Type ii CAS proteins and applications thereof

Abstract

Type II Cas proteins, for example Type II Cas proteins referred to as DGHJ, CUAZ, AHWY, CBGI, ASDR, BCZZ, DRCY, DRPY, EAJR, and CFDE Type II Cas proteins; gRNAs for Type II Cas proteins; systems comprising Type II Cas proteins and gRNAs; nucleic acids encoding the Type II 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.

Description

TYPE II CAS PROTEINS AND APPLICATIONS THEREOF 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application no.63 / 510,713, filed June 28, 2023 the contents of which are incorporated herein in their entireties by reference thereto. 2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on June 21, 2024, is named ALA-012WO_SL.xml and is 689,481 bytes in size. 3. BACKGROUND

[0003] CRISPR-Cas genome editing with Type II Cas proteins and associated guide RNAs (gRNAs) is a powerful tool with the potential to treat a variety of genetic diseases. Adeno-associated viral vectors (AAVs) are commonly used to deliver Cas proteins, for example Streptococcus pyogenes Cas9 (SpCas9), and their guide RNAs (gRNAs).The discovery of novel nucleases with new PAM specificities can broaden the range of targetable sites in the cell genome, making genome editing more flexible and efficient. 4. SUMMARY

[0004] This disclosure is based, in part, on the discovery of a Type II Cas protein from an unclassified Firmicutes bacterium ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type DGHJ Type II &DV´^, a Type II Cas protein from an unclassified bacterium from the Prochlorococcus genus ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type CUAZ Type II &DV´^, a Type II Cas protein from an unclassified bacterium from the Eggerthellaceae family (referred to KHUHLQ^DV^³ZLOG-type AHWY Type II &DV´), a Type II Cas protein from an unclassified bacterium from the Clostridia class ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type CBGI Type II &DV´), a Type II Cas protein from an unclassified bacterium from the Eggerthellaceae family ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type ASDR Type II &DV´^^^D^7\SH^,,^&DV^SURWHLQ^IURP^an unclassified bacterium from the Clostridium genus (referred to KHUHLQ^DV^³ZLOG-type BCZZ 7\SH^,,^&DV´^^^D^7\SH^,,^&DV^SURWHLQ^IURP^Ellagibacter isourolithinifaciens ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type DRCY 7\SH^,,^&DV´^^^D^7\SH^,,^&DV^SURWHLQ^IURP a Lachnospiraceae bacterium ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type DRPY 7\SH^,,^&DV´^^^D^7\SH^,,^&DV^SURWHLQ^IURP^an unclassified bacterium from the Ruminococcaceae family ^UHIHUUHG^WR^KHUHLQ^DV^³ZLOG-type EAJR Type II &DV´^^^ and a Type II Cas protein from an unclassified Acholeplasmatales bacterium (referred to herein DV^³ZLOG-type CFDE 7\SH^,,^&DV´^.

[0005] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID 12^^^^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³DGHJ 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^DGHJ Type II Cas protein sequences are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0006] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:7 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³CUAZ 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^CUAZ Type II Cas protein sequences are set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0007] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:13 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³AHWY 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^AHWY Type II Cas protein sequences are set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0008] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:19 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³CBGI 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^CBGI Type II Cas protein sequences are set forth in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

[0009] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:25 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³ASDR 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^ASDR Type II Cas protein sequences are set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0010] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:31 (such proteins referred WR^KHUHLQ^DV^³BCZZ 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^BCZZ Type II Cas protein sequences are set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0011] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:37 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³DRCY Type II Cas prRWHLQV´^^^([HPSODU\^DRCY Type II Cas protein sequences are set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

[0012] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:43 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³DRPY 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^DRPY Type II Cas protein sequences are set forth in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.

[0013] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:49 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³EAJR 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^EAJR Type II Cas protein sequences are set forth in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.

[0014] In one aspect, the disclosure provides Type II Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (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% identical, or more) to SEQ ID NO:55 ^VXFK^SURWHLQV^UHIHUUHG^WR^KHUHLQ^DV^³CFDE 7\SH^,,^&DV^SURWHLQV´^^^([HPSODU\^CFDE Type II Cas protein sequences are set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.

[0015] In another aspect, the disclosure provides Type II Cas proteins comprising 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%, or more) sequence identity to a RuvC-I domain, RuvC-II domain, RuvC-III domain, BH domain, REC1_1 domain, REC2 domain, REC1_2 domain, HNH domain, Topo domain or CTD of a DGHJ Type II Cas protein, CUAZ Type II Cas protein, AHWY Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, or CFDE Type II Cas protein. In some embodiments, a Type II Cas protein of the disclosure is a chimeric Type II Cas protein, for example, comprising one or more domains from a DGHJ, CUAZ, AHWY, CBGI, ASDR, BCZZ, DRCY, DRPY, EAJR, and / or CFDE Type II Cas protein(s) and one or more domains from a different Type II Cas protein such as SpCas9.

[0016] In some embodiments, the Type II Cas proteins of the disclosure are in the form of a fusion protein, for example, comprising a DGHJ Type II Cas protein, CUAZ Type II Cas protein, AHWY Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, or CFDE Type II Cas protein sequence fused to one or more additional amino acid sequences, for example, one or more nuclear localization signals and / or one or more tags. Other exemplary fusion partners can enable base editing (e.g., where the fusion partner is nucleoside deaminase) or prime editing (e.g., where the fusion partner is a reverse transcriptase).

[0017] Exemplary features of Type II Cas proteins of the disclosure are described in Section 6.2 and specific embodiments 1 to 287 and 736 to 737, infra.

[0018] In further aspects, the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs). In various embodiments, the disclosure provides gRNAs that can be used with the DGHJ Type II Cas proteins of the disclosure, gRNAs that can be used with the CUAZ Type II Cas proteins of the disclosure, gRNAs that can be used with the AHWY Type II Cas proteins of the disclosure, gRNAs that can be used with the CBGI Type II Cas proteins of the disclosure, gRNAs that can be used with the ASDR Type II Cas proteins of the disclosure, gRNAs that can be used with the BCZZ Type II Cas proteins of the disclosure, gRNAs that can be used with the DRCY Type II Cas proteins of the disclosure, gRNAs that can be used with the DRPY Type II Cas proteins of the disclosure, gRNAs that can be used with the EAJR Type II Cas proteins of the disclosure, and gRNAs that can be used with the CFDE Type II Cas proteins of the disclosure. Exemplary features of the gRNAs of the disclosure are described in Section 6.3 and specific embodiments 288 to 608, infra.

[0019] In further aspects, the disclosure provides systems comprising a Type II Cas protein of the disclosure and one or more gRNAs, e.g., sgRNAs. For example, a system can comprise aribonucleoprotein (RNP) comprising a Type II Cas protein complexed with a gRNA, e.g., an sgRNA or separate crRNA and tracrRNA. Exemplary features of systems are described in Section 6.4 and specific embodiments 609 to 680, infra.

[0020] In another aspect, the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type II Cas protein of the disclosure and, optionally, a guide RNA, for example a sgRNA. In some embodiments, the nucleic acids comprise a Type II Cas protein of the disclosure operably linked to a heterologous promoter, e.g., a mammalian promoter, for example a human promoter.

[0021] In another aspect, the disclosure provides nucleic acids encoding a gRNA, for example a sgRNA, of the disclosure and, optionally, a Type II Cas protein, for example a DGHJ Type II Cas protein, CUAZ Type II Cas protein, AHWY Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, or CFDE Type II Cas protein.

[0022] Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5 and specific embodiments 681 to 735, infra.

[0023] In further aspects, the disclosure provides particles comprising the Type II Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6 and specific embodiments 741 to 756, infra.

[0024] In another aspect, the disclosure provides cells and populations of cells containing or contacted with a Type II 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 and specific embodiments 758 to 766 and 794, infra.

[0025] In another aspect, the disclosure provides pharmaceutical compositions comprising a Type II 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 and specific embodiment 757, infra.

[0026] In another aspect, the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type II Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure. Cells altered according to the methods of the disclosure can be used, for example, to treat subjects having a disease or disorder, e.g., genetic disease or disorder. Features of exemplary methods of altering cells are described in Section 6.8 and specific embodiments 767 to 793, infra. 5. BRIEF DESCRIPTION OF THE FIGURES

[0027] FIGS.1A-1B schematically show a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of exemplary sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for DGHJ Type II Cas protein. FIG.1A shows a longer version (SEQ ID NO:103) (v1) while FIG.1B shows a shorter version (SEQ ID NO:104) (v2) where the last stem-ORRS^ORFDWHG^DW^WKH^^¶-end of the scaffold is truncated.

[0028] FIG.2 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of exemplary sgRNA scaffold (SEQ ID NO:105) (not including the spacer sequence) designed from crRNA and tracrRNA identified for CUAZ Type II Cas protein. For this particular structure, the folding was obtained by setting the folding temperature to 30°C.

[0029] FIGS.3A-3B schematically show a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of exemplary sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for AHWY Type II Cas protein. FIG. 3A shows a longer version (SEQ ID NO:107) (v1) while FIG.3B shows a shorter version (SEQ ID NO:108) (v2) where the last stem-loop locateG^DW^WKH^^¶-end of the scaffold is truncated.

[0030] FIG.4 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:109) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for CBGI Type II Cas protein.

[0031] FIG.5 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:110) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for ASDR Type II Cas protein.

[0032] FIG.6 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:111) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for BCZZ Type II Cas protein.

[0033] FIG.7 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:112) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for DRCY Type II Cas protein.

[0034] FIG.8 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:113) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for DRPY Type II Cas protein.

[0035] FIG.9 schematically shows a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:114) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for EAJR Type II Cas protein.

[0036] FIG.10 schematically show a hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of an exemplary sgRNA scaffold (SEQ ID NO:115) (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for CFDE Type II Cas protein.

[0037] FIGS.11A-11D show PAM sequence logos for DGHJ Type II Cas obtained using a longer (v1, FIG.11A) or shorter version of the sgRNA scaffold (v2, FIG.11C) in an in vitro PAM discovery assay and PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for DGHJ Type II Cas (positions 1, 2 and 3, 4) in combination with the same long (FIG.11B) and short (FIG.11D) sgRNA scaffolds.

[0038] FIGS.12A-12B show a PAM sequence logo for CUAZ Type II Cas from an in vitro PAM discovery assay (FIG.12A) and the PAM enrichment heatmap calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for CUAZ Type II Cas (positions 2, 3 and 5, 7; FIG.12B).

[0039] FIGS.13A-13D show PAM sequence logos for AHWY Type II Cas obtained using a longer (v1, FIG.13A) or shorter version of the sgRNA scaffold (v2, FIG.13C) in an in vitro PAM discovery assay and PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for AHWY Type II Cas (positions 2, 3 and 4, 6) in combination with the same long (FIG.13B) and short (FIG.13D) sgRNA scaffolds.

[0040] FIGS.14A-14B show a PAM sequence logo for CBGI Type II Cas from an in vitro PAM discovery assay (FIG.14A) and the PAM enrichment heatmap calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for CBGI Type II Cas (positions 3, 4 and 5, 6; FIG.14B).

[0041] FIGS.15A-15B show a PAM sequence logo for ASDR Type II Cas from an in vitro PAM discovery assay (FIG.15A) and the PAM enrichment heatmap calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for ASDR Type II Cas (positions 3, 4 and 5, 6; FIG.15B).

[0042] FIGS.16A-16B show a PAM sequence logo for BCZZ Type II Cas from an in vitro PAM discovery assay (FIG.16A) and the PAM enrichment heatmap calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for BCZZ Type II Cas (positions 1,2 and 3,4; FIG.16B).

[0043] FIGS.17A-C shows a PAM sequence logo for DRCY Type II Cas as predicted in silico (FIG. 17A) and determined in vitro (FIG.17B); and PAM enrichment heatmap (FIG.17C) calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for DRCY Type II Cas

[0044] FIGS.18A-18F show PAM sequence logos for DRPY Type II Cas obtained in combination with its an exemplary DRPY sgRNA scaffold (FIG.18A) or DRCY sgRNA scaffold (FIG.18D) using an in vitro PAM discovery assay. PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for DRPY Type II Cas in combination with its the exemplary DRPY sgRNA (FIG.18B-18C) and DRCY sgRNA (FIG.18E-18F) scaffold.

[0045] FIGS.19A-19B show a PAM sequence logo for EAJR Type II Cas from an in vitro PAM discovery assay (FIG.19A) and the PAM enrichment heatmap calculated from the same in vitro PAMdiscovery assay showing the nucleotide preferences at different positions along the PAM for EAJR Type II Cas (positions 2, 4 and 5, 6; FIG.19B).

[0046] FIGS.20A-20B show a PAM sequence logo for CFDE Type II Cas from an in vitro PAM discovery assay (FIG.20A) and the PAM enrichment heatmap calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for CFDE Type II Cas (positions 2, 3 and 4, 5; FIG.20B).

[0047] FIG.21 shows the activity of Type II Cas proteins against an EGFP reporter in mammalian cells. ³Y^´^DQG^³Y^´^UHIHU^WR^WZR^GLIIHUHQW^VJ51$^VFDIIROGV^HYDOXDWHG^ZLth AHWY and DGHJ Type II Cas proteins. Data presented as mean ± SEM of n=2 biologically independent runs.

[0048] FIGS.22A-22C show the editing activity of AHWY, ASDR, CBGI, CFDE and DRPY Type II Cas in combination with panels of sgRNAs targeting the B2M (FIG.22A), TRAC (FIG.22B), PD-1 (FIG.22C) after transient plasmid transfection in HEK293T cells. Data presented as mean ± SEM for n=2 independent runs.

[0049] FIGS.23A-23C show the editing activity of ASDR, CFDE and DRPY Type II Cas in combination with selected sgRNAs targeting TRAC, B2M, and PD1 (only for DRPY Type II Cas) after transient mRNA transfection in primary T cells. Indels (FIGS. 23A and 23C) and target downregulation as measured by cytofluorimetry (FIG.23B) are shown. Comparative data have been generated for the three loci also for the benchmark nuclease SpCas9 using previously published sgRNAs. Data presented as mean ± SEM for n=2 independent runs, except for the B2M guide for SpCas9 in (FIGS.23A-23B) and for panel (FIG.23C) where n=1.

[0050] FIG.24 shows the editing activity of DRPY Type II Cas while targeting multiple loci simultaneously (TRAC, B2M, and PD1) in primary T cells after transient mRNA electroporation in combination with selected sgRNAs. Comparative data have been generated also for the benchmark nuclease SpCas9 using SUHYLRXVO\^SXEOLVKHG^VJ51$V^^'DWD^SUHVHQWHG^DV^PHDQ^^^6(0^IRU^Q^^^LQGHSHQGHQW^UXQV^

[0051] FIGS. 25A-25D show the genome-wide specificity profile of DRPY Type II Cas for selected guides targeting the B2M (FIG.25A) and PD1 (FIG.25B) loci in HEK293T cells after transient plasmid transfection. Comparative data have been generated also for the benchmark nuclease SpCas9 using previously published sgRNAs targeting B2M (FIG.25C) and PD1 (FIG.25D). Figure discloses SEQ ID NOS 578-581, respectively, in order of appearance. 6. DETAILED DESCRIPTION

[0052] In one aspect, the disclosure provides Type II Cas proteins (e.g., DGHJ Type II Cas proteins, CUAZ Type II Cas proteins, AHWY Type II Cas proteins, CBGI Type II Cas proteins, ASDR Type II Cas proteins, BCZZ Type II Cas proteins, DRCY Type II Cas proteins, DRPY Type II Cas proteins, EAJR Type II Cas proteins, and CFDE Type II Cas proteins). Type II Cas proteins of the disclosure can be in the form of fusion proteins. Unless required otherwise by context, disclosures relating to Type II Cas proteins encompass Type II Cas proteins which are not fusion proteins and Type II Cas proteins which are in the form of fusion proteins (e.g., Type II Cas protein comprising one or more nuclear localization signals and / or one or more tags).

[0053] In some embodiments, a Type II 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%, or more) sequence identity to a RuvC-I domain, RuvC-IIdomain, RuvC-III domain, BH domain, REC1_2 domain, REC2 domain, REC1_2 domain, HNH domain, Topo domain or CTD of a DGHJ Type II Cas protein, CUAZ Type II Cas protein, AHWY Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, or CFDE Type II Cas protein. In some embodiments, a Type II Cas protein of the disclosure is a chimeric Type II Cas protein, for example, comprising one or more domains from a DGHJ Type II Cas protein and / or CUAZ Type II Cas protein and / or AHWY Type II Cas protein and / or CBGI Type II Cas protein and / or ASDR Type II Cas protein and / or BCZZ Type II Cas protein and / or DRCY Type II Cas protein and / or DRPY Type II Cas protein and / or EAJR Type II Cas protein and / or CFDE Type II Cas protein, and one or more domains from a different Type II Cas protein such as SpCas9.

[0054] Exemplary features of Type II Cas proteins of the disclosure are described in Section 6.2.

[0055] In another aspect, the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs). Exemplary features of the gRNAs of the disclosure are described in Section 6.3.

[0056] In further aspects, the disclosure provides systems comprising a Type II Cas protein of the disclosure and one or more gRNAs, e.g., sgRNAs. Exemplary features of systems are described in Section 6.4.

[0057] In further aspects, the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type II Cas protein of the disclosure and, optionally, a guide RNA, for example a sgRNA, and provides nucleic acids encoding a gRNA, for example a sgRNA, of the disclosure and, optionally, a Type II Cas protein. Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5.

[0058] In further aspects, the disclosure provides particles comprising the Type II Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6.

[0059] In another aspect, the disclosure provides cells and populations of cells containing or contacted with a Type II 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.

[0060] In another aspect, the disclosure provides pharmaceutical compositions comprising a Type II 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.

[0061] In another aspect, the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type II 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.

[0062] Those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can beobtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. 6.1. Definitions

[0063] 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.

[0064] $V^XVHG^LQ^WKH^VSHFLILFDWLRQ^DQG^FODLPV^^WKH^VLQJXODU^IRUP^³D^´^³DQ^´^DQG^³WKH´^LQFOXGH^SOXUDO^ references unless the context clearO\^GLFWDWHV^RWKHUZLVH^^)RU^H[DPSOH^^WKH^WHUP^³DQ^DJHQW´^LQFOXGHV^D^ plurality of agents, including mixtures thereof.

