAATAAGATTTTTACTAATGAAAGATTGGAAGAAAAT TGCGCAATTACAGAAAATAATGAAGAGTGTTATGAAAAAATGGTATATAAACAAATA TCCGATTCAAATAAGATGTTTTCAAAAGTGTTTTTTTCAGAAAAAAACAAAAAAATAT ATATGCCTTCAGAAGAAATTAAAAATATTAGAAAAAATAAAACACATTTGAAAGTAG CAAATAATAAAGACTCACAAACAAAATGGATTAAATTTATGATTGAATGCTATTATAA ACATCCTGAATGGAGTAAATATTTTGATATAAATTTTAAAAAGCCTGAAGAATATGA ATCAATAGTTGAATTTTATAATCAAGTAAATGAAAAAATATATAATATAAAATTTGTA AATATTAAATGTGATTATATAAATAGTATGGTTGATAGTGGAGAATTGTATTTGTTTA AAATATATAATAAGGATTTTTCAAAAAATAAGAAAAAATCTGGAACAGATAATTTACA CACTATGTATTGGAAATTATTATTTTCAAAAGAAAATATGAATTGTGGTGTATACAAA TTAAATGGACAAGCAGAAGTGTTTTTTAGGAAAGCTTCTTTACCTGATAAAATTACA CATGAAAGAAATAAAGAAATAGATAATAAAAATCCAATAAAAGATAAAAAAACAAGT ACATTTACTTATGATTTAAAGAAAGATAAAAGATTCATGGAAGATAAATTCTTCTTTC ATTGCCCAATAACAATAAATTATAAAGGATTAAATGCAAAAGATAAAGAAATAAGAA AATATAATGAGAAAATAAACAAATTTATTGCTGGTAACCCAGATATAAATATTATCG GAATAGATCGTGGTGAACGACATTTGCTATATTATACGATAATAAATCAAAAGGGT GAAATATTAAAACAGTCAACATTAAATAATGTTGGTATTGAAGGGCGTGATAAAGAT TATCAAGAATTATTATCTAATAAAGAGAAAGAACGTCACTTAGCTAGAAAAAGTTGG GGAACAATAGGTAATATAAAAGAACTTAAAGAAGGATATTTATCAATTGTAGTACAT GAATTAGCTAAATTAGTAAAGGAATATAATGCAATAATTGTTCTAGAAAATTTGAAT GCTGGATTTAAAAGGGGAAGAACTAAAGTTGAAAAACAAGTATATCAAAAATTTGA ACTTGCATTGATAAAGAAACTTAATTATTTAGTATTTAAAAACGAAAATATTCAAAAT AAAGGTGGTTATTTAAAAGGATTACAATTAACTCAGCCATTTGATACTTTTAAAGAT ATTGGAAATCAATCTGGTATAATTTATTATGTTATTCCATCATATACATCGAAAATAT GTCCTACTACAGGCTTTATAGATGTAATTAAGCCACAATATGAAAGTGTTGAAAAAG CCAAAGAATTATTTTCTAAATTTAAGCGTATATATTTCGATAATAATAAAAAATGTTT TATATTTGAATTTATGTATAAAGACTTTGGTAGAGATTATGGTTTAGATAAAATATGG AGTATATGTACACTTGGAGAAAAAAGATATTATTATGATTCTAAAAATAAAGTATCAA ATGTAATAAATGTAACAGAATCAATAATTAGTATATTACAAGAAAAAAACATAAATTA TATAAATTCAGACAATATCATAGATGAAATTTTACAATATAGTGATGTTAAGTTGTAT AAAGAATTATTATTTAATTTAAAAGTTGTTTTACAAATGAGATATACGAAGAGTGGTA CAAATAATGAAGATGATTTTATTCTATCACCAGTATTAGATGAAAATGATAAGGCAT TTTGTTCACTTAATGCAAAAGAAACAGAACCTCAAAATGCAGATGCAAACGGTGCA TATCATATTGCTATGAAAGGTTTAAATGCAATAATGAGCATTAAGAATGGTAATGTA GATAGAGATATTAACAATTTAGAAAATTGGATAAATTTTATACAAAAGTTTCATATAG GTAAATAA Codon GGCAAGTTCTTTGAAACCGACGAGTTCATCGGACAGTACAGTATCAACAAAACACT 95 optimized GAGGTTCGAGCTCATCCCTCAAGGCAAGACCAAGGAACTGCTGAACAACTATATG coding AACGACAACAGTAAGATCAAGCAGGACATCCTGCGGGCCGACGAGAAGAACAATT sequence(no TCAAGGAAGTGATCGACGAGTATTATAGAGAGTTGATCCACGACGCCCTGACCGA N-terminal CGAGGACATCTTTTCCATCACCCCTCTCGTCAAGGACGCCTACGAGCTGTACATC methionine,no GCCTCCAGAAAAAACACCAGCGACTCCAGCAAGAAGGAGTATCGGGACGTGAAAA stopcodon) ATAAGATTAGAAAAGAGATCGCTAACATCCTGAACAAGTACAAGACAATCTACGGC CTGGACAAGTTCGCCAATATCTACAAGTCTGAGAGCGACAAGAGCGTTGATGATG ACGAATCTGATAACGATGACTTGGACGAGAAGAATACCACCAACGACGATAATGC CAAGTCTGAGGACAAGCGGATCTATAAGTGGCTGAATAAGAAGCTGAGACTGAAG CAGATCTCCAACGAAGAATACGACCGGTACTACAAGTCCCTGAACGAATACCACG GGTTCACAACAGGACTGCAGGGCCTGCAGAACAACAAGGAAAACATGTTCAGCAG CGAGAACAAGAGCACCGCCATCGCCTTTAGAATCATCGATGACAACATGGAAAAG TATTTTTCTAACATCCTGCTCCTGGAGTTCATCAAAAACAAGTACAAAGATCTGTAC GAGAAGATCGAGGAGAAGGCCAACAAGATGAACGTGGAATGCTTCACCAAGTACT TCACCCAGGAGGGCATCGACGAGTACAATCAGATGATTGGCAGAAGCATTGAGGA AGAATACGCCAAGGGCATCAACCAGGAGATCAACCTGTATAAGCAGAGCAAGGGT CTAAACAATAAGGAGATCAGAACACTGAGCCCCCTGTACAAGCAGATCCTGTCCA AGACCAGCCAGAACGAAATCATCGTGTTCAAAAACGACAAGGAAACCCTGGAATA CATCAAGAATATCTGTGATTACATTATCGAGGAGGACATCTTCGGAAAGATGAACC ACCTGATCAAAACCAACCTGATCGACATGTGCACCGGAATCTACATTAAGAGAAAC GAGCTGAGCAACATCTCTTTCAAGCTCTACAACGACTGGGGCCTGCTGGACAGAA TTATCTGTGACTACGCCAACGAGTTCAAGACAAAGAAGGAAAAGAATGAGTTCGAG AAGCTGAACAAAGAGGTGATCTCTCTGAACCTGCTCAACGATATTTTCAACAAATA CAAGGAAACCAGAGGCAATGATACAGACCTGAAGGAAATCGTGGAATACTTTAAAA ACGTCGACGAGAAAATGATTGAGGACGAGTACAGCAAGATCAAGAGCATACTTAA TCTGGAACGCATCGACATCGACCGTAGAGTGCCAAGCAAGGACGAGGAAAAGGG CGGCGAAGGCTTTGAGCAGATCTGCATGATCAAGACGTTCCTGGATCTGCTGTTG GAGAGCATCCACATCTACAAGCCTCTGTCTCTGATCAAGAACGGCGAGAAGGTGG AAATCTACAATTATAACGAGAACTTCTACAACGAGTACGACATCCTGTTCAGCCAG CTGGATAACATTATAAATCTGTACAATAAGGTGCGGAACTACTTCAGCAAGAAAAC CTACAGCAAAGAGAAAATCAAAATCTATTTCTCCAAACCCACCCTGCTGAACGGAT GGGACGTGAACAAGGAGATCAGCAACTACTCTATCATCCTGAGAAAAGACGAAGA GTACTTTCTGGCAATTATGAACAGCGACAACAAGATCTTCACGAATGAGAGGCTGG AAGAAAACTGCGCCATCACCGAGAATAATGAAGAATGTTACGAGAAAATGGTGTAC AAGCAAATCTCTGACTCTAACAAGATGTTCAGCAAGGTGTTTTTCAGCGAGAAAAA CAAGAAGATCTACATGCCCAGCGAAGAGATCAAGAATATCAGAAAGAACAAGACC CATCTCAAGGTGGCCAACAATAAGGATTCTCAAACAAAGTGGATCAAGTTCATGAT CGAGTGCTACTATAAACACCCTGAGTGGAGTAAGTACTTCGATATCAACTTCAAGA AACCTGAAGAATATGAAAGCATCGTGGAATTTTACAACCAGGTGAACGAGAAGATC TACAACATCAAGTTCGTGAATATCAAATGCGACTACATCAACAGCATGGTGGATTC GGGAGAGCTGTACCTGTTCAAGATCTACAACAAGGACTTCTCTAAGAACAAGAAAA AAAGTGGCACAGATAACCTGCACACCATGTATTGGAAGCTGCTGTTTAGCAAAGAA AACATGAATTGCGGCGTGTACAAGCTGAACGGCCAGGCCGAGGTGTTCTTCAGAA AGGCCAGCCTGCCTGATAAGATCACACACGAAAGAAATAAGGAGATCGACAACAA AAATCCTATCAAGGACAAGAAAACCAGCACCTTCACATACGACCTGAAGAAAGATA AGCGGTTCATGGAAGATAAGTTCTTCTTCCACTGCCCCATAACCATCAACTACAAG GGCCTTAACGCCAAGGACAAGGAGATCAGAAAGTACAACGAAAAGATCAACAAAT TCATCGCTGGCAACCCCGACATCAACATCATAGGCATCGACCGGGGCGAACGGC ACCTGCTGTACTACACCATCATCAACCAGAAGGGAGAGATCCTGAAGCAATCTACA CTGAACAACGTGGGCATCGAGGGCAGAGACAAAGATTACCAGGAGCTGCTGAGC AACAAGGAAAAGGAAAGACACCTCGCTAGAAAGAGCTGGGGCACCATCGGCAAC ATAAAAGAACTGAAGGAAGGCTACCTGAGCATCGTGGTGCACGAGCTGGCCAAGC TCGTGAAGGAGTACAACGCCATCATCGTGCTGGAGAATCTGAACGCCGGCTTCAA GAGAGGCAGAACCAAGGTGGAAAAACAGGTCTACCAGAAGTTTGAGCTGGCCCT GATCAAGAAGCTGAACTACCTCGTGTTCAAGAACGAGAACATCCAGAACAAGGGA GGCTACCTGAAGGGACTGCAACTGACACAGCCTTTCGACACCTTTAAGGATATCG GCAACCAGAGCGGCATCATCTACTACGTGATCCCCAGCTACACAAGCAAAATTTGT CCAACAACCGGCTTCATCGACGTGATCAAACCTCAGTACGAGTCTGTGGAAAAGG CCAAGGAGCTGTTCTCCAAATTCAAACGGATTTACTTCGACAACAACAAGAAGTGC TTTATCTTCGAATTTATGTACAAAGATTTCGGCAGAGATTACGGTCTGGACAAGATC TGGAGCATCTGTACCCTGGGCGAGAAGAGATACTACTACGACAGCAAGAACAAGG TTTCCAATGTGATCAACGTGACCGAGAGCATCATCAGCATCCTGCAGGAGAAGAA CATCAACTACATCAACAGCGACAACATCATCGACGAGATCCTGCAGTACAGCGAC GTGAAGCTGTATAAGGAGCTGCTTTTTAACCTGAAGGTGGTGCTGCAGATGCGGT ACACCAAGAGCGGCACCAATAACGAGGACGACTTCATTCTGTCTCCTGTGCTGGA CGAGAACGACAAGGCCTTCTGCAGCCTGAACGCTAAGGAAACAGAGCCTCAGAAT GCTGATGCTAATGGCGCCTATCATATCGCCATGAAGGGACTGAACGCCATCATGT CCATCAAGAACGGCAACGTGGATAGAGATATTAACAACCTGGAAAACTGGATCAA CTTCATCCAGAAATTCCACATCGGGAAG Expression ATGggctccggaGGCAAGTTCTTTGAAACCGACGAGTTCATCGGACAGTACAGTATCA 96 construct(with ACAAAACACTGAGGTTCGAGCTCATCCCTCAAGGCAAGACCAAGGAACTGCTGAA N-terminal CAACTATATGAACGACAACAGTAAGATCAAGCAGGACATCCTGCGGGCCGACGAG methionine AAGAACAATTTCAAGGAAGTGATCGACGAGTATTATAGAGAGTTGATCCACGACGC andstop CCTGACCGACGAGGACATCTTTTCCATCACCCCTCTCGTCAAGGACGCCTACGAG codon, CTGTACATCGCCTCCAGAAAAAACACCAGCGACTCCAGCAAGAAGGAGTATCGGG includesV5- ACGTGAAAAATAAGATTAGAAAAGAGATCGCTAACATCCTGAACAAGTACAAGACA tagandC- ATCTACGGCCTGGACAAGTTCGCCAATATCTACAAGTCTGAGAGCGACAAGAGCG terminalNLS) TTGATGATGACGAATCTGATAACGATGACTTGGACGAGAAGAATACCACCAACGAC GATAATGCCAAGTCTGAGGACAAGCGGATCTATAAGTGGCTGAATAAGAAGCTGA GACTGAAGCAGATCTCCAACGAAGAATACGACCGGTACTACAAGTCCCTGAACGA ATACCACGGGTTCACAACAGGACTGCAGGGCCTGCAGAACAACAAGGAAAACATG TTCAGCAGCGAGAACAAGAGCACCGCCATCGCCTTTAGAATCATCGATGACAACA TGGAAAAGTATTTTTCTAACATCCTGCTCCTGGAGTTCATCAAAAACAAGTACAAAG ATCTGTACGAGAAGATCGAGGAGAAGGCCAACAAGATGAACGTGGAATGCTTCAC CAAGTACTTCACCCAGGAGGGCATCGACGAGTACAATCAGATGATTGGCAGAAGC ATTGAGGAAGAATACGCCAAGGGCATCAACCAGGAGATCAACCTGTATAAGCAGA GCAAGGGTCTAAACAATAAGGAGATCAGAACACTGAGCCCCCTGTACAAGCAGAT CCTGTCCAAGACCAGCCAGAACGAAATCATCGTGTTCAAAAACGACAAGGAAACC CTGGAATACATCAAGAATATCTGTGATTACATTATCGAGGAGGACATCTTCGGAAA GATGAACCACCTGATCAAAACCAACCTGATCGACATGTGCACCGGAATCTACATTA AGAGAAACGAGCTGAGCAACATCTCTTTCAAGCTCTACAACGACTGGGGCCTGCT GGACAGAATTATCTGTGACTACGCCAACGAGTTCAAGACAAAGAAGGAAAAGAAT GAGTTCGAGAAGCTGAACAAAGAGGTGATCTCTCTGAACCTGCTCAACGATATTTT CAACAAATACAAGGAAACCAGAGGCAATGATACAGACCTGAAGGAAATCGTGGAA TACTTTAAAAACGTCGACGAGAAAATGATTGAGGACGAGTACAGCAAGATCAAGAG CATACTTAATCTGGAACGCATCGACATCGACCGTAGAGTGCCAAGCAAGGACGAG GAAAAGGGCGGCGAAGGCTTTGAGCAGATCTGCATGATCAAGACGTTCCTGGATC TGCTGTTGGAGAGCATCCACATCTACAAGCCTCTGTCTCTGATCAAGAACGGCGA GAAGGTGGAAATCTACAATTATAACGAGAACTTCTACAACGAGTACGACATCCTGT TCAGCCAGCTGGATAACATTATAAATCTGTACAATAAGGTGCGGAACTACTTCAGC AAGAAAACCTACAGCAAAGAGAAAATCAAAATCTATTTCTCCAAACCCACCCTGCT GAACGGATGGGACGTGAACAAGGAGATCAGCAACTACTCTATCATCCTGAGAAAA GACGAAGAGTACTTTCTGGCAATTATGAACAGCGACAACAAGATCTTCACGAATGA GAGGCTGGAAGAAAACTGCGCCATCACCGAGAATAATGAAGAATGTTACGAGAAA ATGGTGTACAAGCAAATCTCTGACTCTAACAAGATGTTCAGCAAGGTGTTTTTCAG CGAGAAAAACAAGAAGATCTACATGCCCAGCGAAGAGATCAAGAATATCAGAAAG AACAAGACCCATCTCAAGGTGGCCAACAATAAGGATTCTCAAACAAAGTGGATCAA GTTCATGATCGAGTGCTACTATAAACACCCTGAGTGGAGTAAGTACTTCGATATCA ACTTCAAGAAACCTGAAGAATATGAAAGCATCGTGGAATTTTACAACCAGGTGAAC GAGAAGATCTACAACATCAAGTTCGTGAATATCAAATGCGACTACATCAACAGCAT GGTGGATTCGGGAGAGCTGTACCTGTTCAAGATCTACAACAAGGACTTCTCTAAG AACAAGAAAAAAAGTGGCACAGATAACCTGCACACCATGTATTGGAAGCTGCTGTT TAGCAAAGAAAACATGAATTGCGGCGTGTACAAGCTGAACGGCCAGGCCGAGGT GTTCTTCAGAAAGGCCAGCCTGCCTGATAAGATCACACACGAAAGAAATAAGGAG ATCGACAACAAAAATCCTATCAAGGACAAGAAAACCAGCACCTTCACATACGACCT GAAGAAAGATAAGCGGTTCATGGAAGATAAGTTCTTCTTCCACTGCCCCATAACCA TCAACTACAAGGGCCTTAACGCCAAGGACAAGGAGATCAGAAAGTACAACGAAAA GATCAACAAATTCATCGCTGGCAACCCCGACATCAACATCATAGGCATCGACCGG GGCGAACGGCACCTGCTGTACTACACCATCATCAACCAGAAGGGAGAGATCCTGA AGCAATCTACACTGAACAACGTGGGCATCGAGGGCAGAGACAAAGATTACCAGGA GCTGCTGAGCAACAAGGAAAAGGAAAGACACCTCGCTAGAAAGAGCTGGGGCAC CATCGGCAACATAAAAGAACTGAAGGAAGGCTACCTGAGCATCGTGGTGCACGAG CTGGCCAAGCTCGTGAAGGAGTACAACGCCATCATCGTGCTGGAGAATCTGAACG CCGGCTTCAAGAGAGGCAGAACCAAGGTGGAAAAACAGGTCTACCAGAAGTTTGA GCTGGCCCTGATCAAGAAGCTGAACTACCTCGTGTTCAAGAACGAGAACATCCAG AACAAGGGAGGCTACCTGAAGGGACTGCAACTGACACAGCCTTTCGACACCTTTA AGGATATCGGCAACCAGAGCGGCATCATCTACTACGTGATCCCCAGCTACACAAG CAAAATTTGTCCAACAACCGGCTTCATCGACGTGATCAAACCTCAGTACGAGTCTG TGGAAAAGGCCAAGGAGCTGTTCTCCAAATTCAAACGGATTTACTTCGACAACAAC AAGAAGTGCTTTATCTTCGAATTTATGTACAAAGATTTCGGCAGAGATTACGGTCT GGACAAGATCTGGAGCATCTGTACCCTGGGCGAGAAGAGATACTACTACGACAGC AAGAACAAGGTTTCCAATGTGATCAACGTGACCGAGAGCATCATCAGCATCCTGC AGGAGAAGAACATCAACTACATCAACAGCGACAACATCATCGACGAGATCCTGCA GTACAGCGACGTGAAGCTGTATAAGGAGCTGCTTTTTAACCTGAAGGTGGTGCTG CAGATGCGGTACACCAAGAGCGGCACCAATAACGAGGACGACTTCATTCTGTCTC CTGTGCTGGACGAGAACGACAAGGCCTTCTGCAGCCTGAACGCTAAGGAAACAGA GCCTCAGAATGCTGATGCTAATGGCGCCTATCATATCGCCATGAAGGGACTGAAC GCCATCATGTCCATCAAGAACGGCAACGTGGATAGAGATATTAACAACCTGGAAAA CTGGATCAACTTCATCCAGAAATTCCACATCGGGAAGtctagaAAGCGGACAGCAGA CGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTAT CCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0133] In some embodiments a ZSYN Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 91, SEQ ID NO:92, or SEQ ID NO:93. In some embodiments, a ZSYN Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:91, SEQ ID NO: 92, or SEQ ID NO:93. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D902 substitution, wherein the position of the D902 substitution is defined with respect to the amino acid numbering of SEQ ID NO:92 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E991 substitution, wherein the position of the E991 substitution is defined with respect to the amino acid numbering of SEQ ID NO:92 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1200 substitution, wherein the position of the R1200 substitution is defined with respect to the amino acid numbering of SEQ ID NO:92 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1239 substitution, wherein the position of the D1239 substitution is defined with respect to the amino acid numbering of SEQ ID NO:92 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZSYN Type V Cas protein is catalytically inactive, for example due to a R1200 substitution in combination with a D902 substitution, a E991 substitution, and/or D1239 substitution.

6.2.17. ZRBH Type V Cas Protein

[0134] In one aspect, the disclosure provides ZRBH Type V Cas proteins. ZRBH Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZRBH Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:97. In some embodiments, the ZRBH Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:97. In some embodiments, a ZRBH Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:97.

[0135] Exemplary ZRBH Type V Cas protein sequences and nucleotide sequences encoding exemplary ZRBH Type V Cas proteins are set forth in Table 1Q.

TABLE-US-00018 TABLE1Q ZRBHTypeVCasSequences SEQ ID Name Sequence NO. Wildtype EFDNSFVNRYPLSKTLSFSLLPVGSTEANFEKKLLLQEDEKRAAEYILVKSYIDRYHKAY 97 aminoacid IESVLSKVVLDGINNYAQLYCKNNKTEQDIKRLEQLEGSFRKQISKSLKSDARYKLIYKK sequence EMLEKLLPEFLDNEEEKARVISFENFTTYFTGFHTNRENMYTDEAKSTAVSFRCINDNL (withoutN- PKFLDNISVFKWVTAFLSESDINELKADFSGLLGCSLEEMFTPDYFSFVLSQSGIERYN terminal NVIGGYTCSDGEKVKGLNEYINLYNQKLQHGEKKLPLLKRLFKQILSDTESVSFIPEKLE methionine) NDDAVISAINGFCNIKIENETFFEILDKTKCLFSNLNEFDSAGVYITNGFAVTDISNAVFG TWDVISEAWKKEYAKAIPLKNIAKADAYYEKQGKAYKAIKSFSVSELQRLANTTEGKAA YKHNGDISAYFSETVCFAVQDIFEKYSSSKALFASPYKNEKRLFKNNEAIALIKDFLDSIK NLEKLIKPFNGSGRENDKDESFYGEFTACYERLSKIDLLYDKVRNYMTQKPYSGDKIKL NFENPQFLNGWDRNKERDYRTVLLRKGGYYYLAIMDKSNNRIFEDLPEPKNGEDCYE KIDYKLLPGPNKMLPKVFFAASNIDYFAPSEQILKIRQKETFKKGVNFNIDDCHAFIDFLK ESIEKHDEWCKYGFEFKDTSDYNNIGEFYKDVREQGYSISFRNVPESYINSCVNSGSL YLFQIYNKDFSPYSKGTKSLHTLYFEMLFDERNLKNVVYQLNGGAEMFYRKASIKERD KIVHPANIPIKNKNPDNPKAESVFEYDIIKDRRFTERQFSLHIPVTLNFKGSGGSANLNA DVRRAIRGADENYVIGIDRGERNLLYITVINSKGEIVEQIPGNVIINGKQVVDYHKLLDAK EKERLAARQNWTTVENIKELKEGYLSVIIHNICELVKKYNAVIAMEDLSSGFKNSRVKVE KQVYQKFEKMLTEKLNFLVDKKADVQSRGGLLQAYQLTNSTKDYKRAGSQDGIVFYV PAWLTSKIDPVTGFVDLLKPKYTSVQEAKELFSNFEAVEYIPEEDLFSFTFDYSKFPRC SVAYRNKWTVYSNGERIYTFRDKNSNNEYVSKTVALTTEFKSLFDEYSVYYRDNLKSQ ILCQDKVDFFKQLIRLLSLTMQMRNSISNSAVDYLISPVKDKNGNFFDSRKSIKNLPENA DANGAYNIAKKALWAIGQIKEADENDLMKVKLSVSNKEWLKYVQEVE Wildtype MEFDNSFVNRYPLSKTLSFSLLPVGSTEANFEKKLLLQEDEKRAAEYILVKSYIDRYHK 98 aminoacid AYIESVLSKVVLDGINNYAQLYCKNNKTEQDIKRLEQLEGSFRKQISKSLKSDARYKLIY sequence(with KKEMLEKLLPEFLDNEEEKARVISFENFTTYFTGFHTNRENMYTDEAKSTAVSFRCIND N-terminal NLPKFLDNISVFKWVTAFLSESDINELKADFSGLLGCSLEEMFTPDYFSFVLSQSGIER methionine) YNNVIGGYTCSDGEKVKGLNEYINLYNQKLQHGEKKLPLLKRLFKQILSDTESVSFIPEK LENDDAVISAINGFCNIKIENETFFEILDKTKCLFSNLNEFDSAGVYITNGFAVTDISNAVF GTWDVISEAWKKEYAKAIPLKNIAKADAYYEKQGKAYKAIKSFSVSELQRLANTTEGKA AYKHNGDISAYFSETVCFAVQDIFEKYSSSKALFASPYKNEKRLFKNNEAIALIKDFLDSI KNLEKLIKPFNGSGRENDKDESFYGEFTACYERLSKIDLLYDKVRNYMTQKPYSGDKIK LNFENPQFLNGWDRNKERDYRTVLLRKGGYYYLAIMDKSNNRIFEDLPEPKNGEDCY EKIDYKLLPGPNKMLPKVFFAASNIDYFAPSEQILKIRQKETFKKGVNFNIDDCHAFIDFL KESIEKHDEWCKYGFEFKDTSDYNNIGEFYKDVREQGYSISFRNVPESYINSCVNSGS LYLFQIYNKDFSPYSKGTKSLHTLYFEMLFDERNLKNVVYQLNGGAEMFYRKASIKER DKIVHPANIPIKNKNPDNPKAESVFEYDIIKDRRFTERQFSLHIPVTLNFKGSGGSANLN ADVRRAIRGADENYVIGIDRGERNLLYITVINSKGEIVEQIPGNVIINGKQVVDYHKLLDA KEKERLAARQNWTTVENIKELKEGYLSVIIHNICELVKKYNAVIAMEDLSSGFKNSRVKV EKQVYQKFEKMLTEKLNFLVDKKADVQSRGGLLQAYQLTNSTKDYKRAGSQDGIVFY VPAWLTSKIDPVTGFVDLLKPKYTSVQEAKELFSNFEAVEYIPEEDLFSFTFDYSKFPR CSVAYRNKWTVYSNGERIYTFRDKNSNNEYVSKTVALTTEFKSLFDEYSVYYRDNLKS QILCQDKVDFFKQLIRLLSLTMQMRNSISNSAVDYLISPVKDKNGNFFDSRKSIKNLPEN ADANGAYNIAKKALWAIGQIKEADENDLMKVKLSVSNKEWLKYVQEVE Expression MGSGEFDNSFVNRYPLSKTLSFSLLPVGSTEANFEKKLLLQEDEKRAAEYILVKSYIDR 99 construct(with YHKAYIESVLSKVVLDGINNYAQLYCKNNKTEQDIKRLEQLEGSFRKQISKSLKSDARY N-terminal KLIYKKEMLEKLLPEFLDNEEEKARVISFENFTTYFTGFHTNRENMYTDEAKSTAVSFR methionine, CINDNLPKFLDNISVFKWVTAFLSESDINELKADFSGLLGCSLEEMFTPDYFSFVLSQS V5-tagandC- GIERYNNVIGGYTCSDGEKVKGLNEYINLYNQKLQHGEKKLPLLKRLFKQILSDTESVS terminalNLS) FIPEKLENDDAVISAINGFCNIKIENETFFEILDKTKCLFSNLNEFDSAGVYITNGFAVTDI aasequence SNAVFGTWDVISEAWKKEYAKAIPLKNIAKADAYYEKQGKAYKAIKSFSVSELQRLANT TEGKAAYKHNGDISAYFSETVCFAVQDIFEKYSSSKALFASPYKNEKRLFKNNEAIALIK DFLDSIKNLEKLIKPFNGSGRENDKDESFYGEFTACYERLSKIDLLYDKVRNYMTQKPY SGDKIKLNFENPQFLNGWDRNKERDYRTVLLRKGGYYYLAIMDKSNNRIFEDLPEPKN GEDCYEKIDYKLLPGPNKMLPKVFFAASNIDYFAPSEQILKIRQKETFKKGVNFNIDDCH AFIDFLKESIEKHDEWCKYGFEFKDTSDYNNIGEFYKDVREQGYSISFRNVPESYINSC VNSGSLYLFQIYNKDFSPYSKGTKSLHTLYFEMLFDERNLKNVVYQLNGGAEMFYRKA SIKERDKIVHPANIPIKNKNPDNPKAESVFEYDIIKDRRFTERQFSLHIPVTLNFKGSGGS ANLNADVRRAIRGADENYVIGIDRGERNLLYITVINSKGEIVEQIPGNVIINGKQVVDYHK LLDAKEKERLAARQNWTTVENIKELKEGYLSVIIHNICELVKKYNAVIAMEDLSSGFKNS RVKVEKQVYQKFEKMLTEKLNFLVDKKADVQSRGGLLQAYQLTNSTKDYKRAGSQD GIVFYVPAWLTSKIDPVTGFVDLLKPKYTSVQEAKELFSNFEAVEYIPEEDLFSFTFDYS KFPRCSVAYRNKWTVYSNGERIYTFRDKNSNNEYVSKTVALTTEFKSLFDEYSVYYRD NLKSQILCQDKVDFFKQLIRLLSLTMQMRNSISNSAVDYLISPVKDKNGNFFDSRKSIKN LPENADANGAYNIAKKALWAIGQIKEADENDLMKVKLSVSNKEWLKYVQEVESRKRTA DGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGGAATTCGACAATAGCTTTGTTAACCGATACCCTTTATCAAAAACACTAAGCTTC 100 coding AGTTTGCTTCCTGTTGGCAGTACCGAAGCAAATTTTGAGAAAAAACTGTTGCTGCA sequence(with GGAGGACGAAAAAAGAGCCGCGGAATATATTTTGGTGAAGTCATACATTGACAGA N-terminal TACCATAAAGCCTATATTGAATCGGTTTTATCAAAGGTTGTGCTTGACGGCATAAAT methionine AACTATGCACAGCTGTACTGCAAGAACAACAAAACCGAACAGGATATCAAACGACT andstop GGAGCAGCTTGAAGGTTCATTTAGAAAGCAGATTTCAAAGAGCTTGAAATCCGATG codon) CCCGTTATAAGTTGATTTATAAAAAAGAAATGCTTGAAAAGCTTTTGCCTGAGTTTC TTGATAATGAAGAAGAAAAGGCGAGGGTAATATCTTTTGAAAACTTTACAACATATT TCACAGGCTTTCATACCAATAGAGAAAATATGTATACCGACGAAGCAAAATCCACT GCGGTGTCCTTCAGATGTATAAATGATAATTTACCAAAATTTCTTGATAATATTTCA GTTTTTAAATGGGTTACGGCATTTTTGAGCGAAAGTGATATCAACGAATTAAAGGC GGATTTTTCAGGTCTGTTAGGTTGTTCGCTTGAAGAAATGTTTACACCGGATTATTT TTCCTTTGTGTTATCTCAAAGCGGGATAGAAAGATATAACAATGTTATCGGCGGTT ACACATGTTCTGACGGTGAAAAAGTTAAGGGACTAAATGAATACATAAATTTATACA ACCAAAAGTTACAACACGGTGAAAAAAAGCTCCCGCTTTTAAAACGCTTGTTCAAG CAGATATTGAGTGATACCGAAAGTGTATCCTTTATTCCGGAAAAGCTTGAAAACGA CGATGCTGTTATTTCTGCGATAAACGGATTTTGTAATATCAAAATTGAAAACGAAAC ATTCTTTGAAATTCTTGATAAAACTAAATGCTTGTTTTCAAATTTAAATGAGTTTGAC AGCGCCGGTGTATATATTACCAACGGTTTTGCTGTAACCGATATTTCAAATGCTGT TTTCGGTACTTGGGATGTTATTTCGGAAGCGTGGAAAAAGGAGTATGCGAAAGCA ATCCCGCTTAAAAATATCGCCAAGGCAGATGCATATTACGAAAAGCAGGGCAAGG CGTATAAGGCAATTAAAAGCTTTTCGGTAAGCGAGCTTCAAAGGCTGGCCAACACA ACAGAAGGGAAGGCGGCATATAAGCACAACGGAGATATTTCTGCATATTTTTCGGA AACTGTTTGCTTTGCGGTTCAAGATATATTTGAAAAATACAGTAGTTCAAAAGCCCT TTTTGCGTCGCCCTATAAAAATGAAAAGCGGCTCTTCAAAAACAATGAGGCTATAG CGCTGATTAAGGATTTTCTTGACAGCATCAAAAATCTGGAAAAGCTTATTAAACCAT TTAACGGCTCCGGTAGAGAAAACGATAAGGACGAAAGCTTCTACGGTGAATTTAC CGCTTGCTACGAGAGGCTTTCTAAAATTGACCTGCTATATGATAAGGTTCGCAACT ATATGACACAAAAACCTTATTCCGGGGACAAGATAAAGTTGAATTTTGAAAATCCG CAATTTCTAAATGGTTGGGACAGGAACAAAGAGCGGGATTACAGAACTGTTCTCTT AAGAAAAGGCGGGTATTACTACCTTGCTATTATGGATAAAAGCAACAACAGGATTT TTGAAGATTTGCCGGAGCCCAAAAACGGCGAGGATTGTTATGAAAAAATAGACTAC AAGCTTCTGCCGGGACCGAATAAGATGTTGCCAAAGGTGTTTTTTGCCGCGAGCA ATATTGATTATTTTGCACCCTCTGAGCAAATTTTGAAAATTAGACAGAAAGAAACCT TTAAGAAGGGTGTGAATTTTAATATTGATGATTGCCATGCTTTCATAGACTTCCTTA AAGAGTCTATAGAAAAACACGATGAGTGGTGCAAGTATGGGTTCGAATTTAAAGAT ACTTCAGATTATAACAACATCGGTGAATTTTATAAAGATGTAAGGGAGCAGGGCTA TTCTATCAGCTTTAGAAATGTGCCTGAGTCTTATATAAATTCTTGCGTTAATTCCGG TTCACTTTACCTTTTCCAAATCTACAACAAGGATTTTTCACCTTACAGCAAAGGGAC CAAGAGTTTGCACACATTGTATTTTGAAATGCTTTTTGATGAAAGGAACCTTAAGAA TGTTGTTTATCAGCTTAACGGCGGTGCAGAGATGTTTTACCGCAAAGCAAGTATTA AGGAAAGGGATAAAATAGTACACCCTGCTAATATTCCGATAAAAAATAAAAATCCC GATAACCCAAAAGCTGAAAGTGTTTTTGAGTATGACATCATAAAGGACAGACGCTT TACTGAAAGACAGTTCTCTTTGCATATTCCTGTTACGCTCAATTTTAAAGGCTCGGG CGGCTCTGCAAATCTTAATGCTGATGTGCGCAGAGCCATAAGAGGCGCTGATGAA AACTATGTTATAGGTATAGACAGAGGAGAAAGAAATTTGCTTTACATCACCGTAATA AACAGTAAAGGTGAAATTGTTGAGCAGATTCCGGGCAATGTAATAATCAACGGAAA ACAAGTGGTCGATTATCACAAGCTGCTTGATGCCAAAGAAAAAGAGCGTCTTGCA GCACGGCAAAACTGGACAACGGTTGAAAATATCAAGGAGCTTAAAGAGGGCTATT TGAGCGTAATCATACACAATATTTGTGAACTTGTAAAAAAATACAATGCTGTTATTG CTATGGAGGATCTTTCTTCCGGTTTTAAAAACAGCAGGGTTAAAGTAGAAAAACAG GTTTATCAGAAATTTGAAAAAATGCTTACCGAAAAGCTTAATTTTCTTGTTGATAAAA AAGCTGATGTTCAAAGCAGGGGAGGACTTCTGCAGGCATATCAGTTAACAAACAG CACCAAGGATTATAAGCGGGCAGGCTCACAAGACGGTATTGTTTTCTATGTTCCG GCGTGGCTTACAAGCAAAATCGATCCCGTTACGGGTTTTGTTGATTTGCTTAAGCC TAAGTATACAAGTGTGCAGGAAGCAAAGGAGCTGTTTTCAAATTTTGAAGCTGTTG AATATATCCCTGAGGAGGATTTGTTCAGCTTTACTTTTGATTATTCGAAATTTCCCC GTTGCTCCGTAGCTTACCGTAACAAATGGACTGTATACTCAAACGGCGAAAGAATT TATACATTCAGGGATAAAAACAGCAATAATGAATATGTTAGCAAAACAGTTGCTCTT ACAACGGAGTTTAAATCCTTGTTTGACGAATACAGCGTTTATTACCGCGATAACCTT AAATCGCAGATTCTATGTCAAGATAAAGTCGATTTCTTCAAACAGCTAATTCGGTTA CTGTCTTTGACAATGCAAATGCGAAACAGTATTTCAAATTCAGCAGTAGATTATCTG ATTTCTCCGGTTAAGGATAAAAACGGAAATTTCTTTGACAGCCGGAAAAGTATAAA AAATCTTCCGGAAAATGCAGATGCTAACGGTGCTTACAACATTGCCAAAAAGGCTC TTTGGGCAATCGGGCAAATAAAGGAAGCGGATGAGAATGATTTAATGAAGGTCAA GCTGTCTGTTTCAAACAAGGAATGGCTTAAATATGTGCAGGAGGTAGAATGA Codon GAATTTGATAACTCTTTCGTGAATAGATATCCTCTGAGCAAGACCCTGAGCTTCAG 101 optimized TCTGCTGCCAGTGGGCAGCACCGAAGCCAACTTCGAAAAAAAGCTGCTGCTGCAG coding GAGGACGAAAAGAGAGCCGCCGAGTACATCCTGGTGAAAAGCTACATCGACAGAT sequence(no ACCACAAGGCCTACATCGAGAGCGTGCTGAGCAAGGTGGTGCTGGACGGCATCA N-terminal ACAACTATGCCCAGCTGTACTGCAAGAACAACAAGACCGAACAGGACATCAAGCG methionine,no GCTGGAGCAGCTGGAGGGCAGCTTCAGAAAGCAGATCTCTAAAAGCCTGAAGTCC stopcodon) GACGCCAGATACAAGCTGATCTACAAAAAGGAGATGCTGGAAAAGCTCCTGCCTG AGTTCCTGGACAACGAGGAAGAAAAGGCTAGAGTGATCAGCTTCGAGAACTTTAC AACCTACTTCACTGGCTTCCACACCAACCGGGAAAACATGTACACCGATGAGGCC AAGTCTACGGCCGTTTCCTTTAGGTGTATCAACGATAACCTGCCAAAGTTCCTGGA CAACATCAGCGTATTCAAGTGGGTCACCGCCTTTCTGAGCGAGTCTGACATCAAC GAACTGAAGGCCGATTTCAGCGGCCTGTTGGGCTGCTCCCTGGAAGAGATGTTCA CCCCTGATTACTTCAGCTTCGTGCTGTCTCAGAGCGGCATCGAGAGATACAACAA CGTGATCGGCGGATACACCTGTAGCGATGGCGAGAAAGTCAAAGGACTTAATGAG TACATCAACCTGTATAACCAGAAGCTGCAACACGGCGAAAAGAAACTGCCCCTGC TCAAGCGGCTGTTCAAGCAGATTCTGTCAGACACCGAGAGCGTGTCCTTCATCCC CGAGAAACTGGAAAATGATGACGCCGTGATCTCCGCCATTAACGGATTTTGTAATA TCAAGATCGAGAATGAAACATTCTTCGAGATCCTGGACAAGACCAAGTGCCTGTTC AGCAATCTGAACGAGTTCGACTCTGCCGGAGTGTACATCACCAACGGCTTCGCAG TGACAGACATCAGCAACGCCGTGTTCGGCACCTGGGACGTCATCAGCGAAGCCT GGAAGAAAGAGTACGCCAAAGCTATCCCCCTGAAGAACATCGCTAAGGCCGACGC CTACTATGAGAAGCAGGGCAAGGCCTACAAGGCCATCAAGAGCTTCTCTGTAAGC GAACTGCAGAGACTGGCCAACACCACGGAGGGAAAGGCCGCCTACAAGCACAAC GGCGACATCAGCGCCTATTTCAGCGAGACAGTCTGCTTCGCTGTGCAGGATATCT TCGAGAAGTATAGCAGCAGCAAGGCCCTGTTCGCCAGCCCCTATAAGAACGAGAA GCGGCTGTTCAAGAACAATGAGGCAATCGCTCTGATTAAGGACTTCCTGGATAGC ATCAAGAACCTGGAGAAGCTGATTAAGCCATTCAACGGCAGCGGCAGAGAGAACG ACAAGGACGAGAGCTTTTACGGCGAGTTCACCGCCTGCTACGAGCGGCTGAGCA AAATCGATCTGCTGTACGACAAGGTGCGGAACTACATGACACAGAAACCTTACAG CGGCGATAAGATCAAGCTGAACTTCGAGAATCCTCAGTTCCTGAACGGATGGGAT AGAAACAAGGAGCGGGATTACAGAACAGTGCTGCTGAGAAAGGGAGGTTATTACT ACCTGGCCATCATGGACAAGAGCAACAACCGGATCTTCGAGGATCTGCCTGAGCC TAAGAATGGTGAGGACTGCTACGAAAAAATCGATTACAAGCTGCTGCCTGGCCCT AACAAGATGCTGCCCAAAGTGTTCTTCGCCGCTAGTAACATCGACTACTTCGCCCC TAGCGAACAGATCCTCAAAATCCGGCAGAAGGAAACCTTCAAAAAGGGCGTGAAC TTCAACATTGACGACTGTCACGCCTTCATCGACTTCCTGAAGGAATCTATCGAGAA GCACGACGAGTGGTGCAAGTACGGCTTCGAGTTTAAGGACACCAGCGACTACAAC AATATAGGCGAGTTCTACAAGGACGTGCGGGAACAGGGCTACAGCATCTCTTTTC GGAATGTGCCCGAGTCCTACATCAACAGCTGCGTGAACTCTGGCTCTCTGTACCT GTTTCAGATCTACAACAAAGATTTTAGCCCTTACTCTAAGGGCACAAAGAGCCTGC ACACCCTGTACTTTGAAATGCTGTTTGACGAGCGCAACCTGAAGAACGTGGTGTAT CAGCTGAATGGTGGCGCTGAGATGTTCTACAGAAAGGCCAGCATCAAGGAAAGAG ACAAGATCGTGCACCCCGCCAACATCCCTATCAAGAACAAGAACCCCGACAACCC TAAGGCCGAGAGCGTGTTCGAATACGACATTATCAAGGACAGAAGATTCACCGAA CGGCAGTTCTCCCTGCACATCCCTGTGACCCTGAACTTCAAAGGCTCTGGCGGAT CTGCCAACCTGAACGCCGACGTTAGGCGGGCTATCAGAGGCGCCGATGAGAACT ACGTGATCGGCATCGACCGGGGCGAGAGGAACCTGCTGTACATCACAGTGATCA ATAGCAAGGGCGAGATCGTGGAACAAATCCCAGGCAACGTGATCATCAACGGCAA GCAAGTGGTGGACTACCACAAGCTGCTGGATGCTAAAGAGAAGGAAAGACTGGCT GCCAGACAGAACTGGACAACAGTTGAAAACATCAAGGAACTGAAGGAAGGCTACC TGTCCGTGATCATCCACAACATCTGCGAGCTGGTGAAAAAGTACAACGCTGTGAT CGCTATGGAGGACCTGAGCAGCGGCTTCAAGAACAGCCGCGTGAAGGTGGAAAA GCAGGTATACCAGAAGTTCGAAAAAATGCTGACCGAGAAACTGAACTTCCTGGTG GACAAGAAAGCCGATGTGCAAAGCAGAGGCGGCCTGCTGCAGGCCTACCAGCTG ACAAATAGCACAAAGGATTACAAGCGGGCCGGCAGCCAAGACGGCATCGTGTTCT ACGTGCCTGCCTGGCTGACAAGCAAAATTGACCCTGTGACCGGCTTTGTGGACCT GCTGAAACCTAAATACACCAGCGTTCAGGAGGCCAAAGAGCTGTTCAGCAACTTC GAGGCCGTCGAGTACATCCCCGAGGAGGACCTGTTCAGCTTCACCTTCGACTACA GCAAGTTCCCCAGATGCAGCGTGGCCTACAGAAACAAGTGGACCGTGTACAGTAA CGGAGAGAGAATCTACACATTCAGAGATAAGAACAGCAACAACGAATACGTGTCC AAGACAGTTGCCCTGACCACCGAGTTTAAAAGCCTCTTCGACGAATATAGCGTGTA CTACCGAGACAACCTGAAGAGTCAGATTTTGTGCCAGGATAAGGTGGATTTCTTCA AGCAACTTATCAGACTGCTGTCCCTGACCATGCAGATGAGAAACAGCATCAGCAA CAGCGCCGTGGACTACCTGATCTCCCCTGTGAAGGATAAGAATGGCAATTTTTTC GACAGCAGAAAGAGCATCAAGAACCTGCCTGAGAACGCCGACGCCAACGGCGCC TACAACATTGCTAAGAAGGCTCTGTGGGCCATCGGTCAGATCAAAGAGGCTGATG AGAATGACCTGATGAAGGTGAAGCTGTCCGTGTCTAATAAAGAGTGGCTGAAGTA CGTGCAGGAGGTGGAA Expression ATGggctccggaGAATTTGATAACTCTTTCGTGAATAGATATCCTCTGAGCAAGACCCT 102 construct(with GAGCTTCAGTCTGCTGCCAGTGGGCAGCACCGAAGCCAACTTCGAAAAAAAGCTG N-terminal CTGCTGCAGGAGGACGAAAAGAGAGCCGCCGAGTACATCCTGGTGAAAAGCTAC methionine ATCGACAGATACCACAAGGCCTACATCGAGAGCGTGCTGAGCAAGGTGGTGCTG andstop GACGGCATCAACAACTATGCCCAGCTGTACTGCAAGAACAACAAGACCGAACAGG codon, ACATCAAGCGGCTGGAGCAGCTGGAGGGCAGCTTCAGAAAGCAGATCTCTAAAAG includesV5- CCTGAAGTCCGACGCCAGATACAAGCTGATCTACAAAAAGGAGATGCTGGAAAAG tagandC- CTCCTGCCTGAGTTCCTGGACAACGAGGAAGAAAAGGCTAGAGTGATCAGCTTCG terminalNLS) AGAACTTTACAACCTACTTCACTGGCTTCCACACCAACCGGGAAAACATGTACACC GATGAGGCCAAGTCTACGGCCGTTTCCTTTAGGTGTATCAACGATAACCTGCCAAA GTTCCTGGACAACATCAGCGTATTCAAGTGGGTCACCGCCTTTCTGAGCGAGTCT GACATCAACGAACTGAAGGCCGATTTCAGCGGCCTGTTGGGCTGCTCCCTGGAAG AGATGTTCACCCCTGATTACTTCAGCTTCGTGCTGTCTCAGAGCGGCATCGAGAG ATACAACAACGTGATCGGCGGATACACCTGTAGCGATGGCGAGAAAGTCAAAGGA CTTAATGAGTACATCAACCTGTATAACCAGAAGCTGCAACACGGCGAAAAGAAACT GCCCCTGCTCAAGCGGCTGTTCAAGCAGATTCTGTCAGACACCGAGAGCGTGTCC TTCATCCCCGAGAAACTGGAAAATGATGACGCCGTGATCTCCGCCATTAACGGATT TTGTAATATCAAGATCGAGAATGAAACATTCTTCGAGATCCTGGACAAGACCAAGT GCCTGTTCAGCAATCTGAACGAGTTCGACTCTGCCGGAGTGTACATCACCAACGG CTTCGCAGTGACAGACATCAGCAACGCCGTGTTCGGCACCTGGGACGTCATCAGC GAAGCCTGGAAGAAAGAGTACGCCAAAGCTATCCCCCTGAAGAACATCGCTAAGG CCGACGCCTACTATGAGAAGCAGGGCAAGGCCTACAAGGCCATCAAGAGCTTCTC TGTAAGCGAACTGCAGAGACTGGCCAACACCACGGAGGGAAAGGCCGCCTACAA GCACAACGGCGACATCAGCGCCTATTTCAGCGAGACAGTCTGCTTCGCTGTGCAG GATATCTTCGAGAAGTATAGCAGCAGCAAGGCCCTGTTCGCCAGCCCCTATAAGA ACGAGAAGCGGCTGTTCAAGAACAATGAGGCAATCGCTCTGATTAAGGACTTCCT GGATAGCATCAAGAACCTGGAGAAGCTGATTAAGCCATTCAACGGCAGCGGCAGA GAGAACGACAAGGACGAGAGCTTTTACGGCGAGTTCACCGCCTGCTACGAGCGG CTGAGCAAAATCGATCTGCTGTACGACAAGGTGCGGAACTACATGACACAGAAAC CTTACAGCGGCGATAAGATCAAGCTGAACTTCGAGAATCCTCAGTTCCTGAACGG ATGGGATAGAAACAAGGAGCGGGATTACAGAACAGTGCTGCTGAGAAAGGGAGG TTATTACTACCTGGCCATCATGGACAAGAGCAACAACCGGATCTTCGAGGATCTGC CTGAGCCTAAGAATGGTGAGGACTGCTACGAAAAAATCGATTACAAGCTGCTGCC TGGCCCTAACAAGATGCTGCCCAAAGTGTTCTTCGCCGCTAGTAACATCGACTACT TCGCCCCTAGCGAACAGATCCTCAAAATCCGGCAGAAGGAAACCTTCAAAAAGGG CGTGAACTTCAACATTGACGACTGTCACGCCTTCATCGACTTCCTGAAGGAATCTA TCGAGAAGCACGACGAGTGGTGCAAGTACGGCTTCGAGTTTAAGGACACCAGCG ACTACAACAATATAGGCGAGTTCTACAAGGACGTGCGGGAACAGGGCTACAGCAT CTCTTTTCGGAATGTGCCCGAGTCCTACATCAACAGCTGCGTGAACTCTGGCTCTC TGTACCTGTTTCAGATCTACAACAAAGATTTTAGCCCTTACTCTAAGGGCACAAAG AGCCTGCACACCCTGTACTTTGAAATGCTGTTTGACGAGCGCAACCTGAAGAACG TGGTGTATCAGCTGAATGGTGGCGCTGAGATGTTCTACAGAAAGGCCAGCATCAA GGAAAGAGACAAGATCGTGCACCCCGCCAACATCCCTATCAAGAACAAGAACCCC GACAACCCTAAGGCCGAGAGCGTGTTCGAATACGACATTATCAAGGACAGAAGAT TCACCGAACGGCAGTTCTCCCTGCACATCCCTGTGACCCTGAACTTCAAAGGCTC TGGCGGATCTGCCAACCTGAACGCCGACGTTAGGCGGGCTATCAGAGGCGCCGA TGAGAACTACGTGATCGGCATCGACCGGGGCGAGAGGAACCTGCTGTACATCAC AGTGATCAATAGCAAGGGCGAGATCGTGGAACAAATCCCAGGCAACGTGATCATC AACGGCAAGCAAGTGGTGGACTACCACAAGCTGCTGGATGCTAAAGAGAAGGAAA GACTGGCTGCCAGACAGAACTGGACAACAGTTGAAAACATCAAGGAACTGAAGGA AGGCTACCTGTCCGTGATCATCCACAACATCTGCGAGCTGGTGAAAAAGTACAAC GCTGTGATCGCTATGGAGGACCTGAGCAGCGGCTTCAAGAACAGCCGCGTGAAG GTGGAAAAGCAGGTATACCAGAAGTTCGAAAAAATGCTGACCGAGAAACTGAACT TCCTGGTGGACAAGAAAGCCGATGTGCAAAGCAGAGGCGGCCTGCTGCAGGCCT ACCAGCTGACAAATAGCACAAAGGATTACAAGCGGGCCGGCAGCCAAGACGGCA TCGTGTTCTACGTGCCTGCCTGGCTGACAAGCAAAATTGACCCTGTGACCGGCTT TGTGGACCTGCTGAAACCTAAATACACCAGCGTTCAGGAGGCCAAAGAGCTGTTC AGCAACTTCGAGGCCGTCGAGTACATCCCCGAGGAGGACCTGTTCAGCTTCACCT TCGACTACAGCAAGTTCCCCAGATGCAGCGTGGCCTACAGAAACAAGTGGACCGT GTACAGTAACGGAGAGAGAATCTACACATTCAGAGATAAGAACAGCAACAACGAAT ACGTGTCCAAGACAGTTGCCCTGACCACCGAGTTTAAAAGCCTCTTCGACGAATAT AGCGTGTACTACCGAGACAACCTGAAGAGTCAGATTTTGTGCCAGGATAAGGTGG ATTTCTTCAAGCAACTTATCAGACTGCTGTCCCTGACCATGCAGATGAGAAACAGC ATCAGCAACAGCGCCGTGGACTACCTGATCTCCCCTGTGAAGGATAAGAATGGCA ATTTTTTCGACAGCAGAAAGAGCATCAAGAACCTGCCTGAGAACGCCGACGCCAA CGGCGCCTACAACATTGCTAAGAAGGCTCTGTGGGCCATCGGTCAGATCAAAGAG GCTGATGAGAATGACCTGATGAAGGTGAAGCTGTCCGTGTCTAATAAAGAGTGGC TGAAGTACGTGCAGGAGGTGGAAtctagaAAGCGGACAGCAGACGGCTCCGAATTT GAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTG CTGGGCCTGGACAGCACCTGA