[0065] 8QOHVV^LQGLFDWHG^RWKHUZLVH^^DQ^³RU´^FRQMXQFWLRQ^LV^LQWHQGHG^WR^EH^XVHG^LQ^LWV^FRUUHFW^VHQVH^DV^D^ 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 ERWK^$^DQG^%^DUH^VHOHFWHG^^^,Q^VRPH^SODFHV^LQ^WKH^WH[W^^WKH^WHUP^³DQG^RU´^LV^XVHG^IRU^the same purpose, ZKLFK^VKDOO^QRW^EH^FRQVWUXHG^WR^LPSO\^WKDW^³RU´^LV^XVHG^ZLWK^UHIHUHQFH^WR^PXWXDOO\^H[FOXVLYH^DOWHUQDWLYHV^

[0066] A Type II Cas protein refers to a wild-type or engineered Type II Cas protein. Engineered Type II Cas proteins can also be referred to as Type II Cas variants. For the avoidance of doubt, any GLVFORVXUH^SHUWDLQLQJ^WR^D^³7\SH^,,^&DV´^RU^³7\SH^,,^&DV^SURWHLQ´^SHUWDLQV^WR^ZLOG-type Type II Cas proteins and Type II Cas variants, unless the context dictates otherwise. A Type II Cas protein can have nuclease activity or be catalytically inactive (e.g., as in a dCas).

[0067] 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. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, by manual alignment or using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for achieving maximum alignment.

[0068] Guide RNA molecule (gRNA) refers to an RNA capable of forming a complex with a Type II Cas protein and which can direct the Type II Cas protein to a target DNA. gRNAs typically comprise a spacer of 15 to 30 nucleotides in length. gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically FRPSULVH^D^VSDFHU^DW^WKH^^¶^HQG^RI^WKH^PROHFXOH^DQG^D^^¶^VJ51$^VFDIIROG^^ Various non-OLPLWLQJ^H[DPSOHV^RI^^¶^VJ51$^VFDIIROGV^DUH^GHVFULEHG^LQ^6HFWLRQ^6.3.

[0069] $Q^VJ51$^FDQ^LQ^VRPH^HPERGLPHQWV^FRPSULVH^QR^XUDFLO^EDVH^DW^WKH^^¶^HQG^RI^WKH^VJ51$^ sequence. AlternaWLYHO\^^D^VJ51$^FDQ^FRPSULVH^RQH^RU^PRUH^XUDFLO^EDVHV^DW^WKH^^¶^HQG^RI^WKH^VJ51$^ VHTXHQFH^^)RU^H[DPSOH^^D^VJ51$^FDQ^FRPSULVH^^^XUDFLO^^8^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^^^XUDFLO^^88^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^^^XUDFLO^^888^^DW^WKH^^¶^HQG^Rf the sgRNA sequence, 4 XUDFLO^^8888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^^^XUDFLO^^88888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^ VHTXHQFH^^^^XUDFLO^^888888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^^^XUDFLO^^8888888^^DW^WKH^^¶^HQG^ of the sgRNA sequence, or 8 uracil (UUU88888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^'LIIHUHQW^OHQJWK^ VWUHWFKHV^RI^XUDFLO^FDQ^EH^DSSHQGHG^DW^WKH^^¶^HQG^RI^D^VJ51$^DV^WHUPLQDWRUV^^7KXV^^IRU^H[DPSOH^^WKH^^¶^ sgRNA scaffolds set forth in Section 6.3 can be modified by adding or removing one or more uracils at the end of the sequence.

[0070] 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). 7KH^WHUPV^³SRO\SHSWLGH^VHTXHQFH´^RU^³DPLQR^DFLG^VHTXHQFH´^DUH^DQ^DOSKDEHWLFDO^UHSUHVHQWDWLRQ^RI^D^ polypeptide molecule.

[0071] 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 XUDFLO^^8^^IRU^WK\PLQH^^7^^ZKHQ^WKH^SRO\QXFOHRWLGH^LV^51$^^7KXV^^WKH^WHUP^³QXFOHRWLGH^VHTXHQFH´^LV^WKH^alphabetical representation of a polynucleotide molecule. The letters used in polynucleotide sequences GHVFULEHG^KHUHLQ^FRUUHVSRQG^WR^,83$&^QRWDWLRQ^^)RU^H[DPSOH^^WKH^OHWWHU^³1´^LQ^D^QXFOHRWLGH^VHTXHQFH^ represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA VHTXHQFH^^WKH^OHWWHU^³5´^LQ^D^QXFOHRWLGH^VHTXHQFH^UHSUHVHQWV^D^QXFOHRWLGH^ZKLFK^FDQ^EH^$^RU^*^^DQG^WKH^ OHWWHU^³9´^LQ^D^QXFOHRWLGH^VHTXHQFH^UHSUHVHQWV^D^QXFOHRWLGH^ZKLFK^FDQ^EH^³$^^&^^RU^*^

[0072] Protospacer adjacent motif (PAM) refers to a DNA sequence downstream (e.g., immediately downstream) of a target sequence on the non-target strand recognized by a Type II Cas protein. A PAM VHTXHQFH^LV^ORFDWHG^^¶^RI^WKH^WDUJHW^VHTXHQFH^RQ^WKH^QRQ-target strand.

[0073] 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 II Cas protein, the gRNA directs the Type II Cas to the target sequence in the genomic DNA. A spacer of a Type II 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 II Cas Proteins 6.2.1.1. DGHJ Type II Cas Proteins

[0074] In one aspect, the disclosure provides DGHJ Type II Cas proteins. DGHJ Type II Cas proteins can be further classified as Type IIA Cas proteins. The DGHJ Type II 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 DGHJ Type II 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 DGHJ Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:1.