[0136] In some embodiments a ZRBH Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 97, SEQ ID NO:98, or SEQ ID NO:99. In some embodiments, a ZRBH Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D851 substitution, wherein the position of the D851 substitution is defined with respect to the amino acid numbering of SEQ ID NO:98 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E940 substitution, wherein the position of the E940 substitution is defined with respect to the amino acid numbering of SEQ ID NO:98 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1152 substitution, wherein the position of the R1152 substitution is defined with respect to the amino acid numbering of SEQ ID NO:98 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1189 substitution, wherein the position of the D1189 substitution is defined with respect to the amino acid numbering of SEQ ID NO:98 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZRBH Type V Cas protein is catalytically inactive, for example due to a R1152 substitution in combination with a D851 substitution, a E940 substitution, and/or D1152 substitution.

6.2.18. ZWPU Type V Cas Protein

[0137] In one aspect, the disclosure provides ZWPU Type V Cas proteins. ZWPU Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZWPU Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 103. In some embodiments, the ZWPU Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 103. In some embodiments, a ZWPU Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 103.

[0138] Exemplary ZWPU Type V Cas protein sequences and nucleotide sequences encoding exemplary ZWPU Type V Cas proteins are set forth in Table 1R.

TABLE-US-00019 TABLE1R ZWPUTypeVCasSequences SEQ ID Name Sequence NO. Wildtype KAETNLTELVNLYSLQKTLRFELIPQGKTLENIEKNGILTQDNQRADDYEKVKKLIDEY 103 aminoacid HKHHIEISLDDCRLEGLEEYKELYEKKDDLKKIQENLRKQIVKSLTENERYKDKRLFSD sequence KLFKEDLPNYLKDREQDKALVKKFEKFTTYFTGFNENRKNMYSSEDKPTSIAYRLIHE (withoutN- NLPKFIDNLHIFDKIKETTIKDDFDKIVEKLNKHLKIHIKSFDEIFSIEYFNKTLSQKQIDNY terminal NNIIGGMSFENGTKIQGLNEYINRYNQKQEDKHQKLPCVKTLYKQILSDREKISWIPEQ methionine) FDDDKQMAESISNLYNEMLPIIKDDLLPLMANIGDYDLSKIFISNDSALTTISQRIFGAYN VYTLAIIEKLKSDKPKSKRQSESKYLDEIDKNFKNMKSFSIAKLNNAVKGKYDKTIENYI KVFGAFDEEENLLQRLETAYNEAEPILNNIEDRCKNINQDKDAVEKIKTLLDALKDIQH FAKLLLCDNDETEIDAEFYNKLHDIVVKLDKITPIYNMVRNYVTKKPYSEEKIKLNFEKS TLLGGWDLNKEKNNLSVILRKDNLYYLGIMKKDNNKIFDSTNIKTDGVCFEKMEYKLL PDPKKMLPKVFFSKKCSKDFNPNDKILEIKENESFKKTSSNFNIEQCRKLIDFYKESIN KHKDWQKFNFQFSDTKTYNDINEFYNEVEKQGYKISFCKISEDYINELVKDNKLYLFKI WNKDFSKYSKGTPNTHTLYWKQIFAPENINNVVYKLNGQAEIFFRQASISQKNVIKHL ANKPVKNKNIKNEKKESTFSYDLVKDKRFTMDKFHFHVPITINFKAKGINNTNPIVNNLI RQNKIEHIIGIDRGERHLLYLSLIDLKGNIIEQKSLNEIINNYNGNEYKTDYHTLLDDKEK ERKDARLSWNTIENIKELKDGYMSQWVHIISQMIVKYNAIVVLEDLNHGFVRGRQKIEK QVYEKFEHKLIDKLNYYVDKNADSNAVGGLYNALQLTNPFDSFEKLGKQSGCLFYIPA WKTSKIDPVTGFINMFTNLKYESVEKSKKFFSKFDDIRYNKEKNRFEFDVSFDKFDSD FVRITQESKLHWTLCSVGQRIELVKENNGYKPNEINLTDAFKSVFNTNKIEINTAKLNR EIGKINDTAFFKELMRLMKLLLQMRNSKPNSIEKNDDYIISPVADENGVFFDSSKVEDN GNLPKDADANGAYNIARKGLYVIHQIKQSEDDKKIDFKDFNPRWLKFIQQKLYLND Wildtype MKAETNLTELVNLYSLQKTLRFELIPQGKTLENIEKNGILTQDNQRADDYEKVKKLIDE 104 aminoacid YHKHHIEISLDDCRLEGLEEYKELYEKKDDLKKIQENLRKQIVKSLTENERYKDKRLFS sequence(with DKLFKEDLPNYLKDREQDKALVKKFEKFTTYFTGFNENRKNMYSSEDKPTSIAYRLIH N-terminal ENLPKFIDNLHIFDKIKETTIKDDFDKIVEKLNKHLKIHIKSFDEIFSIEYFNKTLSQKQIDN methionine) YNNIIGGMSFENGTKIQGLNEYINRYNQKQEDKHQKLPCVKTLYKQILSDREKISWIPE QFDDDKQMAESISNLYNEMLPIIKDDLLPLMANIGDYDLSKIFISNDSALTTISQRIFGAY NVYTLAIIEKLKSDKPKSKRQSESKYLDEIDKNFKNMKSFSIAKLNNAVKGKYDKTIEN YIKVFGAFDEEENLLQRLETAYNEAEPILNNIEDRCKNINQDKDAVEKIKTLLDALKDIQ HFAKLLLCDNDETEIDAEFYNKLHDIWVKLDKITPIYNMVRNYVTKKPYSEEKIKLNFEK STLLGGWDLNKEKNNLSVILRKDNLYYLGIMKKDNNKIFDSTNIKTDGVCFEKMEYKL LPDPKKMLPKVFFSKKCSKDFNPNDKILEIKENESFKKTSSNFNIEQCRKLIDFYKESIN KHKDWQKFNFQFSDTKTYNDINEFYNEVEKQGYKISFCKISEDYINELVKDNKLYLFKI WNKDFSKYSKGTPNTHTLYWKQIFAPENINNVVYKLNGQAEIFFRQASISQKNVIKHL ANKPVKNKNIKNEKKESTFSYDLVKDKRFTMDKFHFHVPITINFKAKGINNTNPIVNNLI RQNKIEHIIGIDRGERHLLYLSLIDLKGNIIEQKSLNEIINNYNGNEYKTDYHTLLDDKEK ERKDARLSWNTIENIKELKDGYMSQWVHIISQMIVKYNAIVVLEDLNHGFVRGRQKIEK QVYEKFEHKLIDKLNYYVDKNADSNAVGGLYNALQLTNPFDSFEKLGKQSGCLFYIPA WKTSKIDPVTGFINMFTNLKYESVEKSKKFFSKFDDIRYNKEKNRFEFDVSFDKFDSD FVRITQESKLHWTLCSVGQRIELVKENNGYKPNEINLTDAFKSVFNTNKIEINTAKLNR EIGKINDTAFFKELMRLMKLLLQMRNSKPNSIEKNDDYIISPVADENGVFFDSSKVEDN GNLPKDADANGAYNIARKGLYVIHQIKQSEDDKKIDFKDFNPRWLKFIQQKLYLND Expression MGSGKAETNLTELVNLYSLQKTLRFELIPQGKTLENIEKNGILTQDNQRADDYEKVKK 105 construct(with LIDEYHKHHIEISLDDCRLEGLEEYKELYEKKDDLKKIQENLRKQIVKSLTENERYKDK N-terminal RLFSDKLFKEDLPNYLKDREQDKALVKKFEKFTTYFTGFNENRKNMYSSEDKPTSIAY methionine, RLIHENLPKFIDNLHIFDKIKETTIKDDFDKIVEKLNKHLKIHIKSFDEIFSIEYFNKTLSQK V5-tagandC- QIDNYNNIIGGMSFENGTKIQGLNEYINRYNQKQEDKHQKLPCVKTLYKQILSDREKIS terminalNLS) WIPEQFDDDKQMAESISNLYNEMLPIIKDDLLPLMANIGDYDLSKIFISNDSALTTISQRI aasequence FGAYNVYTLAIIEKLKSDKPKSKRQSESKYLDEIDKNFKNMKSFSIAKLNNAVKGKYDK TIENYIKVFGAFDEEENLLQRLETAYNEAEPILNNIEDRCKNINQDKDAVEKIKTLLDAL KDIQHFAKLLLCDNDETEIDAEFYNKLHDIWVKLDKITPIYNMVRNYVTKKPYSEEKIKL NFEKSTLLGGWDLNKEKNNLSVILRKDNLYYLGIMKKDNNKIFDSTNIKTDGVCFEKM EYKLLPDPKKMLPKVFFSKKCSKDFNPNDKILEIKENESFKKTSSNFNIEQCRKLIDFY KESINKHKDWQKFNFQFSDTKTYNDINEFYNEVEKQGYKISFCKISEDYINELVKDNKL YLFKIWNKDFSKYSKGTPNTHTLYWKQIFAPENINNVVYKLNGQAEIFFRQASISQKN VIKHLANKPVKNKNIKNEKKESTFSYDLVKDKRFTMDKFHFHVPITINFKAKGINNTNPI VNNLIRQNKIEHIIGIDRGERHLLYLSLIDLKGNIIEQKSLNEIINNYNGNEYKTDYHTLLD DKEKERKDARLSWNTIENIKELKDGYMSQVVHIISQMIVKYNAIVVLEDLNHGFVRGR QKIEKQVYEKFEHKLIDKLNYYVDKNADSNAVGGLYNALQLTNPFDSFEKLGKQSGC LFYIPAWKTSKIDPVTGFINMFTNLKYESVEKSKKFFSKFDDIRYNKEKNRFEFDVSFD KFDSDFVRITQESKLHWTLCSVGQRIELVKENNGYKPNEINLTDAFKSVENTNKIEINT AKLNREIGKINDTAFFKELMRLMKLLLQMRNSKPNSIEKNDDYIISPVADENGVFFDSS KVEDNGNLPKDADANGAYNIARKGLYVIHQIKQSEDDKKIDFKDFNPRWLKFIQQKLY LNDSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAAGCAGAAACAAATCTGACAGAATTAGTGAATCTGTATTCATTGCAGAAAAC 106 coding ACTTCGTTTTGAATTAATCCCACAGGGCAAAACATTAGAAAACATTGAGAAAAATG sequence(with GTATTCTTACACAAGATAACCAAAGAGCAGACGATTACGAAAAAGTCAAAAAACTT N-terminal ATTGATGAGTATCATAAGCACCATATTGAAATAAGTCTTGACGATTGTCGCCTTGA methionine AGGTTTAGAGGAATATAAAGAACTCTACGAAAAGAAAGATGATTTGAAAAAAATTC andstop AAGAGAATCTACGAAAACAAATCGTTAAAAGTTTAACGGAGAACGAAAGGTATAA codon) AGACAAACGTCTATTCTCTGATAAACTCTTCAAAGAAGATCTTCCGAATTATCTAA AAGATAGAGAACAAGACAAAGCTCTTGTTAAAAAATTTGAAAAATTCACCACATAT TTTACTGGATTTAACGAAAACAGAAAAAATATGTATTCTTCCGAAGACAAACCTAC CTCAATTGCTTATAGATTAATCCATGAAAATTTACCTAAGTTTATAGACAATTTACA TATTTTTGATAAAATTAAAGAAACAACAATCAAAGATGATTTTGATAAGATTGTTGA AAAATTAAACAAGCATCTAAAAATTCATATCAAATCATTTGACGAAATTTTCTCTAT TGAATATTTCAATAAAACTCTTAGCCAAAAACAAATAGACAATTATAACAATATAAT TGGAGGAATGTCTTTTGAGAATGGTACAAAGATACAAGGCTTAAACGAATATATTA ATCGTTACAATCAAAAGCAGGAAGATAAACATCAAAAACTTCCTTGCGTCAAAACA CTTTATAAGCAAATACTCAGTGATAGAGAAAAAATATCGTGGATTCCAGAACAATT TGATGATGATAAACAAATGGCAGAAAGTATTTCGAATTTGTACAATGAAATGCTTC CAATTATTAAAGATGATCTACTTCCGCTAATGGCTAATATAGGCGATTATGATCTT AGCAAAATATTTATCTCCAACGACTCTGCTTTAACAACAATATCTCAACGAATTTTT GGAGCTTACAACGTTTACACTCTTGCAATAATAGAAAAATTAAAAAGTGATAAACC TAAATCAAAAAGACAATCCGAGTCTAAGTATTTAGACGAAATTGACAAAAACTTCA AAAATATGAAAAGTTTCAGTATTGCAAAACTAAACAATGCCGTAAAAGGCAAATAC GATAAAACAATAGAAAATTATATCAAGGTTTTCGGGGCTTTTGACGAAGAAGAGAA CTTGCTACAACGATTAGAAACAGCCTATAACGAAGCTGAGCCTATACTTAATAATA TAGAAGACAGATGCAAAAATATTAATCAAGACAAAGATGCTGTTGAAAAGATTAAA ACATTATTAGATGCTTTGAAAGATATTCAACATTTTGCAAAACTTCTATTATGTGAT AACGACGAAACTGAAATAGATGCGGAGTTTTATAATAAATTACATGATATATGGGT AAAATTGGACAAGATAACACCTATATATAATATGGTGAGAAATTATGTTACAAAGA AACCTTATTCAGAAGAAAAAATCAAATTGAATTTTGAAAAATCTACACTATTAGGC GGCTGGGATTTGAACAAAGAAAAAAATAATTTATCAGTTATACTCCGCAAAGATAA TTTGTATTACTTAGGGATTATGAAAAAAGATAATAACAAAATCTTTGATAGTACAAA TATCAAAACCGATGGCGTTTGTTTTGAGAAAATGGAATACAAACTACTTCCTGATC CAAAGAAAATGCTGCCAAAGGTATTCTTTTCAAAAAAATGTTCAAAGGACTTTAAC CCGAACGACAAAATATTAGAAATTAAGGAAAATGAAAGTTTCAAGAAAACAAGCA GTAATTTCAATATTGAGCAATGTCGTAAATTAATAGACTTCTATAAAGAATCTATCA ATAAACATAAAGATTGGCAAAAATTTAATTTCCAATTCTCTGACACTAAAACTTACA ATGACATAAACGAATTTTACAACGAAGTTGAAAAACAAGGTTATAAAATATCTTTTT GTAAAATTTCTGAGGATTATATAAATGAGTTGGTGAAAGACAATAAACTTTATTTGT TTAAGATTTGGAACAAAGACTTTTCAAAATATAGCAAAGGAACTCCAAATACGCAC ACTCTTTATTGGAAACAAATATTTGCACCTGAAAATATCAACAATGTCGTATATAAA CTAAACGGACAAGCCGAAATATTTTTTAGGCAAGCAAGTATTTCTCAAAAAAACGT TATCAAACATTTGGCAAACAAACCTGTTAAAAACAAGAATATAAAAAACGAAAAAA AGGAAAGTACGTTCAGTTATGATTTAGTAAAAGATAAACGTTTTACTATGGATAAA TTCCATTTCCACGTACCGATTACTATTAATTTCAAGGCAAAAGGAATAAATAATAC CAATCCTATTGTCAATAATCTAATTCGTCAAAACAAGATAGAACATATTATTGGTAT AGATAGAGGCGAAAGGCATTTGCTTTATCTTTCTCTTATAGATTTGAAAGGAAATA TCATTGAACAAAAGTCGTTGAATGAAATCATAAACAACTACAATGGCAATGAATAT AAAACAGATTACCATACCTTGCTTGATGATAAGGAAAAAGAAAGAAAAGATGCCC GACTTTCGTGGAATACTATTGAAAATATCAAAGAACTCAAAGACGGGTATATGAG CCAAGTTGTGCATATTATCTCACAAATGATTGTGAAGTACAATGCAATAGTTGTTT TGGAAGACCTTAATCATGGCTTTGTTCGTGGTCGCCAGAAGATAGAAAAACAAGT TTATGAAAAATTTGAGCATAAACTTATTGATAAACTAAACTATTATGTCGATAAGAA TGCCGATAGCAATGCCGTTGGAGGACTTTACAATGCTTTGCAACTAACAAATCCA TTTGATAGTTTTGAAAAATTAGGAAAACAAAGCGGCTGTTTATTCTATATCCCTGC TTGGAAAACAAGTAAGATTGATCCCGTTACTGGATTTATTAATATGTTTACAAATCT CAAATACGAATCAGTGGAAAAATCAAAGAAGTTCTTTTCAAAGTTTGACGATATTA GATACAATAAAGAAAAAAATAGGTTTGAATTTGATGTTTCATTTGATAAATTCGATA GTGATTTTGTCCGTATTACACAGGAAAGTAAATTACATTGGACGCTTTGCAGTGTT GGTCAGCGTATAGAATTAGTAAAAGAGAATAATGGTTATAAACCTAATGAAATAAA TTTAACTGATGCTTTCAAATCAGTGTTTAATACTAATAAAATAGAGATAAACACTGC TAAACTGAATAGAGAGATTGGTAAAATCAATGATACAGCGTTTTTCAAGGAACTTA TGCGTTTAATGAAATTGTTATTACAAATGAGAAATAGTAAGCCAAATTCAATAGAG AAGAACGACGATTATATTATCTCTCCTGTTGCAGACGAAAATGGAGTATTCTTTGA CAGCAGTAAAGTTGAAGACAATGGCAATTTGCCAAAAGATGCCGATGCCAACGG AGCATACAATATTGCTCGCAAAGGCTTGTATGTAATACACCAAATAAAGCAAAGC GAAGATGATAAAAAAATCGATTTCAAAGATTTCAACCCACGTTGGTTAAAATTCAT TCAGCAAAAACTATATTTGAATGATTGA Codon AAGGCCGAGACAAACCTGACAGAACTCGTGAACCTGTACAGCCTGCAAAAAACC 107 optimized CTGAGATTTGAGCTCATCCCCCAGGGCAAGACCCTTGAGAACATCGAGAAGAAC coding GGTATCCTGACCCAGGACAATCAGAGAGCCGACGACTACGAGAAGGTGAAAAAA sequence(no CTGATCGACGAGTACCACAAGCACCACATCGAGATCAGCCTGGACGATTGCAGA N-terminal CTGGAAGGCCTGGAAGAATACAAGGAACTGTATGAGAAGAAGGATGACCTAAAG methionine,no AAAATCCAGGAAAACCTGAGAAAGCAGATCGTGAAGTCCCTCACTGAGAACGAA stopcodon) CGGTACAAGGACAAAAGACTCTTCTCAGATAAGCTGTTCAAGGAAGATCTGCCTA ATTACCTGAAGGACAGAGAACAGGACAAGGCCCTGGTAAAAAAGTTCGAGAAGT TCACCACCTACTTCACCGGCTTCAACGAAAACCGCAAAAACATGTACAGCAGCGA GGATAAGCCCACCAGCATCGCTTATAGACTGATCCACGAGAACCTGCCTAAGTTC ATCGACAACCTGCACATCTTTGATAAGATCAAGGAAACCACCATCAAGGACGATT TCGATAAGATCGTGGAAAAGCTGAATAAACACCTGAAGATCCACATCAAATCCTT CGACGAGATCTTTTCTATTGAATACTTCAACAAGACACTGAGTCAAAAGCAAATCG ACAACTACAACAACATCATCGGCGGAATGAGCTTCGAGAATGGCACCAAGATCCA GGGCCTGAATGAGTACATCAACAGATACAACCAGAAACAAGAGGACAAGCATCA AAAGCTGCCTTGCGTGAAAACCCTGTACAAGCAGATCCTGAGCGACAGAGAGAA GATTTCCTGGATTCCTGAACAGTTCGATGACGACAAACAGATGGCCGAGAGCATC AGCAATCTGTACAACGAGATGCTGCCAATCATCAAGGACGACCTGCTGCCTCTGA TGGCCAACATTGGCGACTACGACCTGAGCAAAATCTTCATCAGCAATGACAGCG CCCTGACAACCATCTCGCAGCGGATCTTCGGAGCTTACAACGTGTACACCCTGG CCATCATTGAGAAGCTGAAGTCTGATAAGCCTAAGAGCAAGCGGCAGTCTGAGT CTAAGTACCTGGACGAGATCGACAAGAACTTCAAGAACATGAAGTCTTTTAGCAT CGCCAAGCTGAACAACGCCGTGAAGGGCAAGTATGACAAGACAATCGAAAATTA CATCAAGGTGTTTGGCGCCTTTGATGAGGAGGAGAATCTCCTGCAGAGGCTGGA AACAGCCTATAACGAGGCCGAGCCTATCCTGAACAACATCGAGGACAGATGCAA AAACATCAATCAAGACAAGGATGCCGTGGAAAAGATCAAGACCTTACTGGACGCT CTGAAAGATATCCAGCACTTTGCCAAGTTACTGCTGTGCGACAATGACGAAACCG AGATTGACGCCGAGTTCTACAACAAGCTGCACGACATCTGGGTGAAGCTGGACA AAATCACACCAATCTACAACATGGTGCGGAACTACGTGACCAAGAAGCCCTACTC TGAAGAGAAAATCAAGCTGAACTTCGAAAAGTCTACACTGCTGGGCGGCTGGGA TCTGAACAAGGAAAAGAACAATCTGAGCGTGATCCTGAGAAAGGACAACCTGTAC TACCTGGGCATCATGAAGAAAGACAACAACAAGATCTTCGACTCCACAAACATCA AGACCGACGGCGTTTGTTTCGAGAAGATGGAATATAAGCTGTTACCTGACCCTAA AAAGATGCTGCCCAAGGTGTTCTTCTCAAAGAAATGCAGCAAGGATTTCAATCCT AACGACAAGATCCTGGAGATCAAAGAGAACGAATCTTTCAAGAAAACCTCTAGCA ACTTTAATATCGAGCAGTGCAGAAAACTGATCGACTTTTACAAGGAGTCCATCAAT AAGCACAAAGACTGGCAGAAATTCAACTTTCAGTTCAGCGATACCAAGACCTACA ACGATATCAACGAGTTCTACAACGAGGTGGAAAAACAGGGCTACAAAATTAGCTT CTGCAAGATCAGCGAGGACTACATCAATGAGCTGGTTAAGGACAACAAACTGTAC CTGTTTAAGATCTGGAACAAGGATTTCAGTAAGTACAGCAAGGGGACCCCTAACA CCCACACCCTGTACTGGAAGCAGATCTTCGCCCCTGAGAACATCAACAACGTCG TGTACAAGCTGAACGGACAGGCCGAGATCTTCTTCAGACAAGCATCTATCTCCCA GAAGAACGTCATCAAGCACCTAGCTAATAAGCCAGTGAAAAACAAGAACATCAAG AACGAGAAGAAGGAGAGCACCTTCAGCTACGATCTTGTTAAGGACAAGCGGTTTA CAATGGACAAGTTCCACTTCCACGTGCCAATCACCATAAACTTTAAGGCCAAGGG CATCAACAACACCAATCCTATTGTCAACAACCTGATCCGGCAGAACAAGATTGAA CACATCATCGGCATCGACAGAGGCGAGAGACACCTGCTGTATCTGAGCCTGATC GATCTGAAGGGCAACATCATAGAACAGAAGAGCCTGAACGAGATCATCAACAATT ACAACGGCAATGAGTACAAGACCGATTACCATACCTTGCTGGATGACAAGGAAAA GGAGAGAAAGGATGCTAGACTGAGCTGGAACACCATCGAAAATATCAAGGAACT GAAAGATGGCTACATGAGCCAGGTGGTGCACATCATCAGTCAGATGATCGTGAA ATACAACGCCATTGTGGTCCTGGAGGATCTCAACCACGGCTTCGTGCGGGGCAG ACAGAAGATCGAGAAGCAGGTGTATGAAAAATTTGAACACAAGCTGATCGACAAG CTGAATTACTACGTGGACAAGAATGCTGACAGCAACGCCGTGGGAGGACTGTAC AATGCCCTGCAGCTGACAAACCCCTTCGACAGCTTCGAGAAGCTGGGCAAGCAG AGCGGCTGTCTGTTTTACATCCCCGCCTGGAAAACAAGTAAGATCGATCCTGTGA CCGGATTCATCAACATGTTCACCAACCTGAAGTACGAATCTGTGGAAAAGAGCAA AAAGTTCTTCAGCAAGTTCGATGACATCAGATACAACAAGGAGAAAAACCGATTC GAGTTCGACGTGTCCTTCGACAAGTTCGACTCCGACTTCGTGCGGATCACCCAG GAGAGCAAACTGCATTGGACCTTGTGTAGCGTGGGCCAGAGAATCGAACTGGTC AAGGAAAACAACGGATACAAGCCTAACGAAATCAACCTGACAGATGCTTTCAAGA GCGTGTTCAACACAAACAAGATCGAGATCAACACCGCCAAACTGAATCGGGAAAT CGGAAAAATCAACGACACAGCTTTCTTCAAGGAACTGATGCGGCTGATGAAGCTG CTCCTGCAGATGAGAAACAGCAAGCCCAACTCCATCGAAAAGAACGATGATTACA TCATCAGCCCTGTGGCCGATGAGAACGGCGTGTTCTTTGACAGCAGCAAAGTGG AGGACAATGGCAACCTGCCAAAGGACGCCGATGCCAACGGCGCCTACAACATCG CCAGGAAGGGCCTGTATGTGATCCACCAGATTAAGCAGTCTGAGGACGACAAGA AGATCGACTTTAAGGACTTCAACCCCAGATGGCTGAAGTTCATCCAGCAGAAGCT GTACCTGAACGAT Expression ATGggctccggaAAGGCCGAGACAAACCTGACAGAACTCGTGAACCTGTACAGCCTG 108 construct(with CAAAAAACCCTGAGATTTGAGCTCATCCCCCAGGGCAAGACCCTTGAGAACATC N-terminal GAGAAGAACGGTATCCTGACCCAGGACAATCAGAGAGCCGACGACTACGAGAAG methionine GTGAAAAAACTGATCGACGAGTACCACAAGCACCACATCGAGATCAGCCTGGAC andstop GATTGCAGACTGGAAGGCCTGGAAGAATACAAGGAACTGTATGAGAAGAAGGAT codon, GACCTAAAGAAAATCCAGGAAAACCTGAGAAAGCAGATCGTGAAGTCCCTCACTG includesV5- AGAACGAACGGTACAAGGACAAAAGACTCTTCTCAGATAAGCTGTTCAAGGAAGA tagandC- TCTGCCTAATTACCTGAAGGACAGAGAACAGGACAAGGCCCTGGTAAAAAAGTTC terminalNLS) GAGAAGTTCACCACCTACTTCACCGGCTTCAACGAAAACCGCAAAAACATGTACA GCAGCGAGGATAAGCCCACCAGCATCGCTTATAGACTGATCCACGAGAACCTGC CTAAGTTCATCGACAACCTGCACATCTTTGATAAGATCAAGGAAACCACCATCAA GGACGATTTCGATAAGATCGTGGAAAAGCTGAATAAACACCTGAAGATCCACATC AAATCCTTCGACGAGATCTTTTCTATTGAATACTTCAACAAGACACTGAGTCAAAA GCAAATCGACAACTACAACAACATCATCGGCGGAATGAGCTTCGAGAATGGCAC CAAGATCCAGGGCCTGAATGAGTACATCAACAGATACAACCAGAAACAAGAGGA CAAGCATCAAAAGCTGCCTTGCGTGAAAACCCTGTACAAGCAGATCCTGAGCGA CAGAGAGAAGATTTCCTGGATTCCTGAACAGTTCGATGACGACAAACAGATGGCC GAGAGCATCAGCAATCTGTACAACGAGATGCTGCCAATCATCAAGGACGACCTG CTGCCTCTGATGGCCAACATTGGCGACTACGACCTGAGCAAAATCTTCATCAGCA ATGACAGCGCCCTGACAACCATCTCGCAGCGGATCTTCGGAGCTTACAACGTGT ACACCCTGGCCATCATTGAGAAGCTGAAGTCTGATAAGCCTAAGAGCAAGCGGC AGTCTGAGTCTAAGTACCTGGACGAGATCGACAAGAACTTCAAGAACATGAAGTC TTTTAGCATCGCCAAGCTGAACAACGCCGTGAAGGGCAAGTATGACAAGACAATC GAAAATTACATCAAGGTGTTTGGCGCCTTTGATGAGGAGGAGAATCTCCTGCAGA GGCTGGAAACAGCCTATAACGAGGCCGAGCCTATCCTGAACAACATCGAGGACA GATGCAAAAACATCAATCAAGACAAGGATGCCGTGGAAAAGATCAAGACCTTACT GGACGCTCTGAAAGATATCCAGCACTTTGCCAAGTTACTGCTGTGCGACAATGAC GAAACCGAGATTGACGCCGAGTTCTACAACAAGCTGCACGACATCTGGGTGAAG CTGGACAAAATCACACCAATCTACAACATGGTGCGGAACTACGTGACCAAGAAGC CCTACTCTGAAGAGAAAATCAAGCTGAACTTCGAAAAGTCTACACTGCTGGGCGG CTGGGATCTGAACAAGGAAAAGAACAATCTGAGCGTGATCCTGAGAAAGGACAA CCTGTACTACCTGGGCATCATGAAGAAAGACAACAACAAGATCTTCGACTCCACA AACATCAAGACCGACGGCGTTTGTTTCGAGAAGATGGAATATAAGCTGTTACCTG ACCCTAAAAAGATGCTGCCCAAGGTGTTCTTCTCAAAGAAATGCAGCAAGGATTT CAATCCTAACGACAAGATCCTGGAGATCAAAGAGAACGAATCTTTCAAGAAAACC TCTAGCAACTTTAATATCGAGCAGTGCAGAAAACTGATCGACTTTTACAAGGAGT CCATCAATAAGCACAAAGACTGGCAGAAATTCAACTTTCAGTTCAGCGATACCAA GACCTACAACGATATCAACGAGTTCTACAACGAGGTGGAAAAACAGGGCTACAAA ATTAGCTTCTGCAAGATCAGCGAGGACTACATCAATGAGCTGGTTAAGGACAACA AACTGTACCTGTTTAAGATCTGGAACAAGGATTTCAGTAAGTACAGCAAGGGGAC CCCTAACACCCACACCCTGTACTGGAAGCAGATCTTCGCCCCTGAGAACATCAAC AACGTCGTGTACAAGCTGAACGGACAGGCCGAGATCTTCTTCAGACAAGCATCTA TCTCCCAGAAGAACGTCATCAAGCACCTAGCTAATAAGCCAGTGAAAAACAAGAA CATCAAGAACGAGAAGAAGGAGAGCACCTTCAGCTACGATCTTGTTAAGGACAA GCGGTTTACAATGGACAAGTTCCACTTCCACGTGCCAATCACCATAAACTTTAAG GCCAAGGGCATCAACAACACCAATCCTATTGTCAACAACCTGATCCGGCAGAACA AGATTGAACACATCATCGGCATCGACAGAGGCGAGAGACACCTGCTGTATCTGA GCCTGATCGATCTGAAGGGCAACATCATAGAACAGAAGAGCCTGAACGAGATCA TCAACAATTACAACGGCAATGAGTACAAGACCGATTACCATACCTTGCTGGATGA CAAGGAAAAGGAGAGAAAGGATGCTAGACTGAGCTGGAACACCATCGAAAATAT CAAGGAACTGAAAGATGGCTACATGAGCCAGGTGGTGCACATCATCAGTCAGAT GATCGTGAAATACAACGCCATTGTGGTCCTGGAGGATCTCAACCACGGCTTCGT GCGGGGCAGACAGAAGATCGAGAAGCAGGTGTATGAAAAATTTGAACACAAGCT GATCGACAAGCTGAATTACTACGTGGACAAGAATGCTGACAGCAACGCCGTGGG AGGACTGTACAATGCCCTGCAGCTGACAAACCCCTTCGACAGCTTCGAGAAGCT GGGCAAGCAGAGCGGCTGTCTGTTTTACATCCCCGCCTGGAAAACAAGTAAGAT CGATCCTGTGACCGGATTCATCAACATGTTCACCAACCTGAAGTACGAATCTGTG GAAAAGAGCAAAAAGTTCTTCAGCAAGTTCGATGACATCAGATACAACAAGGAGA AAAACCGATTCGAGTTCGACGTGTCCTTCGACAAGTTCGACTCCGACTTCGTGCG GATCACCCAGGAGAGCAAACTGCATTGGACCTTGTGTAGCGTGGGCCAGAGAAT CGAACTGGTCAAGGAAAACAACGGATACAAGCCTAACGAAATCAACCTGACAGAT GCTTTCAAGAGCGTGTTCAACACAAACAAGATCGAGATCAACACCGCCAAACTGA ATCGGGAAATCGGAAAAATCAACGACACAGCTTTCTTCAAGGAACTGATGCGGCT GATGAAGCTGCTCCTGCAGATGAGAAACAGCAAGCCCAACTCCATCGAAAAGAA CGATGATTACATCATCAGCCCTGTGGCCGATGAGAACGGCGTGTTCTTTGACAG CAGCAAAGTGGAGGACAATGGCAACCTGCCAAAGGACGCCGATGCCAACGGCG CCTACAACATCGCCAGGAAGGGCCTGTATGTGATCCACCAGATTAAGCAGTCTG AGGACGACAAGAAGATCGACTTTAAGGACTTCAACCCCAGATGGCTGAAGTTCAT CCAGCAGAAGCTGTACCTGAACGATtctagaAAGCGGACAGCAGACGGCTCCGAAT TTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCC CTGCTGGGCCTGGACAGCACCTGA

[0139] In some embodiments a ZWPU Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105. In some embodiments, a ZWPU Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D845 substitution, wherein the position of the D845 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 104 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E938 substitution, wherein the position of the E938 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 104 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1153 substitution, wherein the position of the R1153 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 104 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1195 substitution, wherein the position of the D1195 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 104 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZWPU Type V Cas protein is catalytically inactive, for example due to a R1153 substitution in combination with a D845 substitution, a E938 substitution, and/or D1195 substitution.

6.2.19. ZZQE Type V Cas Protein

[0140] In one aspect, the disclosure provides ZZQE Type V Cas proteins. ZZQE Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZQE Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 109. In some embodiments, the ZZQE Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109. In some embodiments, a ZZQE Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 109.

[0141] Exemplary ZZQE Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZQE Type V Cas proteins are set forth in Table 1S.

TABLE-US-00020 TABLE1S ZZQETypeVCasSequences SEQ ID Name Sequence NO. Wildtype DMKSLNSFQNQYSLSKTLRFQLIPQGKTLDNINESRILEEDQHRSESYKLVKKIIDDYH 109 aminoacid KAYIEQALGSFELKIASDSKNDSLEEFYSQYIAERKEDKAKKLFEKTQDNLRKQISKKL sequence KQGEAYKRLFGKELIQEDLLEFVATDPEADSKKRLIEEFKDFTTYFIGFHENRKNMYA (withoutN- EEAQSTAIAYRIIHENLPKFIDNIRTFEELAKSSIADVLPQVYEDFKAYLKVESVKELFSL terminal DYFNTVLTQKQLDIYNAVIGGKSLDENSRIQGLNEYINLYNQQHKDKKLPFLKPLFKQI methionine) LSDRNSLSWLPEAFDNDKQVLQAVHDCYTSLLESVFHKDGLQQLLQSLPTYNLKGIY LRNDLSMTNVSQKLLGDWGAITRAVKEKLQKENPAKKRESDEAYQERINKIFKQAGS YSLDYINQALEATDQTNIKVEDYFINMGVDNEQKEPLFQRVAQAYNQASDLLEKEYPA NKNLMQDKESIEHIKFLLDNLKAVQHFIKPLLGDGNEADKDNRFYGELTALWNELDQV TRLYNKVRNYMTRKPYSVDKIKINFKNSTLLNGWDRNKERDNTAVILRKDGKFYLAIM HKEHNKVFEKFPVGTKDSDFEKMEYKLLPGANKMLPKVFFSKSRIDEFKPSAELLQK YQMGTHKKGELFSLNDCHSLIDFFKASIEKHDDWKQFNFHFSPTSSYEDLSGFYREV EQQGYKLTFKSVDADYINKMVDEGKIFLFQIYNKDFSEHSKGTPNLHTLYWKMLFDE RNLQNVVYKLNGEAEVFFRKKSLTYTRPTHPKKEPIKNKNVQNAKKESIFDYDLIKNK RFTVDSFQFHVPITMNFKSEGRSNLNERVNEFLRQNNDAHIIGIDRGERHLLYLVVIDR HGNIVEQFSLNSIINEYQGNTYATNYHDLLDKREKEREEARESWQSIENIKELKEGYL SQVVHKIADLMVKYHAIVVLEDLNMGFMRGRQKVEKQVYQKFEKMLIDKLNYLVDKK QDAETDGGLLKAYQLTNQFESFQKLGKQSGFLFYVPAWNTSKIDPCTGFTNLLDTRY ESIEKAKKFFQTFNAIRYNAAQGYFEFELDYNKFNKRADGTQTLWTLCTYGPRIETLR STEDNNKWTSKEVDLTDELKKHFYHYGIKLDADLKEAIGQQTDKPFFTNLLHLLKLTL QMRNSKIGTEVDYLISPIRNEDGTFYDSRQGNKSLPANADANGAYNIARKGLWVINQI KQTPQDQKPKLAITNKEWLQFAQEKPYLKD Wildtype MDMKSLNSFQNQYSLSKTLRFQLIPQGKTLDNINESRILEEDQHRSESYKLVKKIIDDY 110 aminoacid HKAYIEQALGSFELKIASDSKNDSLEEFYSQYIAERKEDKAKKLFEKTQDNLRKQISKK sequence(with LKQGEAYKRLFGKELIQEDLLEFVATDPEADSKKRLIEEFKDFTTYFIGFHENRKNMYA N-terminal EEAQSTAIAYRIIHENLPKFIDNIRTFEELAKSSIADVLPQVYEDFKAYLKVESVKELFSL methionine) DYFNTVLTQKQLDIYNAVIGGKSLDENSRIQGLNEYINLYNQQHKDKKLPFLKPLFKQI LSDRNSLSWLPEAFDNDKQVLQAVHDCYTSLLESVFHKDGLQQLLQSLPTYNLKGIY LRNDLSMTNVSQKLLGDWGAITRAVKEKLQKENPAKKRESDEAYQERINKIFKQAGS YSLDYINQALEATDQTNIKVEDYFINMGVDNEQKEPLFQRVAQAYNQASDLLEKEYPA NKNLMQDKESIEHIKFLLDNLKAVQHFIKPLLGDGNEADKDNRFYGELTALWNELDQV TRLYNKVRNYMTRKPYSVDKIKINFKNSTLLNGWDRNKERDNTAVILRKDGKFYLAIM HKEHNKVFEKFPVGTKDSDFEKMEYKLLPGANKMLPKVFFSKSRIDEFKPSAELLQK YQMGTHKKGELFSLNDCHSLIDFFKASIEKHDDWKQFNFHFSPTSSYEDLSGFYREV EQQGYKLTFKSVDADYINKMVDEGKIFLFQIYNKDFSEHSKGTPNLHTLYWKMLFDE RNLQNVVYKLNGEAEVFFRKKSLTYTRPTHPKKEPIKNKNVQNAKKESIFDYDLIKNK RFTVDSFQFHVPITMNFKSEGRSNLNERVNEFLRQNNDAHIIGIDRGERHLLYLVVIDR HGNIVEQFSLNSIINEYQGNTYATNYHDLLDKREKEREEARESWQSIENIKELKEGYL SQVVHKIADLMVKYHAIVVLEDLNMGFMRGRQKVEKQVYQKFEKMLIDKLNYLVDKK QDAETDGGLLKAYQLTNQFESFQKLGKQSGFLFYVPAWNTSKIDPCTGFTNLLDTRY ESIEKAKKFFQTFNAIRYNAAQGYFEFELDYNKFNKRADGTQTLWTLCTYGPRIETLR STEDNNKWTSKEVDLTDELKKHFYHYGIKLDADLKEAIGQQTDKPFFTNLLHLLKLTL QMRNSKIGTEVDYLISPIRNEDGTFYDSRQGNKSLPANADANGAYNIARKGLWVINQI KQTPQDQKPKLAITNKEWLQFAQEKPYLKD Expression MGSGDMKSLNSFQNQYSLSKTLRFQLIPQGKTLDNINESRILEEDQHRSESYKLVKKII 111 construct(with DDYHKAYIEQALGSFELKIASDSKNDSLEEFYSQYIAERKEDKAKKLFEKTQDNLRKQI N-terminal SKKLKQGEAYKRLFGKELIQEDLLEFVATDPEADSKKRLIEEFKDFTTYFIGFHENRKN methionine, MYAEEAQSTAIAYRIIHENLPKFIDNIRTFEELAKSSIADVLPQVYEDFKAYLKVESVKE V5-tagandC- LFSLDYFNTVLTQKQLDIYNAVIGGKSLDENSRIQGLNEYINLYNQQHKDKKLPFLKPL terminalNLS) FKQILSDRNSLSWLPEAFDNDKQVLQAVHDCYTSLLESVFHKDGLQQLLQSLPTYNL aasequence KGIYLRNDLSMTNVSQKLLGDWGAITRAVKEKLQKENPAKKRESDEAYQERINKIFKQ AGSYSLDYINQALEATDQTNIKVEDYFINMGVDNEQKEPLFQRVAQAYNQASDLLEK EYPANKNLMQDKESIEHIKFLLDNLKAVQHFIKPLLGDGNEADKDNRFYGELTALWNE LDQVTRLYNKVRNYMTRKPYSVDKIKINFKNSTLLNGWDRNKERDNTAVILRKDGKF YLAIMHKEHNKVFEKFPVGTKDSDFEKMEYKLLPGANKMLPKVFFSKSRIDEFKPSAE LLQKYQMGTHKKGELFSLNDCHSLIDFFKASIEKHDDWKQFNFHFSPTSSYEDLSGF YREVEQQGYKLTFKSVDADYINKMVDEGKIFLFQIYNKDFSEHSKGTPNLHTLYWKM LFDERNLQNVVYKLNGEAEVFFRKKSLTYTRPTHPKKEPIKNKNVQNAKKESIFDYDLI KNKRFTVDSFQFHVPITMNFKSEGRSNLNERVNEFLRQNNDAHIIGIDRGERHLLYLV VIDRHGNIVEQFSLNSIINEYQGNTYATNYHDLLDKREKEREEARESWQSIENIKELKE GYLSQVVHKIADLMVKYHAIVVLEDLNMGFMRGRQKVEKQVYQKFEKMLIDKLNYLV DKKQDAETDGGLLKAYQLTNQFESFQKLGKQSGFLFYVPAWNTSKIDPCTGFTNLLD TRYESIEKAKKFFQTFNAIRYNAAQGYFEFELDYNKFNKRADGTQTLWTLCTYGPRIE TLRSTEDNNKWTSKEVDLTDELKKHFYHYGIKLDADLKEAIGQQTDKPFFTNLLHLLK LTLQMRNSKIGTEVDYLISPIRNEDGTFYDSRQGNKSLPANADANGAYNIARKGLWVI NQIKQTPQDQKPKLAITNKEWLQFAQEKPYLKDSRKRTADGSEFESPKKKRKVGSG KPIPNPLLGLDST Wildtype ATGGATATGAAAAGTTTAAACAGCTTTCAGAACCAGTATTCCCTATCCAAGACCCT 112 coding CCGGTTTCAGCTAATACCCCAGGGTAAAACTTTGGATAACATTAACGAGAGCAGA sequence(with ATATTGGAGGAAGACCAACACCGAAGCGAAAGCTACAAGTTGGTCAAGAAAATCA N-terminal TTGACGACTATCACAAGGCCTACATCGAACAAGCCCTGGGCAGTTTCGAACTCAA methionine AATTGCCAGTGACTCTAAAAACGATTCGTTAGAGGAGTTCTACTCGCAGTATATTG andstop CCGAACGGAAAGAAGATAAAGCCAAAAAACTTTTCGAAAAGACGCAAGACAACTT codon) GCGAAAGCAAATCTCCAAGAAATTAAAGCAGGGCGAAGCCTACAAGCGGTTGTTT GGCAAGGAACTCATTCAAGAAGACCTGCTGGAGTTTGTAGCTACCGACCCTGAG GCTGATAGCAAAAAGCGTCTGATTGAAGAATTCAAGGACTTCACCACCTACTTTAT CGGATTCCACGAGAACCGAAAGAACATGTATGCTGAGGAAGCCCAATCCACAGC AATTGCCTACCGCATCATTCACGAGAACCTGCCGAAGTTCATTGATAACATACGC ACCTTCGAAGAACTTGCTAAAAGTTCCATTGCCGACGTCCTGCCACAGGTTTATG AAGATTTCAAAGCGTACTTAAAGGTCGAATCGGTCAAAGAACTTTTCAGTCTGGA CTATTTCAATACCGTCTTGACCCAAAAGCAGCTTGACATTTACAATGCGGTTATCG GCGGTAAGTCGTTAGATGAGAACAGCCGCATCCAGGGGCTCAACGAGTATATCA ACCTGTACAACCAGCAGCACAAGGACAAAAAGTTACCCTTCTTAAAACCCTTGTT CAAGCAAATTCTGAGCGACCGCAACAGCCTTTCGTGGTTGCCCGAAGCTTTCGA CAATGACAAGCAGGTACTTCAGGCTGTACACGACTGCTACACCTCGCTATTGGAG AGCGTATTCCACAAAGACGGCCTGCAACAGTTGCTACAGTCACTGCCTACCTACA ACCTGAAGGGCATTTACCTGCGCAACGACCTTTCCATGACCAACGTTTCTCAAAA ACTATTGGGCGATTGGGGAGCTATTACACGTGCCGTTAAAGAAAAACTACAAAAA GAAAATCCTGCCAAAAAACGAGAGTCGGACGAAGCCTACCAAGAACGCATCAAC AAGATATTCAAGCAAGCCGGCAGCTACTCTTTAGATTACATCAACCAAGCGCTCG AAGCAACAGACCAGACCAATATCAAAGTCGAAGACTACTTCATCAACATGGGCGT AGACAACGAGCAAAAAGAGCCCCTGTTCCAGCGTGTAGCGCAAGCCTACAATCA GGCCAGCGATTTGCTTGAAAAGGAATATCCCGCAAACAAAAATCTGATGCAGGAT AAAGAAAGCATCGAGCACATCAAATTCTTGCTCGATAACCTCAAAGCCGTTCAAC ACTTTATAAAGCCCCTGCTCGGCGATGGTAACGAGGCTGATAAAGATAATCGTTT TTACGGAGAACTTACAGCGCTGTGGAACGAATTAGACCAGGTAACGCGCCTGTA TAACAAGGTGCGAAACTACATGACCCGCAAGCCCTACTCGGTTGATAAAATCAAG ATTAACTTTAAGAACTCAACTCTACTTAATGGCTGGGACAGAAATAAGGAACGTGA CAATACCGCTGTTATTCTGCGCAAAGACGGCAAGTTCTATCTGGCCATTATGCAT AAAGAACACAATAAGGTGTTCGAAAAATTCCCGGTCGGAACAAAGGATTCTGACT TCGAGAAAATGGAGTATAAGTTACTTCCGGGCGCCAATAAAATGCTTCCGAAGGT TTTCTTCTCTAAATCGCGTATCGATGAGTTTAAGCCCAGCGCCGAACTTCTCCAAA AGTACCAGATGGGTACCCACAAAAAGGGCGAACTCTTCAGTCTGAACGACTGCC ATTCTCTGATTGACTTCTTTAAGGCTTCTATTGAAAAGCATGACGATTGGAAACAG TTTAACTTCCATTTCTCACCCACTTCGAGCTACGAAGACTTGAGCGGATTTTACAG AGAGGTTGAACAGCAGGGGTACAAACTGACCTTCAAATCCGTTGACGCCGACTA TATCAACAAAATGGTTGACGAGGGCAAAATCTTTCTCTTCCAGATTTACAATAAAG ACTTCTCGGAACATAGCAAAGGCACCCCCAACCTGCATACGCTCTACTGGAAAAT GCTCTTTGACGAACGCAACCTGCAGAACGTGGTCTACAAACTGAACGGCGAGGC CGAAGTCTTCTTCCGGAAGAAGAGTCTTACCTACACCCGTCCTACGCACCCCAAG AAAGAGCCTATCAAGAACAAGAACGTTCAGAATGCCAAAAAGGAAAGCATCTTCG ACTACGACCTGATTAAAAACAAACGCTTTACGGTCGACTCCTTCCAGTTCCACGT TCCCATCACGATGAACTTCAAGAGCGAAGGACGCTCCAACCTGAACGAGCGGGT CAACGAGTTTTTACGCCAGAACAACGATGCCCACATCATTGGCATTGACCGGGG CGAACGCCATTTGCTCTACCTGGTGGTTATTGACCGGCACGGAAACATTGTGGAA CAATTTTCGCTCAACTCTATCATCAACGAATATCAGGGTAATACGTACGCCACCAA CTACCACGACTTGTTGGATAAGCGCGAAAAGGAAAGAGAGGAAGCACGCGAAAG CTGGCAGAGTATTGAGAATATTAAAGAACTGAAAGAAGGATACTTGAGCCAGGTG GTGCATAAAATTGCCGACCTCATGGTAAAGTATCATGCCATCGTGGTGCTCGAAG ACTTGAATATGGGCTTCATGCGCGGACGCCAGAAGGTAGAAAAGCAGGTCTATC AGAAGTTTGAAAAAATGCTGATAGACAAGTTAAACTATCTGGTTGACAAGAAGCAA GATGCCGAAACCGACGGCGGTCTGCTCAAGGCATACCAACTGACCAACCAGTTC GAAAGTTTCCAGAAGTTAGGCAAGCAGAGCGGTTTCCTCTTCTATGTGCCTGCCT GGAACACCAGCAAAATTGACCCCTGCACCGGATTTACCAACCTGCTCGACACTC GATACGAGAGCATCGAAAAGGCCAAAAAGTTCTTTCAAACTTTCAATGCCATCCG CTACAATGCTGCGCAGGGGTACTTTGAGTTCGAACTGGATTACAATAAATTCAAC AAGCGGGCCGATGGTACACAAACCCTATGGACGCTCTGCACCTACGGCCCACGC ATCGAAACACTCCGAAGCACCGAGGATAATAACAAGTGGACAAGCAAAGAGGTT GATTTGACCGACGAATTGAAAAAGCACTTCTACCACTATGGCATTAAGCTGGATG CCGACCTGAAGGAAGCCATCGGCCAACAAACCGACAAACCTTTCTTCACCAACTT GCTCCATCTGCTCAAACTAACACTGCAAATGCGAAACAGCAAAATCGGCACGGA GGTTGACTACCTCATTTCGCCAATTCGCAATGAAGACGGAACGTTCTACGACAGC CGACAAGGCAACAAATCATTGCCTGCCAATGCCGATGCCAATGGTGCCTACAAC ATTGCCCGAAAGGGTTTATGGGTAATTAACCAGATAAAACAAACACCTCAAGACC AAAAGCCCAAGTTAGCTATTACCAACAAGGAATGGCTGCAATTTGCTCAAGAGAA GCCCTACCTTAAGGATTGA Codon GACATGAAGAGCCTGAACTCTTTTCAGAACCAATACTCTCTGAGCAAAACCCTGC 113 optimized GGTTCCAGCTGATCCCTCAGGGCAAGACACTGGATAATATCAACGAGAGCAGAA coding TCCTGGAAGAGGATCAGCACAGAAGCGAGTCATATAAACTGGTGAAGAAGATCAT sequence(no TGACGACTATCACAAGGCCTACATCGAGCAGGCCCTGGGCAGCTTCGAGCTGAA N-terminal AATTGCCTCCGATAGCAAGAACGACAGCCTGGAGGAGTTCTACTCTCAGTACATT methionine,no GCGGAGAGAAAGGAGGACAAGGCCAAGAAGCTGTTCGAAAAGACCCAGGACAA stopcodon) TCTGAGAAAGCAGATCTCCAAGAAGCTGAAACAGGGTGAAGCCTACAAACGGCT GTTCGGCAAAGAACTGATCCAGGAGGACCTGCTGGAGTTCGTGGCCACAGATCC TGAGGCCGACTCTAAGAAGAGACTGATCGAAGAGTTCAAGGACTTTACCACCTAC TTCATCGGATTTCACGAAAATAGAAAGAACATGTACGCCGAGGAGGCTCAGAGCA CAGCTATTGCCTACAGAATCATCCACGAGAACCTGCCAAAGTTTATCGATAATATC AGAACCTTCGAGGAACTGGCCAAGAGCAGCATCGCCGACGTGCTGCCCCAGGT CTACGAGGACTTTAAGGCCTACCTGAAGGTGGAAAGCGTGAAAGAACTGTTCTCT CTGGATTATTTCAACACCGTGCTGACACAGAAACAACTGGACATCTACAATGCCG TGATCGGCGGAAAAAGCCTGGACGAGAACAGCAGAATCCAGGGCCTGAACGAG TACATCAACCTCTACAACCAGCAGCATAAGGACAAGAAGCTGCCTTTCCTGAAGC CCCTGTTCAAGCAAATCCTGTCCGATAGAAACAGCCTGTCCTGGCTGCCTGAGG CCTTCGACAACGACAAGCAGGTGCTGCAGGCCGTGCACGACTGCTACACCAGCC TGCTGGAATCTGTGTTCCACAAGGACGGCCTGCAACAGCTGCTGCAGAGCCTCC CAACCTACAACTTAAAAGGCATCTACCTGCGGAACGACCTTAGCATGACCAATGT GTCCCAGAAGCTGCTGGGCGATTGGGGCGCTATCACCAGAGCCGTGAAGGAAA AGCTGCAGAAGGAAAACCCTGCCAAGAAGAGAGAGTCGGACGAGGCCTACCAG GAGCGGATCAACAAGATCTTCAAGCAGGCCGGCTCATATTCACTGGATTACATCA ACCAGGCCCTCGAAGCCACAGACCAGACAAACATCAAAGTGGAGGACTACTTTA TCAACATGGGCGTGGATAATGAGCAGAAAGAGCCTCTGTTTCAAAGGGTGGCCC AGGCCTATAACCAGGCCAGCGACCTGCTGGAAAAAGAATACCCCGCTAACAAGA ATCTGATGCAGGACAAGGAGAGCATCGAGCACATCAAATTCCTGCTCGACAACCT TAAGGCCGTGCAGCACTTCATCAAGCCTCTGCTGGGAGATGGCAACGAAGCCGA CAAGGACAACAGATTCTACGGCGAGCTAACCGCCCTGTGGAACGAACTTGACCA GGTGACCCGCCTGTACAACAAGGTGCGGAATTACATGACCAGGAAGCCTTACAG CGTGGACAAGATCAAAATCAACTTCAAGAACAGCACCCTGCTGAACGGATGGGA CAGAAACAAGGAACGGGACAACACAGCTGTCATCCTGAGAAAGGACGGCAAGTT CTACCTCGCCATCATGCACAAGGAACACAACAAGGTCTTTGAGAAGTTTCCTGTG GGCACTAAGGATTCTGACTTCGAGAAGATGGAATACAAGCTGCTGCCCGGCGCC AACAAGATGCTGCCTAAGGTTTTCTTTAGCAAGAGCAGAATCGACGAGTTCAAGC CATCTGCCGAGCTGCTGCAGAAGTACCAGATGGGAACTCACAAGAAGGGAGAAC TGTTCAGCCTGAACGATTGCCACAGCCTGATCGACTTCTTCAAAGCCTCTATCGA GAAGCACGATGATTGGAAGCAGTTCAACTTCCATTTCAGCCCTACCAGCAGCTAC GAGGACCTGAGCGGCTTCTACCGGGAGGTGGAACAGCAGGGCTACAAGCTGAC CTTCAAGAGCGTGGACGCTGATTACATCAATAAGATGGTCGATGAAGGCAAAATC TTCCTGTTCCAGATCTACAACAAGGATTTTAGCGAGCACAGCAAGGGCACACCTA ACCTGCACACCCTGTACTGGAAGATGCTGTTCGACGAGAGAAACCTGCAGAACG TGGTGTACAAGCTGAACGGCGAAGCTGAGGTGTTCTTTCGGAAGAAGAGCCTGA CCTACACACGCCCCACCCACCCTAAGAAGGAGCCTATCAAGAACAAAAACGTGC AGAACGCTAAAAAGGAAAGCATCTTCGATTACGACCTGATCAAGAACAAAAGATT CACAGTGGATTCTTTCCAGTTCCACGTGCCTATCACAATGAACTTCAAATCTGAG GGCAGAAGCAACCTGAATGAGAGGGTGAACGAGTTCCTGAGACAAAACAACGAT GCCCACATCATCGGAATCGACAGAGGCGAAAGGCATCTGCTGTACCTGGTGGTG ATTGATAGACACGGCAACATCGTGGAACAATTTAGCCTGAACAGCATAATCAATG AGTACCAAGGCAATACCTACGCCACAAACTATCACGACCTCCTGGACAAGAGAG AGAAGGAGCGGGAAGAGGCCAGAGAGTCCTGGCAGTCTATCGAGAACATCAAG GAGCTCAAAGAAGGCTACCTGAGTCAGGTGGTGCACAAAATCGCCGACCTGATG GTGAAGTATCACGCCATCGTGGTGCTGGAGGACCTGAACATGGGCTTCATGAGA GGCCGACAGAAGGTAGAGAAGCAGGTTTACCAGAAATTCGAGAAGATGCTGATT GACAAGCTGAACTATCTGGTGGACAAAAAGCAAGATGCTGAAACCGACGGCGGC CTGCTCAAGGCCTACCAACTGACCAACCAGTTCGAGAGCTTCCAGAAGCTGGGC AAACAGTCTGGCTTCCTGTTTTACGTGCCCGCCTGGAACACCAGCAAGATCGATC CCTGTACAGGCTTCACCAACCTGCTGGACACCCGATACGAGAGCATCGAAAAAG CAAAGAAGTTCTTCCAAACATTCAACGCCATAAGATACAACGCTGCTCAGGGGTA TTTTGAGTTCGAGCTCGACTACAACAAGTTTAACAAGCGGGCCGATGGCACCCA GACCCTGTGGACACTGTGCACCTACGGACCTAGAATCGAAACCCTGCGGAGCAC AGAGGACAACAACAAGTGGACCAGCAAAGAGGTGGACCTGACAGACGAGCTGAA GAAACACTTCTACCACTACGGCATCAAGTTGGATGCCGACCTGAAAGAGGCCAT CGGCCAGCAAACAGACAAGCCCTTCTTCACCAACCTGCTGCACCTGCTGAAGCT GACACTGCAGATGAGAAACAGCAAGATCGGAACCGAGGTGGACTACCTGATTAG CCCCATCAGAAACGAAGATGGCACCTTCTACGACAGCAGACAGGGAAACAAGAG CCTGCCTGCTAATGCGGACGCCAATGGCGCCTACAACATCGCTAGAAAAGGCCT CTGGGTCATCAACCAGATCAAACAGACCCCTCAGGATCAGAAACCTAAGCTGGC CATCACCAATAAGGAGTGGCTGCAGTTCGCCCAGGAGAAACCATACCTGAAAGA C Expression ATGggctccggaGACATGAAGAGCCTGAACTCTTTTCAGAACCAATACTCTCTGAGCA 114 construct(with AAACCCTGCGGTTCCAGCTGATCCCTCAGGGCAAGACACTGGATAATATCAACG N-terminal AGAGCAGAATCCTGGAAGAGGATCAGCACAGAAGCGAGTCATATAAACTGGTGA methionine AGAAGATCATTGACGACTATCACAAGGCCTACATCGAGCAGGCCCTGGGCAGCT andstop TCGAGCTGAAAATTGCCTCCGATAGCAAGAACGACAGCCTGGAGGAGTTCTACT codon, CTCAGTACATTGCGGAGAGAAAGGAGGACAAGGCCAAGAAGCTGTTCGAAAAGA includesV5- CCCAGGACAATCTGAGAAAGCAGATCTCCAAGAAGCTGAAACAGGGTGAAGCCT tagandC- ACAAACGGCTGTTCGGCAAAGAACTGATCCAGGAGGACCTGCTGGAGTTCGTGG terminalNLS) CCACAGATCCTGAGGCCGACTCTAAGAAGAGACTGATCGAAGAGTTCAAGGACT TTACCACCTACTTCATCGGATTTCACGAAAATAGAAAGAACATGTACGCCGAGGA GGCTCAGAGCACAGCTATTGCCTACAGAATCATCCACGAGAACCTGCCAAAGTTT ATCGATAATATCAGAACCTTCGAGGAACTGGCCAAGAGCAGCATCGCCGACGTG CTGCCCCAGGTCTACGAGGACTTTAAGGCCTACCTGAAGGTGGAAAGCGTGAAA GAACTGTTCTCTCTGGATTATTTCAACACCGTGCTGACACAGAAACAACTGGACA TCTACAATGCCGTGATCGGCGGAAAAAGCCTGGACGAGAACAGCAGAATCCAGG GCCTGAACGAGTACATCAACCTCTACAACCAGCAGCATAAGGACAAGAAGCTGC CTTTCCTGAAGCCCCTGTTCAAGCAAATCCTGTCCGATAGAAACAGCCTGTCCTG GCTGCCTGAGGCCTTCGACAACGACAAGCAGGTGCTGCAGGCCGTGCACGACT GCTACACCAGCCTGCTGGAATCTGTGTTCCACAAGGACGGCCTGCAACAGCTGC TGCAGAGCCTCCCAACCTACAACTTAAAAGGCATCTACCTGCGGAACGACCTTAG CATGACCAATGTGTCCCAGAAGCTGCTGGGCGATTGGGGCGCTATCACCAGAGC CGTGAAGGAAAAGCTGCAGAAGGAAAACCCTGCCAAGAAGAGAGAGTCGGACG AGGCCTACCAGGAGCGGATCAACAAGATCTTCAAGCAGGCCGGCTCATATTCAC TGGATTACATCAACCAGGCCCTCGAAGCCACAGACCAGACAAACATCAAAGTGG AGGACTACTTTATCAACATGGGCGTGGATAATGAGCAGAAAGAGCCTCTGTTTCA AAGGGTGGCCCAGGCCTATAACCAGGCCAGCGACCTGCTGGAAAAAGAATACCC CGCTAACAAGAATCTGATGCAGGACAAGGAGAGCATCGAGCACATCAAATTCCT GCTCGACAACCTTAAGGCCGTGCAGCACTTCATCAAGCCTCTGCTGGGAGATGG CAACGAAGCCGACAAGGACAACAGATTCTACGGCGAGCTAACCGCCCTGTGGAA CGAACTTGACCAGGTGACCCGCCTGTACAACAAGGTGCGGAATTACATGACCAG GAAGCCTTACAGCGTGGACAAGATCAAAATCAACTTCAAGAACAGCACCCTGCTG AACGGATGGGACAGAAACAAGGAACGGGACAACACAGCTGTCATCCTGAGAAAG GACGGCAAGTTCTACCTCGCCATCATGCACAAGGAACACAACAAGGTCTTTGAGA AGTTTCCTGTGGGCACTAAGGATTCTGACTTCGAGAAGATGGAATACAAGCTGCT GCCCGGCGCCAACAAGATGCTGCCTAAGGTTTTCTTTAGCAAGAGCAGAATCGA CGAGTTCAAGCCATCTGCCGAGCTGCTGCAGAAGTACCAGATGGGAACTCACAA GAAGGGAGAACTGTTCAGCCTGAACGATTGCCACAGCCTGATCGACTTCTTCAAA GCCTCTATCGAGAAGCACGATGATTGGAAGCAGTTCAACTTCCATTTCAGCCCTA CCAGCAGCTACGAGGACCTGAGCGGCTTCTACCGGGAGGTGGAACAGCAGGGC TACAAGCTGACCTTCAAGAGCGTGGACGCTGATTACATCAATAAGATGGTCGATG AAGGCAAAATCTTCCTGTTCCAGATCTACAACAAGGATTTTAGCGAGCACAGCAA GGGCACACCTAACCTGCACACCCTGTACTGGAAGATGCTGTTCGACGAGAGAAA CCTGCAGAACGTGGTGTACAAGCTGAACGGCGAAGCTGAGGTGTTCTTTCGGAA GAAGAGCCTGACCTACACACGCCCCACCCACCCTAAGAAGGAGCCTATCAAGAA CAAAAACGTGCAGAACGCTAAAAAGGAAAGCATCTTCGATTACGACCTGATCAAG AACAAAAGATTCACAGTGGATTCTTTCCAGTTCCACGTGCCTATCACAATGAACTT CAAATCTGAGGGCAGAAGCAACCTGAATGAGAGGGTGAACGAGTTCCTGAGACA AAACAACGATGCCCACATCATCGGAATCGACAGAGGCGAAAGGCATCTGCTGTA CCTGGTGGTGATTGATAGACACGGCAACATCGTGGAACAATTTAGCCTGAACAG CATAATCAATGAGTACCAAGGCAATACCTACGCCACAAACTATCACGACCTCCTG GACAAGAGAGAGAAGGAGCGGGAAGAGGCCAGAGAGTCCTGGCAGTCTATCGA GAACATCAAGGAGCTCAAAGAAGGCTACCTGAGTCAGGTGGTGCACAAAATCGC CGACCTGATGGTGAAGTATCACGCCATCGTGGTGCTGGAGGACCTGAACATGGG CTTCATGAGAGGCCGACAGAAGGTAGAGAAGCAGGTTTACCAGAAATTCGAGAA GATGCTGATTGACAAGCTGAACTATCTGGTGGACAAAAAGCAAGATGCTGAAACC GACGGCGGCCTGCTCAAGGCCTACCAACTGACCAACCAGTTCGAGAGCTTCCAG AAGCTGGGCAAACAGTCTGGCTTCCTGTTTTACGTGCCCGCCTGGAACACCAGC AAGATCGATCCCTGTACAGGCTTCACCAACCTGCTGGACACCCGATACGAGAGC ATCGAAAAAGCAAAGAAGTTCTTCCAAACATTCAACGCCATAAGATACAACGCTG CTCAGGGGTATTTTGAGTTCGAGCTCGACTACAACAAGTTTAACAAGCGGGCCGA TGGCACCCAGACCCTGTGGACACTGTGCACCTACGGACCTAGAATCGAAACCCT GCGGAGCACAGAGGACAACAACAAGTGGACCAGCAAAGAGGTGGACCTGACAG ACGAGCTGAAGAAACACTTCTACCACTACGGCATCAAGTTGGATGCCGACCTGAA AGAGGCCATCGGCCAGCAAACAGACAAGCCCTTCTTCACCAACCTGCTGCACCT GCTGAAGCTGACACTGCAGATGAGAAACAGCAAGATCGGAACCGAGGTGGACTA CCTGATTAGCCCCATCAGAAACGAAGATGGCACCTTCTACGACAGCAGACAGGG AAACAAGAGCCTGCCTGCTAATGCGGACGCCAATGGCGCCTACAACATCGCTAG AAAAGGCCTCTGGGTCATCAACCAGATCAAACAGACCCCTCAGGATCAGAAACCT AAGCTGGCCATCACCAATAAGGAGTGGCTGCAGTTCGCCCAGGAGAAACCATAC CTGAAAGACtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAA AAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACA GCACCTGA

[0142] In some embodiments a ZZQE Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111. In some embodiments, a ZZQE Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 109, SEQ ID NO:110, or SEQ ID NO: 111. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D859 substitution, wherein the position of the D859 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 110 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E952 substitution, wherein the position of the E952 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 110 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1164 substitution, wherein the position of the R1164 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 110 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1201 substitution, wherein the position of the D1201 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 110 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZZQE Type V Cas protein is catalytically inactive, for example due to a R1164 substitution in combination with a D859 substitution, a E952 substitution, and/or D1201 substitution.

6.2.20. ZRXE Type V Cas Protein

[0143] In one aspect, the disclosure provides ZRXE Type V Cas proteins. ZRXE Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZRXE Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 115. In some embodiments, the ZRXE Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 115. In some embodiments, a ZRXE Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO: 115.

[0144] Exemplary ZRXE Type V Cas protein sequences and nucleotide sequences encoding exemplary ZRXE Type V Cas proteins are set forth in Table 1T.