[0075] Exemplary DGHJ Type II Cas protein sequences and nucleotide sequences encoding exemplary DGHJ Type II Cas proteins are set forth in Table 1A. TABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO DGHJ Type II RKDADYNIGLDIGVASVGWCVTNDENNILKRNGKNMWGSRLFSEASTAK 1 Cas amino NTRMLRSSRRRLERRKNRILYLQQIFQDDMEKEYPNFFPMLKETANIPED acid KRITKLLDGKKYNLFSEQNFTDVQYYNQKTIYHLRKYLIESNKKADLRLVY sequence LALHHIIKYRGNFLYEGDFNNQVDEIENDILIVNNFVKENYFINSKIEEKEIIN (without N- ILKDKTKTKAEKKEILIKVFEFDSASKPVITNIVKAIIGYNFDVSKIFEIDYEK terminal GNISFAKEIENEEEIEELLGNQVEIYEAMKNIYSWFTLQDILKGNKYISDAFI methionine) EKYDKYKGDLILLKNIYKKYYSNEYKSFFKEENINNYVHYNGKQIRKLDKS CDNNEFFKKVSQKIKSLPEDYEKLYDEVNKKVKIENEKNGSNIELDDRYR DKENILQDIESGNFICKLNVTDNGAIPHQLHEIELKKIIENQKQYYEFLNEN AEKIIKIFETRIPYYVGPLIEGKKGSRWSWLIKNNEQEKAHIRPWNMEETIN KNETAEKFINELTNYCTYLQDEKVMPKQSILYSKYCVLNELNNIRINGKHIA KDFKQKIINEYFERKTKVTKKDIISFYDKEGINVSSFEGLSDVNNFNSNMK SYKDMIEILGKVDDSNIEMCEKIIYYVTIFEDKKILREKIKNEYSGINDEQLR RIMKLKYSGWSRLSRKFLTGIKSYNNETIMDRLMNTHENLMQIINNKELG FDKIIENNIPKKSGRITYEDVDEIQTSPANKRAIWQTICVVNEIKKIMKKEPKTABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO NIYIEFARSEDRNKRLKDSRLKNLLKIYEDIKEQIDEIKKFDPDVYKELKKH QDDKDVTEKMYLYFIQNGKCLYSGEKLDIDELDKYEVDHIIPRSYIKDDSI DNKALVKRRENQRKKDSLLLTEDVIRKNISWWKNLYDNNLISAKKYHNLT RRKMFETESEKNKFVQRQLVETRQITKYVTNLLKDGEENVEIYALRAELT HWFREKYEIYKNRNVNNCHHAQDAYIISTIGNIISKEWPKKEDFQYGQYV DNYLREAKAKNEKYGMVIGFISKRVEISKIKQAMNYKDYFISRMLEEQTG EFYNQTIQKAGQGKIKLKPNKDTNRYGGYTNENEAYSVIYKYIDKSGKQQ YKMIGIPIRIASDIKRKRTNLQEYITDVDFNNNDNIELQIIRNKIMKNQIYLDQ NDEPLMLCSSKEIRPAKELILNEEMSKLVYMMNSSENKLTDEEKEEVKRS YSKMYDYLMSKLEKEYKIHKNSYMRLINKKEEFETLEDEDKKKAINGIIDL MFKGQGNLKVLGMSDREGRMSGKTFKTDKLIHMTFVDKSVTGMYERRQ KINGMENNSSK DGHJ Type II MRKDADYNIGLDIGVASVGWCVTNDENNILKRNGKNMWGSRLFSEAST 2 Cas amino AKNTRMLRSSRRRLERRKNRILYLQQIFQDDMEKEYPNFFPMLKETANIP acid EDKRITKLLDGKKYNLFSEQNFTDVQYYNQKTIYHLRKYLIESNKKADLRL sequence VYLALHHIIKYRGNFLYEGDFNNQVDEIENDILIVNNFVKENYFINSKIEEKE IINILKDKTKTKAEKKEILIKVFEFDSASKPVITNIVKAIIGYNFDVSKIFEIDYE KGNISFAKEIENEEEIEELLGNQVEIYEAMKNIYSWFTLQDILKGNKYISDA FIEKYDKYKGDLILLKNIYKKYYSNEYKSFFKEENINNYVHYNGKQIRKLDK SCDNNEFFKKVSQKIKSLPEDYEKLYDEVNKKVKIENEKNGSNIELDDRY RDKENILQDIESGNFICKLNVTDNGAIPHQLHEIELKKIIENQKQYYEFLNE NAEKIIKIFETRIPYYVGPLIEGKKGSRWSWLIKNNEQEKAHIRPWNMEETI NKNETAEKFINELTNYCTYLQDEKVMPKQSILYSKYCVLNELNNIRINGKH IAKDFKQKIINEYFERKTKVTKKDIISFYDKEGINVSSFEGLSDVNNFNSNM KSYKDMIEILGKVDDSNIEMCEKIIYYVTIFEDKKILREKIKNEYSGINDEQL RRIMKLKYSGWSRLSRKFLTGIKSYNNETIMDRLMNTHENLMQIINNKEL GFDKIIENNIPKKSGRITYEDVDEIQTSPANKRAIWQTICVVNEIKKIMKKEP KNIYIEFARSEDRNKRLKDSRLKNLLKIYEDIKEQIDEIKKFDPDVYKELKK HQDDKDVTEKMYLYFIQNGKCLYSGEKLDIDELDKYEVDHIIPRSYIKDDS IDNKALVKRRENQRKKDSLLLTEDVIRKNISWWKNLYDNNLISAKKYHNLT RRKMFETESEKNKFVQRQLVETRQITKYVTNLLKDGEENVEIYALRAELT HWFREKYEIYKNRNVNNCHHAQDAYIISTIGNIISKEWPKKEDFQYGQYV DNYLREAKAKNEKYGMVIGFISKRVEISKIKQAMNYKDYFISRMLEEQTG EFYNQTIQKAGQGKIKLKPNKDTNRYGGYTNENEAYSVIYKYIDKSGKQQ YKMIGIPIRIASDIKRKRTNLQEYITDVDFNNNDNIELQIIRNKIMKNQIYLDQ NDEPLMLCSSKEIRPAKELILNEEMSKLVYMMNSSENKLTDEEKEEVKRS YSKMYDYLMSKLEKEYKIHKNSYMRLINKKEEFETLEDEDKKKAINGIIDL MFKGQGNLKVLGMSDREGRMSGKTFKTDKLIHMTFVDKSVTGMYERRQ KINGMENNSSK DGHJ Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSRKDADYNIGLDIGVA 3 Cas SVGWCVTNDENNILKRNGKNMWGSRLFSEASTAKNTRMLRSSRRRLER mammalian RKNRILYLQQIFQDDMEKEYPNFFPMLKETANIPEDKRITKLLDGKKYNLF expression SEQNFTDVQYYNQFKTIYHLRKYLIESNKKADLRLVYLALHHIIKYRGNFL construct YEGDFNNQVDEIENDILIVNNFVKENYFINSKIEEKEIINILKDKTKTKAEKK (with N- EILIKVFEFDSASKPVITNIVKAIIGYNFDVSKIFEIDYEKGNISFAKEIENEEE terminal IEELLGNQVEIYEAMKNIYSWFTLQDILKGNKYISDAFIEKYDKYKGDLILLK methionine, NIYKKYYSNEYKSFFKEENINNYVHYNGKQIRKLDKSCDNNEFFKKVSQK stop codon, IKSLPEDYEKLYDEVNKKVKIENEKNGSNIELDDRYRDKENILQDIESGNFI V5 tag, N- CKLNVTDNGAIPHQLHEIELKKIIENQKQYYEFLNENAEKIIKIFETRIPYYV terminal NLS GPLIEGKKGSRWSWLIKNNEQEKAHIRPWNMEETINKNETAEKFINELTN and C- YCTYLQDEKVMPKQSILYSKYCVLNELNNIRINGKHIAKDFKQKIINEYFER terminal NLS) KTKVTKKDIISFYDKEGINVSSFEGLSDVNNFNSNMKSYKDMIEILGKVDD (aa) SNIEMCEKIIYYVTIFEDKKILREKIKNEYSGINDEQLRRIMKLKYSGWSRL SRKFLTGIKSYNNETIMDRLMNTHENLMQIINNKELGFDKIIENNIPKKSGR ITYEDVDEIQTSPANKRAIWQTICVVNEIKKIMKKEPKNIYIEFARSEDRNK RLKDSRLKNLLKIYEDIKEQIDEIKKFDPDVYKELKKHQDDKDVTEKMYLY FIQNGKCLYSGEKLDIDELDKYEVDHIIPRSYIKDDSIDNKALVKRRENQRTABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO KKDSLLLTEDVIRKNISWWKNLYDNNLISAKKYHNLTRRKMFETESEKNK FVQRQLVETRQITKYVTNLLKDGEENVEIYALRAELTHWFREKYEIYKNR NVNNCHHAQDAYIISTIGNIISKEWPKKEDFQYGQYVDNYLREAKAKNEK YGMVIGFISKRVEISKIKQAMNYKDYFISRMLEEQTGEFYNQTIQKAGQG KIKLKPNKDTNRYGGYTNENEAYSVIYKYIDKSGKQQYKMIGIPIRIASDIK RKRTNLQEYITDVDFNNNDNIELQIIRNKIMKNQIYLDQNDEPLMLCSSKEI RPAKELILNEEMSKLVYMMNSSENKLTDEEKEEVKRSYSKMYDYLMSKL EKEYKIHKNSYMRLINKKEEFETLEDEDKKKAINGIIDLMFKGQGNLKVLG MSDREGRMSGKTFKTDKLIHMTFVDKSVTGMYERRQKINGMENNSSKS RKRTADGSEFESPKKKRKV DGHJ Type II ATGAGAAAAGACGCAGATTATAATATAGGATTAGATATAGGAGTAGCT 4 Cas coding TCTGTTGGTTGGTGTGTAACAAATGATGAAAATAATATATTGAAAAGAA sequence (nt) ATGGAAAAAACATGTGGGGGTCACGATTGTTTAGTGAAGCATCAACA (not codon GCTAAAAATACGCGAATGTTAAGATCATCTAGAAGGAGACTAGAAAGA optimized) AGAAAAAATAGAATTTTATATTTGCAGCAAATATTTCAAGATGACATGG AAAAAGAATATCCTAATTTCTTTCCTATGCTTAAAGAAACTGCGAATAT CCCAGAAGACAAAAGAATAACAAAATTATTGGATGGAAAAAAATATAA CTTATTTTCAGAACAGAATTTTACAGATGTACAATATTATAATCAATTCA AAACTATTTATCATCTTAGAAAATATTTAATTGAATCAAACAAAAAAGC CGATTTAAGGCTTGTTTATCTTGCTCTTCACCATATCATAAAATACAGA GGAAATTTCTTATATGAGGGAGATTTTAACAATCAAGTAGACGAAATT GAAAATGATATTTTAATAGTTAATAACTTTGTAAAAGAGAACTATTTTAT AAATTCAAAAATAGAAGAAAAAGAAATAATAAATATTTTAAAAGATAAA ACTAAAACTAAGGCTGAAAAGAAAGAGATATTGATAAAAGTATTCGAA TTTGATAGTGCATCAAAACCTGTGATAACAAATATTGTAAAAGCTATTA TTGGCTATAATTTTGATGTAAGTAAAATATTTGAAATTGATTATGAGAA AGGAAATATTTCATTTGCAAAAGAAATAGAAAACGAAGAAGAAATTGA AGAACTATTAGGAAATCAAGTAGAAATCTATGAAGCAATGAAAAACAT ATATAGTTGGTTTACATTACAAGATATCTTAAAGGGAAATAAATATATA TCAGATGCATTTATAGAAAAATATGATAAATATAAAGGAGATCTAATAC TACTAAAAAATATATACAAAAAATATTATTCTAATGAATATAAATCTTTT TTCAAAGAAGAAAACATAAATAATTATGTACATTATAATGGAAAACAAA TAAGAAAATTAGACAAGTCTTGTGATAACAATGAATTTTTCAAAAAAGT ATCTCAAAAAATAAAAAGCCTTCCAGAGGACTATGAAAAATTATATGAT GAAGTAAATAAAAAAGTAAAAATAGAAAATGAAAAAAACGGAAGTAAT ATTGAGCTGGATGATAGATACAGGGATAAAGAAAATATTTTACAAGAC ATAGAAAGCGGAAACTTCATATGTAAATTAAATGTGACAGATAATGGT GCAATACCACATCAATTACATGAAATCGAATTAAAGAAAATTATTGAGA ATCAAAAACAATATTATGAATTTTTAAATGAAAATGCAGAAAAAATTATA AAAATATTTGAGACAAGAATCCCATATTATGTTGGACCATTAATTGAAG GAAAAAAAGGAAGCAGATGGTCATGGCTTATAAAGAATAATGAACAAG AAAAGGCACACATTAGACCTTGGAACATGGAAGAAACAATTAATAAAA ATGAAACAGCAGAGAAATTCATAAATGAATTAACAAACTATTGCACTTA TTTGCAAGATGAAAAAGTAATGCCAAAGCAATCAATCTTATATTCCAAA TATTGTGTATTGAATGAATTGAACAATATAAGAATAAATGGAAAGCATA TAGCGAAAGACTTTAAACAGAAGATAATTAACGAATATTTTGAGAGAA AAACCAAGGTTACTAAGAAAGACATAATAAGTTTTTATGATAAAGAGG GAATAAATGTTTCAAGCTTTGAAGGATTGTCAGATGTAAACAACTTTAA CTCAAATATGAAATCATATAAAGACATGATAGAGATATTGGGCAAAGT AGATGATAGTAACATAGAAATGTGCGAAAAAATAATATATTATGTAACA ATATTTGAAGATAAAAAAATACTAAGAGAAAAGATAAAAAATGAATATT CAGGAATAAATGATGAACAATTAAGAAGAATAATGAAGTTAAAATATTC TGGCTGGTCAAGACTTTCTAGAAAATTCTTGACAGGAATTAAATCATA TAACAATGAAACTATAATGGATAGACTAATGAATACTCATGAAAATCTT ATGCAAATAATAAACAATAAAGAACTTGGTTTTGATAAAATAATTGAAA ATAATATACCTAAAAAAAGCGGAAGGATTACTTATGAGGATGTTGATG AAATTCAGACGTCACCAGCAAATAAAAGAGCAATTTGGCAAACAATATTABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO GTGTTGTTAATGAAATAAAAAAGATAATGAAAAAAGAACCTAAAAATAT TTATATAGAGTTTGCGAGAAGTGAGGATAGAAATAAAAGGCTAAAAGA TTCTAGATTAAAAAATCTTTTAAAAATTTATGAGGACATAAAAGAACAA ATAGACGAAATAAAGAAATTTGATCCAGATGTATATAAAGAGCTAAAG AAACATCAAGACGATAAAGATGTTACTGAAAAAATGTATTTGTATTTTA TTCAAAATGGAAAATGTTTATATAGTGGAGAAAAGTTGGATATAGACG AACTAGACAAGTATGAAGTTGACCATATCATACCTCGTTCTTATATAAA AGATGATAGCATCGATAATAAGGCTTTGGTTAAGAGAAGAGAAAATCA AAGGAAGAAAGATAGCTTACTTTTAACAGAAGATGTTATCAGAAAAAA TATTTCGTGGTGGAAAAACTTATATGACAATAATTTAATTAGTGCAAAA AAATATCATAATTTAACAAGAAGAAAAATGTTTGAAACTGAAAGTGAAA AAAACAAATTTGTTCAAAGACAGCTTGTCGAAACAAGGCAGATAACTA AGTATGTTACAAATTTACTAAAAGATGGGGAAGAAAATGTTGAAATTTA TGCATTAAGAGCAGAGTTAACACATTGGTTCAGAGAAAAATACGAAAT ATATAAAAATAGAAATGTAAATAATTGTCACCATGCTCAGGATGCATAT ATAATTTCGACTATTGGAAATATAATAAGTAAAGAATGGCCAAAAAAAG AAGATTTCCAATATGGACAATATGTGGATAATTATTTAAGAGAAGCAAA AGCTAAGAATGAAAAATATGGCATGGTTATAGGGTTCATTAGCAAAAG AGTTGAAATAAGCAAAATAAAGCAAGCTATGAACTATAAAGATTACTTC ATAAGTAGAATGTTAGAAGAGCAAACTGGAGAATTTTATAATCAAACA ATTCAAAAAGCTGGGCAAGGAAAAATCAAACTCAAACCTAATAAAGAT ACAAACAGATATGGGGGATATACCAACGAAAATGAGGCATATAGTGTT ATATACAAATATATTGACAAATCTGGAAAACAACAATATAAGATGATAG GAATTCCAATTAGAATTGCAAGTGATATTAAGAGAAAAAGGACTAATTT ACAAGAGTATATTACAGATGTGGATTTCAACAATAATGATAATATTGAA CTGCAAATTATAAGAAACAAAATAATGAAGAATCAAATATATCTTGACC AGAATGATGAGCCATTAATGCTATGTAGTTCAAAAGAAATAAGACCTG CAAAAGAACTGATATTAAATGAAGAAATGTCAAAACTAGTATATATGAT GAACAGTTCGGAAAATAAATTAACAGATGAAGAAAAAGAAGAAGTAAA AAGATCGTATTCAAAAATGTATGACTATTTAATGAGCAAACTAGAAAAA GAATATAAAATCCATAAAAACAGTTATATGAGACTAATAAACAAAAAAG AAGAATTTGAAACACTAGAAGACGAGGATAAGAAAAAAGCAATCAATG GGATTATTGATTTGATGTTCAAAGGACAAGGAAATTTAAAAGTTTTAG GGATGTCAGACAGAGAAGGAAGAATGAGTGGCAAAACATTCAAAACT GATAAATTAATACATATGACGTTTGTTGATAAATCGGTAACAGGAATGT ACGAAAGAAGGCAAAAGATAAATGGGATGGAGAACAATAGTAGTAAG TAA DGHJ Type II AGAAAAGATGCTGACTACAACATCGGTCTGGACATCGGCGTGGCCTC 5 Cas coding CGTGGGCTGGTGCGTGACCAACGACGAGAACAATATCCTGAAACGG sequence (nt) AACGGCAAGAACATGTGGGGCTCTCGGCTGTTCAGCGAAGCGTCTA (human CCGCCAAGAATACCAGAATGCTCAGATCCAGCAGACGTAGACTGGAA codon- AGACGGAAGAACAGAATCCTGTACCTGCAGCAGATCTTTCAGGACGA optimized; CATGGAAAAGGAGTACCCCAACTTCTTTCCAATGCTGAAGGAAACCG lacking N- CCAACATCCCAGAGGACAAGAGAATCACAAAGCTCCTGGACGGCAAA terminal AAGTACAACCTGTTCAGCGAGCAGAACTTCACCGATGTCCAGTATTAC methionine; AACCAGTTCAAGACCATCTACCACCTGCGGAAGTACCTGATCGAGAG no stop CAACAAGAAGGCCGATCTGAGACTGGTTTACCTGGCCCTCCATCACA codon) TCATCAAGTACAGAGGCAACTTCCTGTACGAGGGCGACTTCAACAAC CAAGTGGACGAAATCGAGAATGACATCCTGATCGTGAACAACTTCGT GAAAGAGAACTACTTCATCAACTCTAAGATCGAGGAAAAGGAAATCAT CAATATCCTGAAGGACAAGACCAAAACCAAAGCTGAGAAAAAGGAAA TCCTGATCAAGGTGTTCGAGTTTGACAGCGCCTCTAAACCTGTGATCA CCAACATCGTGAAGGCTATCATTGGCTACAACTTCGACGTGTCCAAG ATCTTCGAGATCGACTACGAAAAAGGCAATATCAGCTTCGCCAAAGAA ATCGAAAATGAAGAAGAGATCGAAGAGCTGCTTGGCAACCAGGTGGA AATCTATGAAGCCATGAAGAACATCTACTCCTGGTTCACCCTGCAGGA CATCCTGAAGGGCAACAAGTACATCAGCGACGCCTTCATAGAGAAGTTABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO ACGACAAGTACAAGGGCGATCTGATCCTGCTGAAGAACATCTATAAA AAGTACTACTCTAACGAGTACAAGAGCTTTTTTAAGGAAGAAAACATT AACAATTACGTGCACTACAATGGAAAGCAGATCAGAAAGCTGGACAA GTCATGCGACAACAATGAGTTCTTCAAGAAGGTTTCTCAGAAAATCAA GAGTCTGCCTGAGGACTACGAGAAGCTGTACGACGAGGTGAACAAG AAGGTGAAGATCGAGAATGAGAAGAACGGCAGCAACATCGAGCTGG ACGACAGATACAGAGACAAGGAAAACATCCTGCAGGACATTGAGAGC GGCAACTTTATTTGTAAACTGAACGTGACAGACAACGGCGCCATCCC CCACCAGCTGCACGAGATCGAGCTGAAGAAGATCATCGAGAACCAAA AGCAGTACTACGAGTTCCTGAACGAGAATGCCGAGAAGATTATTAAG ATATTCGAAACCAGAATCCCTTACTATGTGGGACCTCTGATCGAGGG AAAAAAGGGCAGCAGATGGAGCTGGCTGATCAAAAACAACGAGCAAG AGAAGGCCCACATCAGACCTTGGAACATGGAAGAAACCATCAATAAA AACGAAACAGCTGAAAAGTTCATCAACGAATTAACTAACTACTGCACC TACCTGCAAGACGAAAAGGTGATGCCCAAACAGAGCATCCTGTACAG CAAGTATTGTGTGCTGAACGAGCTGAACAACATTAGAATCAACGGAAA GCACATCGCCAAGGATTTCAAGCAGAAGATAATCAACGAATACTTCGA AAGAAAGACCAAGGTCACCAAGAAGGACATTATCAGCTTTTACGACAA GGAAGGCATCAACGTGTCCTCTTTCGAAGGCCTGAGCGACGTGAACA ACTTCAACTCAAATATGAAGTCCTACAAGGATATGATAGAGATTCTGG GAAAGGTGGACGATAGCAACATCGAGATGTGCGAGAAAATCATCTAC TACGTGACAATCTTCGAGGATAAAAAGATTCTGAGGGAAAAGATCAAG AATGAGTATAGCGGCATCAACGACGAACAGTTGAGAAGAATCATGAA ACTTAAGTACAGCGGGTGGTCCAGACTGAGCAGAAAATTCCTGACCG GCATCAAGTCTTACAACAATGAAACGATTATGGATCGACTGATGAACA CCCATGAGAACCTGATGCAGATCATCAACAACAAGGAGTTAGGATTT GATAAGATCATCGAGAACAACATCCCAAAGAAGAGCGGCAGAATCAC CTACGAGGACGTGGACGAGATTCAGACCAGCCCTGCTAACAAAAGG GCCATCTGGCAGACCATCTGCGTGGTGAACGAGATCAAGAAAATCAT GAAAAAGGAGCCTAAGAACATCTACATCGAGTTCGCCAGAAGCGAGG ATAGAAACAAGAGACTTAAAGATTCCAGACTGAAGAACCTGCTGAAGA TCTACGAGGACATCAAGGAGCAGATCGACGAGATCAAGAAATTCGAT CCTGACGTGTACAAAGAACTGAAGAAGCACCAGGATGATAAGGACGT GACCGAGAAGATGTACCTGTATTTCATCCAGAACGGCAAGTGTCTGT ACAGCGGAGAGAAGCTGGACATCGACGAGCTGGACAAGTACGAGGT GGACCACATAATACCCAGATCATACATCAAGGACGACAGCATCGATA ACAAGGCCCTGGTGAAGCGGAGAGAGAATCAGAGAAAGAAGGATTC TCTGCTGCTGACCGAGGACGTGATCAGGAAGAACATCAGCTGGTGG AAGAATCTGTATGACAACAACCTGATAAGCGCCAAGAAGTACCACAA CCTGACCAGACGGAAAATGTTCGAGACAGAGAGCGAGAAAAACAAAT TCGTGCAGCGGCAGCTGGTCGAGACAAGACAGATCACAAAGTACGT GACAAACCTGCTGAAGGATGGCGAAGAGAACGTGGAAATCTACGCC CTGCGGGCCGAGCTGACCCACTGGTTTAGAGAGAAATACGAGATATA TAAGAATCGGAACGTGAACAACTGCCACCACGCCCAGGACGCCTACA TCATCAGCACAATCGGCAATATCATCAGCAAGGAGTGGCCTAAGAAG GAGGATTTCCAGTACGGGCAGTACGTGGACAACTACCTGAGAGAGG CCAAGGCCAAGAACGAGAAGTACGGCATGGTGATCGGCTTCATCTCT AAAAGAGTGGAAATCAGCAAGATCAAACAGGCCATGAACTACAAGGA TTACTTTATCAGCAGAATGCTGGAAGAGCAAACCGGCGAGTTCTACA ACCAGACAATCCAAAAGGCCGGCCAGGGAAAGATCAAGCTGAAACCT AACAAGGACACCAATAGATACGGCGGATACACCAACGAAAACGAAGC ATATTCTGTGATCTACAAGTACATCGACAAGAGCGGCAAACAGCAGTA TAAGATGATCGGCATCCCTATCAGAATCGCCTCTGACATCAAGAGAAA GCGGACAAATCTGCAGGAGTACATCACAGATGTGGATTTCAATAACA ACGACAACATAGAGCTGCAGATCATCCGCAACAAAATCATGAAAAAC CAGATCTACCTGGATCAGAACGACGAACCCCTGATGCTGTGCAGCAG CAAGGAAATCAGACCCGCTAAAGAACTGATCCTGAATGAAGAGATGATABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO GCAAGCTCGTATACATGATGAACAGCAGCGAGAACAAGCTGACAGAT GAAGAAAAGGAAGAGGTGAAGCGGAGCTATAGCAAGATGTACGACTA CCTGATGTCCAAGCTGGAGAAGGAGTACAAAATCCACAAGAACTCCT ACATGAGACTGATCAACAAAAAGGAGGAATTTGAGACACTGGAGGAC GAGGATAAGAAAAAGGCCATCAACGGCATCATCGACCTGATGTTCAA GGGCCAGGGCAACCTGAAGGTCCTGGGCATGAGCGATCGGGAAGG CAGGATGAGTGGAAAGACCTTCAAGACCGACAAGCTGATCCACATGA CATTCGTGGACAAAAGCGTCACCGGCATGTACGAGCGACGGCAGAA GATCAACGGCATGGAAAATAACAGCTCCAAG DGHJ Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 6 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGggatccAGAAAAGATGCTGACTACAACATCGGTCTGGACATCG expression GCGTGGCCTCCGTGGGCTGGTGCGTGACCAACGACGAGAACAATAT construct CCTGAAACGGAACGGCAAGAACATGTGGGGCTCTCGGCTGTTCAGC (with N- GAAGCGTCTACCGCCAAGAATACCAGAATGCTCAGATCCAGCAGACG terminal TAGACTGGAAAGACGGAAGAACAGAATCCTGTACCTGCAGCAGATCT methionine, TTCAGGACGACATGGAAAAGGAGTACCCCAACTTCTTTCCAATGCTG stop codon, AAGGAAACCGCCAACATCCCAGAGGACAAGAGAATCACAAAGCTCCT V5 tag, N- GGACGGCAAAAAGTACAACCTGTTCAGCGAGCAGAACTTCACCGATG terminal NLS TCCAGTATTACAACCAGTTCAAGACCATCTACCACCTGCGGAAGTACC and C- TGATCGAGAGCAACAAGAAGGCCGATCTGAGACTGGTTTACCTGGCC terminal NLS) CTCCATCACATCATCAAGTACAGAGGCAACTTCCTGTACGAGGGCGA (nt) CTTCAACAACCAAGTGGACGAAATCGAGAATGACATCCTGATCGTGA ACAACTTCGTGAAAGAGAACTACTTCATCAACTCTAAGATCGAGGAAA AGGAAATCATCAATATCCTGAAGGACAAGACCAAAACCAAAGCTGAG AAAAAGGAAATCCTGATCAAGGTGTTCGAGTTTGACAGCGCCTCTAAA CCTGTGATCACCAACATCGTGAAGGCTATCATTGGCTACAACTTCGAC GTGTCCAAGATCTTCGAGATCGACTACGAAAAAGGCAATATCAGCTTC GCCAAAGAAATCGAAAATGAAGAAGAGATCGAAGAGCTGCTTGGCAA CCAGGTGGAAATCTATGAAGCCATGAAGAACATCTACTCCTGGTTCA CCCTGCAGGACATCCTGAAGGGCAACAAGTACATCAGCGACGCCTTC ATAGAGAAGTACGACAAGTACAAGGGCGATCTGATCCTGCTGAAGAA CATCTATAAAAAGTACTACTCTAACGAGTACAAGAGCTTTTTTAAGGAA GAAAACATTAACAATTACGTGCACTACAATGGAAAGCAGATCAGAAAG CTGGACAAGTCATGCGACAACAATGAGTTCTTCAAGAAGGTTTCTCAG AAAATCAAGAGTCTGCCTGAGGACTACGAGAAGCTGTACGACGAGGT GAACAAGAAGGTGAAGATCGAGAATGAGAAGAACGGCAGCAACATC GAGCTGGACGACAGATACAGAGACAAGGAAAACATCCTGCAGGACAT TGAGAGCGGCAACTTTATTTGTAAACTGAACGTGACAGACAACGGCG CCATCCCCCACCAGCTGCACGAGATCGAGCTGAAGAAGATCATCGAG AACCAAAAGCAGTACTACGAGTTCCTGAACGAGAATGCCGAGAAGAT TATTAAGATATTCGAAACCAGAATCCCTTACTATGTGGGACCTCTGAT CGAGGGAAAAAAGGGCAGCAGATGGAGCTGGCTGATCAAAAACAAC GAGCAAGAGAAGGCCCACATCAGACCTTGGAACATGGAAGAAACCAT CAATAAAAACGAAACAGCTGAAAAGTTCATCAACGAATTAACTAACTA CTGCACCTACCTGCAAGACGAAAAGGTGATGCCCAAACAGAGCATCC TGTACAGCAAGTATTGTGTGCTGAACGAGCTGAACAACATTAGAATCA ACGGAAAGCACATCGCCAAGGATTTCAAGCAGAAGATAATCAACGAA TACTTCGAAAGAAAGACCAAGGTCACCAAGAAGGACATTATCAGCTTT TACGACAAGGAAGGCATCAACGTGTCCTCTTTCGAAGGCCTGAGCGA CGTGAACAACTTCAACTCAAATATGAAGTCCTACAAGGATATGATAGA GATTCTGGGAAAGGTGGACGATAGCAACATCGAGATGTGCGAGAAAA TCATCTACTACGTGACAATCTTCGAGGATAAAAAGATTCTGAGGGAAA AGATCAAGAATGAGTATAGCGGCATCAACGACGAACAGTTGAGAAGA ATCATGAAACTTAAGTACAGCGGGTGGTCCAGACTGAGCAGAAAATT CCTGACCGGCATCAAGTCTTACAACAATGAAACGATTATGGATCGACT GATGAACACCCATGAGAACCTGATGCAGATCATCAACAACAAGGAGTTABLE 1A DGHJ Type II Cas Sequences Name Sequence SEQ ID NO TAGGATTTGATAAGATCATCGAGAACAACATCCCAAAGAAGAGCGGC AGAATCACCTACGAGGACGTGGACGAGATTCAGACCAGCCCTGCTAA CAAAAGGGCCATCTGGCAGACCATCTGCGTGGTGAACGAGATCAAGA AAATCATGAAAAAGGAGCCTAAGAACATCTACATCGAGTTCGCCAGAA GCGAGGATAGAAACAAGAGACTTAAAGATTCCAGACTGAAGAACCTG CTGAAGATCTACGAGGACATCAAGGAGCAGATCGACGAGATCAAGAA ATTCGATCCTGACGTGTACAAAGAACTGAAGAAGCACCAGGATGATA AGGACGTGACCGAGAAGATGTACCTGTATTTCATCCAGAACGGCAAG TGTCTGTACAGCGGAGAGAAGCTGGACATCGACGAGCTGGACAAGT ACGAGGTGGACCACATAATACCCAGATCATACATCAAGGACGACAGC ATCGATAACAAGGCCCTGGTGAAGCGGAGAGAGAATCAGAGAAAGAA GGATTCTCTGCTGCTGACCGAGGACGTGATCAGGAAGAACATCAGCT GGTGGAAGAATCTGTATGACAACAACCTGATAAGCGCCAAGAAGTAC CACAACCTGACCAGACGGAAAATGTTCGAGACAGAGAGCGAGAAAAA CAAATTCGTGCAGCGGCAGCTGGTCGAGACAAGACAGATCACAAAGT ACGTGACAAACCTGCTGAAGGATGGCGAAGAGAACGTGGAAATCTAC GCCCTGCGGGCCGAGCTGACCCACTGGTTTAGAGAGAAATACGAGA TATATAAGAATCGGAACGTGAACAACTGCCACCACGCCCAGGACGCC TACATCATCAGCACAATCGGCAATATCATCAGCAAGGAGTGGCCTAA GAAGGAGGATTTCCAGTACGGGCAGTACGTGGACAACTACCTGAGA GAGGCCAAGGCCAAGAACGAGAAGTACGGCATGGTGATCGGCTTCA TCTCTAAAAGAGTGGAAATCAGCAAGATCAAACAGGCCATGAACTACA AGGATTACTTTATCAGCAGAATGCTGGAAGAGCAAACCGGCGAGTTC TACAACCAGACAATCCAAAAGGCCGGCCAGGGAAAGATCAAGCTGAA ACCTAACAAGGACACCAATAGATACGGCGGATACACCAACGAAAACG AAGCATATTCTGTGATCTACAAGTACATCGACAAGAGCGGCAAACAG CAGTATAAGATGATCGGCATCCCTATCAGAATCGCCTCTGACATCAAG AGAAAGCGGACAAATCTGCAGGAGTACATCACAGATGTGGATTTCAA TAACAACGACAACATAGAGCTGCAGATCATCCGCAACAAAATCATGAA AAACCAGATCTACCTGGATCAGAACGACGAACCCCTGATGCTGTGCA GCAGCAAGGAAATCAGACCCGCTAAAGAACTGATCCTGAATGAAGAG ATGAGCAAGCTCGTATACATGATGAACAGCAGCGAGAACAAGCTGAC AGATGAAGAAAAGGAAGAGGTGAAGCGGAGCTATAGCAAGATGTACG ACTACCTGATGTCCAAGCTGGAGAAGGAGTACAAAATCCACAAGAAC TCCTACATGAGACTGATCAACAAAAAGGAGGAATTTGAGACACTGGA GGACGAGGATAAGAAAAAGGCCATCAACGGCATCATCGACCTGATGT TCAAGGGCCAGGGCAACCTGAAGGTCCTGGGCATGAGCGATCGGGA AGGCAGGATGAGTGGAAAGACCTTCAAGACCGACAAGCTGATCCACA TGACATTCGTGGACAAAAGCGTCACCGGCATGTACGAGCGACGGCA GAAGATCAACGGCATGGAAAATAACAGCTCCAAGtctagaAAGCGGACA GCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGTG A