TABLE-US-00021 TABLE1T ZRXETypeVCasSequences SEQID Name Sequence NO. Wildtype KAFENFTGLYPLSKTLRFELKPIGKTLEYIEKHGILDKDKHRANSYVKVKDIIDRYHK 115 aminoacid QFIEDSLSDSDFKLKYENKGKKESLEEYFYYYKLRNRDDKQKKDFDEIQKNLRKQI sequence ASQLKKQDRFKRIDKKELIKEDLLEFVSDDNERNLINEFKDFTTYFTGFHENRQNMY (withoutN- SDEAKSTAIAYRLIHENLPKFIDNISVFERVAATDVADCFAQIYSDFEEYLNVNDISEI terminal FRLDYYTEILTQTQIDAYNLIIGGRSEGNIKIKGLNEYINLYNQQQKDKSQRLPKLKSL methionine) FKQILSDRNAISWLPESFENDNQLLEKLESCYQSFNETYDDKKSIFVRFRELLLTISD YEMDKIFLRNDLQLTDISQKMFGSYSIISRSLLEDLKRGTSRKSKKETDESFEERLR NIIKNQDSFAIGTIDSSLQQMDVEEYKKSICDYFPNLSVDDKGDDIFDRIVKAYSEVK DLLNSPYPSDKNLAQEDDDIDKIKNLLESMKDLQKFVKPLCGKGNESDKDERFYGE FTALYEELDKITPLYNMVRNYLTRKPYSTEKIKLNFDNAQLLNGWDLNKESDNTSVI LRKDGLYYLAIMNKKHNKVFEKNKLQSDGVCFEKMEYKLLPGANKMLPKVFFSKS RIDEFGPSQRLLDSYQNETHKKGDKFNIEDCHELIDFFKRSIDKHEDWSKFSFSFS DTKTYEDLSGFYREVEHQGYILSFVNVSVDYVNSLVDEGKIYLFQIYNKDFSPFSKG TPNMHTLYWKMLFDEENLKDVVYKLNGQAEVFFRKSSIKYDKPTHPANLPIDNKNV SNHKKRSVFEYDLVKDKRYTVDKFQFHVPVTINFKSDGNGNINPLVNDYIKKSDDL HVIGIDRGERHLLYLTVIDMKGNIKKQFSLNEIVNEYKGNTYSTNYHDLLEKREDKR DKERKEWKTIETIKELKEGYLSQVIHKITELMVEYNAIIVLEDLNLGFMRGRQKVEKS VYQKFEKMLIDKLNYLADKKKEPEDLGGVLKAYQLANKFESFQKMGKQSGFLFYT QAWNTSKIDPVTGFVNLFDTHYENILKSKNFFSKFDLIKYNSDKDWFEFSFDYNNF TTKAEGTKTKWTLCTFGNRIISFRNPDNNMQWDGKEINLTEEFKLFFEKFGININSD LHAEILKQDKKDFFEGLLHLLKLTLQMRNSKTRTDIDYMQSPVADENGVLYNSNKC GKSLPENADANGAYNIARKGLMIIDKIKKSDNLNKIDLTISNKEWLVFAQNKPYLKN Wildtype MKAFENFTGLYPLSKTLRFELKPIGKTLEYIEKHGILDKDKHRANSYVKVKDIIDRYH 116 aminoacid KQFIEDSLSDSDFKLKYENKGKKESLEEYFYYYKLRNRDDKQKKDFDEIQKNLRKQ sequence(with IASQLKKQDRFKRIDKKELIKEDLLEFVSDDNERNLINEFKDFTTYFTGFHENRQNM N-terminal YSDEAKSTAIAYRLIHENLPKFIDNISVFERVAATDVADCFAQIYSDFEEYLNVNDISE methionine) IFRLDYYTEILTQTQIDAYNLIIGGRSEGNIKIKGLNEYINLYNQQQKDKSQRLPKLKS LFKQILSDRNAISWLPESFENDNQLLEKLESCYQSFNETYDDKKSIFVRFRELLLTIS DYEMDKIFLRNDLQLTDISQKMFGSYSIISRSLLEDLKRGTSRKSKKETDESFEERL RNIIKNQDSFAIGTIDSSLQQMDVEEYKKSICDYFPNLSVDDKGDDIFDRIVKAYSEV KDLLNSPYPSDKNLAQEDDDIDKIKNLLESMKDLQKFVKPLCGKGNESDKDERFYG EFTALYEELDKITPLYNMVRNYLTRKPYSTEKIKLNFDNAQLLNGWDLNKESDNTS VILRKDGLYYLAIMNKKHNKVFEKNKLQSDGVCFEKMEYKLLPGANKMLPKVFFSK SRIDEFGPSQRLLDSYQNETHKKGDKFNIEDCHELIDFFKRSIDKHEDWSKFSFSFS DTKTYEDLSGFYREVEHQGYILSFVNVSVDYVNSLVDEGKIYLFQIYNKDFSPFSKG TPNMHTLYWKMLFDEENLKDVVYKLNGQAEVFFRKSSIKYDKPTHPANLPIDNKNV SNHKKRSVFEYDLVKDKRYTVDKFQFHVPVTINFKSDGNGNINPLVNDYIKKSDDL HVIGIDRGERHLLYLTVIDMKGNIKKQFSLNEIVNEYKGNTYSTNYHDLLEKREDKR DKERKEWKTIETIKELKEGYLSQVIHKITELMVEYNAIIVLEDLNLGFMRGRQKVEKS VYQKFEKMLIDKLNYLADKKKEPEDLGGVLKAYQLANKFESFQKMGKQSGFLFYT QAWNTSKIDPVTGFVNLFDTHYENILKSKNFFSKFDLIKYNSDKDWFEFSFDYNNF TTKAEGTKTKWTLCTFGNRIISFRNPDNNMQWDGKEINLTEEFKLFFEKFGININSD LHAEILKQDKKDFFEGLLHLLKLTLQMRNSKTRTDIDYMQSPVADENGVLYNSNKC GKSLPENADANGAYNIARKGLMIIDKIKKSDNLNKIDLTISNKEWLVFAQNKPYLKN Expression MGSGKAFENFTGLYPLSKTLRFELKPIGKTLEYIEKHGILDKDKHRANSYVKVKDIID 117 construct(with RYHKQFIEDSLSDSDFKLKYENKGKKESLEEYFYYYKLRNRDDKQKKDFDEIQKNL N-terminal RKQIASQLKKQDRFKRIDKKELIKEDLLEFVSDDNERNLINEFKDFTTYFTGFHENR methionine, QNMYSDEAKSTAIAYRLIHENLPKFIDNISVFERVAATDVADCFAQIYSDFEEYLNVN V5-tagandC- DISEIFRLDYYTEILTQTQIDAYNLIIGGRSEGNIKIKGLNEYINLYNQQQKDKSQRLP terminalNLS) KLKSLFKQILSDRNAISWLPESFENDNQLLEKLESCYQSFNETYDDKKSIFVRFREL aasequence LLTISDYEMDKIFLRNDLQLTDISQKMFGSYSIISRSLLEDLKRGTSRKSKKETDESF EERLRNIIKNQDSFAIGTIDSSLQQMDVEEYKKSICDYFPNLSVDDKGDDIFDRIVKA YSEVKDLLNSPYPSDKNLAQEDDDIDKIKNLLESMKDLQKFVKPLCGKGNESDKDE RFYGEFTALYEELDKITPLYNMVRNYLTRKPYSTEKIKLNFDNAQLLNGWDLNKES DNTSVILRKDGLYYLAIMNKKHNKVFEKNKLQSDGVCFEKMEYKLLPGANKMLPKV FFSKSRIDEFGPSQRLLDSYQNETHKKGDKFNIEDCHELIDFFKRSIDKHEDWSKFS FSFSDTKTYEDLSGFYREVEHQGYILSFVNVSVDYVNSLVDEGKIYLFQIYNKDFSP FSKGTPNMHTLYWKMLFDEENLKDVVYKLNGQAEVFFRKSSIKYDKPTHPANLPID NKNVSNHKKRSVFEYDLVKDKRYTVDKFQFHVPVTINFKSDGNGNINPLVNDYIKK SDDLHVIGIDRGERHLLYLTVIDMKGNIKKQFSLNEIVNEYKGNTYSTNYHDLLEKR EDKRDKERKEWKTIETIKELKEGYLSQVIHKITELMVEYNAIIVLEDLNLGFMRGRQK VEKSVYQKFEKMLIDKLNYLADKKKEPEDLGGVLKAYQLANKFESFQKMGKQSGF LFYTQAWNTSKIDPVTGFVNLFDTHYENILKSKNFFSKFDLIKYNSDKDWFEFSFDY NNFTTKAEGTKTKWTLCTFGNRIISFRNPDNNMQWDGKEINLTEEFKLFFEKFGINI NSDLHAEILKQDKKDFFEGLLHLLKLTLQMRNSKTRTDIDYMQSPVADENGVLYNS NKCGKSLPENADANGAYNIARKGLMIIDKIKKSDNLNKIDLTISNKEWLVFAQNKPYL KNSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype ATGAAAGCATTTGAGAATTTTACAGGATTGTATCCTCTTTCTAAAACATTAAGAT 118 coding TTGAGCTGAAACCGATTGGAAAGACATTGGAATATATTGAGAAGCATGGTATTC sequence(with TTGATAAGGATAAACACAGAGCAAATAGTTATGTTAAGGTCAAGGATATAATTG N-terminal ACAGATATCATAAACAATTTATTGAAGACTCGTTAAGTGATAGTGATTTTAAACT methionine TAAATATGAAAACAAAGGAAAGAAAGAATCATTAGAAGAATATTTCTATTATTAT andstop AAATTAAGAAATAGAGACGACAAACAGAAGAAAGATTTTGATGAAATTCAAAAG codon) AATCTTAGAAAACAGATTGCAAGTCAATTAAAGAAACAAGATCGTTTTAAAAGAA TTGATAAAAAGGAACTTATAAAGGAAGATCTTTTAGAATTTGTTAGTGATGATAA TGAAAGGAATCTTATTAATGAATTTAAAGATTTCACGACATATTTTACAGGTTTT CACGAAAACAGACAAAATATGTATTCTGATGAAGCCAAATCAACTGCGATAGCG TATAGACTGATACATGAGAATCTTCCTAAATTTATAGATAACATTTCAGTTTTTGA AAGAGTTGCTGCTACAGATGTGGCTGATTGTTTTGCACAAATCTATTCTGATTTT GAGGAATATCTGAATGTAAATGATATATCTGAAATTTTTAGATTAGACTATTATA CGGAAATATTAACTCAGACACAGATTGATGCTTATAATCTGATAATTGGAGGAC GTTCTGAGGGCAATATTAAAATAAAAGGTTTGAACGAATATATTAATCTGTATAA TCAACAGCAGAAAGACAAGTCTCAACGGTTGCCAAAACTGAAGTCTTTGTTTAA ACAGATTTTGAGTGATAGAAATGCTATATCTTGGTTGCCAGAATCGTTTGAAAAT GATAATCAACTCTTGGAAAAGTTGGAGAGTTGTTATCAGTCTTTTAATGAAACAT ATGACGATAAGAAGTCAATATTTGTAAGGTTTAGAGAATTATTGTTGACTATATC TGATTATGAAATGGATAAAATATTTCTTCGTAATGATTTGCAGTTGACAGATATT TCACAAAAGATGTTCGGTAGTTATAGTATTATTTCAAGGTCTTTATTGGAAGATT TAAAGAGAGGTACATCTCGTAAATCAAAGAAGGAAACTGATGAAAGTTTTGAAG AAAGGTTGAGAAATATTATCAAAAACCAAGATAGTTTTGCCATTGGAACAATAG ATTCGTCTTTGCAACAAATGGATGTTGAAGAATACAAGAAATCTATTTGTGATTA TTTCCCTAATTTATCTGTTGATGACAAAGGAGATGATATTTTTGATAGAATAGTA AAAGCGTATTCGGAGGTTAAAGACTTGTTGAATTCTCCGTATCCGTCAGATAAA AACCTTGCTCAAGAAGATGATGATATTGATAAGATTAAAAATCTTTTAGAGTCAA TGAAAGATCTTCAGAAGTTTGTGAAACCTCTCTGTGGAAAAGGAAATGAATCTG ATAAAGATGAGCGTTTCTATGGTGAGTTTACGGCTTTATATGAAGAATTAGACA AGATAACACCATTATATAATATGGTGAGAAATTATCTTACTCGCAAACCGTATTC TACGGAAAAGATAAAGTTAAACTTTGACAATGCTCAACTTTTGAATGGATGGGA TTTAAATAAAGAAAGTGATAATACGAGTGTCATATTGCGTAAAGACGGATTGTAT TATCTTGCCATCATGAACAAGAAGCATAATAAAGTCTTCGAGAAAAATAAATTAC AGTCAGATGGTGTTTGCTTTGAAAAAATGGAGTATAAATTACTTCCTGGTGCAA ACAAGATGCTTCCAAAAGTTTTCTTCTCTAAATCAAGGATAGATGAGTTTGGAC CTTCTCAAAGATTGTTGGACAGTTATCAGAATGAAACTCATAAAAAAGGTGATA AATTCAATATTGAAGATTGCCATGAATTGATAGATTTTTTCAAAAGGTCTATTGA TAAACATGAGGATTGGAGTAAATTTAGCTTTAGTTTCTCAGATACTAAGACATAT GAAGATTTAAGCGGATTTTACAGAGAAGTTGAGCATCAGGGTTATATACTTTCT TTTGTAAATGTTTCTGTAGATTATGTAAATAGTTTGGTAGATGAAGGAAAGATAT ATTTATTTCAAATTTATAATAAAGATTTCTCGCCATTTAGCAAAGGAACTCCAAAT ATGCATACTTTGTATTGGAAAATGCTTTTTGATGAAGAAAATCTGAAAGATGTGG TGTATAAATTGAATGGTCAGGCAGAAGTGTTTTTCAGGAAATCCAGTATAAAGT ATGATAAACCGACTCATCCTGCTAATTTGCCTATTGATAATAAAAATGTATCTAA CCATAAGAAACGGAGTGTCTTTGAGTATGATTTGGTCAAAGATAAGAGATATAC GGTTGATAAATTCCAGTTTCATGTTCCTGTAACAATCAATTTTAAAAGTGATGGA AATGGAAATATCAATCCTCTCGTCAATGATTATATCAAAAAGTCTGATGATTTGC ATGTGATTGGTATCGACAGGGGAGAGCGTCATCTTTTGTATCTTACGGTCATAG ATATGAAAGGTAATATCAAGAAGCAGTTTTCATTGAATGAAATCGTCAATGAATA TAAAGGAAATACATATAGTACCAATTATCATGATTTGTTGGAAAAACGCGAGGA CAAACGTGATAAGGAAAGAAAAGAATGGAAAACTATAGAAACCATCAAGGAGTT GAAAGAAGGTTATCTCAGCCAGGTTATTCATAAAATAACGGAATTGATGGTTGA ATATAATGCAATCATTGTGCTGGAGGATCTTAATTTAGGATTTATGCGTGGGCG ACAAAAGGTGGAGAAGTCTGTTTATCAAAAGTTTGAAAAGATGTTGATTGATAA ACTGAATTATCTTGCTGATAAAAAGAAAGAACCGGAAGATTTGGGTGGTGTGTT GAAGGCATATCAACTGGCAAATAAGTTTGAAAGTTTTCAAAAAATGGGAAAACA ATCAGGTTTCTTATTCTATACCCAAGCATGGAATACAAGTAAGATAGATCCGGT TACTGGTTTTGTTAATCTTTTTGACACACATTATGAGAATATCTTAAAGTCTAAAA ATTTCTTCTCTAAGTTTGATTTGATAAAGTATAATTCTGATAAAGATTGGTTCGA GTTTTCTTTTGATTATAATAATTTTACAACTAAAGCAGAAGGTACAAAAACAAAAT GGACATTATGTACCTTTGGAAATAGAATAATATCATTCCGTAATCCTGATAATAA TATGCAATGGGATGGAAAAGAAATTAATCTTACTGAAGAATTCAAGTTATTCTTT GAGAAATTTGGAATCAATATTAATTCTGATTTGCATGCGGAAATATTAAAACAAG ATAAAAAAGACTTCTTTGAAGGTCTTTTGCATTTGTTGAAATTGACATTGCAGAT GCGTAATAGTAAGACTCGCACTGATATAGATTATATGCAGTCTCCTGTAGCAGA CGAAAACGGAGTGTTATACAATAGTAATAAATGTGGTAAATCCTTGCCAGAAAA TGCTGATGCTAACGGTGCGTATAATATTGCAAGAAAAGGTCTTATGATAATTGA CAAAATAAAGAAGTCTGATAATCTGAATAAAATAGATCTTACGATCTCTAATAAG GAGTGGTTGGTATTCGCACAAAATAAACCATATTTGAAGAATTGA Codon AAGGCCTTCGAGAACTTCACCGGCCTGTATCCCCTCTCTAAAACCCTGAGATTT 119 optimized GAGCTGAAGCCAATCGGCAAGACCCTCGAATACATTGAGAAGCACGGCATCCT coding GGACAAGGACAAGCACAGAGCCAATAGCTACGTGAAGGTGAAGGACATCATC sequence(no GACAGATACCACAAACAGTTCATCGAGGACTCTCTGTCTGATAGCGACTTCAA N-terminal GCTAAAGTACGAGAACAAAGGCAAGAAGGAGAGCCTGGAAGAGTACTTCTACT methionine,no ACTACAAGCTGCGGAACCGGGATGATAAGCAAAAGAAAGATTTTGATGAGATC stopcodon) CAGAAGAACCTGAGAAAACAAATCGCCAGCCAGCTCAAAAAACAGGACAGATT CAAGCGGATCGACAAGAAAGAACTGATCAAGGAAGATCTGCTGGAGTTCGTGA GCGACGACAATGAAAGAAACCTGATCAACGAGTTCAAGGATTTTACTACATACT TTACCGGCTTCCACGAGAACCGGCAGAACATGTACTCTGATGAGGCCAAGTCC ACCGCCATCGCTTATAGACTGATTCACGAGAATCTGCCTAAGTTCATCGATAAC ATAAGCGTGTTCGAGCGGGTCGCAGCTACAGATGTGGCCGACTGCTTCGCCC AGATCTACTCCGATTTCGAGGAATACCTGAACGTGAACGACATCAGCGAGATC TTCAGACTGGACTACTATACAGAAATCCTGACCCAGACCCAGATCGACGCCTA CAATCTGATCATTGGCGGCAGAAGCGAGGGCAACATCAAAATTAAAGGCTTGA ACGAGTACATCAATCTGTACAACCAGCAGCAGAAAGACAAGAGCCAAAGACTG CCCAAGCTGAAGAGCCTGTTTAAACAGATCCTGAGCGACAGAAATGCCATATC TTGGTTGCCTGAGTCTTTCGAGAACGATAACCAGCTGCTGGAGAAGCTGGAGA GCTGCTACCAGAGCTTCAACGAAACCTACGACGACAAGAAGTCTATCTTTGTTA GATTTAGAGAACTGCTGCTGACAATCTCTGACTACGAGATGGACAAAATCTTCC TGAGAAATGACCTGCAGCTGACCGACATCTCCCAAAAAATGTTCGGATCTTACA GCATCATCTCCCGGAGCCTGTTAGAGGATCTCAAGAGAGGAACCAGCCGGAA GTCAAAGAAGGAAACAGACGAGAGCTTCGAAGAACGGCTGCGCAACATTATCA AGAATCAGGACTCCTTTGCCATCGGCACCATCGATAGCAGCCTGCAGCAGATG GACGTGGAAGAGTACAAGAAATCCATCTGCGACTATTTCCCTAATCTGAGTGTT GACGACAAGGGCGATGACATATTTGACAGAATCGTGAAAGCCTATAGCGAGGT GAAGGACCTGCTGAACTCCCCTTACCCTAGCGACAAGAACCTGGCTCAGGAG GACGACGACATCGACAAGATCAAAAACCTGCTGGAAAGCATGAAGGACCTGCA GAAGTTCGTCAAGCCTCTGTGTGGCAAGGGCAACGAGAGCGATAAGGATGAA AGGTTCTACGGCGAGTTCACAGCCCTGTACGAGGAACTGGACAAGATCACCCC TCTGTACAATATGGTGCGGAACTACCTGACAAGAAAGCCATACTCTACCGAGA AGATCAAACTGAACTTCGACAACGCCCAGCTGCTGAACGGATGGGACCTGAAT AAAGAGAGCGACAACACCAGCGTCATCCTGCGTAAGGATGGCCTGTACTACCT GGCCATCATGAACAAGAAGCACAACAAGGTGTTCGAGAAGAACAAGCTCCAAA GCGATGGCGTGTGCTTCGAGAAGATGGAGTACAAGCTGCTGCCTGGCGCCAA CAAGATGCTGCCAAAGGTGTTCTTCTCTAAGAGCAGAATCGATGAGTTCGGCC CTTCTCAGAGACTGCTGGACAGCTACCAGAACGAAACCCACAAGAAGGGCGA CAAATTCAACATCGAGGACTGTCACGAGCTGATCGACTTTTTCAAAAGAAGCAT CGACAAACATGAAGATTGGAGCAAGTTTTCTTTTAGCTTCAGCGACACCAAGAC CTACGAGGACCTGAGCGGCTTCTACAGAGAAGTAGAACACCAGGGCTACATCC TGAGCTTTGTGAACGTGAGCGTGGATTACGTGAACAGCCTGGTGGACGAGGG AAAGATCTACTTATTTCAGATCTACAACAAGGATTTCAGCCCTTTCTCTAAGGG CACCCCTAACATGCACACACTGTACTGGAAGATGCTGTTCGACGAGGAAAACC TGAAGGATGTGGTGTACAAGCTGAATGGCCAGGCCGAAGTGTTCTTCAGAAAG TCCTCTATCAAGTACGACAAACCTACCCATCCTGCCAATCTCCCCATCGATAAC AAGAACGTGAGCAACCACAAGAAGCGGAGCGTGTTCGAGTACGACCTGGTGA AGGACAAACGTTACACCGTGGATAAGTTCCAGTTCCACGTGCCCGTGACCATC AACTTCAAGAGCGATGGCAACGGCAATATCAACCCCCTGGTGAACGACTACAT CAAGAAGAGCGACGATCTACACGTGATCGGCATCGACAGAGGAGAACGGCAC CTGCTGTACCTGACGGTGATCGACATGAAGGGCAACATCAAGAAACAATTTAG CCTGAACGAGATCGTGAACGAATATAAGGGCAATACCTACAGCACCAACTACC ACGACCTGCTGGAGAAACGGGAAGATAAGAGAGATAAGGAGAGAAAGGAATG GAAAACCATTGAAACAATCAAGGAACTGAAAGAAGGATATCTGAGCCAGGTGA TCCACAAGATCACCGAGCTGATGGTGGAGTACAACGCCATCATCGTCCTGGAG GACCTGAACCTGGGCTTCATGAGAGGGAGACAGAAGGTGGAGAAGTCCGTAT ACCAGAAATTTGAAAAGATGCTGATCGACAAGCTGAACTACCTGGCTGACAAG AAAAAGGAACCTGAGGACCTTGGAGGCGTCCTGAAGGCCTACCAGCTGGCCA ACAAATTCGAATCTTTCCAAAAGATGGGCAAACAGAGCGGCTTTCTGTTTTACA CCCAGGCTTGGAACACCAGCAAGATCGACCCCGTGACGGGCTTCGTGAACCT CTTCGATACACATTACGAGAACATCCTGAAGAGCAAGAATTTCTTCAGCAAGTT CGATCTCATCAAATATAACAGCGATAAAGATTGGTTCGAGTTCTCGTTCGACTA CAACAATTTCACCACCAAGGCCGAGGGCACCAAAACAAAGTGGACACTGTGCA CCTTCGGAAACAGAATCATCAGCTTTAGAAACCCTGACAACAACATGCAGTGG GATGGCAAGGAGATCAACCTGACAGAGGAGTTCAAGCTGTTCTTCGAGAAGTT CGGCATCAACATCAACTCCGACCTGCACGCTGAGATCCTGAAGCAAGACAAGA AGGACTTCTTCGAGGGCCTGCTGCACCTGCTGAAACTGACACTCCAGATGCGG AACAGCAAGACGAGGACCGATATCGACTACATGCAGAGCCCCGTGGCCGACG AGAATGGGGTGCTGTACAACTCCAACAAATGCGGCAAGAGCCTGCCCGAGAA CGCCGATGCCAACGGAGCCTACAACATCGCTAGAAAGGGACTGATGATCATTG ACAAGATCAAGAAGTCTGACAACCTGAACAAGATCGATCTGACTATCTCTAACA AGGAATGGCTGGTGTTCGCCCAGAACAAGCCTTACCTGAAAAAT Expression ATGggctccggaAAGGCCTTCGAGAACTTCACCGGCCTGTATCCCCTCTCTAAAAC 120 construct(with CCTGAGATTTGAGCTGAAGCCAATCGGCAAGACCCTCGAATACATTGAGAAGC N-terminal ACGGCATCCTGGACAAGGACAAGCACAGAGCCAATAGCTACGTGAAGGTGAA methionine GGACATCATCGACAGATACCACAAACAGTTCATCGAGGACTCTCTGTCTGATA andstop GCGACTTCAAGCTAAAGTACGAGAACAAAGGCAAGAAGGAGAGCCTGGAAGA codon, GTACTTCTACTACTACAAGCTGCGGAACCGGGATGATAAGCAAAAGAAAGATTT includesV5- TGATGAGATCCAGAAGAACCTGAGAAAACAAATCGCCAGCCAGCTCAAAAAAC tagandC- AGGACAGATTCAAGCGGATCGACAAGAAAGAACTGATCAAGGAAGATCTGCTG terminalNLS) GAGTTCGTGAGCGACGACAATGAAAGAAACCTGATCAACGAGTTCAAGGATTT TACTACATACTTTACCGGCTTCCACGAGAACCGGCAGAACATGTACTCTGATGA GGCCAAGTCCACCGCCATCGCTTATAGACTGATTCACGAGAATCTGCCTAAGT TCATCGATAACATAAGCGTGTTCGAGCGGGTCGCAGCTACAGATGTGGCCGAC TGCTTCGCCCAGATCTACTCCGATTTCGAGGAATACCTGAACGTGAACGACAT CAGCGAGATCTTCAGACTGGACTACTATACAGAAATCCTGACCCAGACCCAGA TCGACGCCTACAATCTGATCATTGGCGGCAGAAGCGAGGGCAACATCAAAATT AAAGGCTTGAACGAGTACATCAATCTGTACAACCAGCAGCAGAAAGACAAGAG CCAAAGACTGCCCAAGCTGAAGAGCCTGTTTAAACAGATCCTGAGCGACAGAA ATGCCATATCTTGGTTGCCTGAGTCTTTCGAGAACGATAACCAGCTGCTGGAG AAGCTGGAGAGCTGCTACCAGAGCTTCAACGAAACCTACGACGACAAGAAGTC TATCTTTGTTAGATTTAGAGAACTGCTGCTGACAATCTCTGACTACGAGATGGA CAAAATCTTCCTGAGAAATGACCTGCAGCTGACCGACATCTCCCAAAAAATGTT CGGATCTTACAGCATCATCTCCCGGAGCCTGTTAGAGGATCTCAAGAGAGGAA CCAGCCGGAAGTCAAAGAAGGAAACAGACGAGAGCTTCGAAGAACGGCTGCG CAACATTATCAAGAATCAGGACTCCTTTGCCATCGGCACCATCGATAGCAGCCT GCAGCAGATGGACGTGGAAGAGTACAAGAAATCCATCTGCGACTATTTCCCTA ATCTGAGTGTTGACGACAAGGGCGATGACATATTTGACAGAATCGTGAAAGCC TATAGCGAGGTGAAGGACCTGCTGAACTCCCCTTACCCTAGCGACAAGAACCT GGCTCAGGAGGACGACGACATCGACAAGATCAAAAACCTGCTGGAAAGCATG AAGGACCTGCAGAAGTTCGTCAAGCCTCTGTGTGGCAAGGGCAACGAGAGCG ATAAGGATGAAAGGTTCTACGGCGAGTTCACAGCCCTGTACGAGGAACTGGAC AAGATCACCCCTCTGTACAATATGGTGCGGAACTACCTGACAAGAAAGCCATA CTCTACCGAGAAGATCAAACTGAACTTCGACAACGCCCAGCTGCTGAACGGAT GGGACCTGAATAAAGAGAGCGACAACACCAGCGTCATCCTGCGTAAGGATGG CCTGTACTACCTGGCCATCATGAACAAGAAGCACAACAAGGTGTTCGAGAAGA ACAAGCTCCAAAGCGATGGCGTGTGCTTCGAGAAGATGGAGTACAAGCTGCTG CCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTCTCTAAGAGCAGAATCGA TGAGTTCGGCCCTTCTCAGAGACTGCTGGACAGCTACCAGAACGAAACCCACA AGAAGGGCGACAAATTCAACATCGAGGACTGTCACGAGCTGATCGACTTTTTC AAAAGAAGCATCGACAAACATGAAGATTGGAGCAAGTTTTCTTTTAGCTTCAGC GACACCAAGACCTACGAGGACCTGAGCGGCTTCTACAGAGAAGTAGAACACCA GGGCTACATCCTGAGCTTTGTGAACGTGAGCGTGGATTACGTGAACAGCCTGG TGGACGAGGGAAAGATCTACTTATTTCAGATCTACAACAAGGATTTCAGCCCTT TCTCTAAGGGCACCCCTAACATGCACACACTGTACTGGAAGATGCTGTTCGAC GAGGAAAACCTGAAGGATGTGGTGTACAAGCTGAATGGCCAGGCCGAAGTGT TCTTCAGAAAGTCCTCTATCAAGTACGACAAACCTACCCATCCTGCCAATCTCC CCATCGATAACAAGAACGTGAGCAACCACAAGAAGCGGAGCGTGTTCGAGTAC GACCTGGTGAAGGACAAACGTTACACCGTGGATAAGTTCCAGTTCCACGTGCC CGTGACCATCAACTTCAAGAGCGATGGCAACGGCAATATCAACCCCCTGGTGA ACGACTACATCAAGAAGAGCGACGATCTACACGTGATCGGCATCGACAGAGGA GAACGGCACCTGCTGTACCTGACGGTGATCGACATGAAGGGCAACATCAAGAA ACAATTTAGCCTGAACGAGATCGTGAACGAATATAAGGGCAATACCTACAGCA CCAACTACCACGACCTGCTGGAGAAACGGGAAGATAAGAGAGATAAGGAGAG AAAGGAATGGAAAACCATTGAAACAATCAAGGAACTGAAAGAAGGATATCTGA GCCAGGTGATCCACAAGATCACCGAGCTGATGGTGGAGTACAACGCCATCATC GTCCTGGAGGACCTGAACCTGGGCTTCATGAGAGGGAGACAGAAGGTGGAGA AGTCCGTATACCAGAAATTTGAAAAGATGCTGATCGACAAGCTGAACTACCTGG CTGACAAGAAAAAGGAACCTGAGGACCTTGGAGGCGTCCTGAAGGCCTACCA GCTGGCCAACAAATTCGAATCTTTCCAAAAGATGGGCAAACAGAGCGGCTTTC TGTTTTACACCCAGGCTTGGAACACCAGCAAGATCGACCCCGTGACGGGCTTC GTGAACCTCTTCGATACACATTACGAGAACATCCTGAAGAGCAAGAATTTCTTC AGCAAGTTCGATCTCATCAAATATAACAGCGATAAAGATTGGTTCGAGTTCTCG TTCGACTACAACAATTTCACCACCAAGGCCGAGGGCACCAAAACAAAGTGGAC ACTGTGCACCTTCGGAAACAGAATCATCAGCTTTAGAAACCCTGACAACAACAT GCAGTGGGATGGCAAGGAGATCAACCTGACAGAGGAGTTCAAGCTGTTCTTCG AGAAGTTCGGCATCAACATCAACTCCGACCTGCACGCTGAGATCCTGAAGCAA GACAAGAAGGACTTCTTCGAGGGCCTGCTGCACCTGCTGAAACTGACACTCCA GATGCGGAACAGCAAGACGAGGACCGATATCGACTACATGCAGAGCCCCGTG GCCGACGAGAATGGGGTGCTGTACAACTCCAACAAATGCGGCAAGAGCCTGC CCGAGAACGCCGATGCCAACGGAGCCTACAACATCGCTAGAAAGGGACTGAT GATCATTGACAAGATCAAGAAGTCTGACAACCTGAACAAGATCGATCTGACTAT CTCTAACAAGGAATGGCTGGTGTTCGCCCAGAACAAGCCTTACCTGAAAAATtct agaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAA GGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTG A

[0145] In some embodiments a ZRXE Type V Cas protein comprises an amino acid sequence of SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117. In some embodiments, a ZRXE Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D862 substitution, wherein the position of the D862 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 116 (corresponding to amino acid 908 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E955 substitution, wherein the position of the E955 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 116 (corresponding to amino acid 993 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1167 substitution, wherein the position of the R1167 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 116 (corresponding to amino acid 1226 of SEQ ID NO: 121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1204 substitution, wherein the position of the D1204 substitution is defined with respect to the amino acid numbering of SEQ ID NO: 116 (corresponding to amino acid 1263 of SEQ ID NO: 121). In some embodiments, a ZRXE Type V Cas protein is catalytically inactive, for example due to a R1167 substitution in combination with a D862 substitution, a E955 substitution, and/or D1204 substitution.

6.2.21. Fusion and Chimeric Proteins

[0146] The disclosure provides Type V Cas proteins, e.g., a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, and a ZRXE Type V Cas protein, which are in the form of fusion proteins comprising a Type V Cas protein sequence fused with one or more additional amino acid sequences, such as one or more nuclear localization signals and/or one or more non-native tags. Fusion proteins can also comprise an amino acid sequence of, for example, a nucleoside deaminase, a reverse transcriptase, a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., Krppel associated box (KRAB)), a histone-modifying protein, an integrase, or a recombinase. Fusion proteins can include linker sequences joining different portions of the fusion protein. For example, glycine-serine linkers such as GS, SG, or GS or SG repeats, (e.g., GSGS (SEQ ID NO:259)). In some embodiments, one or more fusion partners (e.g., an adenosine deaminase or cytidine deaminase) is/are positioned N-terminal to a Type V Cas protein sequence. In some embodiments, one or more fusion partners (e.g., an adenosine deaminase or cytidine deaminase) is/are positioned C-terminal to a Type V Cas protein sequence.

[0147] In some embodiments, a fusion protein of the disclosure comprises a means for localizing the Type V Cas protein to the nucleus, for example a nuclear localization signal.

[0148] Non-limiting examples of nuclear localization signals include KRTADGSEFESPKKKRKV (SEQ ID NO: 122), PKKKRKV (SEQ ID NO: 123), PKKKRRV (SEQ ID NO: 124), KRPAATKKAGQAKKKK (SEQ ID NO: 125), YGRKKRRQRRR (SEQ ID NO: 126), RKKRRQRRR (SEQ ID NO: 127), PAAKRVKLD (SEQ ID NO: 128), RQRRNELKRSP (SEQ ID NO: 129), VSRKRPRP (SEQ ID NO: 130), PPKKARED (SEQ ID NO: 131), PQPKKKPL (SEQ ID NO: 132), SALIKKKKKMAP (SEQ ID NO: 133), PKQKKRK (SEQ ID NO: 134), RKLKKKIKKL (SEQ ID NO: 135), REKKKFLKRR (SEQ ID NO: 136), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 137), RKCLQAGMNLEARKTKK (SEQ ID NO:138), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 139), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:140), and SSDDEATADSQHAAPPKKKRKV (SEQ ID NO: 178). Additional non-limiting examples of nuclear localization signals include PKKKRKVG (SEQ ID NO:179) and GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO:180).

[0149] Exemplary fusion partners include protein tags (e.g., V5-tag (e.g., having the sequence GKPIPNPLLGLDST (SEQ ID NO:141) or IPNPLLGLD (SEQ ID NO:142)), FLAG-tag, myc-tag, HA-tag, GST-tag, polyHis-tag, MBP-tag), protein domains, transcription modulators, enzymes acting on small molecule substrates, DNA, RNA and protein modification enzymes (e.g., adenosine deaminase, cytidine deaminase, guanosyl transferase, DNA methyltransferase, RNA methyltransferases, DNA demethylases, RNA demethylases, dioxygenases, polyadenylate polymerases, pseudouridine synthases, acetyltransferases, deacetylase, ubiquitin-ligases, deubiquitinases, kinases, phosphatases, NEDD8-ligases, de-NEDDylases, SUMO-ligases, deSUMOylases, histone deacetylases, reverse transcriptases, histone acetyltransferases histone methyltransferases, histone demethylases), protein DNA binding domains, RNA binding proteins, polypeptide sequences with specific biological functions (e.g., nuclear localization signals, mitochondrial localization signals, plastid localization signals, subcellular localization signals, destabilizing signals, Geminin destruction box motifs), and biological tethering domains (e.g., MS2, Csy4 and lambda N protein). Various Type V Cas fusion proteins are described in Ribeiro et al., 2018, In. J. Genomics, Article ID: 1652567; Jayavaradhan, et al., 2019, Nat Commun 10:2866; Xiao et al., 2019, The CRISPR Journal, 2 (1): 51-63; Mali et al., 2013, Nat Methods. 10 (10): 957-63; U.S. Pat. Nos. 9,322,037, and 9,388,430. In some embodiments, a fusion partner is an adenosine deaminase. An exemplary adenosine deaminase is the tRNA adenosine deaminase (TadA) moiety contained in the adenine base editor ABE8e (Richter, 2020, Nature Biotechnology 38:883-891). The TadA moiety of ABE8e comprises the following amino acid sequence:

TABLE-US-00022 (SEQIDNO:143) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS RIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALL CDFYRMPRQVFNAQKKAQSSIN

[0150] In some embodiments, an adenosine deaminase fusion partner comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% amino acid sequence identity with SEQ ID NO: 143.

[0151] Type V Cas proteins of the disclosure in the form of a fusion protein comprising an adenosine deaminase can be used, for example, as an adenine base editor (ABE) to change an A to a G in DNA. Type V Cas proteins of the disclosure in the form of a fusion protein comprising a cytidine deaminase can be used, for example, as a cytosine base editor (CBE) to change a C to a T in DNA.

[0152] In some embodiments, a fusion protein of the disclosure comprises a means for deaminating adenosine, for example an adenosine deaminase, e.g., a TadA variant. In some embodiments, a fusion protein of the disclosure comprises a means for deaminating cytidine, for example a cytidine deaminase, e.g., cytidine deaminase 1 (CDA1) or an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase (see, e.g., Cheng et al., 2019, Nat Commun. 10 (1): 3612; Gehrke et al., 2018, Nat Biotechnol. 36 (10): 977-982; Komor et al., 2016, Nature 533 (7603): 420-424, Porto and Komor, 2023, PLOS Biol 21 (4): e3002071, the contents of each of which are incorporated herein by reference in their entireties).

[0153] Exemplary deaminases that can be used in fusion proteins of the disclosure are set forth in Table 2.

TABLE-US-00023 TABLE2 Addgene catalog#/ SEQID DOI Name AminoAcidSequence NO Note reference APOBEC1 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKET 214 #87437 CLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFT TERYFCPNTRCSITWFLSWSPCGECSRAITEFLSR YPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQI MTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQS CHYQRLPPHILWATGLK evoAPOBEC SSKTGPVAVDPTLRRRIEPHEFEVFFDPRELRKET 215 APOBEC1 #122611 CLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFT E4KH109N TERYFCPNTRCSITWFLSWSPCGECSRAITEFLSR H122L YPNVTLFIYIARLYHLANPRNRQGLRDLISSGVTIQI D124N MTEQESGYCWHNFVNYSPSNESHWPRYPHLWV R154H RLYVLELYCIILGLPPCLNILRRKQSQLTSFTIALQS A165SP201S CHYQRLPPHILWATGLK F205S YE1 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKET 216 APOBEC1 #138155 CLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFT W90Y TERYFCPNTRCSITWFLSYSPCGECSRAITEFLSR R126E YPHVTLFIYIARLYHHADPENRQGLRDLISSGVTIQI MTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQS CHYQRLPPHILWATGLK FERNY SFERNYDPRELRKETYLLYEIKWGKSGKLWRHWC 217 #157944 QNNRTQHAEVYFLENIFNARRFNPSTHCSITWYLS WSPCAECSQKIVDFLKEHPNVNLEIYVARLYYHED ERNRQGLRDLVNSGVTIRIMDLPDYNYCWKTFVS DQGGDEDYWPGHFAPWIKQYSLKL ppAPOBEC1 TSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKE 218 #138349 TCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKF TSERRFHSSISCSITWFLSWSPCWECSQAIREFLS QHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSG VTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPP LWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRL HLQNCHYQTIPPHILLATGLIHPSVTWRLK amAPOBEC1 ADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCE 219 #138342 IKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFK AKKHPVHCYVTWYLSWSPCADCASKIVKFLEERP YLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVM DISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKE NYSRLLDIFWESKCRSPNPW Anc689 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKET 220 #163526 CLLYEIKWGTSHKIWRHSSKNTTKHVEVNFIEKFT SERHFCPSTSCSITWFLSWSPCGECSKAITEFLSQ HPNVTLVIYVARLYHHMDQQNRQGLRDLVNSGVT IQIMTAPEYDYCWRNFVNYPPGKEAHWPRYPPLW MKLYALELHAGILGLPPCLNILRRKQPQLTFFTIALQ SCHYQRLPPHILWATGLK APOBEC EASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYE 221 #113410 A3A VERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGR HAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW GCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEA LQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCP FQPWDGLDEHSQALSGRLRAILQNQGN APOBEC3 EASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYE 222 APOBEC #131315 eA3A VERLDNGTSVKMDQHRGFLHGQAKNLLCGFYGR A3AN57G HAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW GCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEA LQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCP FQPWDGLDEHSQALSGRLRAILQNQGN APOBEC NPQIRNPMERMYRDTFYDNFENEPILYGRSYTWL 223 #113411 A3B CYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEM CFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAK LAEFLSEHPNVTLTISAARLYYYWERDYRRALCRL SQAGARVKIMDYEEFAYCWENFVYNEGQQFMPW YKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDP LVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLC NEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYR VTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIF AARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEY CWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAI LQNQGN APOBEC NPQIRNPMKAMYPGTFYFQFKNLWEANDRNETW 224 #113412 A3C LCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAE #119136 RCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCA GEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLR SLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKP WKGLKTNFRLLKRRLRESLQ APOBEC NPQIRNPMERMYRDTFYDNFENEPILYGRSYTWL 225 #119137 A3D CYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQ EVYFRFENHAEMCFLSWFCGNRLPANRRFQITWF VSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYY RDRDWRWVLLRLHKAGARVKIMDYEDFAYCWEN FVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPM EAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTK HHSAVFRKRGVFRNQVDPETHCHAERCFLSWFC DDILSPNTNYEVTWYTSWSPCPECAGEVAEFLAR HSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGAS VKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNF RLLKRRLREILQ APOBEC KPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWL 226 #119138 A3F CYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMC FLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKL AEFLAEHPNVTLTISAARLYYYWERDYRRALCRLS QAGARVKIMDDEEFAYCWENFVYSEGQPFMPWY KFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKN LRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFR NQVDPETHCHAERCFLSWFCDDILSPNTNYEVTW YTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYY FWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWE NFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE APOBEC KPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWL 227 #119139 A3G CYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMR FFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTR DMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRS LCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRE LFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNF NNEPWVRGRHETYLCYEVERMHNDTWVLLNQRR GFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDL DQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSL CIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEF KHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRL RAILQNQEN APOBEC ALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTP 228 #119140 A3H QNGSTPTRGYFENKKKCHAEICFINEIKSMGLDET QCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLG IFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFP EFADCWENFVDHEKPLSFNPYKMLEELDKNSRAI KRRLERIKQS RrA3F KPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRN 229 #138340 ETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHC HAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCP ECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQE GLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEP FKRWKGLKYNFQSLTRRLREILQ ss-APOBEC- DPQRLRQWPGPGPASRGGYGQRPRIRNPEEWF 230 #138343 3b HELSPRTFSFHFRNLRFASGRNRSYICCQVEGKN CFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSP DEHYYVTWFISWSPCCECAAKVAQFLEENRNVSL SLSAARLYYFWKSESREGLRRLSDLGAQVGIMSF QDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVT ELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSH SHTRSEAHLRPNHSSRQHRILNPPREARARTCVL VDASWICYR AID DSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKR 231 #100803 RDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPN LSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIM TFKDYFYCWNTFVENHERTFKAWEGLHENSVRLS RQLRRILLPLYEVDDLRDAFRTLGL AIDmono DPATFTYQFKNVRWAKGRRETYLCYVVKRRDSAT 232 DOI: SFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGR 10.1016/j. CYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRI celrep.2018. FTARLYFCEDRKAEPEGLRRLAEAGVQIAIMTFKD 09.090 YFYCWNTFVENHERTFKAWEGLHENSVRLSRQL RRILQ AID-3c DPATFTYQFKNVRWAKGRRETYLCYVVKRRDSAT 233 DOI: SFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGR 10.1016/j. CYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRI celrep.2018. FTARLYYFQYPCYQEGLRRLHRAGVQIAIMTFKDY 09.090 FYCWNTFVENHERTFKAWEGLHENSVRLSRQLR RILQ AID-3f DPATFTYQFKNVRWAKGRRETYLCYVVKRRDSAT 234 DOI: SFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGR 10.1016/j. CYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRI celrep.2018. FTARLYYFWDTDYQEGLRRLHRAGVQIAIMTFKDY 09.090 FYCWNTFVENHERTFKAWEGLHENSVRLSRQLR RILQ PmCDA1 TDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLF 235 #100804 ELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSI RKVEEYLRDNPGQFTINWYSSWSPCADCAEKILE WYNQELRGNGHTLKIWACKLYYEKNARNQIGLWN LRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNEN RWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV ABE7.10 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 236 TadA+TadA* #102919 VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL (with VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRV linker) VFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI LADECAALLSDFFRMRRQEIKAQKKAQSSTDSG GSSGGSSGSETPGTSESATPESSGGSSGGSSEV EFSHEYWMRHALTLAKRARDEREVPVGAVLVLNN RVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQN YRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR NAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC AALLCYFFRMPRQVFNAQKKAQSSTD ABE8e SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 237 #138489 LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVF GVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA DECAALLCDFYRMPRQVFNAQKKAQSSIN miniABE7.10 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 238 DOI: LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV 10.1038/ MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVF s41587- GVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILA 019-0236-6 DECAALLCYFFRMPRQVFNAQKKAQSSTD ABE6.3 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 239 TadA+TadA* #102916 VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL (with VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRV linker) VFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI LADECAALLSDFFRMRRQEIKAQKKAQSSTDSG GSSGGSSGSETPGTSESATPESSGGSSGGSSEV EFSHEYWMRHALTLAKRAWDEREVPVGAVLVLN NRVIGEGWNRSIGLHDPTAHAEIMALRQGGLVMQ NYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGV RNAKTGAAGSLMDVLHYPGMNHRVEITEGILADE CAALLCYFFRMRRQVFNAQKKAQSSTD ABE7.8 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 240 TadA+TadA* #102917 VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL (with VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRV linker) VFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI LADECAALLSDFFRMRRQEIKAQKKAQSSTDSG GSSGGSSGSETPGTSESATPESSGGSSGGSSEV EFSHEYWMRHALTLAKRALDEREVPVGAVLVLNN RVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQN YRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR NAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC NALLCYFFRMRRQVFNAQKKAQSSTD ABE7.9 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 241 TadA+TadA* #194843 VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL (with VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRV linker) VFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI LADECAALLSDFFRMRRQEIKAQKKAQSSTDSG GSSGGSSGSETPGTSESATPESSGGSSGGSSEV EFSHEYWMRHALTLAKRALDEREVPVGAVLVLNN RVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQN YRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR NAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC NALLCYFFRMPRQVFNAQKKAQSSTD ABE8.8-m SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 242 ABE8variant DOI: LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV 10.1038/ MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVF s41587-020- GVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA 0491-6 DECAALLCRFFRMPRRVFNAQKKAQSSTD ABE8.8-d SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 243 ABE8variant DOI: VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL 10.1038/ VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV s41587-020- FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGS SGGSSGSETPGTSESATPESSGGSSGGSSEVEF SHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR LIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA KTGAAGSLMDVLHHPGMNHRVEITEGILADECAAL LCRFFRMPRRVFNAQKKAQSSTD ABE8.13-m SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 244 ABE8variant DOI: LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV 10.1038/ MQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVV s41587-020- FGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLCRFFRMPRRVFNAQKKAQSSTD ABE8.13-d SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 245 ABE8variant DOI: VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL 10.1038/ VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV s41587-020- FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGS SGGSSGSETPGTSESATPESSGGSSGGSSEVEF SHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR LYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA KTGAAGSLMDVLHHPGMNHRVEITEGILADECAAL LCRFFRMPRRVFNAQKKAQSSTD ABE8.17-m SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 246 ABE8variant DOI: LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV 10.1038/ MQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVF s41587-020- GVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILA 0491-6 DECAALLCYFFRMPRRVFNAQKKAQSSTD ABE8.17-d SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 247 ABE8variant DOI: VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL 10.1038/ VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV s41587-020- FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGS SGGSSGSETPGTSESATPESSGGSSGGSSEVEF SHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR LIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNA KTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL LCYFFRMPRRVFNAQKKAQSSTD ABE8.20-m SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 248 ABE8variant DOI: LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV 10.1038/ MQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVV s41587-020- FGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLCRFFRMPRRVFNAQKKAQSSTD ABE8.20-d SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVL 249 ABE8variant DOI: VHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGL 10.1038/ VMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV s41587-020- FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGIL 0491-6 ADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGS SGGSSGSETPGTSESATPESSGGSSGGSSEVEF SHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR LYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNA KTGAAGSLMDVLHHPGMNHRVEITEGILADECAAL LCRFFRMPRRVFNAQKKAQSSTD

[0154] In some embodiments, a deaminase fusion partner comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in Table 2. The amino acid sequences shown in Table 2 are shown without an N-terminal methionine; an N-terminal methionine can be added, for example when the deaminase amino acid sequence is at the N-terminal end of the molecule.

[0155] In some embodiments, a fusion protein of the disclosure comprises a deaminase, e.g., as described in Table 2 and a uracil glycosylase inhibitor (UGI) domain (e.g., as described in Wu et al., 2022, Mol. Cell 82 (23): 4487-4502, the contents of which are incorporated herein by reference in their entireties.) An exemplary UGI domain comprises the amino acid sequence

TABLE-US-00024 (SEQIDNO:250) TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDE STDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0156] Type V Cas proteins of the disclosure in the form of a fusion protein comprising a transcriptional repressor or an effector domain thereof can be used, for example, to silence genes via epigenome editing (see, e.g., Cappelluti et al., 2024 Nature 627:416-423, the contents of which are incorporated herein by reference in their entireties). Exemplary effector domains are described in Table 3.

TABLE-US-00025 TABLE3 SEQID Name AminoAcidSequence NO KRAB ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQI 251 VYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKS SV KRAB SRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLE 252 alternative KGEEPWLV cdDNMT3A GTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIAT 253 GLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFD LVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVV AMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLEL QECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPV HYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV DNMT3L AAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQ 254 VHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCY CFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRE SENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDT VRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFW MFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVS EEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL DNMT3A- NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSI 255 DNMT3L TVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEG dimer TGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSN SIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSW SVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEA EPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFE GGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLV GPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFE TVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWG PFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNK EDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQN KQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL

[0157] In some embodiments, an effector domain fusion partner comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in Table 3. The amino acid sequences shown in Table 3 are shown without an N-terminal methionine; an N-terminal methionine can be added, for example when the effector domain amino acid sequence is at the N-terminal end of the molecule.

[0158] In some embodiments, a fusion protein of the disclosure comprises a means for synthesizing DNA from a single-stranded template, for example a reverse transcriptase, e.g., a MMLV reverse transcriptase (see, WO 2021/226558, the contents of which are incorporated herein by reference in their entireties). An exemplary reverse transcriptase comprises the amino acid sequence

TABLE-US-00026 (SEQIDNO:256) TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPP SHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQ ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQ RAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSII HCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP(see,Chenetal.,2021, Cell184(22):5635-5652,thecontentsofwhichareincorporatedhereinby referenceintheirentireties).

[0159] Another exemplary reverse transcriptase comprises the amino acid sequence

TABLE-US-00027 (SEQIDNO:257) ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVK MQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNVYPLPLIEQLLAKIQGST IFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDI LIHSKSESEHVKHVKDVLQKLKNANLIINQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQPKNQ KELRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLE TDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTD HRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQI SIS(see,Domanetal.,2023,Cell186(18):3983-4002,thecontentsof whichareincorporatedhereinbyreferenceintheirentireties).

[0160] In some embodiments, a reverse transcriptase fusion partner comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:256 or SEQ ID NO:257.

[0161] Type V Cas proteins of the disclosure in the form of a fusion protein comprising a reverse transcriptase (RT) can be used as a prime editor to carry out precise DNA editing without double-stranded DNA breaks.

[0162] In some embodiments, a Type V Cas protein described herein can be used for prime editing, e.g., with different Circular RNA-mediated Prime Editors (CPEs) for various editing scenarios: for example a nickase-dependent CPE (niCPE), a nuclease-dependent CPE (nuCPE), a split nickase-dependent CPE (sniCPE), or a split nuclease-dependent CPE (snuCPE) (Liang et al., 2004, Nature Biotechnology doi.org/10.1038/s41587-023-02095-x).

[0163] In some embodiments, a fusion protein of the disclosure comprises one or more nuclear localization signals positioned N-terminal and/or C-terminal to a Type V Cas protein sequence (e.g., a Type V Cas protein comprising an amino acid sequence set forth in Section 6.2). In some embodiments, a fusion protein of the disclosure comprises a C-terminal nuclear localization signal, for example having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 122). In some embodiments, a fusion protein of the disclosure comprises a N-terminal nuclear localization signal, for example having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 122). In some embodiments, a fusion protein of the disclosure comprises a N-terminal and a C-terminal nuclear localization signal, for example each having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO:122).

[0164] The disclosure provides chimeric Type V Cas proteins comprising one or more domains of an ZWGD Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZJHK Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZIKV Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZZFT Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an YYAN Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZZGY Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZKBG Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZZKD Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZXPB Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZPPX Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZXHQ Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZQKH Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZRGM Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZTAE Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZSQQ Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZSYN Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZRBH Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZWPU Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZZQE Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins); a chimeric Type V Cas proteins comprising one or more domains of an ZRXE Type V Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type V Cas proteins).

[0165] The domain structures of the Type V Cas proteins described herein were inferred by multiple alignment with the amino acid sequences of Type V Cas proteins for which the crystal structure is known and for which it is thus possible to define the boundaries of each functional domain. The domains identified in Type V Cas proteins are: wedge (WED) domain (WED-1 domain, WED-II domain, WED-III domain), the RuvC catalytic domain (discontinuous, represented by RuvC-I domain, RuvC-II domain, RuvCIII domain), recognition (REC) domain (REC1 domain, REC2 domain), PAM-interacting domain (PI domain), bridge helix (BH domain), and nuclease (NUC) domain,

[0166] Table 4 below report the amino acid positions corresponding to the boundaries between different functional domains in full-length wild-type ZWGD (SEQ ID NO:2), ZJHK (SEQ ID NO:8), ZIKV (SEQ ID NO: 14), ZZFT (SEQ ID NO:20), YYAN (SEQ ID NO:26), ZZGY (SEQ ID NO:32), ZKBG (SEQ ID NO:38), ZZKD (SEQ ID NO:44), ZXPB (SEQ ID NO:50), ZPPX (SEQ ID NO:56), ZXHQ (SEQ ID NO:62), ZQKH (SEQ ID NO: 68), ZRGM (SEQ ID NO:74), ZTAE (SEQ ID NO:80), ZSQQ (SEQ ID NO:86), ZSYN (SEQ ID NO: 92), ZRBH (SEQ ID NO:98), ZWPU (SEQ ID NO: 104), ZZQE (SEQ ID NO: 110), and ZRXE (SEQ ID NO: 116) Type V Cas proteins.

TABLE-US-00028 TABLE 4 Amino Acid Positions of Domains of Exemplified Type V Cas Proteins Type V Cas WED-I REC1 REC2 WED-II PI WED-III RuvC-I BH RuvC-II NUC RuvC-III ZRGM 1- 25- 292- 507- 575- 700- 867- 927- 944- 1054- 1236- 24 291 506 574 699 866 926 943 1053 1235 1284 ZZGY 1- 24- 308- 519- 591- 711- 881- 945- 962- 1071- 1253- 23 307 518 590 710 880 944 961 1070 1252 1302 ZRXE 1- 24- 305- 546- 616- 707- 839- 902- 919- 1027- 1203- 23 304 545 615 706 838 901 918 1026 1202 1252 ZRBH 1- 24- 295- 532- 603- 694- 828- 887- 904- 1012- 1188- 23 294 531 602 693 827 886 903 1011 1187 1235 ZSYN 1- 27- 341- 574- 650- 741- 874- 938- 955- 1063- 1238- 26 340 573 649 740 873 937 954 1062 1237 1283 ZKBG 1- 24- 303- 531- 600- 724- 858- 925- 942- 1054- 1233- 23 302 530 599 723 857 924 941 1053 1232 1271 ZXHQ 1- 27- 290- 525- 601- 692- 812- 910- 927- 1040- 1210- 26 289 524 600 691 811 909 926 1039 1209 1262 ZZQE 1- 26- 308- 543- 613- 704- 836- 899- 916- 1024- 1200- 25 307 542 612 703 835 898 915 1023 1199 1249 YYAN 1- 23- 292- 518- 590- 678- 815- 875- 892- 998- 1169- 22 291 517 589 677 814 874 891 997 1168 1215 ZQKH 1- 26- 249- 444- 505- 610- 721- 778- 795- 905- 1090- 25 248 443 504 609 720 777 794 904 1089 1133 ZZFT 1- 24- 297- 525- 596- 699- 830- 896- 913- 1025- 1202- 23 296 524 595 698 829 895 912 1024 1201 1245 ZIKV 1- 24- 282- 497- 565- 668- 791- 846- 863- 971- 1147- 23 281 496 564 667 790 845 862 970 1146 1195 ZWPU 1- 27- 297- 527- 597- 689- 822- 885- 902- 1010- 1194- 26 296 526 596 688 821 884 901 1009 1193 1243 ZPPX 1- 21- 300- 537- 607- 720- 854- 916- 933- 1041- 1216- 20 299 536 606 719 853 915 932 1040 1215 1264 ZZKD 1- 25- 291- 514- 583- 674- 805- 872- 889- 997- 1175- 24 290 513 582 673 804 871 888 996 1174 1220 ZSQQ 1- 27- 310- 549- 618- 721- 888- 953- 970- 1078- 1263- 26 309 548 617 720 887 952 969 1077 1262 1310 ZJHK 1- 25- 286- 516- 586- 711- 877- 934- 951- 1062- 1243- 24 285 515 585 710 876 933 950 1061 1242 1294 ZWGD 1- 31- 311- 564- 639- 733- 868- 937- 954- 1061- 1247- 30 310 563 638 732 867 936 953 1060 1246 1292 ZTAE 1- 23- 323- 551- 625- 716- 882- 937- 954- 1062- 1242- 22 322 550 624 715 881 936 953 1061 1241 1289 ZXPB 1- 23- 276- 505- 575- 666- 798- 853- 870- 978- 1152- 22 275 504 574 665 797 852 869 977 1151 1201

[0167] A chimeric Type V Cas protein can comprise one of more of the following domains (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more) from a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, and/or a ZRXE Type V Cas protein, and one or more domains from one or more other proteins, for example Cas12a: WED-1 domain, REC1 domain, REC2 domain, WED-II domain, PI domain, WED-III domain, RuvC-I domain, BH domain, RuvC-II domain, NUC domain, or RuvC-III domain. For example, the PID domain can be swapped between different Type V Cas proteins to change the PAM specificity of the resulting chimeric protein (which is given by the donor PID domain). Swapping of other domains or portions of them is also within the scope of the disclosure (e.g., through protein shuffling).

[0168] In some embodiments, a Type V Cas protein of the disclosure comprises one, two, three, four, five, six, seven, or eight of a WED-1 domain, REC1 domain, REC2 domain, WED-II domain, PI domain, WED-III domain, RuvC-I domain, BH domain, RuvC-II domain, NUC domain, or RuvC-III domain arranged in the N-terminal to C-terminal direction. In some embodiments, all domains are from one Type

[0169] V Cas protein as described herein, e.g., ZWGD, ZJHK, ZIKV), ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, or ZRXE. In other embodiments, one or more domains (e.g., one domain), e.g., a PID domain, is from another Type V Cas protein, for example a Cas12a protein from Alicyclobacillus acidoterrestris, Bacillus thermoamylovorans, Lachnospiraceae bacterium (e.g., LbCas12a, NCBI Reference Sequence WP_051666128.1), Acidaminococcus sp. BV3L6 (e.g., AsCas12a, NCBI Reference Sequence WP_021736722.1), Arcobacter butzleri L348 (e.g., AbCas12a, GeneBank ID: JAIQ01000039.1), Agathobacter rectalisstrain 2789STDY5834884 (e.g., ArCas12a, GeneBank ID: CZAJ01000001.1), Bacteroidetes oraltaxon 274 str. F0058 (e.g., BoCas12a, GeneBank ID: NZ_GG774890.1), Butyrivibrio sp. NC3005 (e.g., BsCas12a, GeneBank ID: NZ_AUKC01000013.1), Candidate division WS6 bacterium GW2011_GWA2_37_6 US52_C0007 (e.g., C6Cas12a, GeneBank ID: LBTH01000007.1), Helcococcus kunzii ATCC 51366 (e.g., HkCas12a, GeneBank ID: JH601088.1/AGEI01000022.1), Lachnospira pectinoschiza strain 2789STDY5834836 (e.g., LpCas12a, GeneBank ID: CZAK01000004), Oribacterium sp. NK2B42 (e.g., OsCas12a, GeneBank ID: NZ_KE384190.1), Pseudobutyrivibrio ruminis CF1b (e.g., PrCas12a, GeneBank ID: NZ_KE384121.1), Proteocatella sphenisci DSM 23131 (e.g., PsCas12a, GeneBank ID: NZ_KE384028.1), Pseudobutyrivibrio xylanivoransstrain DSM 10317 (e.g., PxCas12a, GeneBank ID: FMWK01000002.1), Sneathia amniistrain SN35 (e.g., SaCas12a, GeneBank ID: CP011280.1), Francisella novicida, or Leptotrichia shahii. In addition, one or more amino acid substitutions can be introduced in one or more domains to modify the properties of the resulting nuclease in terms of editing activity, targeting specificity or PAM recognition specificity. For example, one or more amino acid substitutions can be introduced to provide nickase activity. Exemplary amino acid substitutions in Cas12a providing nickase activity are the D908, E993, R1226 and D1263. Corresponding substitutions can be introduced into the Type V Cas nucleases of the disclosure to provide nickases and catalytically inactive Cas proteins. Positions corresponding to such Cas12a positions for Type V Cas proteins of the disclosure as shown in Table 5. Nickases and catalytically inactive Type V Cas proteins of the disclosure can be used, for example, in base editors comprising a cytosine or adenosine deaminase fusion partner. Catalytically inactive Type V Cas proteins can also be used, for example, as fusion partners for transcriptional activators or repressors.

TABLE-US-00029 TABLE 5 Reference Position Position Position Position SEQ ID NO corresponding corresponding corresponding corresponding defining Type V Cas to D908 of to E993 of to R1226 of to D1263 of amino acid Protein AsCas12a AsCas12a AsCas12a AsCas12a numbering ZWGD 891 990 1200 1248 2 ZJHK 900 987 1203 1244 8 ZIKV 814 899 1111 1148 14 ZZFT 856 949 1166 1203 20 YYAN 838 928 1135 1170 26 ZZGY 905 998 1214 1254 32 ZKBG 885 978 1194 1234 38 ZZKD 828 925 1138 1176 44 ZXPB 821 906 1116 1153 50 ZPPX 877 969 1181 1217 56 ZXHQ 836 963 1172 1211 62 ZQKH 744 831 1048 1091 68 ZRGM 890 980 1194 1237 74 ZTAE 905 990 1206 1243 80 ZSQQ 913 1006 1219 1264 86 ZSYN 902 991 1200 1239 92 ZRBH 851 940 1152 1189 98 ZWPU 845 938 1153 1195 104 ZZQE 859 952 1164 1201 110 ZRXE 862 955 1167 1204 116

6.3. Guide RNAs

[0170] The disclosure provides crRNA scaffolds and gRNA molecules that can be used with Type V Cas proteins of the disclosure to edit genomic DNA, for example mammalian DNA, e.g., human DNA. gRNAs of the disclosure typically comprise a spacer of 15 to 30 nucleotides in length. The spacer can be positioned 3 of a crRNA scaffold to form a full gRNA.

TABLE-US-00030 AnexemplarycrRNAscaffoldsequencethatcanbeusedforZWGDTypeVCasgRNAs comprises. (SEQIDNO:144) ACGAUUAGAAAUAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZJHKTypeVCasgRNAs comprises. (SEQIDNO:145) CUUUGAAAGAAUAUAAUUUCUACUGAAAGUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZIKVTypeVCasgRNAs comprises. (SEQIDNO:146) GUUUAAUAAUAAUACAUAAUUUCUACUAUUGUAGAU. AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZFTTypeVCasgRNAs comprises. (SEQIDNO:147) GUCUAUAAGACUAAUUUAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforYYANTypeVCasgRNAs comprises. (SEQIDNO:148) GUUUAUAAACCUUAUCUAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZGYTypeVCasgRNAs comprises. (SEQIDNO:149) UCUAAAGCUCUUUAAGAAUUUCUACUUUCGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZKBGTypeVCasgRNAs comprises. (SEQIDNO:150) CUAAGAGGCUCAAAUAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZKDTypeVCasgRNAs comprises. (SEQIDNO:151) CCUUUGGAAGUACUAAGAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZKDTypeVCasgRNAs comprises. (SEQIDNO:211) GAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZXPBTypeVCasgRNAs comprises. (SEQIDNO:152) GGCUAUAAAAGCCAUAUAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZPPXTypeVCasgRNAs comprises. (SEQIDNO:153) GACUAUUAAGUCUUUUGAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZXHQTypeVCasgRNAs comprises. (SEQIDNO:154) UCUAGAAUAUAUAGGUAAUUUCUACUUAUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZQKHTypeVCasgRNAs comprises. (SEQIDNO:155) GGCAAUAAGCCAUAUACAAUUUCUACUGUAUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZRGMTypeVCasgRNAs comprises. (SEQIDNO:156) GUCUGAAAGACUAUAUAAUUUCUACUUCGUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZRGMTypeVCasgRNAs comprises. (SEQIDNO:213) AAUUUCUACUUCGUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZTAETypeVCasgRNAs comprises. (SEQIDNO:157) GUCUACGGAACGUCUGUAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZSQQTypeVCasgRNAs comprises. (SEQIDNO:158) UUUAAACGAACUAUUAAAUUUCUACUGUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZSYNTypeVCasgRNAs comprises. (SEQIDNO:159) GUUUAAUACUUAUAUAUAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZRBHTypeVCasgRNAs comprises. (SEQIDNO:160) AAUAAUAAUCCCUUAUAAUUUCUACUUUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZWPUTypeVCasgRNAs comprises. (SEQIDNO:161) GUCUAUAAGACGAACUAAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZQETypeVCasgRNAs comprises. (SEQIDNO:162) GGCUACUAAGCCUUUAUAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZZQETypeVCasgRNAs comprises. (SEQIDNO:212) UAAUUUCUACUAUUGUAGAU AnexemplarycrRNAscaffoldsequencethatcanbeusedforZRXETypeVCasgRNAs comprises. (SEQIDNO:163) GUCUAUAAAGACGAAUGAAUUUCUACUAUUGUAGAU

[0171] Type V Cas gRNAs of the disclosure are generally 40-70 nucleotides long (e.g., 50 to 60 nucleotides long, 55 to 65 nucleotides long, or 55 to 60 nucleotides long), but gRNAs of other lengths are also contemplated. For example, a crRNA scaffold described herein can be trimmed to a shorter length or extended at the 5 end (e.g., as described in Park et al., 2018, Nature Communications, 9:3313), which can be helpful for enhancing gene editing efficacy. Additionally, gRNAs of the disclosure can optionally be chemically modified, which can be useful, for example, to enhance serum stability of a gRNA (see, e.g., Park et al., 2018, Nature Communications, 9:3313). Chemical modifications are further discussed in Section 6.3.2.

[0172] Further optimization of the structure can be obtained by introducing targeted base changes into the stems of the gRNA to increase their stability and folding. Such base changes will preferably correspond to the introduction of G: C couples, which are known to generate the strongest Watson-Crick pairing. For the sake of clarity, these substitutions can consist in the introduction of a G or a C in a specific position of a stem together with a complementary substitution in another position of the gRNA sequence which is predicted to base pair with the former, for example according to available bioinformatic tools for RNA folding such as UNAfold or RNAfold.

[0173] Stem-loop trimming can also be exploited to stabilize desired secondary structures by removing portions of the guide RNA producing unwanted secondary structures through annealing with other regions of the RNA molecule

6.3.1. Spacers

[0174] The spacer sequence is partially or fully complementary to a target sequence found in a genomic DNA sequence, for example a human genomic DNA sequence. For example, a spacer sequence can be partially or fully complementary to a nucleotide sequence in a gene having a disease causing mutation. A spacer that is partially complementary to a target sequence can have, for example, one, two, or three mismatches with the target sequence.

[0175] gRNAs of the disclosure can comprise a spacer that is 15 to 30 nucleotides in length (e.g., 15 to 25, 16 to 24, 17 to 23, 18 to 22, 19 to 21, 18 to 30, 20 to 28, 22 to 26, or 23 to 25 nucleotides in length). In some embodiments, a spacer is 15 nucleotides in length. In other embodiments, a spacer is 16 nucleotides in length. In other embodiments, a spacer is 17 nucleotides in length. In other embodiments, a spacer is 18 nucleotides in length. In other embodiments, a spacer is 19 nucleotides in length. In other embodiments, a spacer is 20 nucleotides in length. In other embodiments, a spacer is 21 nucleotides in length. In other embodiments, a spacer is 22 nucleotides in length. In other embodiments, a spacer is 23 nucleotides in length. In other embodiments, a spacer is 24 nucleotides in length. In other embodiments, a spacer is 25 nucleotides in length. In other embodiments, a spacer is 26 nucleotides in length. In other embodiments, a spacer is 27 nucleotides in length. In other embodiments, a spacer is 28 nucleotides in length. In other embodiments, a spacer is 29 nucleotides in length. In other embodiments, a spacer is 30 nucleotides in length.

[0176] Type V Cas endonucleases require a specific sequence, called a protospacer adjacent motif (PAM) that is upstream (e.g., directly upstream) of the target sequence on the non-target strand. Thus, spacer sequences for targeting a gene of interest can be identified by scanning the gene for PAM sequences recognized by the Type V Cas protein. Exemplary PAM sequences for Type V Cas proteins of the disclosure are shown in Table 6A-4B. In addition, TTTV is a canonical PAM sequence for Type V-A Cas proteins, and it expected that Type V Cas proteins of the disclosure can recognize the TTTV PAM.

TABLE-US-00031 TABLE6A ExemplaryTypeVCasProteinPAMSequences (insilicodetermined) CasProtein PAMSequence ZWGD TTN ZJHK TTTN ZIKV TTTR ZZFT TTTN,TTTR YYAN TTTN ZZGY TTTN,TTTR ZKBG YTTN ZZKD TTTN ZXPB TTTN ZPPX YTTN,TTN ZZQE YTTV

TABLE-US-00032 TABLE6B ExemplaryTypeVCasProteinPAMSequences (invitrodetermined) Casprotein PAMSequence ZZKD NTTV,VTTV,NCTV,TTTT ZRGM YTTV ZZQE NYYN,NTTN,NCTV

[0177] Section 7 describes exemplary sequences that can be used to target B2M, TRAC and PD1 genes. Section 7 further describes exemplary sequences that can be used to target AAVS1, BCL11A, EMX1, PCSK9, VEGFA, and Match6 genomic sequences. Exemplary spacer sequences that can be used in gRNAs of the disclosure are set forth in Table 7. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting TRAC. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting B2M. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting PD1. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting AAVS1. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting BCL11A. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting EMX1. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting PCSK9. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting VEGFA. In some embodiments, a gRNA of the disclosure comprises a spacer sequence targeting Match6.

TABLE-US-00033 TABLE7 ExemplarySpacerSequencesTargetingEndogenousGenomicLoci GuideID Target Spacer(5.fwdarw.3) SEQIDNO. B2M-g1 B2M UGGCCUGGAGGCUAUCCAGCGUG 164 B2M-g2 B2M CUCACGUCAUCCAGCAGAGAAUG 165 B2M-g3 B2M ACUUUCCAUUCUCUGCUGGAUGA 166 B2M-g4 B2M CUGAAUUGCUAUGUGUCUGGGUU 167 B2M-g5 B2M AAUUCUCUCUCCAUUCUUCAGUA 168 B2M-g8 B2M GUGUCAAGCUAUAUCAGGCACCA 181 B2M-g9 B2M AUGUGUCUUUUCCCGAUAUUCCU 182 B2M-g1_21nt B2M UGGCCUGGAGGCUAUCCAGCG 183 TRAC-g1 TRAC AGAAUCAAAAUCGGUGAAUAGGC 169 TRAC-g2 TRAC UGACACAUUUGUUUGAGAAUCAA 170 TRAC-g3 TRAC GAGUCUCUCAGCUGGUACACGGC 171 TRAC-g4 TRAC UCUGUGAUAUACACAUCAGAAUC 172 TRAC-g5 TRAC AUUCUCAAACAAAUGUGUCACAA 173 TRAC-g6 TRAC UCACUGGAUUUAGAGUCUCUCAG 184 TRAC-g9 TRAC GAUUCUCAAACAAAUGUGUCACA 185 TRAC-g11 TRAC AAGAGGGAAAUGAGAUCAUGUCC 186 TRAC-g13 TRAC ACCGAUUUUGAUUCUCAAACAAA 187 TRAC-g15 TRAC GUCUGUGAUAUACACAUCAGAAU 188 TRACg3_20nt TRAC GAGUCUCUCAGCUGGUACAC 189 TRACg3_21nt TRAC GAGUCUCUCAGCUGGUACACG 190 TRACg3_22nt TRAC GAGUCUCUCAGCUGGUACACGG 191 TRACg3_24nt TRAC GAGUCUCUCAGCUGGUACACGGCA 192 PD1-g1 PD1 CCUUCCGCUCACCUCCGCCUGAG 174 PD1-g2 PD1 GCACGAAGCUCUCCGAUGUGUUG 175 PD1-g3 PD1 AUCUGCGCCUUGGGGGCCAGGGA 176 PD1-g4 PD1 GAACUGGCCGGCUGGCCUGGGUG 177 AAVS1-g1 AAVS1 AUUUGGGCAGCUCCCCUACCCCC 193 AAVS1-g2 AAVS1 GGCAGCUCCCCUACCCCCCUUAC 194 AAVS1-g6 AAVS1 CAGGGGUCCGAGAGCUCAGCUAG 195 AAVS1-g7 AAVS1 AUCUGUCCCCUCCACCCCACAGU 196 EMX1-g2 EMX1 UACUUUGUCCUCCGGUUCUGGAA 197 EMX1-g3 EMX1 UCCUCCGGUUCUGGAACCACACC 198 BCL11A-g1 BCL11A AGCCAUCUCACUACAGAUAACUC 199 BCL11A-g2 BCL11A AAGCUAGUCUAGUGCAAGCUAAC 200 BCL11A-g3 BCL11A GCCUCUGAUUAGGGUGGGGGCGU 201 BCL11A-g4 BCL11A UCACAGGCUCCAGGAAGGGUU 202 PCSK9-g1 PCSK9 UCUGCCACCCACCUCCUCACCUU 203 PCSK9-g2 PSCK9 CAGGUCAUCACAGUUGGGGCCAC 204 VEGFA-g1 VEGFA GAGAGUGAGGACGUGUGUGUC 205 Match6_20nt Match6 GGGUGAUCAGACCCAACAGC 206 Match6_21nt Match6 GGGUGAUCAGACCCAACAGCA 207 Match6_22nt Match6 GGGUGAUCAGACCCAACAGCAG 208 Match6_23nt Match6 GGGUGAUCAGACCCAACAGCAGG 209 Match6_24nt Match6 GGGUGAUCAGACCCAACAGCAGGU 210

[0178] In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 17 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 18 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 19 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 20 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 21 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 22 or more consecutive nucleotides from a sequence shown in Table 7. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 23 or more consecutive nucleotides from a sequence shown in Table 5. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises a sequence shown in Table 7.

6.3.2. Modified gRNA Molecules

[0179] Guide RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated, as described in the art. The disclosed gRNA (e.g., sgRNA) molecules can be unmodified or can contain any one or more of an array of chemical modifications.

[0180] While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach that can be used for generating chemically modified RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Type V Cas endonuclease, are more readily generated enzymatically. While fewer types of modifications are available for use in enzymatically produced RNAs, there are still modifications that can be used to, for instance, enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described herein and in the art.

[0181] By way of illustration of various types of modifications, especially those used frequently with smaller chemically synthesized RNAs, modifications can comprise one or more nucleotides modified at the 2 position of the sugar, for instance a 2-O-alkyl, 2-O-alkyl-O-alkyl, or 2-fluoro-modified nucleotide. In some examples, RNA modifications can comprise 2-fluoro, 2-amino or 2-O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3 end of the RNA. Such modifications can be routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (thus, higher target binding affinity) than 2-deoxyoligonucleotides against a given target.

[0182] A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligonucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH.sub.2NHOCH.sub.2, CH, N(CH.sub.3)OCH.sub.2 (known as a methylene (methylimino) or MMI backbone), CH.sub.2ON(CH.sub.3)CH.sub.2, CH.sub.2N(CH.sub.3)N(CH.sub.3)CH.sub.2 and ON(CH.sub.3)CH.sub.2CH.sub.2 backbones, wherein the native phosphodiester backbone is represented as OPOCH); amide backbones (see De Mesmaeker et al. 1995, Ace. Chem. Res., 28:366-374); morpholino backbone structures (see U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., 1991, Science 254:1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3-5 linkages, 2-5 linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3-5 to 5-3 or 2-5 to 5-2; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0183] Morpholino-based oligomeric compounds are described in Braasch and David Corey, 2002, Biochemistry, 41 (14): 4503-4510; Genesis, Volume 30, Issue 3, (2001); Heasman, 2002, Dev. Biol., 243:209-214; Nasevicius et al., 2000, Nat. Genet., 26:216-220; Lacerra et al., 2000, Proc. Natl. Acad. Sci., 97:9591-9596; and U.S. Pat. No. 5,034,506.

[0184] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., 2000, J. Am. Chem. Soc., 122:8595-8602.

[0185] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH.sub.2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0186] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2 position: OH, SH, SCH.sub.3, F, OCN, OCH.sub.3, OCH.sub.3O(CH.sub.2)n CH.sub.3, O(CH.sub.2)n NH.sub.2, or O(CH.sub.2)n CH.sub.3, where n is from 1 to about 10; C.sub.1 to C.sub.10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF.sub.3; OCF.sub.3; O-, S-, or bi-alkyl; O-, S-, or N-alkenyl; SOCH.sub.3; SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. In some aspects, a modification includes 2-methoxyethoxy (2-OCH.sub.2CH.sub.2OCH.sub.3, also known as 2-O-(2-methoxyethyl)) (Martin et al., 1995, Helv. Chim. Acta, 78, 486). Other modifications include 2-methoxy (2-OCH.sub.3), 2-propoxy (2-OCH.sub.2 CH.sub.2CH.sub.3) and 2-fluoro (2-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3 position of the sugar on the 3 terminal nucleotide and the 5 position of 5 terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.

[0187] In some examples, both a sugar and an internucleoside linkage (in the backbone) of the nucleotide units can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al., 1991, Science, 254:1497-1500.

[0188] RNAs such as guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as base) modifications or substitutions. As used herein, unmodified or natural nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2 deoxy cytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino) adenine, 2-(imidazolylalkyl) adenine, 2-(aminoalklyamino) adenine or other heterosub stituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine. Komberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A universal base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by about 0.6-1.2 C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions.

[0189] Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0190] Further, nucleobases can comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, p. 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases can be useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by about 0.6-1.2 C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, Antisense Research and Applications, CRC Press, Boca Raton, 1993, 276-278) and are aspects of base substitutions, even more particularly when combined with 2-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication 2003/0158403.

[0191] Thus, a modified gRNA can include, for example, one or more non-natural sugars, internucleotide linkages and/or bases. It is not necessary for all positions in a given gRNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.

[0192] The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al. 1989, Proc. Natl. Acad. Sci. USA, 86:6553-6556); cholic acid (Manoharan et al, 1994, Bioorg. Med. Chem. Let., 4:1053-1060); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, 1992, Ann. N. Y. Acad. Sci., 660:306-309; Manoharan et al., 1993, Bioorg. Med. Chem. Let., 3:2765-2770); a thiocholesterol (Oberhauser et al., 1992, Nucl. Acids Res., 20:533-538); an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al, 1990, FEBS Lett., 259:327-330; Svinarchuk et al, 1993, Biochimie, 75:49-54); a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995, Tetrahedron Lett., 36:3651-3654; and Shea et al, 1990, Nucl. Acids Res., 18:3777-3783); a polyamine or a polyethylene glycol chain (Mancharan et al, 1995, Nucleosides & Nucleotides, 14:969-973); adamantane acetic acid (Manoharan et al, 1995, Tetrahedron Lett., 36:3651-3654); a palmityl moiety (Mishra et al., 1995, Biochim. Biophys. Acta, 1264:229-237); or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al, 1996, J. Pharmacol. Exp. Ther., 277:923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5, 109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

[0193] Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites. For example, hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., 2014, Protein Pept Lett. 21 (10): 1025-30. Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and/or complexes thereof to particular target cells of interest.