[0076] In some embodiments a DGHJ Type II 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 DGHJ Type II 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 D12A substitution, wherein the position of the D12A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H868A substitution, wherein the position of the H868A substitution is defined with respect to the amino acid numbering ofSEQ ID NO:2. In some embodiments, a DGHJ Type II Cas protein is catalytically inactive, for example due to a D12A substitution in combination with an H868A substitution. 6.2.1.2. CUAZ Type II Cas Proteins

[0077] In one aspect, the disclosure provides CUAZ Type II Cas proteins. CUAZ Type II Cas proteins can be further classified as Type IIA Cas proteins. The CUAZ Type II 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 CUAZ Type II 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 CUAZ Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:7.

[0078] Exemplary CUAZ Type II Cas protein sequences and nucleotide sequences encoding exemplary CUAZ Type II Cas proteins are set forth in Table 1B. TABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO CUAZ Type II KTNKKIYKDNQYTLSIDPGLNNVGWALFDAKNKKMVEMGVRKFEPAKEA 7 Cas amino KISRLKRSARRNNGRKKWRKQQLKQAFIDFNLIGEDEFKNSQFYEFHPE acid QYKAAGVKNLMDLRCYALQNKVTYQELFQCIYNILKYRGNFLRDDIDFEN sequence GESLTEELLIQEITNFMIKSLQLDIDNLDKLKESISTMCNQNIDLKEIPYTEIS (without N- DDKVSQKSIENVFKALKDQKFNPFMIEDDPILIDEEEGKSIIPEAKTKTIYM terminal KSVQKCGDLSNRFEQLLNITDSYRLYKVLNGEKYICTAYKKKLDRVADYY methionine) SFKDSPEGCSKYSDIESAKIEEEITYITNNMNATKGKPEKYGYTRKNPRG PIHVIHNVLNRYPNGLLVKEAVDILRTQQNYYGKEVISDEFIEVIRYILKGRI PYYIGPLSETAKNQWCTKTGKFKYSYNYTIEHMDGVDEERSIKDWKDRM RGHCTYLPEAFALPKGSFVAETLLILNELNVLTAIDKNNNTYYFTTNDKIKV FDYLFLDENHYGDVKYKEICDLLDLAYFGPKNQKSQVKTFKQNYTLYKSI TSICTALKLHSIIDLFSTKFQENQLKTIEEIVLNLNLYDEKKHKYQALTKMGI SDENSKKLSALNSKKFYGLSEKFVLKTSLNKDGRCLLEILFEDNQPGYTN EMMTAITNACDMDGNPVSFIANKYEAAFKELEKNGELELNFNSLLEIMNK TGNGFQIPASRPVLRSINQMMKVIQAVYTYYGVPKRIVLEIPRDMSGKNT NHEMPAKHYKTMEALHQDIMKQCKDKDHPRKTCIDQKWNDLKDFELNQ KNRIKLELYLRQNGMDMITGEKINLNELENYEIDHILPRGFGDDSMDDKILI RFVSNALKKDRTPLMWITDPTDPVINKSGKEITADQYINRVNELFDMKLIS ENKKKRLLLKDTEDLDRFINQNLVDTRYITREMIGILKAYNRVKGYPTKVIA LKPAFTKNYSNAFDIYKNRDFGLQHHALDAAVLGITDIMLNRLFPNYESG RISTSFAKHHQMLSEVGNDKKLKDSTKYVIRKMYEKVFGNSPLDYGSLV YVCKNTMPLYSQKSNRNWKGEKFPASRRLPYQKYDIKNGNIVGVYDDK SEENIGKQNSEVLRLFEINNKNHAFDSVNCVAYDIYRIPEKKNNKVIWKNI GIHIPKCIVDEDGTIQKDKYIKLIKKYYGFDDNLIDEKGDLITAYFKLRLYKN DLVGNTEKHEIRDLVLGSIADKKLELNKIDCFVYQKIAQKKASLFKMIIDNQ GSSKYDIHNYKEYQECECKEEILRDMIFDDMNIKNQEFASLLYAKIINDKK LDLENINKICWILAYYQQFLNEITGKKEFNTGRGIGALKKDESINYFKLKTS PLGVRFNTEKNQWTGPVGYENAFKIIKKEAFTWKITV CUAZ Type II MKTNKKIYKDNQYTLSIDPGLNNVGWALFDAKNKKMVEMGVRKFEPAKE 8 Cas amino AKISRLKRSARRNNGRKKWRKQQLKQAFIDFNLIGEDEFKNSQFYEFHP acid EQYKAAGVKNLMDLRCYALQNKVTYQELFQCIYNILKYRGNFLRDDIDFE sequence NGESLTEELLIQEITNFMIKSLQLDIDNLDKLKESISTMCNQNIDLKEIPYTEI SDDKVSQKSIENVFKALKDQKFNPFMIEDDPILIDEEEGKSIIPEAKTKTIY MKSVQKCGDLSNRFEQLLNITDSYRLYKVLNGEKYICTAYKKKLDRVADY YSFKDSPEGCSKYSDIESAKIEEEITYITNNMNATKGKPEKYGYTRKNPR GPIHVIHNVLNRYPNGLLVKEAVDILRTQQNYYGKEVISDEFIEVIRYILKG RIPYYIGPLSETAKNQWCTKTGKFKYSYNYTIEHMDGVDEERSIKDWKDTABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO RMRGHCTYLPEAFALPKGSFVAETLLILNELNVLTAIDKNNNTYYFTTNDK IKVFDYLFLDENHYGDVKYKEICDLLDLAYFGPKNQKSQVKTFKQNYTLY KSITSICTALKLHSIIDLFSTKFQENQLKTIEEIVLNLNLYDEKKHKYQALTK MGISDENSKKLSALNSKKFYGLSEKFVLKTSLNKDGRCLLEILFEDNQPG YTNEMMTAITNACDMDGNPVSFIANKYEAAFKELEKNGELELNFNSLLEI MNKTGNGFQIPASRPVLRSINQMMKVIQAVYTYYGVPKRIVLEIPRDMSG KNTNHEMPAKHYKTMEALHQDIMKQCKDKDHPRKTCIDQKWNDLKDFE LNQKNRIKLELYLRQNGMDMITGEKINLNELENYEIDHILPRGFGDDSMD DKILIRFVSNALKKDRTPLMWITDPTDPVINKSGKEITADQYINRVNELFDM KLISENKKKRLLLKDTEDLDRFINQNLVDTRYITREMIGILKAYNRVKGYPT KVIALKPAFTKNYSNAFDIYKNRDFGLQHHALDAAVLGITDIMLNRLFPNY ESGRISTSFAKHHQMLSEVGNDKKLKDSTKYVIRKMYEKVFGNSPLDYG SLVYVCKNTMPLYSQKSNRNWKGEKFPASRRLPYQKYDIKNGNIVGVYD DKSEENIGKQNSEVLRLFEINNKNHAFDSVNCVAYDIYRIPEKKNNKVIWK NIGIHIPKCIVDEDGTIQKDKYIKLIKKYYGFDDNLIDEKGDLITAYFKLRLYK NDLVGNTEKHEIRDLVLGSIADKKLELNKIDCFVYQKIAQKKASLFKMIIDN QGSSKYDIHNYKEYQECECKEEILRDMIFDDMNIKNQEFASLLYAKIINDK KLDLENINKICWILAYYQQFLNEITGKKEFNTGRGIGALKKDESINYFKLKT SPLGVRFNTEKNQWTGPVGYENAFKIIKKEAFTWKITV CUAZ Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSKTNKKIYKDNQYTLS 9 Cas IDPGLNNVGWALFDAKNKKMVEMGVRKFEPAKEAKISRLKRSARRNNG mammalian RKKWRKQQLKQAFIDFNLIGEDEFKNSQFYEFHPEQYKAAGVKNLMDLR expression CYALQNKVTYQELFQCIYNILKYRGNFLRDDIDFENGESLTEELLIQEITNF construct MIKSLQLDIDNLDKLKESISTMCNQNIDLKEIPYTEISDDKVSQKSIENVFK (with N- ALKDQKFNPFMIEDDPILIDEEEGKSIIPEAKTKTIYMKSVQKCGDLSNRFE terminal QLLNITDSYRLYKVLNGEKYICTAYKKKLDRVADYYSFKDSPEGCSKYSDI methionine, ESAKIEEEITYITNNMNATKGKPEKYGYTRKNPRGPIHVIHNVLNRYPNGL stop codon, LVKEAVDILRTQQNYYGKEVISDEFIEVIRYILKGRIPYYIGPLSETAKNQW V5 tag, N- CTKTGKFKYSYNYTIEHMDGVDEERSIKDWKDRMRGHCTYLPEAFALPK terminal NLS GSFVAETLLILNELNVLTAIDKNNNTYYFTTNDKIKVFDYLFLDENHYGDV and C- KYKEICDLLDLAYFGPKNQKSQVKTFKQNYTLYKSITSICTALKLHSIIDLF terminal NLS) STKFQENQLKTIEEIVLNLNLYDEKKHKYQALTKMGISDENSKKLSALNSK (aa) KFYGLSEKFVLKTSLNKDGRCLLEILFEDNQPGYTNEMMTAITNACDMD GNPVSFIANKYEAAFKELEKNGELELNFNSLLEIMNKTGNGFQIPASRPVL RSINQMMKVIQAVYTYYGVPKRIVLEIPRDMSGKNTNHEMPAKHYKTME ALHQDIMKQCKDKDHPRKTCIDQKWNDLKDFELNQKNRIKLELYLRQNG MDMITGEKINLNELENYEIDHILPRGFGDDSMDDKILIRFVSNALKKDRTPL MWITDPTDPVINKSGKEITADQYINRVNELFDMKLISENKKKRLLLKDTED LDRFINQNLVDTRYITREMIGILKAYNRVKGYPTKVIALKPAFTKNYSNAFD IYKNRDFGLQHHALDAAVLGITDIMLNRLFPNYESGRISTSFAKHHQMLSE VGNDKKLKDSTKYVIRKMYEKVFGNSPLDYGSLVYVCKNTMPLYSQKSN RNWKGEKFPASRRLPYQKYDIKNGNIVGVYDDKSEENIGKQNSEVLRLF EINNKNHAFDSVNCVAYDIYRIPEKKNNKVIWKNIGIHIPKCIVDEDGTIQK DKYIKLIKKYYGFDDNLIDEKGDLITAYFKLRLYKNDLVGNTEKHEIRDLVL GSIADKKLELNKIDCFVYQKIAQKKASLFKMIIDNQGSSKYDIHNYKEYQE CECKEEILRDMIFDDMNIKNQEFASLLYAKIINDKKLDLENINKICWILAYYQ QFLNEITGKKEFNTGRGIGALKKDESINYFKLKTSPLGVRFNTEKNQWTG PVGYENAFKIIKKEAFTWKITVSRKRTADGSEFESPKKKRKV CUAZ Type II ATGAAAACAAATAAAAAAATCTATAAAGATAATCAATATACATTATCAAT 10 Cas coding TGATCCAGGTTTAAATAATGTAGGATGGGCATTATTTGATGCAAAAAA sequence (nt) TAAAAAAATGGTCGAGATGGGTGTAAGAAAGTTTGAACCTGCTAAAGA (not codon AGCTAAGATATCTCGATTAAAGCGAAGTGCTAGAAGAAATAATGGAAG optimized) AAAAAAATGGAGAAAGCAGCAGCTTAAGCAGGCATTTATAGATTTTAA TTTGATAGGGGAAGATGAATTTAAGAATAGTCAGTTTTATGAATTTCAT CCTGAGCAATATAAAGCAGCCGGTGTTAAAAATTTAATGGATTTAAGA TGTTATGCACTTCAGAATAAAGTTACTTATCAAGAATTATTTCAATGCA TATATAACATATTAAAGTATCGTGGTAATTTTTTAAGAGATGATATTGATABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO TTTTGAAAATGGAGAATCTTTAACAGAAGAGCTTTTGATCCAGGAAAT TACAAACTTTATGATTAAGTCATTGCAATTGGATATTGATAATCTAGAT AAATTAAAAGAATCTATTAGTACAATGTGTAATCAAAATATTGATTTAAA AGAAATTCCATATACAGAAATATCAGATGATAAAGTATCTCAAAAGAGT ATTGAAAATGTGTTTAAGGCACTTAAAGATCAAAAATTCAATCCTTTTA TGATTGAAGATGATCCGATTCTGATTGATGAGGAAGAGGGTAAATCTA TTATTCCAGAGGCAAAAACAAAGACAATTTATATGAAATCGGTACAAA AATGTGGTGATTTATCGAATCGATTTGAACAGTTATTAAATATTACAGA TTCATATCGACTTTATAAAGTGTTGAACGGGGAAAAATATATTTGCACT GCGTATAAAAAGAAGCTTGATCGAGTTGCGGATTATTATTCTTTTAAA GATAGCCCAGAAGGGTGCTCTAAATATTCAGATATTGAATCCGCAAAG ATTGAAGAAGAAATAACATATATTACGAATAATATGAATGCTACTAAGG GAAAACCAGAAAAGTATGGTTATACTCGTAAGAATCCACGAGGTCCTA TTCATGTTATACATAATGTTTTGAACAGATATCCTAATGGTCTGCTTGT TAAAGAGGCTGTTGATATTTTAAGAACACAACAGAACTATTATGGTAA AGAAGTGATTTCTGATGAATTTATTGAGGTAATTCGATATATCTTAAAA GGTCGAATTCCATATTATATTGGGCCATTGTCAGAAACGGCAAAAAAT CAGTGGTGTACAAAGACTGGAAAATTCAAATATAGCTACAACTATACA ATTGAACACATGGATGGTGTGGACGAAGAACGTTCAATAAAAGATTG GAAAGATCGTATGCGTGGACATTGTACATATCTTCCTGAGGCATTTGC ACTTCCAAAAGGATCGTTTGTAGCTGAAACATTGCTTATCTTAAATGAA TTAAATGTGCTTACGGCGATAGACAAAAATAATAACACGTATTATTTCA CAACGAATGACAAAATTAAAGTATTTGATTATCTATTTTTAGATGAGAA TCATTACGGGGATGTTAAATATAAAGAAATTTGTGATCTACTGGATTTG GCATATTTTGGACCGAAAAATCAAAAATCACAGGTCAAAACATTTAAG CAGAATTATACGTTATATAAAAGTATAACAAGTATTTGTACTGCTTTAA AATTGCATTCAATTATTGATTTGTTTAGTACAAAATTTCAAGAAAATCAA CTAAAGACAATAGAAGAAATTGTTCTGAATCTTAACCTTTACGATGAG AAGAAGCACAAGTATCAGGCATTAACAAAAATGGGAATATCTGATGAA AATTCAAAAAAACTTTCTGCATTGAACTCAAAGAAATTTTATGGATTGT CAGAAAAATTTGTTTTAAAAACATCATTAAATAAAGATGGACGATGTCT TCTAGAAATATTATTTGAAGATAACCAGCCCGGTTATACAAATGAAAT GATGACAGCTATTACGAATGCTTGTGATATGGATGGAAATCCTGTTTC GTTTATTGCCAATAAATATGAGGCAGCTTTTAAAGAGTTAGAAAAAAAT GGAGAGTTAGAATTAAATTTTAATAGTCTTTTAGAAATAATGAATAAAA CTGGAAATGGTTTCCAAATTCCTGCTTCTCGTCCAGTACTTCGTTCTA TTAATCAAATGATGAAAGTGATTCAGGCTGTATATACATATTACGGGG TTCCTAAACGCATTGTATTAGAAATTCCTAGAGATATGTCTGGAAAAAA TACTAATCATGAAATGCCTGCAAAACACTATAAAACTATGGAAGCTTTA CATCAAGATATTATGAAGCAGTGTAAGGATAAAGATCATCCTAGAAAA ACTTGCATCGATCAAAAATGGAATGATTTAAAAGATTTTGAGCTAAATC AAAAAAATAGAATAAAACTTGAATTGTATCTTCGCCAAAATGGAATGG ATATGATAACAGGAGAAAAAATCAATTTGAATGAGCTTGAAAACTATG AAATTGACCATATTCTTCCAAGAGGATTTGGGGATGATTCAATGGATG ATAAGATACTAATACGTTTTGTATCTAATGCATTGAAAAAGGATAGAAC ACCTCTTATGTGGATTACAGATCCAACGGATCCAGTTATAAATAAATC AGGTAAAGAAATTACAGCCGATCAGTATATAAATCGAGTGAACGAGCT CTTTGATATGAAGTTAATATCTGAAAATAAAAAGAAGAGATTGTTATTA AAAGATACAGAAGATTTGGATCGATTTATTAATCAGAATCTTGTTGATA CTCGATATATCACAAGAGAAATGATTGGAATTTTAAAAGCATATAATCG TGTAAAGGGTTATCCTACAAAAGTGATCGCTTTAAAACCTGCTTTTAC AAAGAATTATTCAAATGCATTCGACATTTATAAGAACAGAGATTTTGGT TTACAACACCACGCGTTGGACGCAGCGGTATTAGGTATTACGGATAT TATGTTAAATAGATTATTTCCAAATTATGAAAGTGGACGAATATCTACA TCATTCGCTAAGCATCATCAGATGTTGTCAGAAGTTGGAAATGATAAA AAGTTAAAAGATTCGACTAAATATGTAATACGTAAAATGTATGAAAAAG TATTTGGTAATTCGCCATTAGATTATGGTTCACTTGTATATGTTTGTAATABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO AAATACCATGCCTTTATATTCTCAAAAATCAAATAGAAACTGGAAGGG TGAAAAGTTTCCGGCTTCTAGAAGATTGCCTTATCAAAAATATGACAT AAAAAATGGAAATATAGTTGGAGTATATGATGATAAAAGTGAAGAAAA TATAGGTAAACAAAACTCTGAAGTATTACGATTATTTGAAATCAACAAT AAAAATCATGCGTTTGATAGTGTGAATTGTGTTGCCTATGATATATATA GAATTCCTGAAAAGAAAAATAATAAAGTGATTTGGAAAAATATAGGTAT ACATATTCCAAAATGTATTGTAGATGAAGATGGCACTATACAAAAGGA TAAGTATATTAAGCTAATTAAGAAGTATTATGGTTTTGATGATAATCTT ATAGATGAAAAAGGTGACCTTATTACAGCTTATTTCAAACTACGTCTTT ATAAAAATGATTTAGTAGGAAATACTGAAAAACATGAAATTAGAGATTT