[0194] Targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups. Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this present disclosure, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this disclosure, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application Publication WO1993007883, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-trityl thiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109, 124; 5,118,802; 5, 138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214, 136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0195] A large variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and/or achieve other benefits that can be useful in connection with the introduction of polynucleotides into human cells, as described herein; see, e.g., the reviews by Whitehead K A et al., 2011, Annual Review of Chemical and Biomolecular Engineering, 2:77-96; Gaglione and Messere, 2010, Mini Rev Med Chem, 10 (7): 578-95; Chernolovskaya et al, 2010, Curr Opin Mol Ther., 12 (2): 158-67; Deleavey et al., 2009, Curr Protoc Nucleic Acid Chem Chapter 16: Unit 16.3; Behlke, 2008, Oligonucleotides 18 (4): 305-19; Fucini et al, 2012, Nucleic Acid Ther 22 (3): 205-210; Bremsen et al, 2012, Front Genet 3:154.

6.4. Systems

[0196] The disclosure provides systems comprising a Type V Cas protein of the disclosure (e.g., as described in Section 6.2) and a means for targeting the Type V Cas protein to a target genomic sequence. The means for targeting the Type V Cas protein to a target genomic sequence can be a guide RNA (gRNA) (e.g., as described in Section 6.3).

[0197] The disclosure also provides systems comprising a Type V Cas protein of the disclosure (e.g., as described in Section 6.2) and a gRNA (e.g., as described in Section 6.3). The systems can comprise a ribonucleoprotein particle (RNP) in which a Type V Cas protein is complexed with a gRNA. Systems of the disclosure can in some embodiments further comprise genomic DNA complexed with the Type V Cas protein and the gRNA. Accordingly, the disclosure provides systems comprising a Type V Cas protein, a genomic DNA, and gRNA, all complexed with one another.

[0198] The systems of the disclosure can exist within a cell (whether the cell is in vivo, ex vivo, or in vitro) or outside a cell (e.g., in a particle our outside of a particle).

6.5. Nucleic Acids

[0199] The disclosure provides nucleic acids (e.g., DNA or RNA) encoding Type V Cas proteins (e.g., a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein), nucleic acids encoding gRNAs of the disclosure (e.g., a single gRNA or combination of gRNAs), nucleic acids encoding both Type V Cas proteins and gRNAs, and pluralities of nucleic acids, for example comprising a nucleic acid encoding a Type V Cas protein and a gRNA.

[0200] A nucleic acid encoding a Type V Cas protein and/or gRNA can be, for example, a plasmid or a viral genome (e.g., a lentivirus, retrovirus, adenovirus, or adeno-associated virus genome). Plasmids can be, for example, plasmids for producing virus particles, e.g., lentivirus particles, or plasmids for propagating the Type V Cas and gRNA coding sequences in bacterial (e.g., E. coli) or eukaryotic (e.g., yeast) cells.

[0201] A nucleic acid encoding a Type V Cas protein can, in some embodiments, further encode a gRNA. Alternatively, a gRNA can be encoded by a separate nucleic acid (e.g., DNA or mRNA).

[0202] Nucleic acids encoding a Type V Cas protein can be codon optimized, e.g., where at least one non-common codon or less-common codon has been replaced by a codon that is common in a host cell. For example, a codon optimized nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system. As an example, if the intended target nucleic acid is within a human cell, a human codon-optimized polynucleotide encoding Type V Cas can be used for producing a Type V Cas polypeptide. Exemplary codon-optimized sequences are shown in Tables 1A to 1T.

[0203] Nucleic acids of the disclosure, e.g., plasmids and viral vectors, can comprise one or more regulatory elements such as promoters, enhancers, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, 1990, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest or in particular cell types. Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a nucleic acid of the disclosure comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof, e.g., to express a Type V Cas protein and a gRNA separately. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous Sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, 1985, Cell 41:521-530), the SV40 promoter, the dihydrofolate reductase promoter, the -actin promoter, the phosphoglycerol kinase (PGK) promoter, and EF1 promoters (for example, full length EF1 promoter and the EFS promoter, which is a short, intron-less form of the full EF1 promoter). Exemplary enhancer elements include WPRE; CMV enhancers; the R-U5 segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit -globin. It will be appreciated by those skilled in the art that the design of an expression vector can depend on such factors as the choice of the host cell, the level of expression desired, etc.

[0204] The term vector refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked. One type of polynucleotide vector includes a plasmid, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated. Another type of polynucleotide vector is a viral vector; wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0205] In some examples, vectors can be capable of directing the expression of nucleic acids to which they are operably linked. Such vectors can be referred to herein as recombinant expression vectors, or more simply expression vectors, which serve equivalent functions.

[0206] The term operably linked means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term regulatory sequence is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0207] Vectors can include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (e.g., AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, AAVrh10), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-I, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.

[0208] In some examples, a vector can comprise one or more transcription and/or translation control elements. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. The vector can be a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.

[0209] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-I promoters (for example, the full EF1 promoter and the EFS promoter), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-l.

[0210] An expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also comprise appropriate sequences for amplifying expression. The expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.

[0211] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, for example a human RHO promoter or human rhodopsin kinase promoter (hGRK), a cell type specific promoter, etc.).

6.6. Particles and Cells

[0212] The disclosure further provides particles comprising a Type V Cas protein of the disclosure (e.g., a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein), particles comprising a gRNA of the disclosure, particles comprising a system of the disclosure, and particles comprising a nucleic acid or plurality of nucleic acids of the disclosure. The particles can in some embodiments comprise or further comprise a gRNA, or a nucleic acid encoding the gRNA (e.g., DNA or mRNA). For example, the particles can comprise a RNP of the disclosure. Exemplary particles include lipid nanoparticles, vesicles, viral-like particles (VLPs) and gold nanoparticles. See, e.g., WO 2020/012335, the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Type V Cas proteins to cells (e.g., complexed together as a RNP).

[0213] The disclosure provides particles (e.g., virus particles) comprising a nucleic acid encoding a Type V Cas protein of the disclosure. The particles can further comprise a nucleic acid encoding a gRNA. Alternatively, a nucleic acid encoding a Type V Cas protein can further encode a gRNA.

[0214] The disclosure further provides pluralities of particles (e.g., pluralities of virus particles). Such pluralities can include a particle encoding a Type V Cas protein and a different particle encoding a gRNA. For example, a plurality of particles can comprise a virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 virus particle) encoding a Type V Cas protein and a second virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 virus particle) encoding a gRNA. Alternatively, a plurality of particles can comprise a plurality of virus particles where each particle encodes a Type V Cas protein and a gRNA.

[0215] The disclosure further provides cells and populations of cells (e.g., ex vivo cells and populations of cells) that can comprise a Type V Cas protein (e.g., introduced to the cell as a RNP) or a nucleic acid encoding the Type V Cas protein (e.g., DNA or mRNA) (optionally also encoding a gRNA). The disclosure further provides cells and populations of cells comprising a gRNA of the disclosure (optionally complexed with a Type V Cas protein) or a nucleic acid encoding the gRNA (e.g., DNA or mRNA) (optionally also encoding a Type V Cas protein). The cells and populations of cells can be, for example, human cells such as a stem cell, e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS), or an embryonic stem cell. In some embodiments, the cells and populations of cells are T cells. Methods for introducing proteins and nucleic acids to cells are known in the art. For example, a RNP can be produced by mixing a Type V Cas protein and one or more guide RNAs in an appropriate buffer. An RNP can be introduced to a cell, for example, via electroporation and other methods known in the art.

[0216] The cell populations of the disclosure can be cells in which gene editing by the systems of the disclosure has taken place, or cells in which the components of a system of the disclosure have been introduced or expressed but gene editing has not taken place, or a combination thereof. A cell population can comprise, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have undergone gene editing by a system of the disclosure.

6.7. Pharmaceutical Compositions

[0217] Also disclosed herein are pharmaceutical formulations and medicaments comprising a Type V Cas protein, gRNA, nucleic acid or plurality of nucleic acids, system, particle, or plurality of particles of the disclosure together with a pharmaceutically acceptable excipient.

[0218] Suitable excipients include, but are not limited to, salts, diluents, (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof. Suitable pharmaceutically acceptable excipients can be selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions.

[0219] The components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

[0220] In some cases, formulations can include one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. In some cases, the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

[0221] In some embodiments, the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration. In some embodiments, the formulations can comprise a guide RNA and a Type V Cas protein in a pharmaceutically effective amount sufficient to edit a gene in a cell. The pharmaceutical compositions can be formulated for medical and/or veterinary use.

6.8. Methods of Altering a Cell

[0222] The disclosure further provides methods of using the Type V Cas proteins, gRNAs, nucleic acids (including pluralities of nucleic acids), systems, and particles (including pluralities of particles) of the disclosure for altering cells.

[0223] In one aspect, a method of altering a cell comprises contacting a eukaryotic cell (e.g., a human cell) with a nucleic acid, particle, system or pharmaceutical composition described herein.

[0224] Contacting a cell with a disclosed nucleic acid, particle, system or pharmaceutical composition can be achieved by any method known in the art and can be performed in vivo, ex vivo, or in vitro. In some embodiments, the methods can include obtaining one or more cells from a subject prior to contacting the cell(s) with a herein disclosed nucleic acid, particle, system or pharmaceutical composition. In some embodiments, the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.

[0225] Type V Cas and gRNA, as well as nucleic acids encoding Type V Cas and gRNAs can be delivered to a cell by any means known in the art, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.

[0226] A polynucleotide encoding Type V Cas and a gRNA, can be delivered to a cell (ex vivo or in vivo) by a lipid nanoparticle (LNP). LNPs can have, for example, a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm. LNPs can be made from cationic, anionic, neutral lipids, and combinations thereof. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as helper lipids to enhance transfection activity and nanoparticle stability.

[0227] LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Lipids and combinations of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerCI4, and PEG-CerC20. Lipids can be combined in any number of molar ratios to produce a LNP. In addition, the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.

[0228] Type V Cas and/or gRNAs can be delivered to a cell via an adeno-associated viral vector (e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 serotype), or by another viral vector. Other viral vectors include, but are not limited to lentivirus, adenovirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus. In some embodiments, a Type V Cas mRNA is formulated in a lipid nanoparticle, while a sgRNA is delivered to a cell in an AAV or other viral vector. In some embodiments, one or more AAV vectors (e.g., one or more AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 serotype) are used to deliver both a sgRNA and a Type V Cas. In some embodiments, a Type V Cas and a sgRNA are delivered using separate vectors. In other embodiments, a Type V Cas and a sgRNA are delivered using a single vector. BNK Type V Cas and AIK Type V Cas, with their relatively small size, can be delivered with a gRNA (e.g., sgRNA) using a single AAV vector.

[0229] Compositions and methods for delivering Type V Cas and gRNAs to a cell and/or subject are further described in PCT Patent Application Publications WO 2019/102381, WO 2020/012335, and WO 2020/053224, each of which is incorporated by reference herein in its entirety.

[0230] DNA cleavage can result in a single-strand break (SSB) or double-strand break (DSB) at particular locations within the DNA molecule. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-dependent repair (HDR) and non-homologous end-joining (NHEJ). These repair processes can edit the targeted polynucleotide by introducing a mutation, thereby resulting in a polynucleotide having a sequence which differs from the polynucleotide's sequence prior to cleavage by a Type V Cas.

[0231] NHEJ and HDR DNA repair processes consist of a family of alternative pathways. Non-homologous end-joining (NHEJ) refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments. See, e.g. Cahill et al., 2006, Front. Biosci. 11:1958-1976. DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. Thus, NHEJ repair mechanisms can introduce mutations into the coding sequence which can disrupt gene function. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with a modification of the polynucleotide sequence such as a loss of or addition of nucleotides in the polynucleotide sequence. The modification of the polynucleotide sequence can disrupt (or perhaps enhance) gene expression.

[0232] Homology-dependent repair (HDR) utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.

[0233] A third repair mechanism includes microhomology-mediated end joining (MMEJ), also referred to as Alternative NHEJ (ANHEJ), in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0234] Modifications of a cleaved polynucleotide by HDR, NHEJ, and/or ANHEJ can result in, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation. The aforementioned process outcomes are examples of editing a polynucleotide.

[0235] When performing prime editing, e.g., with a prime editor comprising a Type V Cas protein of the disclosure that comprises a reverse transcriptase, a DNA mismatch repair (MMR) inhibitor can be used in conjunction with the prime editor. Use of MMR inhibitors have been reported to enhance efficiency of prime editing (see, e.g., Chen et al., 2021 Cell 184 (22): 5635-5652, the contents of which are incorporated herein by reference in their entireties). An exemplary MMR inhibitor is MLH1dn, having the amino acid sequence

TABLE-US-00034 (SEQIDNO:258) SFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDLDI VCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQ GTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRS IFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFL YLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKS TTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEML ELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRII NLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFG VLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLID NYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWT VEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVF.

[0236] In some embodiments, an MMR inhibitor is provided in trans with a prime editor.

[0237] Advantages of ex vivo cell therapy approaches include the ability to conduct a comprehensive analysis of the therapeutic prior to administration. Nuclease-based therapeutics can have some level of off-target effects. Performing gene correction ex vivo allows a method user to characterize the corrected cell population prior to implantation, including identifying any undesirable off-target effects. Where undesirable effects are observed, a method user may opt not to implant the cells or cell progeny, may further edit the cells, or may select new cells for editing and analysis. Other advantages include ease of genetic correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells that will be required for a cell-based therapy. Furthermore, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic correction, without risking a decrease in viability.

[0238] Although certain cells present an attractive target for ex vivo treatment and therapy, increased efficacy in delivery may permit direct in vivo delivery to such cells. Ideally the targeting and editing is directed to the relevant cells. Cleavage in other cells can also be prevented by the use of promoters only active in certain cell types and/or developmental stages.

[0239] Additional promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid. The amount of time that delivered protein and RNA remain in the cell can also be adjusted using treatments or domains added to change the half-life. In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing. In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment.

[0240] An advantage of in vivo gene therapy can be the ease of therapeutic production and administration. The same therapeutic approach and therapy has the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele. In contrast, ex vivo cell therapy typically requires using a subject's own cells, which are isolated, manipulated and returned to the same patient.

[0241] Progenitor cells (also referred to as stem cells herein) are capable of both proliferation and giving rise to more progenitor cells, which in turn have the ability to generate a large number of cells that can in turn give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term stem cell refers then to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one aspect, the term progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell can derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types that each can give rise to can vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors. In many biological instances, stem cells can also be multipotent because they can produce progeny of more than one distinct cell type, but this is not required.

[0242] Human cells described herein can be induced pluripotent stem cells (IPSCs). An advantage of using iPSCs in the methods of the disclosure is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then differentiated into a progenitor cell to be administered to the subject (e.g., an autologous cell). Because progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.

[0243] Methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Pluripotent stem cells generated by such methods can be used in the method of the disclosure.

[0244] Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described. Mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, 2006, Cell 126 (4): 663-76. iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape. In addition, mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3 (6): 595-605), and tetraploid complementation.

[0245] Human iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., 2014, Budniatzky and Gepstein, Stem Cells Transl Med. 3 (4): 448-57; Barrett et al, 2014, Stem Cells Trans Med 3:1-6 sctm.2014-0121; Focosi et al, 2014, Blood Cancer Journal 4: e211. The production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.

[0246] iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell. Further, reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., 2010, Cell Stem Cell, 7 (5): 618-30. Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3/4 or Pouf51), SoxI, Sox2, Sox3, Sox 15, Sox 18, NANOG, KIfI, KIf2, KIf4, KIf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming using the methods and compositions described herein can further comprise introducing one or more of Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell. The methods and compositions described herein can further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from the reprogrammed cells are to be used in, e.g., human therapy, in one aspect the reprogramming is not affected by a method that alters the genome. Thus, in such examples, reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.

[0247] Efficiency of reprogramming (the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al., 2008, Cell-Stem Cell 2:525-528; Huangfu et al., 2008, Nature Biotechnology 26 (7): 795-797; and Marson et al., 2008, Cell-Stem Cell 3:132-135. Thus, an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HD AC) inhibitors, valproic acid, 5-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others. Other non-limiting examples of reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., ()-Depudecin), HC Toxin, Nullscript (4-(1,3-Dioxo-IH,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9, 10-epoxy decanoic acid), CHAP31 and CHAP 50. Other reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g, catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs. Such inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.

[0248] To confirm the induction of pluripotent stem cells, isolated clones can be tested for the expression of a stem cell marker. Such expression in a cell derived from a somatic cell identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, FbxI5, EcatI, EsgI, Eras, Gdfi, Fgf4, Cripto, DaxI, Zpf296, Slc2a3, RexI, UtfI, and NatI. In one case, for example, a cell that expresses Oct4 or Nanog is identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. Detection can involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be best identified via RT-PCR, or protein detection methods such as immunocytochemistry, while cell surface markers are readily identified, e.g., by immunocytochemistry.

[0249] Pluripotency of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells can be introduced into nude mice and histology and/or immunohistochemistry can be performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0250] Patient-specific iPS cells or cell line can be created. There are many established methods in the art for creating patient specific iPS cells, e.g., as described in Takahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example, the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell. The set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

[0251] In some aspects, a biopsy or aspirate of a subject's bone marrow can be performed. A biopsy or aspirate is a sample of tissue or fluid taken from the body. There are many different kinds of biopsies or aspirates. Nearly all of them involve using a sharp tool to remove a small amount of tissue. If the biopsy will be on the skin or other sensitive area, numbing medicine can be applied first. A biopsy or aspirate can be performed according to any of the known methods in the art. For example, in a bone marrow aspirate, a large needle is used to enter the pelvis bone to collect bone marrow.

[0252] In some aspects, a mesenchymal stem cell can be isolated from a subject. Mesenchymal stem cells can be isolated according to any method known in the art, such as from a subject's bone marrow or peripheral blood. For example, marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on a Percoll density gradient. Cells, such as blood cells, liver cells, interstitial cells, macrophages, mast cells, and thymocytes, can be separated using density gradient centrifugation media, Percoll. The cells can then be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger et. al., 1999, Science 284:143-147).

6.8.1. Exemplary Genomic Targets

[0253] The Type V Cas proteins and gRNAs of the disclosure can be used to alter various genomic targets. In some aspects, the methods of altering a cell are methods for altering a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence. In some aspects, the methods of altering a cell are methods of altering a TRAC, B2M, PD1, or LAG3 genomic sequence. Reference sequences of RHO, TRAC, B2M, PD1, and LAG3 are available in public databases, for example those maintained by NCBI. For example, RHO has the NCBI gene ID: 6010; TRAC has the NCBI gene ID: 28755; B2M has the NCBI gene ID: 567; PD1 has the NCBI gene ID: 5133; and LAG3 has the NCBI gene ID: 3902.

[0254] In some embodiments, the methods of altering a cell are methods for altering a hemoglobin subunit beta (HBB) gene. HBB mutations are associated with B-thalassemia and SCD. Dever et al., 2016 Nature 539 (7629): 384-389.

[0255] In some embodiments, the methods of altering a cell are methods for altering a CCR5 gene. CCR5 has demonstrated involvement in several different disease states including, but not limited to, human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS). WO 2018/119359 describes CCR5 editing by CRISPR-Cas to make loss of function CCR5 in order to provide protection against HIV infection, decrease one or more symptoms of HIV infection, halt or delay progression of HIV to AIDS, and/or decrease one or more symptoms of AIDS.

[0256] In some embodiments, the methods of altering a cell are methods for altering a PD1, B2M gene, TRAC gene, or a combination thereof. CAR-T cells having PD1, B2M and TRAC genes disrupted by CRISPR-Type V Cas have demonstrated enhanced activity in preclinical glioma models. Choi et al., 2019, Journal for Immuno Therapy of Cancer 7:309.

[0257] In some embodiments, the methods of altering a cell are methods for altering an USH2A gene. Mutations in the USH2A gene can cause Usher syndrome type 2A, which is characterized by progressive hearing and vision loss.

[0258] In some embodiments, the methods of altering a cell are methods for altering a RHO gene. Mutations in the RHO gene can cause retinitis pigmentosa (RP).

[0259] Targeting of (one or more of) human TRAC, human B2M, human PD1, and human LAG3 genes can be used, for example, in the engineering of chimeric antigen receptor (CAR) T cells. For example, CRISPR/Cas technology has been used to deliver CAR-encoding DNA sequences to loci such as TRAC and PD1 (see, e.g., Eyquem et al., 2017, Nature 543 (7643): 113-117; Hu et al., 2023, eClinicalMedicine 60:102010), while TRAC, B2M, PD1, and LAG3 knockout CAR T-cells have been reported (see, e.g., Dimitri et al., 2022, Molecular Cancer 21:78; Liu et al., 2016, Cell Research 27:154-157; Ren et al., 2017, Clin Cancer Res. 23 (9): 2255-2266; Zhang et al., 2017, Front Med. 11 (4): 554-562). Thus, the Type V Cas proteins and TRAC, B2M, PD1, and LAG3 guides of the disclosure can be used for targeted knock-in of an exogenous DNA sequence to a desired genomic site in a human cell and/or knock-out of TRAC, B2M, PD1, or LAG3 in a human cell, for example a human T cell. In some embodiments, T cells are edited ex vivo to produce CAR-T cells and subsequently administered to a subject in need of CAR-T cell therapy.

[0260] In some embodiments, the methods of altering a cell are methods for altering a DNMT1 gene. Mutations in the DNMT1 gene can cause DNMT1-related disorder, which is a degenerative disorder of the central and peripheral nervous systems. DNMT1-related disorder is characterized by sensory impairment, loss of sweating, dementia, and hearing loss.

[0261] Additional exemplary targets include AVS1, BCL11A, PCSK9, and VEGFA. In some embodiments, the methods of altering a cell are methods for altering an AVS1 gene. AVS1 can be used as a safe harbor locus to insert an transgene of interest (see, e.g., Gu et al., 2022, Methods Mol Biol. 2495:99-114). In some embodiments, the methods of altering a cell are methods for altering a BCL11A gene. Editing BCL11A has been identified in the art a target for treatment of sickle cell disease and -Thalassemia (see, e.g., Frangoul et al., 2021, N Eng J Med 384:252-260). In some embodiments, the methods of altering a cell are methods for altering a PCSK9 gene. PCSK9 has been identified in the art as a target for treatment of hypercholesterolemia (see, e.g., Hoekstra & Van Eck, 2024, Current Atherosclerosis Reports, 26:139-146). In some embodiments, the methods of altering a cell are methods for altering a VEGFA gene. VEGFA has been identified in the art as a target for treatment of eye diseases such as age-related macular degeneration (see, e.g., Park et al., 2023, Scientific Reports 13:3715).

6.9. Methods of Detecting Target Nucleic Acids

[0262] The disclosure further provides methods of using the Type V Cas proteins, gRNAs, and systems of the disclosure for detecting target nucleic acids (e.g., nucleic acids from pathogens, for example viruses, bacteria, or parasites). Nucleic acid detection methods using Cas12a are described in the art (see, e.g., Kaminski et al., 2021, Nature Biomedical Engineering 5:643-656; Sashital, 2018, Genome Med. 10:32, each of which is incorporated herein by reference in its entirety), and such methods can be extended to the Type V Cas proteins of the disclosure. Nucleic acid detection methods typically take advantage of collateral cleavage activity of Type V Cas proteins. For example, target binding of Type V Cas proteins such as Cas12a activates collateral cleavage activity toward single-stranded DNA, and this activity can be exploited in a detection assay by supplying a single-stranded reporter nucleic acid, for example a reporter nucleic acid comprising a quenched fluorescent reporter. Type V Cas protein binding to the target nucleic acid leads to cleavage of the reporter nucleic acid. Detection of the fluorescent reporter following cleavage of the reporter nucleic acid allows for detection and, optionally, quantification of the target nucleic acid.

7. EXAMPLES

7.1. Materials and Methods

7.1.1. Plasmids and Cell Lines

[0263] Plasmids: Type V-A Cas proteins were expressed in mammalian cells from a plasmid vector characterized by a EF1alpha-driven cassette. Each Type V-A Cas protein coding sequence was human codon-optimized and modified by the addition of an SV5 tag and a bipartite nuclear localization signal at the C-terminus. Additional constructs containing different NLS configurations (discussed in Section 7.4.2) were generated using standard cloning techniques. The crRNA were expressed from a U6-driven cassette located on an independent plasmid construct. The human codon-optimized coding sequence of the Type V-A Cas proteins, as well as their crRNA scaffolds, were obtained by synthesis from Twist Bioscience. Spacer sequences (20-24 nt long) were cloned into the crRNA plasmid as annealed DNA oligonucleotides (Eurofins Genomics) using a double BsaI site present in the plasmid. The list of spacer sequences and relative cloning oligonucleotides used in the present example is reported in Table 8. In all cases in which the crRNA scaffold did not contain a matching native 5-G, this nucleotide was appended upstream the scaffold sequence in order to allow efficient transcription from a U6 promoter. Unless otherwise stated in all studies, full-length crRNAs were used.

TABLE-US-00035 TABLE8 SpacersequencesandoligonucleotidesrelativetocrRNAsforTypeV-ACasproteins SEQ SEQ SEQ Spacer ID PAM Oligo1 ID Oligo2 ID GuideID Target (5.fwdarw.3) NO: (5.fwdarw.3) (5.fwdarw.3) NO: (5.fwdarw.3) NO: EGFP-g1 EGFP CGUCGCCGUCCA 260 TTTA agatCGTCGCCGTC 262 AaaaCCTGGTCGAG 308 GCUCGACCAGG CAGCTCGACCAGG CTGGACGGCGACG EGFP-g2 EGFP CUCAGGGGGGA 261 TTTG agatCTCAGGGCGG 263 AaaaCTGAGCACCC 309 CUGGGUGCUCA ACTGGGTGCTCAG AGTCCGCCCTGAG G B2M-g1 B2M UGGCCUGGAGG 164 TTTC agatTGGCCTGGAG 264 aaaaCACGCTGGATA 310 CUAUCCAGCGUG GCTATCCAGCGTG GCCTCCAGGCCA B2M-g2 B2M CUCACGUCAUCC 165 TTTC agatCTCACGTCATC 265 aaaaCATTCTCTGCT 311 AGCAGAGAAUG CAGCAGAGAATG GGATGACGTGAG B2M-g3 B2M ACUUUCCAUUCU 166 TTTG agatACTTTCCATTC 266 aaaaTCATCCAGCAG 312 CUGCUGGAUGA TCTGCTGGATGA AGAATGGAAAGT B2M-g4 B2M CUGAAUUGCUAU 167 TTTC agatCTGAATTGCTA 267 aaaaAACCCAGACAC 313 GUGUCUGGGUU TGTGTCTGGGTT ATAGCAATTCAG B2M-g5 B2M AAUUCUCUCUCC 168 TTTC agatAATTCTCTCTC 268 aaaaTACTGAAGAAT 314 AUUCUUCAGUA CATTCTTCAGTA GGAGAGAGAATT TRAC-g1 TRAC AGAAUCAAAAUC 169 TTTA agatAGAATCAAAAT 269 aaaaGCCTATTCACC 315 GGUGAAUAGGC CGGTGAATAGGC GATTTTGATTCT TRAC-g2 TRAC UGACACAUUUGU 170 TTTG agatTGACACATTTG 270 aaaaTTGATTCTCAA 316 UUGAGAAUCAA TTTGAGAATCAA ACAAATGTGTCA TRAC-g3 TRAC GAGUCUCUCAGC 171 TTTA agatGAGTCTCTCA 271 aaaaGCCGTGTACCA 317 UGGUACACGGC GCTGGTACACGGC GCTGAGAGACTC TRAC-g4 TRAC UCUGUGAUAUAC 172 TTTG agatTCTGTGATATA 272 aaaaGATTCTGATGT 318 ACAUCAGAAUC CACATCAGAATC GTATATCACAGA TRAC-g5 TRAC AUUCUCAAACAA 173 TTTG agatATTCTCAAACA 273 aaaaTTGTGACACAT 319 AUGUGUCACAA AATGTGTCACAA TTGTTTGAGAAT PD1-g1 PD1 CCUUCCGCUCAC 174 TTTC agatCCTTCCGCTC 274 aaaaCTCAGGCGGA 320 CUCCGCCUGAG ACCTCCGCCTGAG GGTGAGCGGAAGG PD1-g2 PD1 GCACGAAGCUCU 175 TTTA agatGCACGAAGCT 275 aaaaCAACACATCGG 321 CCGAUGUGUUG CTCCGATGTGTTG AGAGCTTCGTGC PD1-g3 PD1 AUCUGCGCCUUG 176 TTTG agatATCTGCGCCTT 276 aaaaTCCCTGGCCCC 322 GGGGCCAGGGA GGGGGCCAGGGA CAAGGCGCAGAT PD1-g4 PD1 GAACUGGCCGG 177 TTTG agatGAACTGGCCG 277 aaaaCACCCAGGCC 323 CUGGCCUGGGU GCTGGCCTGGGTG AGCCGGCCAGTTC G AAVS1- AAVS1 CAGGGGUCCGA 195 CTTC agatCAGGGGTCCG 278 aaaaCTAGCTGAGCT 324 g6 GAGCUCAGCUAG AGAGCTCAGCTAG CTCGGACCCCTG AAVS1- AAVS1 AUCUGUCCCCUC 196 TTTT agatATCTGTCCCCT 279 aaaaACTGTGGGGT 325 g7 CACCCCACAGU CCACCCCACAGT GGAGGGGACAGAT AAVS1- AAVS1 GGCAGCUCCCCU 194 TTTG agatGGCAGCTCCC 280 aaaaGTAAGGGGGG 326 g2 ACCCCCCUUAC CTACCCCCCTTAC TAGGGGAGCTGCC B2M-g8 B2M GUGUCAAGCUAU 181 CTTG agatGTGTCAAGCT 281 aaaaTGGTGCCTGAT 327 AUCAGGCACCA ATATCAGGCACCA ATAGCTTGACAC B2M-g9 B2M AUGUGUCUUUUC 182 ATTA agatATGTGTCTTTT 282 aaaaAGGAATATCGG 328 CCGAUAUUCCU CCCGATATTCCT GAAAAGACACAT TRAC-g6 TRAC UCACUGGAUUUA 184 CTTG agatTCACTGGATTT 283 aaaaCTGAGAGACTC 329 GAGUCUCUCAG AGAGTCTCTCAG TAAATCCAGTGA TRAC-g9 TRAC GAUUCUCAAACA 185 TTTT agatGATTCTCAAAC 284 aaaaTCACTGGATTT 330 AAUGUGUCACA AAATGTGTCACA AGAGTCTCTCAG TRAC- TRAC AAGAGGGAAAUG 186 GTTA agatAAGAGGGAAA 285 aaaaGGACATGATCT 331 g11 AGAUCAUGUCC TGAGATCATGTCC CATTTCCCTCTT TRAC- TRAC ACCGAUUUUGAU 187 ATTC agatACCGATTTTGA 286 aaaaTTTGTTTGAGA 332 g13 UCUCAAACAAA TTCTCAAACAAA ATCAAAATCGGT TRAC- TRAC GUCUGUGAUAUA 188 TTTT agatGTCTGTGATAT 287 aaaaATTCTGATGTG 333 g15 CACAUCAGAAU ACACATCAGAAT TATATCACAGAC BCL11A- BCL11A AGCCAUCUCACU 199 TTTC agatAGCCATCTCA 288 aaaaGAGTTATCTGT 334 g1 ACAGAUAACUC CTACAGATAACTC AGTGAGATGGCT AAVS1- AAVS1 AUUUGGGCAGCU 193 TTTC agatATTTGGGCAG 289 aaaaGGGGGTAGGG 335 g1 CCCCUACCCCC CTCCCCTACCCCC GAGCTGCCCAAAT EMX1-g2 EMX1 UACUUUGUCCUC 197 TTTG agatTACTTTGTCCT 290 aaaaTTCCAGAACCG 336 CGGUUCUGGAA CCGGTTCTGGAA GAGGACAAAGTA EMX1-g3 EMX1 UCCUCCGGUUCU 198 TTTG agatTCCTCCGGTT 291 aaaaGGTGTGGTTCC 337 GGAACCACACC CTGGAACCACACC AGAACCGGAGGA BCL11A- BCL11A AAGCUAGUCUAG 200 TTTG agatAAGCTAGTCTA 292 aaaaGTTAGCTTGCA 338 g2 UGCAAGCUAAC GTGCAAGCTAAC CTAGACTAGCTT BCL11A- BCL11A GCCUCUGAUUAG 201 TTTG agatGCCTCTGATTA 293 aaaaACGCCCCCAC 339 g3 GGUGGGGGCGU GGGTGGGGGCGT CCTAATCAGAGGC PCSK9- PCSK9 UCUGCCACCCAC 203 TTTC agatTCTGCCACCC 294 aaaaAAGGTGAGGA 340 g1 CUCCUCACCUU ACCTCCTCACCTT GGTGGGTGGCAGA PCSK9- PSCK9 CAGGUCAUCACA 204 TTTC agatCAGGTCATCA 295 aaaaGTGGCCCCAA 341 g2 GUUGGGGCCAC CAGTTGGGGCCAC CTGTGATGACCTG BCL11A- BCL11A UCACAGGCUCCA 202 TTTA agatTCACAGGCTC 296 aaaaAACCCTTCCTG 342 g4 GGAAGGGUU CAGGAAGGGTT GAGCCTGTGA VEGFA- VEGFA GAGAGUGAGGAC 205 CTTC agatGAGAGTGAGG 297 aaaaGACACACACGT 343 g1 GUGUGUGUC ACGTGTGTGTC CCTCACTCTC B2M- B2M UGGCCUGGAGG 183 TTTC agatTGGCCTGGAG 298 aaaaCGCTGGATAGC 344 g1_21nt CUAUCCAGCG GCTATCCAGCG CTCCAGGCCA TRAC TRAC GAGUCUCUCAGC 189 TTTA AGATGAGTCTCTC 299 AAAAGTGTACCAGC 345 g3_20nt UGGUACAC AGCTGGTACAC TGAGAGACTC TRAC TRAC GAGUCUCUCAGC 190 TTTA AGATGAGTCTCTC 300 AAAACGTGTACCAG 346 g3_21nt UGGUACACG AGCTGGTACACG CTGAGAGACTC TRAC TRAC GAGUCUCUCAGC 191 TTTA AGATGAGTCTCTC 301 AAAACCGTGTACCA 347 g3_22nt UGGUACACGG AGCTGGTACACGG GCTGAGAGACTC TRAC TRAC GAGUCUCUCAGC 192 TTTA AGATGAGTCTCTC 302 AAAATGCCGTGTAC 348 g3_24nt UGGUACACGGCA AGCTGGTACACGG CAGCTGAGAGACTC CA Match6_ Match6 GGGUGAUCAGAC 206 TTTG AGATGGGTGATCA 303 AAAAGCTGTTGGGT 349 20nt CCAACAGC GACCCAACAGC CTGATCACCC Match6_ Match6 GGGUGAUCAGAC 207 TTTG AGATGGGTGATCA 304 AAAATGCTGTTGGG 350 21nt CCAACAGCA GACCCAACAGCA TCTGATCACCC Match6 Match6 GGGUGAUCAGAC 208 TTTG AGATGGGTGATCA 305 AAAACTGCTGTTGG 351 22nt CCAACAGCAG GACCCAACAGCAG GTCTGATCACCC Match6_ Match6 GGGUGAUCAGAC 209 TTTG AGATGGGTGATCA 306 AAAATGCTGTTGGG 350 23nt CCAACAGCAGG GACCCAACAGCAG TCTGATCACCC G Match6_ Match6 GGGUGAUCAGAC 210 TTTG AGATGGGTGATCA 307 AAAAACCTGCTGTT 352 24nt CCAACAGCAGGU GACCCAACAGCAG GGGTCTGATCACCC GT

[0264] Cell lines: U2OS-EGFP cells, harboring a single integrated copy of an EGFP reporter gene, and wild-type U2OS and HEK293T cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 2 mM L-Glutamine (Life Technologies) and penicillin/streptomycin (Thermo Fisher). All cells were incubated at 37 C. and 5% CO.sub.2 in a humidified atmosphere. All cells tested mycoplasma negative (PlasmoTest, Invivogen).

7.1.2. Identification of Novel Type V-A Cas Molecules from Metagenomic Samples

[0265] Type V CRISPR-Cas loci were predicted using CRISPRCasTyper (Russel, J., Pinilla-Redondo, R., Mayo-Muoz, D., Shah, S. A. & Srensen, S. J. CRISPRCasTyper: Automated Identification, Annotation, and Classification of CRISPR-Cas Loci. CRISPR J 3, 462-469 (2020)) version 1.8.0, starting from a collection of >1M metagenome-assembled genomes (MAGs) and reference genomes (Blanco-Mguez, A. et al. Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4. Nat. Biotechnol. 41, 1633-1644 (2023)). A total of 14,568 Type V Cas proteins were recovered. Type V Cas proteins were clustered at 60% sequence identity and 60% sequence coverage using MMseq2 (Steinegger, M. & Sding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026-1028 (2017)) version 13.45111 (-c 0.6--cov-mode 5--min-seq-id 0.6--cluster-reassign) and aligned using mafft (Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772-780 (2013)) version 7.490 (--maxiterate 100). The resulting alignment was trimmed using TrimAl (Capella-Gutirrez, S., Silla-Martnez, J. M. & Gabaldn, T. trimAI: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972-1973 (2009)) version 1.4.rev15 (-gappyout) and used to generate a phylogenetic tree using IQ-TREE 2 (Minh, B. Q. et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 37, 1530-1534 (2020)) version 2.0.3 (-B 1000) and automatic model selection (Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587-589 (2017)), which was visualized using GraPhIAn (Asnicar, F., Weingart, G., Tickle, T. L., Huttenhower, C. & Segata, N. Compact graphical representation of phylogenetic data and metadata with GraPhIAn. PeerJ 3, e1029 (2015)) version 1.1.3. PAM predictions were performed using PAMpredict (Ciciani, M. et al. Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data. Nat. Commun. 13, 6474 (2022)), clustering Type V-A Cas proteins at 90% sequence identity. For selected Type V-A Cas proteins, crRNAs resulting from MinCED predictions (Bland, C. et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8, 209 (2007)) were manually checked for conservation of the 3 end sequence. The structure of the 3 end was checked by aligning the crRNAs using Clustal Omega (Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)) version 1.2.4, generating a consensus secondary structure with RNAalifold version 2.4. 17 (-p-r-d2--noLP) (Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011)) and analyzing the resulting structure with R2R (Weinberg, Z. & Breaker, R. R. R2R--software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinformatics 12, 3 (2011)) version 1.0.6.

7.1.3. PAM Assay

[0266] An in vitro PAM evaluation of the novel Type V-A Cas proteins was performed according to a modified version of the protocol from Karvelis, Young and Siksnys (Karvelis et al., 2019, Methods in Enzymology 616:219-240). The gRNAs to perform the assay were obtained by in vitro transcription using the HighYield T7 RNA Synthesis Kit (Jena Bioscience) starting from a PCR template generated by amplification from each gRNA expression construct. The primers used to generate the IVT templates are reported in Table 9. In vitro transcribed gRNAs were subsequently purified using the MEGAClear Transcription Clean-up kit (Thermo Fisher Scientific). HEK293T cells were transfected 48 hours before the study with nuclease-expressing plasmids, and protein lysates were collected and used for RNP complex formation. The complex was assembled by combining 20 L of the supernatant containing the soluble Type V-A Cas proteins with 1 L of RiboLock RNase Inhibitor (Thermo Fisher Scientific) and 2 ug of guide RNAs (previously transcribed in vitro). The RNP complex was used to digest 1 g of a PAM plasmid DNA library (containing a defined target sequence flanked at the 5-end by a randomized 8 nucleotide PAM sequence) for 1 hour at 37 C.

[0267] A double stranded DNA adapter (Table 10) was ligated to the DNA ends generated by the targeted Type V-A Cas protein cleavage and the final ligation product was purified using CleanNGS SPRI beads.

[0268] One round of a two-step PCR (Phusion HF DNA polymerase, Thermo Fisher Scientific) was performed to enrich the sequences that were cut using a set of forward primers annealing on the adapter and a reverse primer designed on the plasmid backbone downstream of the PAM (Table 11). A second round of PCR was performed to attach the Illumina indexes and adapters. PCR products were purified using the GeneJet PCR Purification Kit (Thermo Fisher Scientific).

[0269] The library was analysed with a 71-bp single read sequencing, using a flow cell v2 micro, on an Illumina MiSeq sequencer.

[0270] PAM sequences were extracted from Illumina MiSeq reads and used to generate PAM sequence logos, using Logomaker version 0.8. PAM heatmaps were used to display PAM enrichment, computed dividing the frequency of PAM sequences in the cleaved library by the frequency of the same sequences in a control uncleaved library.