AGTATTGGGAAGTATTGCTGATAAAAAATTAGAATTAAATAAAATTGAT TGTTTTGTATATCAGAAAATTGCGCAGAAAAAAGCAAGTTTATTTAAAA TGATTATCGATAATCAAGGATCTTCAAAATACGATATTCATAATTATAA GGAATATCAGGAGTGTGAATGTAAAGAAGAGATTCTACGAGATATGAT TTTTGATGATATGAATATCAAAAATCAGGAGTTTGCTTCTCTTCTATAC GCTAAGATTATTAATGATAAAAAATTAGATTTGGAAAATATAAACAAGA TTTGTTGGATACTTGCCTATTATCAGCAATTTTTAAATGAAATTACTGG TAAAAAAGAATTTAATACAGGACGAGGAATAGGTGCTTTAAAGAAAGA TGAGAGTATTAATTACTTCAAGCTAAAAACATCACCGTTGGGGGTGCG ATTTAATACAGAAAAAAACCAATGGACCGGACCTGTTGGATATGAAAA TGCCTTTAAAATTATAAAAAAAGAAGCATTTACTTGGAAAATTACAGTT TAA CUAZ Type II AAAACCAATAAAAAGATCTACAAGGACAACCAGTATACCCTGTCTATC 11 Cas coding GATCCTGGACTGAACAACGTGGGCTGGGCCCTGTTCGATGCCAAGA sequence (nt) ACAAGAAAATGGTGGAGATGGGCGTGCGGAAGTTCGAGCCTGCCAA (human GGAGGCTAAGATCTCCAGACTGAAGAGAAGCGCACGGAGGAACAAC codon- GGCCGCAAGAAGTGGCGGAAGCAGCAGCTGAAGCAGGCCTTTATCG optimized; ACTTCAACCTTATCGGGGAGGATGAGTTCAAGAACAGCCAGTTCTAC lacking N- GAGTTCCACCCTGAGCAGTATAAGGCCGCTGGCGTGAAGAATCTGAT terminal GGACCTGCGCTGCTACGCTCTCCAGAACAAGGTGACCTACCAAGAG methionine; CTGTTCCAGTGTATCTACAACATCCTGAAGTACCGGGGAAACTTCCTG no stop AGGGATGACATCGACTTTGAAAATGGAGAGTCCCTGACAGAAGAACT codon) GTTGATCCAGGAAATTACCAATTTCATGATCAAGTCCCTGCAGTTGGA TATTGACAACTTGGACAAGCTGAAGGAGTCCATCAGCACCATGTGCA ACCAGAACATAGACCTGAAGGAAATCCCCTACACCGAAATCAGCGAC GACAAGGTGAGCCAGAAGAGCATTGAGAACGTGTTCAAGGCCCTGAA GGACCAGAAGTTCAACCCCTTCATGATCGAGGATGATCCTATCCTGA TCGACGAGGAAGAAGGCAAGAGCATCATCCCTGAGGCCAAGACCAA GACTATCTACATGAAATCTGTGCAGAAATGCGGCGACCTGAGCAACA GATTCGAGCAACTGCTGAACATCACCGACTCCTACAGACTGTACAAG GTGCTGAACGGCGAGAAGTACATCTGCACCGCTTACAAGAAGAAACT GGACCGGGTGGCCGATTACTACTCCTTCAAGGACAGCCCTGAGGGT TGTAGCAAATACAGCGACATCGAGTCCGCCAAGATCGAGGAAGAAAT CACCTATATCACCAACAACATGAACGCCACAAAAGGAAAACCCGAGA AGTACGGATACACAAGAAAGAATCCTAGAGGCCCCATCCACGTGATC CACAACGTGCTGAACAGATACCCTAACGGCCTTCTCGTGAAGGAAGC GGTGGACATCCTGAGAACCCAGCAAAACTACTACGGCAAGGAGGTTA TAAGCGACGAGTTCATCGAGGTGATCCGCTACATCTTGAAGGGCAGA ATCCCATACTACATCGGCCCTCTGTCTGAAACAGCCAAGAACCAGTG GTGCACCAAGACCGGCAAATTCAAGTACAGCTACAACTACACCATCG AGCACATGGATGGCGTGGACGAAGAGAGATCTATCAAGGACTGGAA GGATAGAATGAGAGGCCACTGTACCTACCTGCCTGAGGCCTTCGCCC TGCCAAAGGGCAGCTTCGTGGCCGAGACACTGCTGATCCTGAACGA GCTGAACGTGCTGACCGCCATCGACAAGAACAACAACACCTATTACT TCACAACAAACGACAAGATCAAAGTGTTCGATTATCTGTTTCTGGATG AGAATCATTACGGCGATGTGAAGTACAAGGAAATCTGTGATCTCCTG GACCTGGCCTACTTCGGCCCTAAGAACCAGAAATCTCAGGTGAAAACTABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO CTTCAAGCAAAACTACACCCTGTACAAAAGCATCACAAGTATCTGCAC AGCCCTGAAGCTGCACAGCATTATCGATCTGTTCAGCACAAAGTTCC AGGAAAACCAGCTGAAAACCATCGAGGAAATCGTGCTGAATCTGAAT CTGTACGACGAAAAGAAGCACAAGTATCAAGCCTTGACCAAGATGGG AATCAGCGACGAGAATAGCAAGAAGCTGAGTGCCCTGAATAGCAAGA AATTTTACGGCCTGAGCGAGAAGTTTGTGCTGAAAACAAGCCTGAAC AAGGACGGCAGATGCCTGCTGGAAATCCTGTTTGAAGATAATCAACC TGGCTATACCAATGAAATGATGACCGCCATCACCAATGCTTGTGACAT GGATGGCAACCCTGTATCCTTCATCGCTAATAAATACGAGGCCGCTTT TAAAGAACTGGAAAAAAACGGAGAGCTGGAACTGAACTTCAATAGCC TTCTGGAAATCATGAACAAGACCGGCAACGGATTCCAGATCCCGGCT TCCAGACCCGTCCTGAGAAGCATCAACCAGATGATGAAGGTGATCCA GGCCGTTTACACCTACTACGGCGTGCCCAAGCGGATCGTGCTGGAA ATCCCTAGAGATATGAGCGGCAAGAATACCAACCATGAGATGCCCGC CAAGCACTACAAGACGATGGAAGCCCTGCACCAGGACATTATGAAGC AGTGTAAAGACAAGGATCACCCCAGAAAGACATGCATCGATCAGAAG TGGAACGACCTGAAGGACTTCGAGCTGAACCAGAAGAACCGGATCAA GCTCGAACTGTACCTGAGGCAGAACGGCATGGACATGATCACCGGC GAGAAGATCAATCTGAACGAGCTGGAAAACTACGAGATCGACCACAT CCTGCCTAGAGGCTTCGGAGATGACAGCATGGATGACAAGATTCTGA TCAGATTCGTGAGCAATGCCCTGAAGAAGGATAGAACACCTCTGATG TGGATCACCGACCCTACCGATCCTGTGATCAATAAGTCCGGCAAAGA GATCACTGCCGACCAGTACATCAACCGGGTCAACGAACTGTTCGACA TGAAACTGATAAGCGAGAATAAGAAGAAGCGGCTGCTGCTGAAGGAC ACAGAGGATCTGGACAGATTTATCAACCAGAACCTGGTCGACACCAG ATACATCACTAGAGAAATGATCGGCATCCTGAAGGCCTACAACAGAG TTAAGGGTTATCCTACAAAGGTGATCGCTCTGAAGCCCGCCTTTACCA AGAACTACTCTAACGCCTTCGACATCTACAAGAACCGGGATTTCGGC CTGCAGCACCACGCCCTGGATGCTGCTGTGCTGGGCATCACAGACA TCATGCTGAACAGGCTGTTCCCTAATTACGAGAGCGGCAGAATCAGC ACCAGCTTCGCCAAGCACCATCAAATGCTGAGCGAGGTGGGCAACG ACAAGAAGTTAAAAGACTCCACCAAGTACGTGATCAGAAAGATGTAC GAGAAGGTGTTCGGCAACAGCCCTCTGGACTACGGCTCTCTGGTGTA CGTGTGCAAGAACACCATGCCACTGTATTCACAAAAGTCTAACAGAAA TTGGAAGGGCGAAAAGTTCCCCGCCAGCAGACGGCTGCCCTACCAG AAATACGATATTAAGAATGGCAACATCGTCGGTGTCTACGACGACAA GTCCGAGGAGAACATCGGCAAGCAGAACAGCGAAGTGTTACGGCTG TTCGAGATCAACAATAAGAATCACGCCTTTGACAGCGTGAACTGCGT GGCCTACGACATCTACAGAATCCCCGAGAAGAAAAACAACAAGGTGA TCTGGAAGAACATCGGCATTCACATCCCTAAGTGCATCGTGGACGAG GACGGCACAATCCAGAAGGACAAATACATAAAGCTGATCAAGAAGTA CTACGGCTTCGACGACAACCTGATCGATGAGAAAGGCGACCTTATTA CCGCCTACTTTAAACTGCGGCTGTACAAAAATGATCTGGTGGGCAAC ACCGAAAAGCACGAGATCAGAGATCTTGTGCTGGGAAGCATCGCCGA TAAAAAACTGGAGTTAAACAAGATTGACTGCTTTGTGTACCAGAAGAT CGCCCAGAAGAAGGCCAGCCTGTTTAAAATGATCATCGACAACCAGG GCAGCTCTAAGTACGACATCCACAACTACAAAGAGTACCAGGAATGC GAGTGCAAGGAAGAGATCCTGAGAGACATGATCTTCGACGACATGAA TATCAAAAATCAGGAGTTCGCCAGCCTGCTGTACGCCAAGATTATCAA CGACAAGAAGCTGGACCTGGAAAACATCAACAAGATTTGCTGGATTC TGGCCTACTACCAGCAGTTTCTGAATGAGATCACAGGCAAGAAGGAA TTTAACACCGGCCGGGGCATCGGCGCCCTGAAAAAGGACGAGTCTA TCAACTACTTCAAGCTGAAAACCTCTCCACTGGGAGTGCGGTTCAAC ACCGAGAAGAACCAGTGGACCGGCCCTGTGGGCTACGAGAACGCCT TCAAGATCATCAAGAAAGAGGCCTTCACCTGGAAAATTACAGTG CUAZ Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 12 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGGTABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO mammalian AAAGTGGGATCCAAAACCAATAAAAAGATCTACAAGGACAACCAGTAT expression ACCCTGTCTATCGATCCTGGACTGAACAACGTGGGCTGGGCCCTGTT construct CGATGCCAAGAACAAGAAAATGGTGGAGATGGGCGTGCGGAAGTTC (with N- GAGCCTGCCAAGGAGGCTAAGATCTCCAGACTGAAGAGAAGCGCAC terminal GGAGGAACAACGGCCGCAAGAAGTGGCGGAAGCAGCAGCTGAAGCA methionine, GGCCTTTATCGACTTCAACCTTATCGGGGAGGATGAGTTCAAGAACA stop codon, GCCAGTTCTACGAGTTCCACCCTGAGCAGTATAAGGCCGCTGGCGTG V5 tag, N- AAGAATCTGATGGACCTGCGCTGCTACGCTCTCCAGAACAAGGTGAC terminal NLS CTACCAAGAGCTGTTCCAGTGTATCTACAACATCCTGAAGTACCGGG and C- GAAACTTCCTGAGGGATGACATCGACTTTGAAAATGGAGAGTCCCTG terminal NLS) ACAGAAGAACTGTTGATCCAGGAAATTACCAATTTCATGATCAAGTCC (nt) CTGCAGTTGGATATTGACAACTTGGACAAGCTGAAGGAGTCCATCAG CACCATGTGCAACCAGAACATAGACCTGAAGGAAATCCCCTACACCG AAATCAGCGACGACAAGGTGAGCCAGAAGAGCATTGAGAACGTGTTC AAGGCCCTGAAGGACCAGAAGTTCAACCCCTTCATGATCGAGGATGA TCCTATCCTGATCGACGAGGAAGAAGGCAAGAGCATCATCCCTGAGG CCAAGACCAAGACTATCTACATGAAATCTGTGCAGAAATGCGGCGAC CTGAGCAACAGATTCGAGCAACTGCTGAACATCACCGACTCCTACAG ACTGTACAAGGTGCTGAACGGCGAGAAGTACATCTGCACCGCTTACA AGAAGAAACTGGACCGGGTGGCCGATTACTACTCCTTCAAGGACAGC CCTGAGGGTTGTAGCAAATACAGCGACATCGAGTCCGCCAAGATCGA GGAAGAAATCACCTATATCACCAACAACATGAACGCCACAAAAGGAA AACCCGAGAAGTACGGATACACAAGAAAGAATCCTAGAGGCCCCATC CACGTGATCCACAACGTGCTGAACAGATACCCTAACGGCCTTCTCGT GAAGGAAGCGGTGGACATCCTGAGAACCCAGCAAAACTACTACGGC AAGGAGGTTATAAGCGACGAGTTCATCGAGGTGATCCGCTACATCTT GAAGGGCAGAATCCCATACTACATCGGCCCTCTGTCTGAAACAGCCA AGAACCAGTGGTGCACCAAGACCGGCAAATTCAAGTACAGCTACAAC TACACCATCGAGCACATGGATGGCGTGGACGAAGAGAGATCTATCAA GGACTGGAAGGATAGAATGAGAGGCCACTGTACCTACCTGCCTGAG GCCTTCGCCCTGCCAAAGGGCAGCTTCGTGGCCGAGACACTGCTGA TCCTGAACGAGCTGAACGTGCTGACCGCCATCGACAAGAACAACAAC ACCTATTACTTCACAACAAACGACAAGATCAAAGTGTTCGATTATCTG TTTCTGGATGAGAATCATTACGGCGATGTGAAGTACAAGGAAATCTGT GATCTCCTGGACCTGGCCTACTTCGGCCCTAAGAACCAGAAATCTCA GGTGAAAACCTTCAAGCAAAACTACACCCTGTACAAAAGCATCACAAG TATCTGCACAGCCCTGAAGCTGCACAGCATTATCGATCTGTTCAGCA CAAAGTTCCAGGAAAACCAGCTGAAAACCATCGAGGAAATCGTGCTG AATCTGAATCTGTACGACGAAAAGAAGCACAAGTATCAAGCCTTGACC AAGATGGGAATCAGCGACGAGAATAGCAAGAAGCTGAGTGCCCTGAA TAGCAAGAAATTTTACGGCCTGAGCGAGAAGTTTGTGCTGAAAACAA GCCTGAACAAGGACGGCAGATGCCTGCTGGAAATCCTGTTTGAAGAT AATCAACCTGGCTATACCAATGAAATGATGACCGCCATCACCAATGCT TGTGACATGGATGGCAACCCTGTATCCTTCATCGCTAATAAATACGAG GCCGCTTTTAAAGAACTGGAAAAAAACGGAGAGCTGGAACTGAACTT CAATAGCCTTCTGGAAATCATGAACAAGACCGGCAACGGATTCCAGA TCCCGGCTTCCAGACCCGTCCTGAGAAGCATCAACCAGATGATGAAG GTGATCCAGGCCGTTTACACCTACTACGGCGTGCCCAAGCGGATCGT GCTGGAAATCCCTAGAGATATGAGCGGCAAGAATACCAACCATGAGA TGCCCGCCAAGCACTACAAGACGATGGAAGCCCTGCACCAGGACATT ATGAAGCAGTGTAAAGACAAGGATCACCCCAGAAAGACATGCATCGA TCAGAAGTGGAACGACCTGAAGGACTTCGAGCTGAACCAGAAGAACC GGATCAAGCTCGAACTGTACCTGAGGCAGAACGGCATGGACATGATC ACCGGCGAGAAGATCAATCTGAACGAGCTGGAAAACTACGAGATCGA CCACATCCTGCCTAGAGGCTTCGGAGATGACAGCATGGATGACAAGA TTCTGATCAGATTCGTGAGCAATGCCCTGAAGAAGGATAGAACACCT CTGATGTGGATCACCGACCCTACCGATCCTGTGATCAATAAGTCCGGTABLE 1B CUAZ Type II Cas Sequences Name Sequence SEQ ID NO CAAAGAGATCACTGCCGACCAGTACATCAACCGGGTCAACGAACTGT TCGACATGAAACTGATAAGCGAGAATAAGAAGAAGCGGCTGCTGCTG AAGGACACAGAGGATCTGGACAGATTTATCAACCAGAACCTGGTCGA CACCAGATACATCACTAGAGAAATGATCGGCATCCTGAAGGCCTACA ACAGAGTTAAGGGTTATCCTACAAAGGTGATCGCTCTGAAGCCCGCC TTTACCAAGAACTACTCTAACGCCTTCGACATCTACAAGAACCGGGAT TTCGGCCTGCAGCACCACGCCCTGGATGCTGCTGTGCTGGGCATCA CAGACATCATGCTGAACAGGCTGTTCCCTAATTACGAGAGCGGCAGA ATCAGCACCAGCTTCGCCAAGCACCATCAAATGCTGAGCGAGGTGG GCAACGACAAGAAGTTAAAAGACTCCACCAAGTACGTGATCAGAAAG ATGTACGAGAAGGTGTTCGGCAACAGCCCTCTGGACTACGGCTCTCT GGTGTACGTGTGCAAGAACACCATGCCACTGTATTCACAAAAGTCTAA CAGAAATTGGAAGGGCGAAAAGTTCCCCGCCAGCAGACGGCTGCCC TACCAGAAATACGATATTAAGAATGGCAACATCGTCGGTGTCTACGAC GACAAGTCCGAGGAGAACATCGGCAAGCAGAACAGCGAAGTGTTAC GGCTGTTCGAGATCAACAATAAGAATCACGCCTTTGACAGCGTGAAC TGCGTGGCCTACGACATCTACAGAATCCCCGAGAAGAAAAACAACAA GGTGATCTGGAAGAACATCGGCATTCACATCCCTAAGTGCATCGTGG ACGAGGACGGCACAATCCAGAAGGACAAATACATAAAGCTGATCAAG AAGTACTACGGCTTCGACGACAACCTGATCGATGAGAAAGGCGACCT TATTACCGCCTACTTTAAACTGCGGCTGTACAAAAATGATCTGGTGGG CAACACCGAAAAGCACGAGATCAGAGATCTTGTGCTGGGAAGCATCG CCGATAAAAAACTGGAGTTAAACAAGATTGACTGCTTTGTGTACCAGA AGATCGCCCAGAAGAAGGCCAGCCTGTTTAAAATGATCATCGACAAC CAGGGCAGCTCTAAGTACGACATCCACAACTACAAAGAGTACCAGGA ATGCGAGTGCAAGGAAGAGATCCTGAGAGACATGATCTTCGACGACA TGAATATCAAAAATCAGGAGTTCGCCAGCCTGCTGTACGCCAAGATTA TCAACGACAAGAAGCTGGACCTGGAAAACATCAACAAGATTTGCTGG ATTCTGGCCTACTACCAGCAGTTTCTGAATGAGATCACAGGCAAGAA GGAATTTAACACCGGCCGGGGCATCGGCGCCCTGAAAAAGGACGAG TCTATCAACTACTTCAAGCTGAAAACCTCTCCACTGGGAGTGCGGTTC AACACCGAGAAGAACCAGTGGACCGGCCCTGTGGGCTACGAGAACG CCTTCAAGATCATCAAGAAAGAGGCCTTCACCTGGAAAATTACAGTGT CTAGAAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAA AAGAGAAAGGTGTGA

[0079] In some embodiments a CUAZ Type II 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 CUAZ Type II 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 D18A substitution, wherein the position of the D18A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H845A substitution, wherein the position of the H845A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8. In some embodiments, a CUAZ Type II Cas protein is catalytically inactive, for example due to a D18A substitution in combination with a H845A substitution. 6.2.1.3. AHWY Type II Cas Proteins

[0080] In one aspect, the disclosure provides AHWY Type II Cas proteins. AHWY Type II Cas proteins can be further classified as Type IIA Cas proteins. The AHWY Type II Cas proteins typically comprise anamino 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 AHWY Type II 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, an AHWY Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:13.

[0081] Exemplary AHWY Type II Cas protein sequences and nucleotide sequences encoding exemplary AHWY Type II Cas proteins are set forth in Table 1C. TABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO AHWY Type II NLRNVGEYNIGLDIGTGSVGWAVIDSDGEICHFKGKPTWGSRLFPSALPA 13 Cas amino SEARVHRGQRRRYTRRKWRLFLLQELFAGAIEANDPEFFIRLNQSHLLPE acid DRQSDHKDYLFTLFNDSSFDEKAYFKKFPTIYHLRKWLMETDEKADPRLI sequence YLAFHNIVKHRGNFLQQDNTGLNSQNANVRESVEELCAQLQEYCDSLDI (without N- PCTVEDNVDKIVQVLSDTHSVRSHLQEEVQKLLGIGVCDLLDKTSAKKMA terminal KALSGALVGLSAELADVFFLVIEKPEDAKTKIYLSKDEDVESFEEICPDEAL methionine) PLFEAMNKVYSSFVLQEILSSCPGKTLSVNKVKDYEQYGEDLKLLKALVK EYAPEKYDDFFRGEFYRPSSLHPQKYVYNKSVAKGYTRYNEVRGVAYE DFKKDVEKLFRDTPAVEDARYLDMMERFKEEKFLRRLKTSDNGAIPFQL HLEEMDKIIQNQARFYPFLEEKKKELDSLVTFRIPYYVGPLTQKNARKDR KGSNRFSWSERIEGKEHEVIKPWNWEEIIDKDRSATEFILRMTGTCTYLQ GEPVLPRCSLLYEEYCVLNELNGAHFTQDGDKEHRFDYQDRSDMLKEL FRKGKVSYKKAADWMVQRGNSNVRVSGGQGEKGFESKLGSYIFFAKDV FGVDEIPESDYPMIEEIILWNTIFEDRDILKEKLKRNYGDCLSDEQIKKIVRK RFTGWGRLSKKLLCGIKVETDNGRMSIMDVLREGNPNNNGLSKAMVFM EIIRDTGLGFDKRIDELNKEAMIGSTGLAIEDLPGSPALRRGVNQALGIVEE IVHIAGHAPNNIFIEVTREEDGRKKGQRTKKRYESLKEAMSKLKKESPEF WDAATSSQLAEKAKESAALDEKLTLYFLQGGKSLYSGKPLEIGRLSEYQ VDHIIPQSYIKDDSFENKALVLASENQNKSDQMLLPVEMRRKMRPYWDA LLSAGLIGKKKHTNLLRDHIGEKQMKGFIARQLVETSQILKIVQSFLEEQY PETNTLPVKAGLSHELRERIGLVKCREVNDFHHAHDALLAAEIGRFIQKR HPGMYTNPIGYTQVMKQFVKNESDAVRRGHEPGTSPFVIASFMTSGFD EETGELFKDDWSASYEIAKLKSYFNYRQCFISRMPEETSGAFWDQTIYSP RGGKKTPSIPLKKGLSTEKHGGYSGQNPAYFVLIAAYDSKGKPKRLFEG VPIAVAELIKSKKQSIEEWAKQLVESRGFVFGQVLRARLLKYARIDHNGN EYYVPALDCVYSSRELILNENQTTIAAAVCDLARVSDEALVPSEVLELFDA LTEKCLILCTRFKPVYEKLVEGRERFVALSMQDKRKQIVGILKFFNCVTAR VDLSLIGGSPHAGGITNGFLQKPNDSFDIVDQSITGMFERRTHVGL AHWY Type II MNLRNVGEYNIGLDIGTGSVGWAVIDSDGEICHFKGKPTWGSRLFPSAL 14 Cas amino PASEARVHRGQRRRYTRRKWRLFLLQELFAGAIEANDPEFFIRLNQSHLL acid PEDRQSDHKDYLFTLFNDSSFDEKAYFKKFPTIYHLRKWLMETDEKADP sequence RLIYLAFHNIVKHRGNFLQQDNTGLNSQNANVRESVEELCAQLQEYCDS LDIPCTVEDNVDKIVQVLSDTHSVRSHLQEEVQKLLGIGVCDLLDKTSAK KMAKALSGALVGLSAELADVFFLVIEKPEDAKTKIYLSKDEDVESFEEICP DEALPLFEAMNKVYSSFVLQEILSSCPGKTLSVNKVKDYEQYGEDLKLLK ALVKEYAPEKYDDFFRGEFYRPSSLHPQKYVYNKSVAKGYTRYNEVRG VAYEDFKKDVEKLFRDTPAVEDARYLDMMERFKEEKFLRRLKTSDNGAI PFQLHLEEMDKIIQNQARFYPFLEEKKKELDSLVTFRIPYYVGPLTQKNAR KDRKGSNRFSWSERIEGKEHEVIKPWNWEEIIDKDRSATEFILRMTGTCT YLQGEPVLPRCSLLYEEYCVLNELNGAHFTQDGDKEHRFDYQDRSDML KELFRKGKVSYKKAADWMVQRGNSNVRVSGGQGEKGFESKLGSYIFFA KDVFGVDEIPESDYPMIEEIILWNTIFEDRDILKEKLKRNYGDCLSDEQIKKI VRKRFTGWGRLSKKLLCGIKVETDNGRMSIMDVLREGNPNNNGLSKAM VFMEIIRDTGLGFDKRIDELNKEAMIGSTGLAIEDLPGSPALRRGVNQALG IVEEIVHIAGHAPNNIFIEVTREEDGRKKGQRTKKRYESLKEAMSKLKKESTABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO PEFWDAATSSQLAEKAKESAALDEKLTLYFLQGGKSLYSGKPLEIGRLSE YQVDHIIPQSYIKDDSFENKALVLASENQNKSDQMLLPVEMRRKMRPYW DALLSAGLIGKKKHTNLLRDHIGEKQMKGFIARQLVETSQILKIVQSFLEE QYPETNTLPVKAGLSHELRERIGLVKCREVNDFHHAHDALLAAEIGRFIQ KRHPGMYTNPIGYTQVMKQFVKNESDAVRRGHEPGTSPFVIASFMTSG FDEETGELFKDDWSASYEIAKLKSYFNYRQCFISRMPEETSGAFWDQTIY SPRGGKKTPSIPLKKGLSTEKHGGYSGQNPAYFVLIAAYDSKGKPKRLFE GVPIAVAELIKSKKQSIEEWAKQLVESRGFVFGQVLRARLLKYARIDHNG NEYYVPALDCVYSSRELILNENQTTIAAAVCDLARVSDEALVPSEVLELFD ALTEKCLILCTRFKPVYEKLVEGRERFVALSMQDKRKQIVGILKFFNCVTA RVDLSLIGGSPHAGGITNGFLQKPNDSFDIVDQSITGMFERRTHVGL AHWY Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSNLRNVGEYNIGLDIG 15 Cas TGSVGWAVIDSDGEICHFKGKPTWGSRLFPSALPASEARVHRGQRRRY mammalian TRRKWRLFLLQELFAGAIEANDPEFFIRLNQSHLLPEDRQSDHKDYLFTL expression FNDSSFDEKAYFKKFPTIYHLRKWLMETDEKADPRLIYLAFHNIVKHRGN construct FLQQDNTGLNSQNANVRESVEELCAQLQEYCDSLDIPCTVEDNVDKIVQ (with N- VLSDTHSVRSHLQEEVQKLLGIGVCDLLDKTSAKKMAKALSGALVGLSAE terminal LADVFFLVIEKPEDAKTKIYLSKDEDVESFEEICPDEALPLFEAMNKVYSS methionine, FVLQEILSSCPGKTLSVNKVKDYEQYGEDLKLLKALVKEYAPEKYDDFFR stop codon, GEFYRPSSLHPQKYVYNKSVAKGYTRYNEVRGVAYEDFKKDVEKLFRD V5 tag, N- TPAVEDARYLDMMERFKEEKFLRRLKTSDNGAIPFQLHLEEMDKIIQNQA terminal NLS RFYPFLEEKKKELDSLVTFRIPYYVGPLTQKNARKDRKGSNRFSWSERIE and C- GKEHEVIKPWNWEEIIDKDRSATEFILRMTGTCTYLQGEPVLPRCSLLYE terminal NLS) EYCVLNELNGAHFTQDGDKEHRFDYQDRSDMLKELFRKGKVSYKKAAD (aa) WMVQRGNSNVRVSGGQGEKGFESKLGSYIFFAKDVFGVDEIPESDYPM IEEIILWNTIFEDRDILKEKLKRNYGDCLSDEQIKKIVRKRFTGWGRLSKKL LCGIKVETDNGRMSIMDVLREGNPNNNGLSKAMVFMEIIRDTGLGFDKRI DELNKEAMIGSTGLAIEDLPGSPALRRGVNQALGIVEEIVHIAGHAPNNIFI EVTREEDGRKKGQRTKKRYESLKEAMSKLKKESPEFWDAATSSQLAEK AKESAALDEKLTLYFLQGGKSLYSGKPLEIGRLSEYQVDHIIPQSYIKDDS FENKALVLASENQNKSDQMLLPVEMRRKMRPYWDALLSAGLIGKKKHT NLLRDHIGEKQMKGFIARQLVETSQILKIVQSFLEEQYPETNTLPVKAGLS HELRERIGLVKCREVNDFHHAHDALLAAEIGRFIQKRHPGMYTNPIGYTQ VMKQFVKNESDAVRRGHEPGTSPFVIASFMTSGFDEETGELFKDDWSA SYEIAKLKSYFNYRQCFISRMPEETSGAFWDQTIYSPRGGKKTPSIPLKK GLSTEKHGGYSGQNPAYFVLIAAYDSKGKPKRLFEGVPIAVAELIKSKKQ SIEEWAKQLVESRGFVFGQVLRARLLKYARIDHNGNEYYVPALDCVYSS RELILNENQTTIAAAVCDLARVSDEALVPSEVLELFDALTEKCLILCTRFKP VYEKLVEGRERFVALSMQDKRKQIVGILKFFNCVTARVDLSLIGGSPHAG GITNGFLQKPNDSFDIVDQSITGMFERRTHVGLSRKRTADGSEFESPKKK RKV AHWY Type II ATGAATTTAAGAAATGTCGGAGAGTATAATATCGGTCTCGATATTGGT 16 Cas coding ACGGGATCGGTTGGATGGGCTGTCATTGATTCTGATGGCGAGATCTG sequence (nt) CCATTTTAAAGGAAAACCAACATGGGGTAGCCGCTTGTTCCCATCTG (not codon CACTTCCTGCGTCAGAAGCTCGAGTGCACCGTGGGCAACGACGTCG optimized) CTACACCCGTCGTAAATGGCGTTTATTTCTTTTGCAAGAGTTGTTTGC AGGCGCGATCGAAGCAAACGATCCTGAATTCTTCATTCGCTTAAATCA GTCTCATCTGCTTCCTGAAGACCGTCAGTCAGATCATAAAGACTATCT CTTTACGCTCTTCAACGATTCATCGTTTGACGAAAAGGCATATTTCAA GAAATTTCCAACTATCTATCATCTTCGCAAATGGCTTATGGAGACAGA CGAGAAGGCAGATCCGCGTCTTATCTATCTTGCTTTCCACAACATTGT CAAGCACAGAGGGAATTTCCTTCAGCAAGATAATACAGGATTGAATTC GCAAAACGCCAATGTGCGAGAGTCTGTTGAAGAACTCTGCGCGCAGC TTCAAGAGTATTGCGATAGTCTCGATATTCCTTGCACGGTGGAAGATA ACGTCGATAAGATCGTTCAGGTACTGAGTGATACCCATTCAGTTCGCT CTCATCTTCAAGAAGAGGTTCAAAAGCTTCTTGGAATCGGTGTTTGTG ATCTTCTTGATAAAACATCGGCAAAGAAGATGGCGAAAGCCCTTTCAGTABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO GTGCATTGGTGGGACTCTCAGCAGAGCTGGCAGATGTGTTCTTCTTG GTGATAGAGAAGCCAGAGGATGCCAAAACGAAGATATATCTTTCAAA GGATGAGGATGTTGAATCGTTCGAAGAGATCTGTCCAGACGAAGCGC TCCCGCTTTTTGAAGCAATGAACAAGGTTTATTCATCATTCGTTTTGCA GGAGATCTTGAGTAGTTGTCCAGGAAAGACGCTGTCTGTAAACAAGG TCAAAGATTACGAGCAGTATGGTGAAGACCTCAAGCTCCTGAAGGCT CTTGTGAAAGAGTATGCTCCAGAAAAGTACGATGATTTCTTCAGAGGG GAGTTCTACAGGCCAAGCAGCCTGCATCCTCAAAAGTACGTGTACAA CAAGTCAGTAGCAAAAGGGTATACCCGCTACAACGAAGTACGTGGGG TTGCTTATGAAGATTTCAAAAAGGATGTTGAAAAGCTCTTTCGCGATA CACCGGCCGTTGAGGATGCTCGTTACCTTGATATGATGGAGCGATTC AAAGAGGAGAAGTTCCTTCGAAGGCTTAAGACAAGCGACAATGGGGC GATACCCTTTCAACTGCATCTTGAGGAAATGGACAAGATCATTCAGAA TCAAGCACGTTTTTATCCATTCTTGGAAGAGAAGAAAAAAGAGCTTGA TTCGCTCGTGACGTTCCGTATCCCTTATTATGTGGGTCCTCTCACGCA GAAGAACGCACGCAAAGATCGCAAAGGTAGCAATCGGTTTTCGTGGT CGGAGCGCATCGAAGGAAAAGAGCACGAAGTTATCAAACCGTGGAAT TGGGAAGAGATCATCGATAAGGACAGGAGCGCAACCGAGTTCATATT GCGCATGACCGGCACCTGCACCTATCTTCAAGGTGAGCCAGTGCTTC CGCGTTGTTCTCTGCTCTATGAGGAATATTGCGTACTGAACGAACTTA ATGGTGCCCACTTCACACAAGATGGTGACAAAGAGCATCGTTTCGAT TATCAAGATCGTTCAGATATGCTGAAAGAACTCTTCCGAAAAGGAAAG GTGTCGTACAAGAAGGCTGCTGACTGGATGGTGCAGCGCGGCAATT CGAATGTACGTGTTTCGGGCGGCCAAGGCGAAAAGGGTTTCGAGTC GAAGTTAGGATCTTACATCTTCTTTGCAAAAGACGTCTTTGGAGTCGA CGAGATACCTGAGTCTGACTATCCCATGATAGAGGAGATCATTCTTTG GAATACGATCTTTGAAGATCGAGACATCCTTAAGGAAAAGCTAAAGCG CAACTATGGTGATTGTTTGTCGGATGAGCAGATCAAGAAGATCGTTC GAAAGCGCTTTACAGGATGGGGTCGCCTATCGAAGAAGCTTCTTTGT GGGATAAAAGTTGAGACCGATAATGGGCGAATGTCCATTATGGATGT TTTGCGTGAAGGAAATCCGAACAATAATGGATTATCGAAAGCGATGGT CTTTATGGAGATCATCAGAGACACTGGTCTCGGTTTCGATAAGCGAAT CGATGAACTCAACAAAGAAGCGATGATCGGTTCAACCGGTCTTGCTA TCGAGGATCTTCCTGGGTCCCCTGCGCTTCGTCGTGGAGTGAATCAG GCTCTTGGTATCGTGGAAGAGATCGTCCATATTGCTGGGCATGCACC GAACAATATCTTCATCGAGGTAACCCGTGAGGAAGATGGACGAAAAA AGGGACAGAGGACGAAGAAGCGCTATGAGAGCCTCAAAGAGGCTAT GAGCAAGCTGAAGAAAGAATCTCCGGAATTCTGGGATGCAGCAACTT CATCTCAGCTTGCAGAGAAAGCGAAAGAATCGGCTGCTCTTGATGAG AAGCTCACCCTGTATTTCTTGCAAGGCGGAAAATCTCTCTATTCAGGG AAACCGCTTGAGATTGGTCGCCTCTCCGAATATCAGGTCGATCACAT CATTCCGCAGTCGTACATCAAGGATGATAGTTTTGAGAATAAAGCACT TGTTCTTGCGTCGGAGAACCAAAACAAATCTGATCAGATGCTCCTTCC TGTGGAAATGCGTCGCAAGATGCGCCCATATTGGGATGCTCTTTTGA GCGCTGGTCTTATTGGGAAGAAGAAGCACACGAACCTTCTTCGTGAT CATATTGGCGAAAAGCAGATGAAAGGCTTCATAGCGCGTCAGCTTGT AGAAACGAGCCAGATATTGAAGATTGTTCAGAGCTTCTTGGAGGAGC AGTATCCCGAAACGAATACTCTTCCTGTAAAAGCGGGGCTTTCGCAT GAGTTGCGTGAGCGCATTGGTTTGGTGAAGTGCCGCGAAGTGAACG ATTTCCATCATGCTCACGATGCGCTTTTGGCTGCAGAGATCGGCCGT TTCATCCAAAAACGGCATCCTGGTATGTATACGAATCCGATCGGCTAT ACGCAGGTGATGAAGCAATTCGTCAAGAATGAAAGCGATGCTGTTAG AAGAGGCCACGAGCCTGGTACTTCGCCATTCGTTATAGCAAGCTTCA TGACTTCAGGGTTCGATGAAGAGACCGGAGAACTTTTCAAAGATGAC TGGTCGGCATCGTATGAGATTGCAAAGCTAAAGAGCTATTTCAACTAT CGCCAGTGTTTCATATCGAGAATGCCGGAAGAAACATCGGGCGCCTT TTGGGATCAAACCATCTATTCACCACGCGGCGGGAAGAAAACACCAATABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO GTATTCCACTGAAAAAAGGACTCTCTACCGAGAAGCATGGAGGATATT CAGGTCAAAATCCTGCTTACTTTGTCTTGATTGCTGCATATGATTCAAA AGGGAAACCAAAAAGACTATTTGAAGGAGTTCCTATTGCTGTTGCCGA