TABLE-US-00036 TABLE9 SequencesoftheprimersusedforPCRamplificationofgRNAsusedastemplatesfor invitrotranscription SEQID Primername Sequence(5.fwdarw.3) NO: ZZKD_PAMassay_F CCTCTAATACGACTCACTATAGCCTTTGGAAGTACTAAGAATTTCTAC 353 TGTTGTAGATAGGTGAAGTTCGAGGGCGACGAA ZZKD_PAMassay_R TTCGTCGCCCTCGAACTTCACCTATCTACAACAGTAGAAATTCTTAGT 354 ACTTCCAAAGGCTATAGTGAGTCGTATTAGAGG ZZQE_PAMassay_F cctcTAATACGACTCACTATAGGCTACTAAGCCTTTATAATTTCTACTAT 355 TGTAGATAGGTGAAGTTCGAGGGCGACgaa ZZQE_PAMassay_R ttcGTCGCCCTCGAACTTCACCTATCTACAATAGTAGAAATTATAAAGG 356 CTTAGTAGCCTATAGTGAGTCGTATTAgagg ZRGM_PAMassay_F cctcTAATACGACTCACTATAGTCTGAAAGACTATATAATTTCTACTTCG 357 TGTAGATAGGTGAAGTTCGAGGGCGACgaa ZRGM_PAMassay_R ttcGTCGCCCTCGAACTTCACCTATCTACACGAAGTAGAAATTATATAG 358 TCTTTCAGACTATAGTGAGTCGTATTAgagg

TABLE-US-00037 TABLE10 SequencesofthetwooligonucleotidesusedtopreparethedsDNA adapterfortheinvitroPAMassay Name Sequence(5.fwdarw.3) SEQIDNO: OligoUP CGGCATTCCTGCTGAACCGCTCTTCCGATCT 359 OligoBOTTOM GATCGGAAGAGCGGTTCAGCAGGAATGCCG 360

TABLE-US-00038 TABLE11 SequencesoftheprimersusedforNGS librarypreparationintheinvitroPAMassay Primer SEQID name Sequence(5.fwdarw.3) NO: F4a TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGC 361 TGCTGAACCGCTCTTCCGATC F4b TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGT 362 AAGACTGCTGAACCGCTCTTCCGATC F4c TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGG 363 CTAGACCTAATGTGATCTGCTGAACCGCTCTTCC GATC R3 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG 364 TCTGCGTTCTGATTTAATCTGTATCAGGC

7.1.4. In Vitro Cleavage Assays

[0271] In vitro cleavage assays were performed using an RNP complex targeting a PCR product. Briefly, the RNP was assembled combining 105.7 pmol of synthetic RNA with 35 pmol of protein (ratio 3:1) and the complex was incubated 15 min at room temperature (approximately 20-22 C.). Two ug of PCR template was diluted in 90 l of R buffer (10 Mm Tris-HCl PH 7.5; 10 mM NaCl; 1 mM DTT) and mixed with 9 l of RNP complex. The reaction was incubated at 37 C. for 1 hour and then run on 1% agarose gel. Digested bands were gel-extracted and purified using a commercial kit (Macherey-Nagel), and sent for Sanger sequencing using the primers TRAC_ex1 forward and TRAC_ex1 reverse reported in Table 12.

7.1.5. Cell Line Transfections

[0272] For studies in HEK293T cells, 100,000 cells were plated in a 24 well plate. 24 hours later, cells were transfected with 500 ng of nuclease-expressing plasmid and 250 ng of sgRNA-expressing plasmid using Mirus TransIT-LT1 according to the manufacturer's instructions. After 15-30 minutes of incubation at room temperature, the mixture was added drop-wise on HEK293T cultures.

[0273] To perform editing studies, 200,000 U2OS-EGFP cells were nucleofected with 500 ng of nuclease-expressing plasmid and 250 ng of sgRNA-expressing plasmid containing a guide designed to target EGFP using the 4D-Nucleofector SE Kit (Lonza), DN-100 program, according to the manufacturer's protocol. After electroporation, cells were plated in a 24-well plate. EGFP knock-out was analyzed 4 days after nucleofection using a BD FACSymphony A1 (BD) flow cytometer.

7.1.6. RNP Electroporation

[0274] 200,000 U2OS cells were electroporated with RNP complexes (450 pmol of crRNAs+150 pmol of recombinant ZZKD Type V-A Cas protein) pre-formed at room temperature for 20 minutes using the 4D-Nucleofector SE Kit (Lonza), DN-100 program, according to the manufacturer's protocol. For RNP electroporation studies in primary human T cells, commercial lots were purchased from CGT preclinical. Briefly, a vial of 1010.sup.6 T cells, was thawed and incubated in RPMI+100 U/mL IL-2 (ImmunoTools). Four hours later, the T cells were counted, spun down, and resuspended in 5 mL of activation media (RPMI+IL-2 100 U/mL+100 L TransAct T cell activator from Miltenyi Biotech), resulting in 10 million cells at a concentration of 2 million cells/mL. Three days post-activation, activated T cells were electroporated using Lonza 4D-Nucleofector, EO115 program, with a pre-assembled RNP complex generated by mixing 450 pmol of the ZZKD Type V-A Cas protein and 150 pmol of the sgRNA and kept at room temperature for 20 minutes before electroporation. KO efficiency was evaluated 4 days post-electroporation by staining the T cells with an anti-human TCR alpha/beta chain antibody (BioLegend) for 30 minutes at 4 C. and quantifying the percentage of negative cells via flow cytometry. The recombinant ZZKD Type V-A protein was custom-produced by Origene, starting from a 6-His tagged (SEQ ID NO: 365) bacterial expression construct generated by gene synthesis (Twist Bioscience), while synthetic guide RNAs were purchased from IDT.

7.1.7. Evaluation of Gene Editing

[0275] Three days after transfection cells were collected and DNA was extracted using the QuickExtract DNA Extraction Solution (Lucigen) according to the manufacturer's instructions. To amplify the target loci, PCR reactions were performed using the HOT FIREPol polymerase (Solis BioDyne) and the oligonucleotides listed in Table 12. The amplified products were purified, sent for Sanger sequencing (EasyRun service, Microsynth) and analyzed with the TIDE web tool (shinyapps.datacurators.nl/tide/) to quantify indels. The forward primers used for generating the amplicons were also exploited for Sanger sequencing reactions.

TABLE-US-00039 TABLE12 PrimersusedtoamplifytargetlociforSangersequencing SEQID SEQID Target Forwardoligo(5.fwdarw.3) NO: Reverseoligo(5.fwdarw.3) NO: TRAC_ex1 CATCACGAGCAGCTGGTTTC 366 TGGCAATGGATAAGGCCGAG 378 B2M_ex1 CTCTAACCTGGCACTGCGTC 367 GGTGCTAGGACATGCGAACTTAG 379 B2M_ex2 TGGCCAGAGTGGAAATGGAA 368 TGTATTTGTGCAAGTGCTGCT 380 PD1_ex1 CACTGCCTCTGTCACTCTCG 369 TGGGGCTCCCATCCTTA 381 PD1_ex2 CCTCACGTAGAAGGAAGAGGC 370 AGAGATGCCGGTCACCATTC 382 PD1_ex3_F AATGGTGACCGGCATCTCTG 371 AAGGCACAGTGGATCATGCA 383 AAVS1 CCTTATATTCCCAGGGCCGG 372 GAGAAAGGGAGTAGAGGCGG 384 VEGFA_2 ACTTTGATGTCTGCAGGCCA 373 GAGCCTCAGCCCTTCCAC 385 EMX1 ATTTCGGACTACCCTGAGGAG 374 GGAATCTACCACCCCAGGCTCT 386 Match6 TGCTAGACTTGCTGCTCCTT 375 TGAAGGGATTGTGCTGGTGT 387 PCSK9 TGAACTTCAGCTCCTGCACA 376 TGCAGTTCCCAGTACGTTCC 388 BCL11A GCATCACAACAGGCAGAGAAT 377 TATGACGTCAGGGGGAGGCAAG 389 GTC TC

7.2. Example 1: Identification and Characterization of Novel Type V-A Cas Molecules

[0276] This Example describes studies performed to identify and characterize ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, and ZPPX TYPE V-A Cas proteins.

7.2.1. Identification of the crRNAs of Novel Type V-A Cas Proteins

[0277] crRNA sequences for the selected Type V-A Cas proteins were identified in silico by extracting the repeat region of the CRISPR arrays associated with each nuclease, as described in the Materials & Methods (Section 7.1). The secondary structures of the identified cRNAs for each of the Type V-A Cas proteins are reported in FIGS. 1A-1E and FIGS. 2A-2E.

7.2.2. In Silico Prediction of the PAM Specificity of Novel Type V-A Cas Proteins

[0278] An in silico PAM prediction pipeline (as reported above in the Materials & Methods (Section 7.1)) has been used to predict the PAM recognition specificity of the novel Type V-A Cas proteins. Table 13 reported here below contains the PAM preferences as predicted by the algorithm. The predicted PAM logos for each enzyme are reported in FIGS. 3A-3E and 4A-4E.

TABLE-US-00040 TABLE13 InsilicopredictedPAMsequencesforselected TypeV-ACasproteins TypeV-ACasprotein PredictedPAM(5.fwdarw.3) ZWGDTypeV-ACas TTTN,TTN ZJHKTypeV-ACas TTTN,TTTV ZIKVTypeV-ACas TTTR,TNNTTTR,DNNTTTR ZZFTTypeV-ACas TTTR YYANTypeV-ACas TTTN ZZGYTypeV-ACas TTTN,TTTR ZKBGTypeV-ACas YTTN,TTTN ZZKDTypeV-ACas TTTN,TTTV ZXPBTypeV-ACas TTTN,DTTN,DTDN ZPPXTypeV-ACas YTTN,TTTN

7.2.3. Evaluation of Type V-A Cas Proteins Editing Activity Using an EGFP Reporter System

[0279] By exploiting the knowledge on their predicted PAM sequences and their identified crRNAs, the ability to cleave selected targets in mammalian cells of the selected Type V-A Cas proteins was investigated. An EGFP reporter system was used as it allowed an easier readout on the editing activity, based on the loss of fluorescence of treated cells quantitatively measured by cytofluorimetry. Two gRNAs targeting the EGFP coding sequence were designed exploiting PAMs which, based on the in silico prediction, were compatible for all the Type V-A Cas proteins and tested in U2OS cells stably expressing a single copy of an EGFP reporter by transient electroporation. Surprisingly, as reported in FIG. 5, some of the evaluated guides in combination with their respective Type V-A Cas protein were able to significantly downregulate EGFP expression in target cells. In particular, ZZKD Type V-A Cas protein showed very high activity with both of the guides (>70 and >95% EGFP KO); additionally, ZJHK, ZZGY, ZXPB and YYAN Type V-A Cas proteins showed appreciable knock-out activity (>20% EGFP KO) with at least one of the gRNAs. The remaining Type V-A Cas proteins did not show editing levels above the background of the assay against the currently evaluated targets in the EGFP coding sequence. These data clearly demonstrate that several of the selected Type V-A Cas proteins were able to modify very efficiently genetic targets in mammalian cells and can thus be exploited to edit the mammalian genome.

7.2.4. Evaluation of ZZKD Type V-A Cas Protein Editing Activity on Benchmark Genomic Loci in Mammalian Cells

[0280] To further validate the editing activity of the highest performing candidate Type V-A Cas protein in the EGFP assay, ZZKD, guide RNAs were designed to target the B2M, TRAC and PD1 benchmark genomic loci in human cells. U2OS cells were electroporated with plasmids encoding ZZKD Type V-A Cas and the selected gRNAs and indel formation was measured by Sanger chromatogram deconvolution on extracted genomic DNA. Strikingly, for all three target loci it was possible to identify at least one gRNA showing high levels of genomic modification (>40%, see FIG. 6A-C) and except for the B2M target locus more than one well performing guide was identified (g3-g4 for the TRAC locus, g1-g2 for the PD1 locus).

[0281] Overall these data clearly demonstrate that ZZKD Type V-A is proficient in editing the human genome at several target sites.

7.3. Example 2: Further Characterization of Novel Type V-A Cas Molecules

[0282] This Example describes studies performed to further characterize Type V-A Cas proteins identified in Example 1.

7.3.1. Evaluation of Additional Type V-A Cas Proteins Editing Activity Using an EGFP Reporter System

[0283] Leveraging on the conserved nature of PAM preferences among Type V-A Cas proteins, guide RNAs targeting the EGFP coding sequence were designed for novel Type V-A Cas proteins isolated from the human microbiome to evaluate their activity in human cells. An EGFP reporter system was used as it allowed an easier readout on the editing activity, based on the loss of fluorescence of treated cells quantitatively measured by cytofluorimetry. Two gRNAs targeting the EGFP coding sequence were designed and evaluated in U2OS cells stably expressing a single copy of the EGFP reporter by transient electroporation. As reported in FIG. 10, while most of the evaluated nucleases showed relatively low levels of EGFP downregulation in this particular assay (close to the detection limit of the assay), some of the selected enzymes were particularly proficient in editing their target sequence. In particular, ZZQE Type V-A Cas protein showed very high activity with both of the evaluated guides (>60% EGFP KO), followed by ZRGM, ZSQQ and ZRXE Type V-A Cas proteins which showed appreciable knock-out activity (>40% EGFP KO) with at least one of the evaluated gRNAs. These data clearly demonstrate that several of the selected Type V-A Cas proteins were able to very efficiently modify genetic targets in mammalian cells and can thus be exploited to modify the genome of mammalian cells.

7.3.2. Evaluation of Novel Type V-A Cas Proteins Editing Activity on Benchmark Genomic Loci in Mammalian Cells

[0284] The evaluation of the editing activity of the top performing Type V-A Cas proteins from the EGFP reporter assay KO, ZZKD, ZRGM and ZZQE, was extended to endogenous genomic loci. Guide RNAs were designed to target the B2M (g2), TRAC (g3) and PD1 (g2) benchmark genomic loci in human cells. HEK293T cells were lipofected with plasmids encoding ZZKD, ZRGM and ZZQE Type V-A Cas proteins and the selected gRNAs and indel formation was measured by Sanger chromatogram deconvolution on extracted genomic DNA. Strikingly, for all three target loci all evaluated Type V-A Cas proteins were able to produce appreciable levels of indels, with some variability depending on the target (FIG. 11).

[0285] Overall, these data clearly demonstrate that among the selected Type V-A Cas proteins, ZZKD is the most efficient in editing the human genome at several target sites.

7.3.3. In Vitro Determination of the PAM Specificity of Top-Performing Novel Type V-A Cas Proteins

[0286] After a first evaluation of their activity in mammalian cells, the PAM preferences of the top performing Type V-A Cas proteins were determined using a well-established in vitro assay. Briefly, ZZKD, ZRGM and ZZQE Type V-A Cas proteins were expressed in HEK293T cells to generate cell lysates which were then used in an in vitro cleavage reaction where a plasmid library including a known target flanked by a randomized 8 nt sequence was cut based on PAM recognition preferences by ribonucleoprotein complexes generated using the cell-expressed nucleases and an in vitro transcribed gRNA targeting the library. Cleaved plasmids were then recovered by amplification and sequenced to determine which PAM sequences were preferentially cleaved (see Materials and Methods for more details). These results confirmed the predicted PAM preferences for ZZKD and ZZQE (see FIG. 4C and FIG. 9, respectively), and in general confirmed the possibility to recognize the TTTV PAM which was used in the initial editing evaluation studies, but showed also additional recognition capabilities. The PAM logos and heatmaps for all the selected Type V-A Cas proteins are reported in FIG. 12A-12B, and FIGS. 13A-13D, while a summary of the in vitro determined PAMs are included in Table 14.

TABLE-US-00041 TABLE14 InvitrodeterminedPAMsequencesforselected TypeV-ACasproteins TypeV-ACasprotein PAM(5.fwdarw.3) ZZKDTypeV-ACas NTTV,VTTV,NCTV,TTTT ZRGMTypeV-ACas YTTV ZZQETypeV-ACas NYYN,NTTN,NCTV

[0287] To further confirm the PAM preferences determined for ZZKD Type V-A Cas, a panel of guide RNAs targeting loci flanked by a VTTV and TTTT PAMs was selected and the editing efficacy of ZZKD towards these loci was evaluated after transient transfection in HEK293T cells. As shown in FIG. 12C, many of the evaluated guides showed efficient editing of the target locus demonstrating the possibility for ZZKD to recognize such PAMs, as indicated by the in vitro assay.

7.4. Example 3: Further Characterization of ZZKD Type V-A Cas Protein

[0288] This example describes additional studies to characterize ZZKD Type V-A Cas protein.

7.4.1. Evaluation of the Cleavage Profile of ZZKD Type V-A Cas Protein

[0289] To further characterize the enzymatic activity of ZZKD Type V-A Cas protein, its cleavage profile was investigated in vitro. Recombinant ZZKD was used to digest in vitro a dsDNA target obtained by PCR amplification of a known target region (TRAC locus, g3). The digestion products were separated on agarose gel and independently Sanger sequenced. Based on the two chromatographic profiles (FIG. 14A), it was possible to determine where the two DNA strands were cut: a staggered double strand break was produced, with the non-target strand cut 23nt downstream (5>3) of the PAM and the target strand cut 18nt upstream (5>3). This is in line with what was observed for other well characterized Type V-A Cas proteins.

7.4.2. Evaluation of Different Nuclear Localization Signals (NLS) for ZZKD Type V-A Cas Protein

[0290] In order to further improve the editing activity of the ZZKD Type V-A Cas protein, alternative types and positioning of nuclear localization signals were evaluated. The amino acid sequence of the different NLS evaluated as well as the relative position are indicated in Table 15 below.

TABLE-US-00042 TABLE15 Nuclearlocalizationsignalsevaluatedintheexample Name Position Aminoacidsequence SEQID SV40 N-term PKKKRKVG 179 bpNLS C-term KRTADGSEFESPKKKRKV 122 FL-SV40 C-term GRSSDDEATADSQHAAPPKKKRKV 180 npNLS C-term KRPAATKKAGQAKKKK 125

[0291] As shown in FIG. 15, when the effect on editing activity of the different NLS designs was evaluated by transient transfection in HEK293T cells using the TRAC benchmark locus (g3) as a target, most of the constructs showed high editing levels with the exception of the single npNLS at the C-terminus, as indicated on the graph. Among all evaluated constructs, the FL-SV40 C-term performed particularly well and was thus used in subsequent studies.

7.5. Example 4: Novel Type V-A Cas Protein Alternative crRNA Scaffolds

[0292] Alternative trimmed scaffolds were evaluated for the top performing identified Type V-A Cas proteins (ZZKD, ZRGM, ZZQE). The editing activity of these enzymes was evaluated using the standard full length scaffold (36 nt) in comparison to a shorter 20nt scaffold, which nevertheless preserves a conserved stem-loop structure shared among the different crRNAs (FIGS. 16A-16C), using the TRAC locus (g3) as a benchmark. After transient transfection in HEK293T cells, indels were measured at the target locus revealing similar editing levels with both versions of the crRNA for all the evaluated nucleases (FIG. 17A). To further confirm this finding, ZZKD Type V-A Cas protein was evaluated on an extended panel of loci including additional guides on TRAC, BCL11A, AAVS1 and B2M. These studies confirmed a similar activity for both versions of the scaffold (FIG. 17B), in accordance with previously generated data. Overall, this demonstrates that truncating the 5-end of the crRNA scaffold does not negatively influence the editing activity of these Type V-A Cas proteins after transfection in human cells.

7.6. Example 5: Evaluation of ZZKD Type V-A Cas Protein Spacer Length

[0293] With the aim of further improving the editing activity of ZZKD Type V-A Cas, different spacer lengths were evaluated to determine which favored the highest target modification. crRNAs with spacer lengths ranging from 20nt to 24nt were evaluated by targeting the TRAC (g3) and Match6 (see, Kleinstiver et al., 2016, Nat Biotechnol. 34 (8): 869-74) benchmark loci by transient transfection in HEK293T cells. While appreciable editing levels were observed for all the evaluated lengths (FIGS. 18A-18B), shorter spacers were generally offering higher activity, with 21nt being the most preferred length.

7.7. Example 6: Side-by-Side Comparison of ZZKD Type V-A Cas Protein Activity with the Commercially Available Benchmark AsCas12a Ultra

[0294] To characterize in depth the editing activity of ZZKD Type V-A Cas, indel formation was compared to the commercially available benchmark AsCas12a Ultra (Zhang et al., 2021, Nat. comms. 12:3908), on a panel of endogenous loci in HEK293T cells after transient transfection. A total of 17 crRNAs targeting multiple genomic loci (TRAC, PD1, EMX1, AAVS1, BCL11A, PCSK9, Match6, VEGFA) were evaluated. Notably, given the PAM compatibility between ZZKD and AsCas12a Ultra, the crRNAs were fully overlapping in all cases. As shown by the violin plots of FIG. 19, summarizing the editing data, the performance of the two nucleases was generally comparable, with ZZKD outperforming AsCas12a Ultra at some loci. The editing levels for each target site for the two nucleases are reported in Table 16 below.

TABLE-US-00043 TABLE 16 Editing levels on endogenous target loci after transient transfection of HEK293T cells (mean SD) Target site ZZKD Type V-A Cas AsCas12a Ultra B2M_g2 16.50 0.99 22.45 3.3 TRAC_g3 28.45 1.77 28.35 1.6 PD1_g2 28.45 1.22 26.45 3.3 BCL11A_g1 30.85 0.35 26.65 1.1 BCL11A_g2 24.10 2.12 22.7 0.3 BCL11A_g3 12.05 3.04 19.55 1.1 PCSK9_g1 24.60 4.24 11.4 0.1 PCSK9_g3 13.20 4.95 15.7 1.6 AAVS1_g1 12.60 0.71 15.5 5.7 AAVS1_g2 31.55 1.20 20.7 0.8 AAVS1_g3 11.85 0.07 9.05 0.1 Match6 28.70 0.28 28.65 2.5 BCL11A_g4 60.65 8.27 57.65 3.5 VEGFA_g1 33.75 3.18 32.35 0.6 EMX1_g2 0.95 0.78 6 0.4 EMX1_g3 20.35 0.35 14.35 3.5 B2M_g1_21nt 54.50 9.19 61.6

[0295] Further to these editing studies, titration studies, where the amounts of transfected nuclease and guide RNA are progressively lowered to better measure differences in the editing activity, were performed on a selection of target loci (BCL11A-g4, B2M-g1 and B2M-g2, VEGFA-g1) in HEK293T cells. As shown in FIGS. 20A-20D, all titration curves showed generally comparable editing activities of the two proteins, with the general tendency for ZZKD Type V-A Cas to perform better than the AsCas12a Ultra benchmark (see for example VEGFA-g1 in FIG. 20B).

[0296] Overall, these data demonstrate that ZZKD Type V-A Cas protein is able to match or outperform the editing activity of the current state-of-the-art commercial AsCas12 Ultra benchmark.

7.8. Example 7: Type V-A Cas Protein Activity after Direct Protein Delivery in Cells

[0297] To demonstrate the efficacy of ZZKD Type V-A Cas protein using alternative delivery modalities, direct ribonucleoprotein (RNP) complex delivery to target cells by electroporation was performed. To this aim, recombinant ZZKD was produced in bacteria and was purified by multiple rounds of chromatography using standard techniques, while crRNAs were obtained either from IDT (chemical synthesis) or through in vitro transcription using the T7 RNA polymerase. The activity of the RNP was initially evaluated in U2OS cells using guides targeting the TRAC (FIG. 21A) and B2M (FIG. 21B) loci. The observed editing activity was generally higher than that of the corresponding electroporated plasmid and, among the different types of crRNA evaluated, the synthetic crRNAs performed better. An AltR-modified guide (a chemical modification available from IDT) targeting B2M was also included in the panel showing editing levels close to the unmodified synthetic guide. Additionally, a titration study using B2M-g2 crRNA was performed by lowering progressively the amount of either recombinant ZZKD or corresponding crRNA and also changing the protein: crRNA ratio from 1:3 to 1:1.5 in order to more stringently evaluate ZZKD potency. As shown in FIG. 21C, in most of the conditions evaluated ZZKD Type V-A Cas protein preserved high levels of editing activity indicating high potency even at low concentrations.

[0298] To further confirm the activity of ZZKD Type V-A Cas as RNP, human commercial primary T cells were electroporated with the complex including a guide targeting the TRAC locus (g3). As shown in FIG. 22, ZZKD was able to produce approximately 80% of TRAC-negative cells as measured by cytofluorimetry, demonstrating high editing activity.

[0299] Overall, these data show not only that ZZKD Type V-A Cas protein is compatible with direct protein delivery in multiple cell types including hard-to-edit primary T cells but that ZZKD is also highly potent and can be used at low concentrations to obtain efficient target modification.

8. SPECIFIC EMBODIMENTS

[0300] The present disclosure is exemplified by the specific embodiments below.

[0301] 1. A Type V Cas protein comprising an amino acid sequence having at least 50% sequence identity to: [0302] (a) the amino acid sequence of a WED-1 domain of a reference protein sequence; [0303] (b) the amino acid sequence of a REC1 domain of a reference protein sequence; [0304] (c) the amino acid sequence of a REC2 domain of a reference protein sequence; [0305] (d) the amino acid sequence of a WED-II domain of a reference protein sequence; [0306] (e) the amino acid sequence of a PI domain of a reference protein sequence; [0307] (f) the amino acid sequence of a WED-III domain of a reference protein sequence; [0308] (g) the amino acid sequence of a RuvC-I domain of a reference protein sequence; [0309] (h) the amino acid sequence of a BH domain of a reference protein sequence; [0310] (i) the amino acid sequence of a RuvC-II domain of a reference protein sequence; [0311] (j) the amino acid sequence of a NUC domain of a reference protein sequence; [0312] (k) the amino acid sequence of a RuvC-III domain of a reference protein sequence; or [0313] (l) the amino acid sequence of the full length of a reference protein sequence; [0314] wherein the reference protein sequence is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 115, or SEQ ID NO: 116.

[0315] 2. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0316] 3. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0317] 4. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0318] 5. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0319] 6. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0320] 7. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0321] 8. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0322] 9. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0323] 10. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0324] 11. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0325] 12. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0326] 13. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0327] 14. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0328] 15. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0329] 16. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the WED-I domain of the reference protein sequence.

[0330] 17. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0331] 18. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0332] 19. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0333] 20. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0334] 21. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0335] 22. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0336] 23. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0337] 24. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0338] 25. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0339] 26. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0340] 27. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0341] 28. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0342] 29. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0343] 30. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0344] 31. The Type V Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the REC1 domain of the reference protein sequence.

[0345] 32. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0346] 33. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0347] 34. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0348] 35. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0349] 36. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0350] 37. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0351] 38. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0352] 39. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0353] 40. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0354] 41. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0355] 42. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0356] 43. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0357] 44. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0358] 45. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0359] 46. The Type V Cas protein of any one of embodiments 1 to 31, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the REC2 domain of the reference protein sequence.

[0360] 47. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0361] 48. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0362] 49. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0363] 50. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0364] 51. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0365] 52. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0366] 53. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0367] 54. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0368] 55. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0369] 56. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0370] 57. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0371] 58. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0372] 59. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0373] 60. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0374] 61. The Type V Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the WED-II domain of the reference protein sequence.

[0375] 62. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0376] 63. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0377] 64. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0378] 65. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0379] 66. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0380] 67. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0381] 68. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0382] 69. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0383] 70. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0384] 71. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0385] 72. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0386] 73. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0387] 74. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0388] 75. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0389] 76. The Type V Cas protein of any one of embodiments 1 to 61, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the PI domain of the reference protein sequence.

[0390] 77. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0391] 78. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0392] 79. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0393] 80. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0394] 81. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0395] 82. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0396] 83. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0397] 84. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0398] 85. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0399] 86. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0400] 87. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0401] 88. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0402] 89. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0403] 90. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0404] 91. The Type V Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the WED-III domain of the reference protein sequence.

[0405] 92. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0406] 93. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0407] 94. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0408] 95. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0409] 96. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0410] 97. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0411] 98. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0412] 99. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0413] 100. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0414] 101. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0415] 102. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0416] 103. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0417] 104. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0418] 105. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0419] 106. The Type V Cas protein of any one of embodiments 1 to 91, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-I domain of the reference protein sequence.

[0420] 107. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0421] 108. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0422] 109. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0423] 110. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0424] 111. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0425] 112. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0426] 113. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0427] 114. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0428] 115. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0429] 116. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0430] 117. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0431] 118. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0432] 119. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0433] 120. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0434] 121. The Type V Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the BH domain of the reference protein sequence.

[0435] 122. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0436] 123. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0437] 124. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0438] 125. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0439] 126. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0440] 127. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0441] 128. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0442] 129. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0443] 130. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0444] 131. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0445] 132. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0446] 133. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0447] 134. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0448] 135. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0449] 136. The Type V Cas protein of any one of embodiments 1 to 121, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-II domain of the reference protein sequence.

[0450] 137. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0451] 138. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0452] 139. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0453] 140. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0454] 141. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0455] 142. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0456] 143. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0457] 144. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0458] 145. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0459] 146. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0460] 147. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0461] 148. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0462] 149. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0463] 150. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0464] 151. The Type V Cas protein of any one of embodiments 1 to 136, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the NUC domain of the reference protein sequence.

[0465] 152. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0466] 153. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0467] 154. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0468] 155. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0469] 156. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0470] 157. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0471] 158. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0472] 159. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0473] 160. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0474] 161. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0475] 162. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0476] 163. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0477] 164. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0478] 165. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0479] 166. The Type V Cas protein of any one of embodiments 1 to 151, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-III domain of the reference protein sequence.

[0480] 167. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 55% identical to the full length of the reference protein sequence.

[0481] 168. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 60% identical to the full length of the reference protein sequence.

[0482] 169. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 65% identical to the full length of the reference protein sequence.

[0483] 170. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 70% identical to the full length of the reference protein sequence.

[0484] 171. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 75% identical to the full length of the reference protein sequence.

[0485] 172. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 80% identical to the full length of the reference protein sequence.

[0486] 173. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 85% identical to the full length of the reference protein sequence.

[0487] 174. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 90% identical to the full length of the reference protein sequence.

[0488] 175. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical to the full length of the reference protein sequence.

[0489] 176. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 96% identical to the full length of the reference protein sequence.

[0490] 177. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 97% identical to the full length of the reference protein sequence.

[0491] 178. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 98% identical to the full length of the reference protein sequence.

[0492] 179. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 99% identical to the full length of the reference protein sequence.

[0493] 180. The Type V Cas protein of embodiment 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the full length of the reference protein sequence.

[0494] 181. The Type V Cas protein of any one of embodiments 1 to 180, which is a chimeric Type V Cas protein.

[0495] 182. The Type V Cas protein of any one of embodiments 1 to 181, which is a fusion protein.

[0496] 183. The Type V Cas protein of embodiment 182, which comprises one or more nuclear localization signals.

[0497] 184. The Type V Cas protein of embodiment 183, which comprises two or more nuclear localization signals.

[0498] 185. The Type V Cas protein of embodiment 183 or embodiment 184, which comprises an N-terminal nuclear localization signal.

[0499] 186. The Type V Cas protein of any one of embodiments 183 to 185, which comprises a C-terminal nuclear localization signal.

[0500] 187. The Type V Cas protein of any one of embodiments 183 to 186, which comprises an N-terminal nuclear localization signal and a C-terminal nuclear localization signal.

[0501] 188. The Type V Cas protein of any one of embodiments 183 to 187, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00044 (SEQIDNO:122) KRTADGSEFESPKKKRKV, (SEQIDNO:123) PKKKRKV, (SEQIDNO:124) PKKKRRV, (SEQIDNO:125) KRPAATKKAGQAKKKK, (SEQIDNO:126) YGRKKRRQRRR, (SEQIDNO:127) RKKRRQRRR, (SEQIDNO:128) PAAKRVKLD, (SEQIDNO:129) RQRRNELKRSP, (SEQIDNO:130) VSRKRPRP, (SEQIDNO:131) PPKKARED, (SEQIDNO:132) PQPKKKPL, (SEQIDNO:133) SALIKKKKKMAP, (SEQIDNO:134) PKQKKRK, (SEQIDNO:135) RKLKKKIKKL, (SEQIDNO:136) REKKKFLKRR, (SEQIDNO:137) KRKGDEVDGVDEVAKKKSKK, (SEQIDNO:138) RKCLQAGMNLEARKTKK, (SEQIDNO:139) NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, (SEQIDNO:140) RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, or (SEQIDNO:178) SSDDEATADSQHAAPPKKKRKV.

[0502] 189. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 122).

[0503] 190. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRKV (SEQ ID NO: 123).

[0504] 191. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRRV (SEQ ID NO: 124).

[0505] 192. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO: 125).

[0506] 193. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 126).

[0507] 194. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKKRRQRRR (SEQ ID NO: 127).

[0508] 195. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 128).

[0509] 196. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RQRRNELKRSP (SEQ ID NO: 129).

[0510] 197. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence VSRKRPRP (SEQ ID NO: 130).

[0511] 198. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PPKKARED (SEQ ID NO: 131).

[0512] 199. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PQPKKKPL (SEQ ID NO: 132).

[0513] 200. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence SALIKKKKKMAP (SEQ ID NO: 133).

[0514] 201. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKQKKRK (SEQ ID NO: 134).

[0515] 202. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKLKKKIKKL (SEQ ID NO: 135).

[0516] 203. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence REKKKFLKRR (SEQ ID NO: 136).

[0517] 204. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00045 (SEQIDNO:137) KRKGDEVDGVDEVAKKKSKK.

[0518] 205. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKCLQAGMNLEARKTKK (SEQ ID NO:138).

[0519] 206. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00046 (SEQIDNO:139) NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.

[0520] 207. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00047 (SEQIDNO:140) RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV.

[0521] 208. The Type V Cas protein of embodiment 188, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00048 (SEQIDNO:178) SSDDEATADSQHAAPPKKKRKV.

[0522] 209. The Type V Cas protein of any one of embodiments 183 to 187, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00049 (SEQIDNO:179) PKKKRKVG.

[0523] 210. The Type V Cas protein of any one of embodiments 183 to 187, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence

TABLE-US-00050 (SEQIDNO:180) GRSSDDEATADSQHAAPPKKKRKV.

[0524] 211. The Type V Cas protein of any one of embodiments 183 to 210, wherein the amino acid sequence of each nuclear localization signal is the same.

[0525] 212. The Type V Cas protein of any one of embodiments 181 to 211, which comprises a fusion partner which is a DNA, RNA or protein modification enzyme, optionally wherein the DNA, RNA or protein modification enzyme is an adenosine deaminase, a cytidine deaminase, a reverse transcriptase, a guanosyl transferase, a DNA methyltransferase, a RNA methyltransferase, a DNA demethylase, a RNA demethylase, a dioxygenase, a polyadenylate polymerase, a pseudouridine synthase, an acetyltransferase, a deacetylase, a ubiquitin-ligase, a deubiquitinase, a kinase, a phosphatase, a NEDD8-ligase, a de-NEDDylase, a SUMO-ligase, a deSUMOylase, a histone deacetylase, a histone acetyltransferase, a histone methyltransferase, or a histone demethylase.

[0526] 213. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a means for deaminating a nucleobase, optionally wherein the means for deaminating a nucleobase is a deaminase, e.g., an adenosine deaminase or cytidine deaminase.

[0527] 214. The Type V Cas protein of any one of embodiments 181 to 213, which comprises a fusion partner comprising a deaminase, optionally wherein the deaminase is an adenosine deaminase or cytidine deaminase.

[0528] 215. The Type V Cas protein of embodiment 214, wherein the amino acid sequence of the deaminase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 214-249.

[0529] 216. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a means for deaminating adenosine, optionally wherein the means for deaminating adenosine is an adenosine deaminase.

[0530] 217. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a fusion partner which is an adenosine deaminase, optionally wherein the amino acid sequence of the adenosine deaminase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 166, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e.

[0531] 218. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a means for deaminating cytidine, optionally wherein the means for deaminating cytidine is a cytidine deaminase.

[0532] 219. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a fusion partner which is a cytidine deaminase.

[0533] 220. The Type V Cas protein of any one of embodiments 181 to 219, which comprises a fusion partner comprising a UGI domain, optionally wherein the amino acid sequence of the UGI domain comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 250.

[0534] 221. The Type V Cas protein of any one of embodiments 181 to 220, which comprises a means for repressing gene expression, optionally wherein the means for repressing gene expression comprises a transcriptional repressor or effector domain thereof.

[0535] 222. The Type V Cas protein of any one of embodiments 181 to 220, which comprises a fusion partner comprising a transcriptional repressor or effector domain thereof.

[0536] 223. The Type V Cas protein of embodiment 221 or embodiment 222, wherein the amino acid sequence of the transcriptional repressor or effector domain thereof comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 251-255.

[0537] 224. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a means for synthesizing DNA from a single-stranded template, optionally wherein the means for synthesizing DNA from a single-stranded template is a reverse transcriptase.

[0538] 225. The Type V Cas protein of any one of embodiments 181 to 212, which comprises a fusion partner which is a reverse transcriptase.

[0539] 226. The Type V Cas protein of embodiment 224 or embodiment 225, wherein the amino acid sequence of the reverse transcriptase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:256 or SEQ ID NO:257.

[0540] 227. The Type V Cas protein of any one of embodiments 181 to 225, which comprises a tag. 228. The Type V Cas protein of embodiment 226, wherein the tag is a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:141) or IPNPLLGLD (SEQ ID NO:142).

[0541] 229. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:1.

[0542] 230. The Type V Cas protein of embodiment 229, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1.

[0543] 231. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:2.

[0544] 232. The Type V Cas protein of any one of embodiments 229 to 231, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:2.

[0545] 233. The Type V Cas protein of embodiment 229 or embodiment 230, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:3.

[0546] 234. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:7.

[0547] 235. The Type V Cas protein of embodiment 234, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:7.

[0548] 236. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:8.

[0549] 237. The Type V Cas protein of any one of embodiments 234 to 236, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:8.

[0550] 238. The Type V Cas protein of embodiment 234 or embodiment 235, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:9.

[0551] 239. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:13.

[0552] 240. The Type V Cas protein of embodiment 239, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 13.

[0553] 241. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:14.

[0554] 242. The Type V Cas protein of any one of embodiments 239 to 241, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14.

[0555] 243. The Type V Cas protein of embodiment 239 or embodiment 240, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 15.

[0556] 244. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:19.

[0557] 245. The Type V Cas protein of embodiment 244, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:19.

[0558] 246. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:20.

[0559] 247. The Type V Cas protein of any one of embodiments 244 to 246, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:20.

[0560] 248. The Type V Cas protein of embodiment 244 or embodiment 245, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:21.

[0561] 249. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:25.

[0562] 250. The Type V Cas protein of embodiment 249, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:25.

[0563] 251. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:26.

[0564] 252. The Type V Cas protein of any one of embodiments 249 to 251, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:26.

[0565] 253. The Type V Cas protein of embodiment 250 or embodiment 251, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:27.

[0566] 254. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:31.

[0567] 255. The Type V Cas protein of embodiment 254, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:31.

[0568] 256. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:32.

[0569] 257. The Type V Cas protein of any one of embodiments 255 to 256, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:32.

[0570] 258. The Type V Cas protein of embodiment 254 or embodiment 255, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:33.

[0571] 259. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:37.

[0572] 260. The Type V Cas protein of embodiment 259, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:37.

[0573] 261. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:38.

[0574] 262. The Type V Cas protein of any one of embodiments 259 to 261, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:38.

[0575] 263. The Type V Cas protein of embodiment 259 or embodiment 260, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:39.

[0576] 264. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:43.

[0577] 265. The Type V Cas protein of embodiment 264, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:43.

[0578] 266. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:44.

[0579] 267. The Type V Cas protein of any one of embodiments 264 to 266, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:44.

[0580] 268. The Type V Cas protein of embodiment 264 or embodiment 265, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:45.

[0581] 269. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:49.

[0582] 270. The Type V Cas protein of embodiment 269, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:49.

[0583] 271. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:50.

[0584] 272. The Type V Cas protein of any one of embodiments 269 to 271, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:50.

[0585] 273. The Type V Cas protein of embodiment 269 or embodiment 270, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:51.

[0586] 274. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:55.

[0587] 275. The Type V Cas protein of embodiment 274, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:55.

[0588] 276. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:56.

[0589] 277. The Type V Cas protein of any one of embodiments 274 to 276, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:56.

[0590] 278. The Type V Cas protein of embodiment 274 or embodiment 275, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:57.

[0591] 279. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:61.

[0592] 280. The Type V Cas protein of embodiment 279, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:61.

[0593] 281. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:62.

[0594] 282. The Type V Cas protein of any one of embodiments 279 to 281, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:62.

[0595] 283. The Type V Cas protein of embodiment 279 or embodiment 280, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:63.

[0596] 284. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:67.

[0597] 285. The Type V Cas protein of embodiment 284, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:67.

[0598] 286. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:68.

[0599] 287. The Type V Cas protein of any one of embodiments 284 to 286, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:68.

[0600] 288. The Type V Cas protein of embodiment 284 or embodiment 285, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:69.

[0601] 289. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:73.

[0602] 290. The Type V Cas protein of embodiment 289, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:73.

[0603] 291. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:74.

[0604] 292. The Type V Cas protein of any one of embodiments 289 to 291, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:74.

[0605] 293. The Type V Cas protein of embodiment 289 or embodiment 290, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:75.