GCTAATCAAGAGCAAAAAGCAGTCAATAGAAGAGTGGGCGAAACAGC TGGTCGAATCACGTGGGTTTGTTTTTGGCCAAGTTTTAAGAGCCCGAT TGCTTAAATATGCGCGCATTGATCATAACGGGAATGAATACTATGTTC CAGCGCTAGATTGCGTTTACTCGTCACGTGAGTTGATCTTGAACGAAA ACCAGACTACAATTGCTGCAGCAGTTTGCGATCTTGCTCGTGTTTCG GATGAAGCTTTAGTGCCATCCGAAGTACTAGAGCTTTTTGATGCCCTA ACAGAGAAATGCCTGATTCTTTGTACACGATTCAAGCCAGTCTATGAA AAGCTTGTAGAAGGAAGAGAGCGATTCGTAGCTCTATCGATGCAAGA TAAGCGAAAGCAAATCGTTGGAATACTGAAGTTCTTCAATTGCGTTAC AGCTCGTGTTGATTTGTCCTTAATAGGTGGATCGCCACATGCTGGAG GAATTACGAATGGCTTTTTACAAAAGCCCAATGACTCTTTTGATATCGT TGATCAGTCTATAACAGGCATGTTTGAAAGGCGGACTCACGTTGGCC TTTAG AHWY Type II AACCTGCGGAACGTGGGAGAGTACAACATCGGCCTTGACATCGGCA 17 Cas coding CCGGAAGCGTGGGATGGGCAGTGATTGACTCCGACGGCGAGATCTG sequence (nt) CCACTTCAAGGGCAAGCCTACCTGGGGGAGCAGACTGTTCCCTAGC (human GCCCTCCCTGCCTCTGAGGCCAGAGTGCATAGAGGCCAGAGAAGAA codon- GATACACACGTAGAAAGTGGCGGCTGTTCCTGCTGCAAGAGCTGTTC optimized; GCCGGCGCCATCGAGGCGAATGACCCTGAGTTCTTCATCAGACTGAA lacking N- CCAGAGCCACCTGCTCCCTGAGGATCGGCAGTCTGATCACAAGGACT terminal ACCTGTTTACCCTGTTTAACGACAGCTCTTTCGACGAGAAAGCCTATT methionine; TCAAAAAATTCCCAACAATCTATCACCTCAGAAAATGGCTGATGGAAA no stop CAGATGAAAAAGCCGATCCTAGACTGATCTATCTGGCCTTCCACAACA codon) TCGTGAAGCACAGAGGAAATTTCCTGCAGCAGGACAATACCGGCCTG AACAGCCAGAACGCCAACGTGAGGGAGTCTGTTGAAGAGCTGTGCG CTCAGCTGCAGGAGTATTGCGACTCGCTGGATATCCCCTGCACCGTG GAGGATAATGTGGATAAGATCGTGCAGGTGCTGTCTGATACCCACAG CGTGCGGAGCCACCTGCAGGAGGAGGTGCAGAAGCTGCTGGGAATC GGTGTCTGCGACCTGCTTGATAAGACATCCGCAAAGAAGATGGCCAA GGCCCTGAGCGGCGCCCTGGTGGGCCTGAGCGCTGAACTGGCCGA TGTGTTTTTCCTGGTGATCGAAAAACCCGAAGATGCCAAGACGAAGA TCTACCTGAGCAAGGATGAGGACGTGGAGAGCTTCGAAGAGATCTGC CCTGATGAGGCTCTTCCACTGTTCGAAGCCATGAACAAGGTGTACAG CAGCTTTGTGCTGCAGGAGATCCTGAGCTCATGCCCCGGCAAGACAC TGTCTGTGAACAAGGTGAAGGACTACGAGCAGTATGGCGAGGACCT GAAGCTGCTGAAGGCCCTGGTCAAGGAATACGCCCCTGAAAAGTAC GATGATTTCTTTCGGGGCGAGTTCTACAGACCTAGCTCTCTGCACCC GCAGAAATACGTGTACAACAAAAGCGTGGCTAAGGGGTACACCCGGT ACAACGAGGTGCGGGGCGTGGCCTACGAGGATTTCAAGAAGGACGT GGAAAAGCTGTTTAGAGACACACCTGCTGTGGAAGATGCTAGATACC TGGACATGATGGAAAGATTCAAGGAGGAAAAATTCCTGCGCAGACTG AAAACAAGCGACAATGGAGCTATTCCTTTCCAGCTGCACCTCGAAGA GATGGACAAAATCATCCAGAACCAGGCCAGATTCTACCCTTTTCTGGA GGAGAAGAAGAAGGAACTGGACTCTCTGGTGACCTTCAGAATCCCTT ACTACGTGGGCCCTCTGACCCAGAAGAACGCCAGAAAGGACAGAAA AGGCAGCAACAGATTCAGCTGGTCCGAGCGGATCGAGGGAAAGGAA CACGAGGTGATCAAGCCTTGGAATTGGGAGGAGATCATCGACAAGGA CCGGTCGGCCACAGAGTTCATCCTGAGAATGACAGGCACCTGTACCT ACCTGCAAGGCGAGCCTGTGCTGCCTAGATGCAGCCTGCTGTATGA GGAATACTGTGTGCTGAACGAGCTGAACGGCGCCCACTTCACCCAG GACGGCGATAAAGAGCACCGGTTTGACTACCAGGATAGAAGCGATAT GCTGAAAGAATTATTCAGAAAGGGAAAGGTGTCTTATAAGAAAGCTGC TGATTGGATGGTGCAGCGGGGCAATAGCAACGTGCGAGTGTCCGGC GGCCAGGGCGAGAAGGGATTTGAATCTAAACTGGGCAGCTACATCTTTABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO CTTCGCCAAGGATGTATTCGGAGTGGACGAGATCCCTGAGAGCGATT ACCCTATGATCGAGGAAATTATCCTGTGGAACACCATCTTCGAGGATC GAGACATCCTGAAGGAGAAGTTAAAGCGCAACTACGGCGACTGTCTG TCTGACGAGCAGATCAAAAAAATCGTGCGCAAACGCTTCACAGGCTG GGGCAGACTCAGCAAGAAGCTCCTGTGCGGCATTAAGGTGGAAACC GACAACGGCAGAATGAGCATCATGGACGTGCTGCGCGAGGGCAACC CCAACAACAACGGCCTGTCTAAGGCTATGGTGTTTATGGAAATCATCA GGGATACCGGCCTGGGCTTCGACAAGAGAATCGACGAGCTGAACAA GGAAGCCATGATCGGATCTACCGGCCTCGCCATCGAGGACCTGCCC GGCAGTCCAGCCCTAAGACGGGGCGTGAACCAAGCCCTGGGCATCG TGGAAGAGATCGTGCACATAGCCGGACACGCCCCTAACAACATCTTC ATTGAAGTGACAAGAGAGGAAGATGGCCGGAAAAAGGGACAAAGAA CCAAGAAGCGGTACGAGTCCCTGAAGGAGGCTATGTCTAAACTGAAG AAAGAGAGCCCCGAGTTCTGGGATGCCGCCACCAGCAGCCAGCTGG CCGAAAAAGCCAAGGAGAGCGCCGCGCTCGACGAAAAGCTGACACT GTACTTCCTGCAGGGAGGCAAGAGCCTGTACTCTGGCAAGCCCCTG GAAATCGGCCGCCTGAGCGAATACCAGGTTGATCATATCATCCCCCA GAGCTATATCAAGGACGATAGCTTCGAGAACAAGGCCCTGGTACTCG CCAGCGAGAACCAAAATAAGAGCGACCAGATGCTGTTACCAGTGGAA ATGCGGAGAAAGATGCGGCCTTACTGGGACGCCCTGCTGAGCGCCG GCCTCATCGGGAAGAAGAAGCATACCAACCTGCTGCGGGACCACAT CGGAGAGAAGCAGATGAAGGGCTTCATCGCAAGACAGCTGGTCGAA ACCTCTCAGATTCTTAAGATCGTGCAGAGCTTCCTGGAAGAACAGTAC CCCGAAACCAACACCCTGCCCGTCAAGGCCGGCTTGTCTCACGAACT GAGAGAGAGAATCGGCCTGGTCAAGTGCAGAGAGGTGAACGACTTC CACCACGCCCACGACGCTCTCCTGGCAGCAGAAATCGGCCGGTTTAT CCAGAAGAGACACCCTGGCATGTACACCAACCCCATCGGCTACACAC AAGTGATGAAGCAGTTCGTGAAAAACGAGTCCGACGCCGTTAGAAGA GGCCACGAGCCTGGAACCAGCCCCTTCGTGATCGCCAGCTTCATGA CAAGCGGGTTCGACGAAGAAACCGGCGAGCTGTTCAAAGACGACTG GTCCGCCAGCTACGAGATCGCCAAACTCAAGTCTTATTTTAACTACCG ACAGTGCTTCATAAGCAGAATGCCTGAAGAAACCAGCGGCGCTTTCT GGGACCAAACCATCTACAGCCCTCGGGGCGGCAAGAAAACCCCTTC TATCCCCCTGAAGAAAGGCCTGAGCACCGAGAAGCACGGCGGCTAC AGCGGACAGAATCCTGCATACTTCGTGCTGATCGCCGCCTACGACAG CAAGGGCAAGCCTAAGCGGCTGTTCGAGGGCGTGCCTATCGCCGTG GCCGAGCTCATCAAGAGTAAGAAACAGAGCATCGAGGAGTGGGCCA AGCAGCTGGTGGAAAGCCGGGGCTTCGTGTTCGGCCAGGTGCTGAG AGCCAGACTGCTGAAGTACGCCAGAATCGATCACAACGGCAACGAGT ACTACGTGCCTGCCCTGGACTGTGTGTACAGCAGCCGGGAACTGATC CTGAACGAGAATCAGACCACAATCGCCGCTGCCGTGTGTGACCTGG CCAGAGTGAGCGACGAGGCCCTGGTGCCTAGCGAAGTGCTGGAACT GTTTGATGCTCTGACCGAGAAGTGCCTGATCCTGTGCACCCGGTTCA AGCCAGTGTACGAGAAGCTGGTAGAAGGCAGAGAAAGATTTGTTGCC CTGTCCATGCAGGATAAGAGAAAGCAGATCGTGGGCATCCTGAAATT TTTCAACTGCGTGACCGCCAGAGTGGACCTGTCCCTGATCGGCGGCT CTCCACACGCCGGCGGAATCACCAATGGCTTTCTGCAAAAGCCTAAC GACAGCTTCGACATCGTTGACCAGAGCATCACAGGCATGTTTGAGCG GCGAACACACGTGGGACTC AHWY Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 18 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCAACCTGCGGAACGTGGGAGAGTACAACATCGGCC expression TTGACATCGGCACCGGAAGCGTGGGATGGGCAGTGATTGACTCCGA construct CGGCGAGATCTGCCACTTCAAGGGCAAGCCTACCTGGGGGAGCAGA (with N- CTGTTCCCTAGCGCCCTCCCTGCCTCTGAGGCCAGAGTGCATAGAG terminal GCCAGAGAAGAAGATACACACGTAGAAAGTGGCGGCTGTTCCTGCTG methionine, CAAGAGCTGTTCGCCGGCGCCATCGAGGCGAATGACCCTGAGTTCTTTABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO stop codon, CATCAGACTGAACCAGAGCCACCTGCTCCCTGAGGATCGGCAGTCTG V5 tag, N- ATCACAAGGACTACCTGTTTACCCTGTTTAACGACAGCTCTTTCGACG terminal NLS AGAAAGCCTATTTCAAAAAATTCCCAACAATCTATCACCTCAGAAAAT and C- GGCTGATGGAAACAGATGAAAAAGCCGATCCTAGACTGATCTATCTG terminal NLS) GCCTTCCACAACATCGTGAAGCACAGAGGAAATTTCCTGCAGCAGGA (nt) CAATACCGGCCTGAACAGCCAGAACGCCAACGTGAGGGAGTCTGTT GAAGAGCTGTGCGCTCAGCTGCAGGAGTATTGCGACTCGCTGGATAT CCCCTGCACCGTGGAGGATAATGTGGATAAGATCGTGCAGGTGCTGT CTGATACCCACAGCGTGCGGAGCCACCTGCAGGAGGAGGTGCAGAA GCTGCTGGGAATCGGTGTCTGCGACCTGCTTGATAAGACATCCGCAA AGAAGATGGCCAAGGCCCTGAGCGGCGCCCTGGTGGGCCTGAGCG CTGAACTGGCCGATGTGTTTTTCCTGGTGATCGAAAAACCCGAAGAT GCCAAGACGAAGATCTACCTGAGCAAGGATGAGGACGTGGAGAGCT TCGAAGAGATCTGCCCTGATGAGGCTCTTCCACTGTTCGAAGCCATG AACAAGGTGTACAGCAGCTTTGTGCTGCAGGAGATCCTGAGCTCATG CCCCGGCAAGACACTGTCTGTGAACAAGGTGAAGGACTACGAGCAG TATGGCGAGGACCTGAAGCTGCTGAAGGCCCTGGTCAAGGAATACG CCCCTGAAAAGTACGATGATTTCTTTCGGGGCGAGTTCTACAGACCT AGCTCTCTGCACCCGCAGAAATACGTGTACAACAAAAGCGTGGCTAA GGGGTACACCCGGTACAACGAGGTGCGGGGCGTGGCCTACGAGGA TTTCAAGAAGGACGTGGAAAAGCTGTTTAGAGACACACCTGCTGTGG AAGATGCTAGATACCTGGACATGATGGAAAGATTCAAGGAGGAAAAA TTCCTGCGCAGACTGAAAACAAGCGACAATGGAGCTATTCCTTTCCA GCTGCACCTCGAAGAGATGGACAAAATCATCCAGAACCAGGCCAGAT TCTACCCTTTTCTGGAGGAGAAGAAGAAGGAACTGGACTCTCTGGTG ACCTTCAGAATCCCTTACTACGTGGGCCCTCTGACCCAGAAGAACGC CAGAAAGGACAGAAAAGGCAGCAACAGATTCAGCTGGTCCGAGCGG ATCGAGGGAAAGGAACACGAGGTGATCAAGCCTTGGAATTGGGAGG AGATCATCGACAAGGACCGGTCGGCCACAGAGTTCATCCTGAGAATG ACAGGCACCTGTACCTACCTGCAAGGCGAGCCTGTGCTGCCTAGATG CAGCCTGCTGTATGAGGAATACTGTGTGCTGAACGAGCTGAACGGCG CCCACTTCACCCAGGACGGCGATAAAGAGCACCGGTTTGACTACCAG GATAGAAGCGATATGCTGAAAGAATTATTCAGAAAGGGAAAGGTGTC TTATAAGAAAGCTGCTGATTGGATGGTGCAGCGGGGCAATAGCAACG TGCGAGTGTCCGGCGGCCAGGGCGAGAAGGGATTTGAATCTAAACT GGGCAGCTACATCTTCTTCGCCAAGGATGTATTCGGAGTGGACGAGA TCCCTGAGAGCGATTACCCTATGATCGAGGAAATTATCCTGTGGAAC ACCATCTTCGAGGATCGAGACATCCTGAAGGAGAAGTTAAAGCGCAA CTACGGCGACTGTCTGTCTGACGAGCAGATCAAAAAAATCGTGCGCA AACGCTTCACAGGCTGGGGCAGACTCAGCAAGAAGCTCCTGTGCGG CATTAAGGTGGAAACCGACAACGGCAGAATGAGCATCATGGACGTGC TGCGCGAGGGCAACCCCAACAACAACGGCCTGTCTAAGGCTATGGT GTTTATGGAAATCATCAGGGATACCGGCCTGGGCTTCGACAAGAGAA TCGACGAGCTGAACAAGGAAGCCATGATCGGATCTACCGGCCTCGC CATCGAGGACCTGCCCGGCAGTCCAGCCCTAAGACGGGGCGTGAAC CAAGCCCTGGGCATCGTGGAAGAGATCGTGCACATAGCCGGACACG CCCCTAACAACATCTTCATTGAAGTGACAAGAGAGGAAGATGGCCGG AAAAAGGGACAAAGAACCAAGAAGCGGTACGAGTCCCTGAAGGAGG CTATGTCTAAACTGAAGAAAGAGAGCCCCGAGTTCTGGGATGCCGCC ACCAGCAGCCAGCTGGCCGAAAAAGCCAAGGAGAGCGCCGCGCTCG ACGAAAAGCTGACACTGTACTTCCTGCAGGGAGGCAAGAGCCTGTAC TCTGGCAAGCCCCTGGAAATCGGCCGCCTGAGCGAATACCAGGTTG ATCATATCATCCCCCAGAGCTATATCAAGGACGATAGCTTCGAGAACA AGGCCCTGGTACTCGCCAGCGAGAACCAAAATAAGAGCGACCAGAT GCTGTTACCAGTGGAAATGCGGAGAAAGATGCGGCCTTACTGGGAC GCCCTGCTGAGCGCCGGCCTCATCGGGAAGAAGAAGCATACCAACC TGCTGCGGGACCACATCGGAGAGAAGCAGATGAAGGGCTTCATCGCTABLE 1C AHWY Type II Cas Sequences Name Sequence SEQ ID NO AAGACAGCTGGTCGAAACCTCTCAGATTCTTAAGATCGTGCAGAGCT TCCTGGAAGAACAGTACCCCGAAACCAACACCCTGCCCGTCAAGGCC GGCTTGTCTCACGAACTGAGAGAGAGAATCGGCCTGGTCAAGTGCA GAGAGGTGAACGACTTCCACCACGCCCACGACGCTCTCCTGGCAGC AGAAATCGGCCGGTTTATCCAGAAGAGACACCCTGGCATGTACACCA ACCCCATCGGCTACACACAAGTGATGAAGCAGTTCGTGAAAAACGAG TCCGACGCCGTTAGAAGAGGCCACGAGCCTGGAACCAGCCCCTTCG TGATCGCCAGCTTCATGACAAGCGGGTTCGACGAAGAAACCGGCGA GCTGTTCAAAGACGACTGGTCCGCCAGCTACGAGATCGCCAAACTCA AGTCTTATTTTAACTACCGACAGTGCTTCATAAGCAGAATGCCTGAAG AAACCAGCGGCGCTTTCTGGGACCAAACCATCTACAGCCCTCGGGG CGGCAAGAAAACCCCTTCTATCCCCCTGAAGAAAGGCCTGAGCACCG AGAAGCACGGCGGCTACAGCGGACAGAATCCTGCATACTTCGTGCT GATCGCCGCCTACGACAGCAAGGGCAAGCCTAAGCGGCTGTTCGAG GGCGTGCCTATCGCCGTGGCCGAGCTCATCAAGAGTAAGAAACAGA GCATCGAGGAGTGGGCCAAGCAGCTGGTGGAAAGCCGGGGCTTCGT GTTCGGCCAGGTGCTGAGAGCCAGACTGCTGAAGTACGCCAGAATC GATCACAACGGCAACGAGTACTACGTGCCTGCCCTGGACTGTGTGTA CAGCAGCCGGGAACTGATCCTGAACGAGAATCAGACCACAATCGCC GCTGCCGTGTGTGACCTGGCCAGAGTGAGCGACGAGGCCCTGGTGC CTAGCGAAGTGCTGGAACTGTTTGATGCTCTGACCGAGAAGTGCCTG ATCCTGTGCACCCGGTTCAAGCCAGTGTACGAGAAGCTGGTAGAAGG CAGAGAAAGATTTGTTGCCCTGTCCATGCAGGATAAGAGAAAGCAGA TCGTGGGCATCCTGAAATTTTTCAACTGCGTGACCGCCAGAGTGGAC CTGTCCCTGATCGGCGGCTCTCCACACGCCGGCGGAATCACCAATG GCTTTCTGCAAAAGCCTAACGACAGCTTCGACATCGTTGACCAGAGC ATCACAGGCATGTTTGAGCGGCGAACACACGTGGGACTCTCTAGAAA GCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAA AGGTGTGA