[0606] 294. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:79.

[0607] 295. The Type V Cas protein of embodiment 294, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:79.

[0608] 296. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:80.

[0609] 297. The Type V Cas protein of any one of embodiments 294 to 296, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:80.

[0610] 298. The Type V Cas protein of embodiment 294 or embodiment 295, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:81.

[0611] 299. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:85.

[0612] 300. The Type V Cas protein of embodiment 299, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:85.

[0613] 301. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:86.

[0614] 302. The Type V Cas protein of any one of embodiments 299 to 301, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:86.

[0615] 303. The Type V Cas protein of embodiment 299 or embodiment 300, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:87.

[0616] 304. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:91.

[0617] 305. The Type V Cas protein of embodiment 304, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:91.

[0618] 306. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:92.

[0619] 307. The Type V Cas protein of any one of embodiments 304 to 306, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:92.

[0620] 308. The Type V Cas protein of embodiment 304 or embodiment 305, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:93.

[0621] 309. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:97.

[0622] 310. The Type V Cas protein of embodiment 309, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:97.

[0623] 311. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:98.

[0624] 312. The Type V Cas protein of any one of embodiments 309 to 311, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:98.

[0625] 313. The Type V Cas protein of embodiment 309 or embodiment 310, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:99.

[0626] 314. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:103.

[0627] 315. The Type V Cas protein of embodiment 314, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 103.

[0628] 316. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:104.

[0629] 317. The Type V Cas protein of any one of embodiments 314 to 316, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 104.

[0630] 318. The Type V Cas protein of embodiment 314 or embodiment 315, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 105.

[0631] 319. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:109.

[0632] 320. The Type V Cas protein of embodiment 319, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 109.

[0633] 321. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:110.

[0634] 322. The Type V Cas protein of any one of embodiments 319 to 321, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 110.

[0635] 323. The Type V Cas protein of embodiment 319 or embodiment 320, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:111.

[0636] 324. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:115.

[0637] 325. The Type V Cas protein of embodiment 324, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 115.

[0638] 326. The Type V Cas protein of any one of embodiments 1 to 228, wherein the reference protein sequence is SEQ ID NO:116.

[0639] 327. The Type V Cas protein of any one of embodiments 324 to 326, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 116.

[0640] 328. The Type V Cas protein of embodiment 324 or embodiment 325, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO: 117.

[0641] 329. A Type V Cas protein whose amino acid sequence is identical to a Type V Cas protein of any one of embodiments 1 to 328 except for one or more amino acid substitutions relative to the reference sequence that provide nickase activity, optionally wherein the one or more amino acid substitutions comprise a substitution (e.g., alanine substitution) at a position corresponding to position D908 of Cas12a, E993 of Cas12a, R1226 of Cas12a, or D1263 of Cas12a (e.g., as shown in Table 5), or a combination thereof.

[0642] 330. A ZWGD Type V Cas guide RNA (gRNA) molecule.

[0643] 331. A ZJHK Type V Cas guide RNA (gRNA) molecule.

[0644] 332. A ZIKV Type V Cas guide RNA (gRNA) molecule.

[0645] 333. A ZZFT Type V Cas guide RNA (gRNA) molecule.

[0646] 334. A YYAN Type V Cas guide RNA (gRNA) molecule.

[0647] 335. A ZZGY Type V Cas guide RNA (gRNA) molecule.

[0648] 336. A ZKBG Type V Cas guide RNA (gRNA) molecule.

[0649] 337. A ZZKD Type V Cas guide RNA (gRNA) molecule.

[0650] 338. A ZXPB Type V Cas guide RNA (gRNA) molecule.

[0651] 339. A ZPPX Type V Cas guide RNA (gRNA) molecule.

[0652] 340. A ZXHQ Type V Cas guide RNA (gRNA) molecule.

[0653] 341. A ZQKH Type V Cas guide RNA (gRNA) molecule.

[0654] 342. A ZRGM Type V Cas guide RNA (gRNA) molecule.

[0655] 343. A ZTAE Type V Cas guide RNA (gRNA) molecule.

[0656] 344. A ZSQQ Type V Cas guide RNA (gRNA) molecule.

[0657] 345. A ZSYN Type V Cas guide RNA (gRNA) molecule.

[0658] 346. A ZRBH Type V Cas guide RNA (gRNA) molecule.

[0659] 347. A ZWPU Type V Cas guide RNA (gRNA) molecule.

[0660] 348. A ZZQE Type V Cas guide RNA (gRNA) molecule.

[0661] 349. A ZRXE Type V Cas guide RNA (gRNA) molecule.

[0662] 350. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human B2M gene.

[0663] 351. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human TRAC gene.

[0664] 352. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human PD1 gene.

[0665] 353. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human AAVS1 genomic sequence.

[0666] 354. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human EMX1 gene.

[0667] 355. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human BCL11A gene.

[0668] 356. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human PCSK9 gene.

[0669] 357. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human VEGF gene.

[0670] 358. The gRNA of any one of embodiments 330 to 349, which is a gRNA for editing a human Match6 genomic sequence.

[0671] 359. A guide RNA (gRNA) molecule for editing a human B2M gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 164-168 and 181-183.

[0672] 360. A guide RNA (gRNA) molecule for editing a human TRAC gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 169-173 and 184-192.

[0673] 361. A guide RNA (gRNA) molecule for editing a human PD1 gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 174-177.

[0674] 362. A guide RNA (gRNA) molecule for editing a human AAVS1 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 193-196.

[0675] 363. A guide RNA (gRNA) molecule for editing a human EMX1 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 197-198.

[0676] 364. A guide RNA (gRNA) molecule for editing a human BCL11A genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 199-202.

[0677] 365. A guide RNA (gRNA) molecule for editing a human PCSK9 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 203-204.

[0678] 366. A guide RNA (gRNA) molecule for editing a human VEGF genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is SEQ ID NO:205.

[0679] 367. A guide RNA (gRNA) molecule for editing a human Match6 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 206-210.

[0680] 368. The gRNA of any one of embodiments 353 to 367, which comprises a spacer that is 15 to 30 nucleotides in length.

[0681] 369. The gRNA of embodiment 368, wherein the spacer is 18 to 30 nucleotides in length.

[0682] 370. The gRNA of embodiment 368, wherein the spacer is 20 to 28 nucleotides in length.

[0683] 371. The gRNA of embodiment 368, wherein the spacer is 22 to 26 nucleotides in length.

[0684] 372. The gRNA of embodiment 368, wherein the spacer is 23 to 25 nucleotides in length.

[0685] 373. The gRNA of embodiment 368, wherein the spacer is 22 to 25 nucleotides in length.

[0686] 374. The gRNA of embodiment 368, wherein the spacer is 15 to 25 nucleotides in length.

[0687] 375. The gRNA of embodiment 368, wherein the spacer is 16 to 24 nucleotides in length.

[0688] 376. The gRNA of embodiment 368, wherein the spacer is 17 to 23 nucleotides in length.

[0689] 377. The gRNA of embodiment 368, wherein the spacer is 18 to 22 nucleotides in length.

[0690] 378. The gRNA of embodiment 368, wherein the spacer is 19 to 21 nucleotides in length.

[0691] 379. The gRNA of embodiment 368, wherein the spacer is 25 nucleotides in length.

[0692] 380. The gRNA of embodiment 368, wherein the spacer is 24 nucleotides in length.

[0693] 381. The gRNA of embodiment 368, wherein the spacer is 23 nucleotides in length.

[0694] 382. The gRNA of embodiment 368, wherein the spacer is 22 nucleotides in length.

[0695] 383. The gRNA of embodiment 368, wherein the spacer is 21 nucleotides in length.

[0696] 384. The gRNA of embodiment 368, wherein the spacer is 20 nucleotides in length.

[0697] 385. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 16 or more consecutive nucleotides of the reference sequence.

[0698] 386. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 17 or more consecutive nucleotides of the reference sequence.

[0699] 387. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 18 or more consecutive nucleotides of the reference sequence.

[0700] 388. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 19 or more consecutive nucleotides of the reference sequence.

[0701] 389. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 20 or more consecutive nucleotides of the reference sequence.

[0702] 390. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 21 or more consecutive nucleotides of the reference sequence.

[0703] 391. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 22 or more consecutive nucleotides of the reference sequence.

[0704] 392. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises 23 consecutive nucleotides of the reference sequence.

[0705] 393. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises a nucleotide sequence that is at least 90% identical to the reference sequence.

[0706] 394. The gRNA of embodiment 393, wherein the spacer comprises a nucleotide sequence that is at least 95% identical to the reference sequence.

[0707] 395. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises a nucleotide sequence that has one mismatch relative to the reference sequence.

[0708] 396. The gRNA of any one of embodiments 359 to 384, wherein the spacer comprises a nucleotide sequence that has two mismatches relative to the reference sequence.

[0709] 397. The gRNA of any one of embodiments 359 to 367, wherein the spacer comprises the reference sequence.

[0710] 398. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 164.

[0711] 399. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 165.

[0712] 400. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 166.

[0713] 401. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 167.

[0714] 402. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 168.

[0715] 403. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 181.

[0716] 404. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 182.

[0717] 405. The gRNA of any one of embodiments 359 and 368 to 397 when depending from embodiment 359, wherein the reference sequence is SEQ ID NO: 183.

[0718] 406. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 169.

[0719] 407. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 170.

[0720] 408. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 171.

[0721] 409. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 172.

[0722] 410. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 173.

[0723] 411. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 184.

[0724] 412. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 185.

[0725] 413. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 186.

[0726] 414. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 187.

[0727] 415. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 188.

[0728] 416. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 189.

[0729] 417. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 190.

[0730] 418. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 191.

[0731] 419. The gRNA of any one of embodiments 360 and 368 to 397 when depending from embodiment 360, wherein the reference sequence is SEQ ID NO: 192.

[0732] 420. The gRNA of any one of embodiments 361 and 368 to 397 when depending from embodiment 361, wherein the reference sequence is SEQ ID NO: 174.

[0733] 421. The gRNA of any one of embodiments 361 and 368 to 397 when depending from embodiment 361, wherein the reference sequence is SEQ ID NO: 175.

[0734] 422. The gRNA of any one of embodiments 361 and 368 to 397 when depending from embodiment 361, wherein the reference sequence is SEQ ID NO: 176.

[0735] 423. The gRNA of any one of embodiments 361 and 368 to 397 when depending from embodiment 361, wherein the reference sequence is SEQ ID NO: 177.

[0736] 424. The gRNA of any one of embodiments 362 and 368 to 397 when depending from embodiment 362, wherein the reference sequence is SEQ ID NO: 193.

[0737] 425. The gRNA of any one of embodiments 362 and 368 to 397 when depending from embodiment 362, wherein the reference sequence is SEQ ID NO: 194.

[0738] 426. The gRNA of any one of embodiments 362 and 368 to 397 when depending from embodiment 362, wherein the reference sequence is SEQ ID NO: 195.

[0739] 427. The gRNA of any one of embodiments 362 and 368 to 397 when depending from embodiment 362, wherein the reference sequence is SEQ ID NO: 196.

[0740] 428. The gRNA of any one of embodiments 363 and 368 to 397 when depending from embodiment 363, wherein the reference sequence is SEQ ID NO: 197.

[0741] 429. The gRNA of any one of embodiments 363 and 368 to 397 when depending from embodiment 363, wherein the reference sequence is SEQ ID NO: 198.

[0742] 430. The gRNA of any one of embodiments 364 and 368 to 397 when depending from embodiment 364, wherein the reference sequence is SEQ ID NO: 199.

[0743] 431. The gRNA of any one of embodiments 364 and 368 to 397 when depending from embodiment 364, wherein the reference sequence is SEQ ID NO:200.

[0744] 432. The gRNA of any one of embodiments 364 and 368 to 397 when depending from embodiment 364, wherein the reference sequence is SEQ ID NO:201.

[0745] 433. The gRNA of any one of embodiments 364 and 368 to 397 when depending from embodiment 364, wherein the reference sequence is SEQ ID NO:202.

[0746] 434. The gRNA of any one of embodiments 365 and 368 to 397 when depending from embodiment 365, wherein the reference sequence is SEQ ID NO:203.

[0747] 435. The gRNA of any one of embodiments 365 and 368 to 397 when depending from embodiment 365, wherein the reference sequence is SEQ ID NO:204.

[0748] 436. The gRNA of any one of embodiments 366 and 368 to 397 when depending from embodiment 366, wherein the reference sequence is SEQ ID NO:205.

[0749] 437. The gRNA of any one of embodiments 367 and 368 to 397 when depending from embodiment 367, wherein the reference sequence is SEQ ID NO:206.

[0750] 438. The gRNA of any one of embodiments 367 and 368 to 397 when depending from embodiment 367, wherein the reference sequence is SEQ ID NO:207.

[0751] 439. The gRNA of any one of embodiments 367 and 368 to 397 when depending from embodiment 367, wherein the reference sequence is SEQ ID NO:208.

[0752] 440. The gRNA of any one of embodiments 367 and 368 to 397 when depending from embodiment 367, wherein the reference sequence is SEQ ID NO:209.

[0753] 441. The gRNA of any one of embodiments 367 and 368 to 397 when depending from embodiment 367, wherein the reference sequence is SEQ ID NO:210.

[0754] 442. A gRNA comprising a spacer and a crRNA scaffold, which is optionally a gRNA according to any one of embodiments 330 to 441, wherein: [0755] (a) the spacer is positioned 3 to the crRNA scaffold; and [0756] (b) the nucleotide sequence of the crRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is selected from SEQ ID NOS: 144-163 and 211-213.

[0757] 443. A gRNA comprising a means for binding a target mammalian genomic sequence and a crRNA scaffold, optionally wherein the means for binding a target mammalian genomic sequence is a spacer, wherein: [0758] (a) the means for binding a target genomic sequence is positioned 3 to the crRNA scaffold; and [0759] (b) the nucleotide sequence of the crRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is selected from SEQ ID NOS: 144-163 and 211-213.

[0760] 444. The gRNA of embodiment 442 or 443, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 55% identical to the reference scaffold sequence.

[0761] 445. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 60% identical to the reference scaffold sequence.

[0762] 446. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 65% identical to the reference scaffold sequence.

[0763] 447. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 70% identical to the reference scaffold sequence.

[0764] 448. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 75% identical to the reference scaffold sequence.

[0765] 449. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 80% identical to the reference scaffold sequence.

[0766] 450. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 85% identical to the reference scaffold sequence.

[0767] 451. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 90% identical to the reference scaffold sequence.

[0768] 452. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 95% identical to the reference scaffold sequence.

[0769] 453. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 96% identical to the reference scaffold sequence.

[0770] 454. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 97% identical to the reference scaffold sequence.

[0771] 455. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 98% identical to the reference scaffold sequence.

[0772] 456. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that is at least 99% identical to the reference scaffold sequence.

[0773] 457. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that has no more than 5 nucleotide mismatches with the reference scaffold sequence.

[0774] 458. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that has no more than 4 nucleotide mismatches with the reference scaffold sequence.

[0775] 459. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that has no more than 3 nucleotide mismatches with the reference scaffold sequence.

[0776] 460. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that has no more than 2 nucleotide mismatches with the reference scaffold sequence.

[0777] 461. The gRNA of embodiment 444, wherein the crRNA scaffold comprises a nucleotide sequence that has no more than 1 nucleotide mismatches with the reference scaffold sequence.

[0778] 462. The gRNA of embodiment 442 or embodiment 443, wherein the crRNA scaffold comprises a nucleotide sequence that is 100% identical to the reference scaffold sequence.

[0779] 463. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 144.

[0780] 464. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 145.

[0781] 465. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 146.

[0782] 466. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 147.

[0783] 467. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 148.

[0784] 468. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 149.

[0785] 469. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 150.

[0786] 470. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 151.

[0787] 471. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 152.

[0788] 472. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 153.

[0789] 473. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 154.

[0790] 474. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 155.

[0791] 475. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 156.

[0792] 476. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 157.

[0793] 477. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 158.

[0794] 478. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 159.

[0795] 479. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 160.

[0796] 480. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 161.

[0797] 481. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 162.

[0798] 482. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO: 163.

[0799] 483. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO:211.

[0800] 484. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO:212.

[0801] 485. The gRNA of any one of embodiments 442 to 462, wherein the reference scaffold sequence is SEQ ID NO:213.

[0802] 486. The gRNA of any one of embodiments 442 to 485, wherein the gRNA comprises 1 to 8 uracils at its 3 end.

[0803] 487. The gRNA of embodiment 486, wherein the gRNA comprises 1 uracil at its 3 end.

[0804] 488. The gRNA of embodiment 486, wherein the gRNA comprises 2 uracils at its 3 end.

[0805] 489. The gRNA of embodiment 486, wherein the gRNA comprises 3 uracils at its 3 end.

[0806] 490. The gRNA of embodiment 486, wherein the gRNA comprises 4 uracils at its 3 end.

[0807] 491. The gRNA of embodiment 486, wherein the gRNA comprises 5 uracils at its 3 end.

[0808] 492. The gRNA of embodiment 486, wherein the gRNA comprises 6 uracils at its 3 end.

[0809] 493. The gRNA of embodiment 486, wherein the gRNA comprises 7 uracils at its 3 end.

[0810] 494. The gRNA of embodiment 486, wherein the gRNA comprises 8 uracils at its 3 end.

[0811] 495. The gRNA of any one of embodiments 442 to 494, which comprises a 5 guanine.

[0812] 496. The gRNA of any one of embodiments 442 to 495, wherein the nucleotide sequence of the spacer is partially or fully complementary to a target mammalian genomic sequence.

[0813] 497. The gRNA of embodiment 496, wherein the target mammalian genomic sequence is downstream of a protospacer adjacent motif (PAM) sequence in the non-target strand recognized by a Type V Cas protein, optionally wherein the Type V Cas protein is a Type V Cas protein according to any one of embodiments 1 to 329.

[0814] 498. The gRNA of embodiment 497, wherein the PAM sequence is TTN.

[0815] 499. The gRNA of embodiment 497, wherein the PAM sequence is TTTN, e.g., TTTA, TTTT, TTTG, or TTTC.

[0816] 500. The gRNA of embodiment 497, wherein the PAM sequence is TTTR.

[0817] 501. The gRNA of embodiment 497, wherein the PAM sequence is YTTN, e.g., CTTC or CTTG.

[0818] 502. The gRNA of embodiment 497, wherein the PAM sequence is YTTV.

[0819] 503. The gRNA of embodiment 497, wherein the PAM sequence is NTTV.

[0820] 504. The gRNA of embodiment 497, wherein the PAM sequence is VTTV, e.g., ATTA, or GTTA, or ATTC.

[0821] 505. The gRNA of embodiment 497, wherein the PAM sequence is NCTV.

[0822] 506. The gRNA of embodiment 497, wherein the PAM sequence is DTTN.

[0823] 507. The gRNA of embodiment 497, wherein the PAM sequence is DTDN.

[0824] 508. The gRNA of embodiment 497, wherein the PAM sequence is TTTT.

[0825] 509. The gRNA of embodiment 497, wherein the PAM sequence is NYYN.

[0826] 510. The gRNA of embodiment 497, wherein the PAM sequence is NTTN.

[0827] 511. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 164.

[0828] 512. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 165.

[0829] 513. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 166.

[0830] 514. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 167.

[0831] 515. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 168.

[0832] 516. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 169.

[0833] 517. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 170.

[0834] 518. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 171.

[0835] 519. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 172.

[0836] 520. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 173.

[0837] 521. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 174.

[0838] 522. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 175.

[0839] 523. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 176.

[0840] 524. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 177.

[0841] 525. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 181.

[0842] 526. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 182.

[0843] 527. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 183.

[0844] 528. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 184.

[0845] 529. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 185.

[0846] 530. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 186.

[0847] 531. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 187.

[0848] 532. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:188.

[0849] 533. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 189.

[0850] 534. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 190.

[0851] 535. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 191.

[0852] 536. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 192.

[0853] 537. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 193.

[0854] 538. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 194.

[0855] 539. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 195.

[0856] 540. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 196.

[0857] 541. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 197.

[0858] 542. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 198.

[0859] 543. A gRNA comprising a spacer whose sequence comprises SEQ ID NO: 199.

[0860] 544. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:200.

[0861] 545. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:201.

[0862] 546. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:202.

[0863] 547. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:203.

[0864] 548. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:204.

[0865] 549. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:205.

[0866] 550. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:206.

[0867] 551. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:207.

[0868] 552. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:208.

[0869] 553. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:209.

[0870] 554. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:210.

[0871] 555. A gRNA comprising a spacer whose sequence comprises SEQ ID NO:211.

[0872] 556. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 144.

[0873] 557. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO:145.

[0874] 558. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 146.

[0875] 559. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 147.

[0876] 560. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 148.

[0877] 561. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 149.

[0878] 562. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO:150.

[0879] 563. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 151.

[0880] 564. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 152.

[0881] 565. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 153.

[0882] 566. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 154.

[0883] 567. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 155.

[0884] 568. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 156.

[0885] 569. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 157.

[0886] 570. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 158.

[0887] 571. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 159.

[0888] 572. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 160.

[0889] 573. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 161.

[0890] 574. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 162.

[0891] 575. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO: 163.

[0892] 576. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO:211

[0893] 577. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO:212.

[0894] 578. The gRNA of any one of embodiments 511 to 555, wherein the spacer is positioned 3 to a scaffold whose sequence comprises the sequence of SEQ ID NO:213.

[0895] 579. A system comprising the Type V Cas protein of any one of embodiments 1 to 329 and a guide RNA (gRNA) comprising a spacer sequence, optionally wherein the gRNA is a gRNA according to any one of embodiments 330 to 578.

[0896] 580. A system comprising the Type V Cas protein of any one of embodiments 1 to 329 and a means for targeting the Type V Cas protein to a target genomic sequence, optionally wherein the means for targeting the Type V Cas protein to a target genomic sequence is a guide RNA (gRNA) molecule, optionally as described in in any one of embodiments 330 to 578, optionally wherein the gRNA molecule comprises a spacer partially or fully complementary to a target mammalian genomic sequence.

[0897] 581. The system of embodiment 580, wherein the spacer sequence is partially or fully complementary to a target mammalian genomic sequence.

[0898] 582. The system of any one of embodiments 580 to 581, wherein the target mammalian genomic sequence is a human genomic sequence.

[0899] 583. The system of embodiment 582, wherein the target mammalian genomic sequence is a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence. 584. The system of embodiment 582, wherein the target mammalian genomic sequence is a RHO genomic sequence.

[0900] 585. The system of embodiment 582, wherein the target mammalian genomic sequence is a TRAC genomic sequence.

[0901] 586. The system of embodiment 582, wherein the target mammalian genomic sequence is a B2M genomic sequence.

[0902] 587. The system of embodiment 582, wherein the target mammalian genomic sequence is a PD1 genomic sequence.

[0903] 588. The system of embodiment 582, wherein the target mammalian genomic sequence is an AAVS 1 genomic sequence.

[0904] 589. The system of embodiment 582, wherein the target mammalian genomic sequence is an EMX1 genomic sequence.

[0905] 590. The system of embodiment 582, wherein the target mammalian genomic sequence is an BCL11A genomic sequence.

[0906] 591. The system of embodiment 582, wherein the target mammalian genomic sequence is an PCSK9 genomic sequence.

[0907] 592. The system of embodiment 582, wherein the target mammalian genomic sequence is an VEGFA genomic sequence.

[0908] 593. The system of embodiment 582, wherein the target mammalian genomic sequence is an Match6 genomic sequence.

[0909] 594. The system of any one of embodiments 579 to 593, which is a ribonucleoprotein (RNP) comprising the Type V Cas protein complexed to the gRNA or means for targeting the Type V Cas protein to a target genomic sequence.

[0910] 595. A nucleic acid encoding the Type V Cas protein of any one of embodiments 1 to 329, optionally wherein the nucleotide sequence encoding the Type V Cas protein is operably linked to a promoter that is heterologous to the Type V Cas protein.

[0911] 596. The nucleic acid of embodiment 595, wherein the nucleotide sequence encoding the Type V Cas protein is codon optimized for expression in human cells.

[0912] 597. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:1 or SEQ ID NO:2, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO: 6.

[0913] 598. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:7 or SEQ ID NO:8, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0914] 599. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO: 13 or SEQ ID NO: 14, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0915] 600. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:19 or SEQ ID NO:20, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:23 or SEQ ID NO: 24.

[0916] 601. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:25 or SEQ ID NO:26, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO: 30.

[0917] 602. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:31 or SEQ ID NO:32, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:35 or SEQ ID NO: 36.

[0918] 603. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:37 or SEQ ID NO:38, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:41 or SEQ ID NO: 42.

[0919] 604. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:43 or SEQ ID NO:44, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or SEQ ID NO: 48.

[0920] 605. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:49 or SEQ ID NO:50, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:53 or SEQ ID NO: 54.

[0921] 606. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:55 or SEQ ID NO:56, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:59 or SEQ ID NO: 60.

[0922] 607. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:61 or SEQ ID NO:62, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:65 or SEQ ID NO: 66.

[0923] 608. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:67 or SEQ ID NO:68, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:71 or SEQ ID NO: 72.

[0924] 609. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:73 or SEQ ID NO:74, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:77 or SEQ ID NO: 78.

[0925] 610. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:79 or SEQ ID NO:80, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:83 or SEQ ID NO: 84.

[0926] 611. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:85 or SEQ ID NO:86, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:89 or SEQ ID NO: 90.

[0927] 612. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:91 or SEQ ID NO:92, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:95 or SEQ ID NO: 96.

[0928] 613. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:97 or SEQ ID NO:98, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 101 or SEQ ID NO: 102.

[0929] 614. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO: 103 or SEQ ID NO: 104, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 107 or SEQ ID NO: 108.

[0930] 615. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO: 109 or SEQ ID NO: 110, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 113 or SEQ ID NO: 114.

[0931] 616. The nucleic acid of embodiment 596, wherein when the reference protein sequence is SEQ ID NO:115 or SEQ ID NO: 116, the nucleotide sequence encoding the Type V Cas protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 119 or SEQ ID NO: 120.

[0932] 617. The nucleic acid of any one of embodiments embodiment 595 to 616, which is a plasmid.

[0933] 618. The nucleic acid of any one of embodiments embodiment 595 to 616, which is a viral genome.

[0934] 619. The nucleic acid of embodiment 618, wherein the viral genome is an adeno-associated virus (AAV) genome.

[0935] 620. The nucleic acid of embodiment 619, wherein the AAV genome is an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 genome.

[0936] 621. The nucleic acid of embodiment 620, wherein the AAV genome is an AAV2 genome.

[0937] 622. The nucleic acid of embodiment 620, wherein the AAV genome is an AAV5 genome.

[0938] 623. The nucleic acid of embodiment 620, wherein the AAV genome is an AAV7m8 genome.

[0939] 624. The nucleic acid of embodiment 620, wherein the AAV genome is an AAV8 genome.

[0940] 625. The nucleic acid of embodiment 620, wherein the AAV genome is an AAV9 genome.

[0941] 626. The nucleic acid of embodiment 620, wherein the AAV genome is an AAVrh8r genome.

[0942] 627. The nucleic acid of embodiment 620, wherein the AAV genome is an AAVrh10 genome.

[0943] 628. The nucleic acid of any one of embodiments 595 to 627, further encoding a gRNA, optionally wherein the gRNA is a gRNA according to any one of embodiments 330 to 578.

[0944] 629. A nucleic acid encoding the gRNA of any one of embodiments 330 to 578.

[0945] 630. The nucleic acid of embodiment 629, which is a plasmid.

[0946] 631. The nucleic acid of embodiment 629, which is a viral genome.

[0947] 632. The nucleic acid of embodiment 631, wherein the viral genome is an adeno-associated virus (AAV) genome.

[0948] 633. The nucleic acid of embodiment 632, wherein the AAV genome is a AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 genome.

[0949] 634. The nucleic acid of embodiment 633, wherein the AAV genome is an AAV2 genome.

[0950] 635. The nucleic acid of embodiment 633, wherein the AAV genome is an AAV5 genome.

[0951] 636. The nucleic acid of embodiment 633, wherein the AAV genome is an AAV7m8 genome.

[0952] 637. The nucleic acid of embodiment 633, wherein the AAV genome is an AAV8 genome.

[0953] 638. The nucleic acid of embodiment 633, wherein the AAV genome is an AAV9 genome.

[0954] 639. The nucleic acid of embodiment 633, wherein the AAV genome is an AAVrh8r genome.

[0955] 640. The nucleic acid of embodiment 633, wherein the AAV genome is an AAVrh10 genome.

[0956] 641. The nucleic acid of any one of embodiments 629 to 640, further encoding a Type V Cas protein, optionally wherein the Type V Cas protein is a Type V Cas protein according to any one of embodiments 1 to 329.

[0957] 642. A nucleic acid encoding the Type V Cas protein and gRNA of the system of any one of embodiments 579 to 594.

[0958] 643. The nucleic acid of embodiment 642, wherein the nucleotide sequence encoding the Type V Cas protein is codon optimized for expression in human cells.

[0959] 644. The nucleic acid of embodiment 642 or embodiment 643, which is a plasmid.

[0960] 645. The nucleic acid of embodiment 642 or embodiment 643, which is a viral genome.

[0961] 646. The nucleic acid of embodiment 645, wherein the viral genome is an adeno-associated virus (AAV) genome.

[0962] 647. The nucleic acid of embodiment 646, wherein the AAV genome is an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 genome.

[0963] 648. The nucleic acid of embodiment 647, wherein the AAV genome is an AAV2 genome.

[0964] 649. The nucleic acid of embodiment 647, wherein the AAV genome is an AAV5 genome.

[0965] 650. The nucleic acid of embodiment 647, wherein the AAV genome is an AAV7m8 genome.

[0966] 651. The nucleic acid of embodiment 647, wherein the AAV genome is an AAV8 genome.

[0967] 652. The nucleic acid of embodiment 647, wherein the AAV genome is an AAV9 genome.

[0968] 653. The nucleic acid of embodiment 647, wherein the AAV genome is an AAVrh8r genome.

[0969] 654. The nucleic acid of embodiment 647, wherein the AAV genome is an AAVrh10 genome.

[0970] 655. A plurality of nucleic acids comprising separate nucleic acids encoding the Type V Cas protein and gRNA of the system of any one of embodiments 579 to 594.

[0971] 656. The plurality of nucleic acid of embodiment 655, wherein the separate nucleic acids encoding the Type V Cas protein and gRNA are plasmids.

[0972] 657. The plurality of nucleic acids of embodiment 655, wherein the separate nucleic acids encoding the Type V Cas protein and gRNA are viral genomes.

[0973] 658. The plurality of nucleic acids of embodiment 657, wherein the viral genomes are adeno-associated virus (AAV) genomes.

[0974] 659. The plurality of nucleic acids of embodiment 658, wherein the AAV genomes the encoding the Type V Cas protein and gRNA are independently an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh10 genome.

[0975] 660. A Type V Cas protein according to any one of embodiments 1 to 329, a gRNA according to any one of embodiments 330 to 578, a system according to of any one of embodiments 579 to 594, a nucleic acid according to any one of embodiments 595 to 654, a plurality of nucleic acids according to of any one of embodiments 655 to 659, particle according to any one of embodiments 672 to 687, or pharmaceutical composition according to embodiment 688 for use in a method of editing a human genomic sequence.

[0976] 661. The Type V Cas protein, gRNA, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence.

[0977] 662. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a RHO genomic sequence, optionally wherein the RHO genomic sequence has a pathogenic mutation.

[0978] 663. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a TRAC genomic sequence, optionally wherein the human genomic sequence is in a T cell.

[0979] 664. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a B2M genomic sequence, optionally wherein the human genomic sequence is in a T cell.

[0980] 665. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a PD1 genomic sequence, optionally wherein the human genomic sequence is in a T cell.

[0981] 666. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a LAG3 genomic sequence, optionally wherein the human genomic sequence is in a T cell.

[0982] 667. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a AAVS1 genomic sequence, optionally wherein the human genomic sequence is in a T cell.

[0983] 668. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is an EMX1 genomic sequence.

[0984] 669. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a BCL11A genomic sequence.

[0985] 670. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a PCSK9 genomic sequence.

[0986] 671. The Type V Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to embodiment 660, wherein the human genomic sequence is a Match6 genomic sequence.

[0987] 672. A particle comprising a Type V Cas protein according to any one of embodiments 1 to 329, a gRNA according to any one of embodiments 330 to 578, a system according to of any one of embodiments 579 to 594, a nucleic acid according to any one of embodiments 595 to 654, or a plurality of nucleic acids according to of any one of embodiments 655 to 659.

[0988] 673. The particle of embodiment 667, which is a lipid nanoparticle, a vesicle, a gold nanoparticle, a viral-like particle (VLP) or a viral particle.

[0989] 674. The particle of embodiment 673, which is a lipid nanoparticle.

[0990] 675. The particle of embodiment 673, which is a vesicle.

[0991] 676. The particle of embodiment 673, which is a gold nanoparticle.

[0992] 677. The particle of embodiment 673, which is a viral-like particle (VLP).

[0993] 678. The particle of embodiment 673, which is a viral particle.

[0994] 679. The particle of embodiment 677, which is an adeno-associated virus (AAV) particle.

[0995] 680. The particle of embodiment 679, wherein the AAV particle is an AAV2, AAV5, AAV7m8,

[0996] AAV8, AAV9, AAVrh8r, or AAVrh10 particle.

[0997] 681. The particle of embodiment 680, wherein the AAV particle is an AAV2 particle.

[0998] 682. The particle of embodiment 680, wherein the AAV particle is an AAV5 particle.

[0999] 683. The particle of embodiment 680, wherein the AAV particle is an AAV7m8 particle.

[1000] 684. The particle of embodiment 680, wherein the AAV particle is an AAV8 particle.

[1001] 685. The particle of embodiment 680, wherein the AAV particle is an AAV9 particle.

[1002] 686. The particle of embodiment 680, wherein the AAV particle is an AAVrh8r particle.

[1003] 687. The particle of embodiment 680, wherein the AAV particle is an AAVrh10 particle.

[1004] 688. A pharmaceutical composition comprising a Type V Cas protein according to any one of embodiments 1 to 329, a gRNA according to any one of embodiments 330 to 578, a system according to of any one of embodiments 579 to 594, a nucleic acid according to any one of embodiments 595 to 654, a plurality of nucleic acids according to of any one of embodiments 655 to 659, or a particle according to any one of embodiments 667 to 687 and at least one pharmaceutically acceptable excipient.

[1005] 689. A cell comprising a Type V Cas protein according to any one of embodiments 1 to 329, a gRNA according to any one of embodiments 330 to 578, a system according to of any one of embodiments 579 to 594, a nucleic acid according to any one of embodiments 595 to 654, a plurality of nucleic acids according to of any one of embodiments 655 to 659, or a particle according to any one of embodiments 667 to 687.

[1006] 690. The cell of embodiment 689, which is a human cell.

[1007] 691. The cell of embodiment 689 or embodiment 690, wherein the cell is a hematopoietic progenitor cell.

[1008] 692. The cell of any one of embodiments 689 to 691, which is a stem cell.

[1009] 693. The cell of embodiment 692, wherein the stem cell is a hematopoietic stem cell (HSC), a pluripotent stem cell, or an induced pluripotent stem cell (iPS).

[1010] 694. The cell of embodiment 693, wherein the stem cell is an embryonic stem cell.

[1011] 695. The cell of embodiment 689 or embodiment 690, which is a T cell.

[1012] 696. The cell of embodiment 689 or embodiment 690, which is a retinal cell.

[1013] 697. The cell of embodiment 689 or embodiment 690, which is a photoreceptor cell.

[1014] 698. The cell of any one of embodiments 689 to 697, which is an ex vivo cell.

[1015] 699. A population of cells according to any one of embodiments 689 to 698.

[1016] 700. A method for altering a cell, the method comprising contacting the cell with a Type V Cas protein according to any one of embodiments 1 to 329, a gRNA according to any one of embodiments 330 to 578, a system according to of any one of embodiments 579 to 594, a nucleic acid according to any one of embodiments 595 to 654, a plurality of nucleic acids according to of any one of embodiments 655 to 659, or a particle according to any one of embodiments 667 to 687, or a pharmaceutical composition according to embodiment 688.

[1017] 701. The method of embodiment 700, which comprises contacting the cell with the Type V Cas protein of any one of embodiments 1 to 329.

[1018] 702. The method of embodiment 700, which comprises contacting the cell with the gRNA of any one of embodiments 330 to 578.

[1019] 703. The method of embodiment 700, which comprises contacting the cell with the system of any one of embodiments 579 to 594.

[1020] 704. The method of embodiment 703, which comprises electroporation of the cell prior to contacting the cell with the system.

[1021] 705. The method of embodiment 703, which comprises lipid-mediated delivery of the system to the cell, optionally wherein the lipid-mediated delivery is cationic lipid-mediated delivery.

[1022] 706. The method of embodiment 703, which comprises polymer-mediated delivery of the system to the cell.

[1023] 707. The method of embodiment 703, which comprises delivery of the system to the cell by lipofection.

[1024] 708. The method of embodiment 703, which comprises delivery of the system to the cell by nucleofection.

[1025] 709. The method of embodiment 700, which comprises contacting the cell with the nucleic acid of any one of embodiments 595 to 654.

[1026] 710. The method of embodiment 700, which comprises contacting the cell with the plurality of nucleic acids of any one of embodiments 655 to 659.

[1027] 711. The method of embodiment 700, which comprises contacting the cell with the particle of any one of embodiments 667 to 687.

[1028] 712. The method of embodiment 700, which comprises contacting the cell with the pharmaceutical composition of embodiment 688.

[1029] 713. The method of any one of embodiments 700 to 712, further comprising contacting the cell with a DNA mismatch repair (MMR) inhibitor or nucleic acid encoding the MMR inhibitor, optionally wherein the MMR inhibitor comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:258.

[1030] 714. The method of any one of embodiments 700 to 713, wherein the contacting alters a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence 715. The method of any one of embodiments 700 to 713, wherein the contacting alters a RHO genomic sequence.

[1031] 716. The method of any one of embodiments 700 to 713, wherein the contacting alters a TRAC genomic sequence.

[1032] 717. The method of any one of embodiments 700 to 713, wherein the contacting alters a B2M genomic sequence.

[1033] 718. The method of any one of embodiments 700 to 713, wherein the contacting alters a PD1 genomic sequence.

[1034] 719. The method of any one of embodiments 700 to 713, wherein the contacting alters a LAG3 genomic sequence.

[1035] 720. The method of any one of embodiments 700 to 713, wherein the contacting alters a AAVS1 genomic sequence. 721. The method of any one of embodiments 700 to 713, wherein the contacting alters an EMX1 genomic sequence.

[1036] 722. The method of any one of embodiments 700 to 713, wherein the contacting alters a BCLA11A genomic sequence.

[1037] 723. The method of any one of embodiments 700 to 713, wherein the contacting alters a PCSK9 genomic sequence.

[1038] 724. The method of any one of embodiments 700 to 713, wherein the contacting alters a VEGFA genomic sequence.

[1039] 725. The method of any one of embodiments 700 to 713, wherein the contacting alters a Match6 genomic sequence.

[1040] 726. The method of any one of embodiments 700 to 725, wherein the cell is a human cell.

[1041] 727. The method of any one of embodiments 700 to 726, wherein the cell is a hematopoietic progenitor cell.

[1042] 728. The method of any one of embodiments 700 to 727, wherein the cell is a stem cell.

[1043] 729. The method of embodiment 728, wherein the stem cell is a hematopoietic stem cell (HSC), a pluripotent stem cell, or an induced pluripotent stem cell (iPS).

[1044] 730. The method of embodiment 729, wherein the stem cell is an embryonic stem cell.

[1045] 731. The method of any one of embodiments 700 to 725, wherein the cell is a retinal cell. 732. The method of any one of embodiments 700 to 725, wherein the cell is a photoreceptor cell.

[1046] 733. The method of any one of embodiments 700 to 725, wherein the cell is a T cell.

[1047] 734. The method of any one of embodiments 700 to 733, wherein the contacting is in vitro.

[1048] 735. The method of embodiment 731, further comprising transplanting the cell to a subject.

[1049] 736. The method of any one of embodiments 700 to 730, wherein the contacting is in vivo in a subject.

[1050] 737. A cell or population of cells produced by the method of any one of embodiments 700 to 734.

[1051] 738. A Type V Cas protein according to any one of embodiments 1 to 329, the gRNA according to any one of embodiments 330 to 578, or the system of any one of embodiments 579 to 594 for use in a nucleic acid detection assay.

[1052] 739. A method of detecting a target nucleic acid, comprising (a) combining a test sample with the Type V Cas protein of any one of embodiments 1 to 329, a gRNA comprising a spacer which is partially or fully complementary to a nucleotide sequence present in the target nucleic acid, and a reporter nucleic acid, and (b) detecting cleavage of the reporter nucleic acid, if any, whereby cleavage of the reporter nucleic acid indicates that the target nucleic acid is present in the test sample.

[1053] 740. The method of embodiment 739, wherein the reporter nucleic acid comprises a quenched fluorescent reporter moiety.

9. CITATION OF REFERENCES

[1054] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.