[0082] In some embodiments an AHWY Type II Cas protein comprises an amino acid sequence of SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, an AHWY Type II 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 D14A substitution, wherein the position of the D14A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H900A substitution, wherein the position of the H900A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14. In some embodiments, an AHWY Type II Cas protein is catalytically inactive, for example due a D14A substitution in combination with an H900A substitution. 6.2.1.4. CBGI Type II Cas Proteins

[0083] In one aspect, the disclosure provides CBGI Type II Cas proteins. CBGI Type II Cas proteins can be further classified as Type IIA Cas proteins. The CBGI Type II 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 CBGI Type II 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 CBGI Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:19.

[0084] Exemplary CBGI Type II Cas protein sequences and nucleotide sequences encoding exemplary CBGI Type II Cas proteins are set forth in Table 1D. TABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO CBGI Type II KLRNIQGDYNIGLDLGTGSVGWAVTGVDGELLTQNGRPTLGSRVFPSGE 19 Cas amino TAADARLKRGQRRRYERRRWRLDLLQRFFENDMADVDRTFFIRLKQAR acid LLREDRDESCRDYHSPLFLTVDAERDYYKRFPTIYHLRVWLMTTEKKADL sequence REIYLALHNIVKHRGNFLHQDNPSLSASAANMEDSVEQLCLALEDRCITL (without N- DIPCSCDAASIQRVLEDSSLSRAEKSESVGKLFGLDKDAQKTMGRGISRA terminal IVGYKVDFAAVLGCEFEDSSFTLSDDEKVDAALAAIPDVAMSLFDAIREVY methionine) SAYVLLGILSSGNGAPISSGSLSSVSGRTISFCKVREYEVYRADLALLKSL VRTYAPSQYDAFFRGELIPGTCNYDPTKAKGYTRYDLVHKVAYADFFKE VKSLLDKTDAVTDERYKDMLGRFEEERFLRRLKTSDNGSIPYQLHLEEM DAILKNQGKYYPFLLENLDKIESLVSFRIPYYLGPLTQKNAALDHHGNPRF AWATRKPGMEDEPVYPWNWEEVIDKGHAAHTFIQRMTSDCTYLIGEGV LPRNSLLYEEFCVLNELNGARYSVDGDDWRRFDYADRMGIMDDLFRQR RSVTYKMVEDWMRANRGWARVHVRGGQGECKFESSLSAYRFFCKDVF KTDELSPSIIPMVETIILWSTLFEDRSILKEQLVRNFSDRLSSEQIKIICKKRL TGWGNLSEKFLAEVEAETDCGPRSIMDILREGSPVGGEQGRTMVLMEV LHDERLGFEDKIEEINAERIADAGRLEVVDLPGSPALRRTVNQAVRVVEEI VHIAGKPPVNIFIENTRDEDASKKGKRTKRRYDTIKEAVNAFKRENADLAR ELKEFKPADFDDERLALYFIQGGKSLYSKAPLDVTRLSEYEVDHIIPQSYI KDDSFENKALVLKSENQTKTNRLLLPQGVRVKMASYWQELHRCGLIGDK KLRNLMCSDVSERRVKGFIARQLVETSQIVKLTKMMLENRLPESRLVPIK ASLSHELREAKHYYKCREINDFHHAHDALLAAEIGRFLLLRHAGMYDNPI GYAHVVKDFVRVQADEAKRTGRLPGSAGFIVSSFLHSGFDKDTGEIFKD TWDAEFECERIRKYLNYRQVYLSRMPEETSGAFWDATIYSPRGKMKLSL PLKEGLDPSKYGGYSSEKYAYFFCYYAKDKKGKRLIDFAPVPVSRAAGG QVDIEAFGREVAEERGYAFESIARAKIAIKQLIEVDGCRLFITGADQVRSA VPLAFGQEETHLVERLLSGAPVGPRVRDSLFNQMMVGIRRFDRRLYDNL KLDDRSAAFERLGEEDKDLILKGLLALSSASNNMADMRPIGGAKTAGQL KIVFRNVLSNQGITFIDQSVTGMFERKTYIGL CBGI Type II MKLRNIQGDYNIGLDLGTGSVGWAVTGVDGELLTQNGRPTLGSRVFPS 20 Cas amino GETAADARLKRGQRRRYERRRWRLDLLQRFFENDMADVDRTFFIRLKQ acid ARLLREDRDESCRDYHSPLFLTVDAERDYYKRFPTIYHLRVWLMTTEKK sequence ADLREIYLALHNIVKHRGNFLHQDNPSLSASAANMEDSVEQLCLALEDRC ITLDIPCSCDAASIQRVLEDSSLSRAEKSESVGKLFGLDKDAQKTMGRGI SRAIVGYKVDFAAVLGCEFEDSSFTLSDDEKVDAALAAIPDVAMSLFDAIR EVYSAYVLLGILSSGNGAPISSGSLSSVSGRTISFCKVREYEVYRADLALL KSLVRTYAPSQYDAFFRGELIPGTCNYDPTKAKGYTRYDLVHKVAYADF FKEVKSLLDKTDAVTDERYKDMLGRFEEERFLRRLKTSDNGSIPYQLHLE EMDAILKNQGKYYPFLLENLDKIESLVSFRIPYYLGPLTQKNAALDHHGNP RFAWATRKPGMEDEPVYPWNWEEVIDKGHAAHTFIQRMTSDCTYLIGE GVLPRNSLLYEEFCVLNELNGARYSVDGDDWRRFDYADRMGIMDDLFR QRRSVTYKMVEDWMRANRGWARVHVRGGQGECKFESSLSAYRFFCK DVFKTDELSPSIIPMVETIILWSTLFEDRSILKEQLVRNFSDRLSSEQIKIICK KRLTGWGNLSEKFLAEVEAETDCGPRSIMDILREGSPVGGEQGRTMVL MEVLHDERLGFEDKIEEINAERIADAGRLEVVDLPGSPALRRTVNQAVRV VEEIVHIAGKPPVNIFIENTRDEDASKKGKRTKRRYDTIKEAVNAFKRENA DLARELKEFKPADFDDERLALYFIQGGKSLYSKAPLDVTRLSEYEVDHIIP QSYIKDDSFENKALVLKSENQTKTNRLLLPQGVRVKMASYWQELHRCGL IGDKKLRNLMCSDVSERRVKGFIARQLVETSQIVKLTKMMLENRLPESRL VPIKASLSHELREAKHYYKCREINDFHHAHDALLAAEIGRFLLLRHAGMY DNPIGYAHVVKDFVRVQADEAKRTGRLPGSAGFIVSSFLHSGFDKDTGEI FKDTWDAEFECERIRKYLNYRQVYLSRMPEETSGAFWDATIYSPRGKMK LSLPLKEGLDPSKYGGYSSEKYAYFFCYYAKDKKGKRLIDFAPVPVSRAA GGQVDIEAFGREVAEERGYAFESIARAKIAIKQLIEVDGCRLFITGADQVRTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO SAVPLAFGQEETHLVERLLSGAPVGPRVRDSLFNQMMVGIRRFDRRLYD NLKLDDRSAAFERLGEEDKDLILKGLLALSSASNNMADMRPIGGAKTAG QLKIVFRNVLSNQGITFIDQSVTGMFERKTYIGL CBGI Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSKLRNIQGDYNIGLDL 21 Cas GTGSVGWAVTGVDGELLTQNGRPTLGSRVFPSGETAADARLKRGQRR mammalian RYERRRWRLDLLQRFFENDMADVDRTFFIRLKQARLLREDRDESCRDY expression HSPLFLTVDAERDYYKRFPTIYHLRVWLMTTEKKADLREIYLALHNIVKHR construct GNFLHQDNPSLSASAANMEDSVEQLCLALEDRCITLDIPCSCDAASIQRV (with N- LEDSSLSRAEKSESVGKLFGLDKDAQKTMGRGISRAIVGYKVDFAAVLG terminal CEFEDSSFTLSDDEKVDAALAAIPDVAMSLFDAIREVYSAYVLLGILSSGN methionine, GAPISSGSLSSVSGRTISFCKVREYEVYRADLALLKSLVRTYAPSQYDAF stop codon, FRGELIPGTCNYDPTKAKGYTRYDLVHKVAYADFFKEVKSLLDKTDAVTD V5 tag, N- ERYKDMLGRFEEERFLRRLKTSDNGSIPYQLHLEEMDAILKNQGKYYPFL terminal NLS LENLDKIESLVSFRIPYYLGPLTQKNAALDHHGNPRFAWATRKPGMEDE and C- PVYPWNWEEVIDKGHAAHTFIQRMTSDCTYLIGEGVLPRNSLLYEEFCVL terminal NLS) NELNGARYSVDGDDWRRFDYADRMGIMDDLFRQRRSVTYKMVEDWM (aa) RANRGWARVHVRGGQGECKFESSLSAYRFFCKDVFKTDELSPSIIPMVE TIILWSTLFEDRSILKEQLVRNFSDRLSSEQIKIICKKRLTGWGNLSEKFLA EVEAETDCGPRSIMDILREGSPVGGEQGRTMVLMEVLHDERLGFEDKIE EINAERIADAGRLEVVDLPGSPALRRTVNQAVRVVEEIVHIAGKPPVNIFIE NTRDEDASKKGKRTKRRYDTIKEAVNAFKRENADLARELKEFKPADFDD ERLALYFIQGGKSLYSKAPLDVTRLSEYEVDHIIPQSYIKDDSFENKALVLK SENQTKTNRLLLPQGVRVKMASYWQELHRCGLIGDKKLRNLMCSDVSE RRVKGFIARQLVETSQIVKLTKMMLENRLPESRLVPIKASLSHELREAKHY YKCREINDFHHAHDALLAAEIGRFLLLRHAGMYDNPIGYAHVVKDFVRVQ ADEAKRTGRLPGSAGFIVSSFLHSGFDKDTGEIFKDTWDAEFECERIRKY LNYRQVYLSRMPEETSGAFWDATIYSPRGKMKLSLPLKEGLDPSKYGGY SSEKYAYFFCYYAKDKKGKRLIDFAPVPVSRAAGGQVDIEAFGREVAEE RGYAFESIARAKIAIKQLIEVDGCRLFITGADQVRSAVPLAFGQEETHLVE RLLSGAPVGPRVRDSLFNQMMVGIRRFDRRLYDNLKLDDRSAAFERLGE EDKDLILKGLLALSSASNNMADMRPIGGAKTAGQLKIVFRNVLSNQGITFI DQSVTGMFERKTYIGLSRKRTADGSEFESPKKKRKV CBGI Type II ATGAAACTGCGCAATATTCAAGGTGACTACAACATTGGTCTTGACCTT 22 Cas coding GGAACCGGCTCTGTGGGGTGGGCGGTTACCGGTGTAGATGGCGAGC sequence (nt) TTCTCACGCAGAACGGTAGACCGACGTTGGGAAGTCGCGTTTTCCCT (not codon TCGGGCGAAACTGCTGCGGACGCTCGGCTCAAGCGCGGTCAGCGCC optimized) GTAGGTATGAGCGTCGTCGTTGGCGTCTGGATTTGCTGCAGCGCTTT TTCGAGAATGATATGGCTGATGTAGATCGGACTTTCTTTATCCGCCTT AAACAGGCTCGCCTCTTGCGTGAAGATAGAGATGAGTCGTGTCGCGA TTACCACAGCCCTCTCTTTCTCACCGTTGATGCGGAGAGAGACTATTA CAAACGCTTCCCCACAATTTACCACCTTCGTGTCTGGCTGATGACCAC CGAGAAAAAGGCCGACCTGCGGGAGATTTATCTCGCGCTGCACAACA TCGTCAAGCATCGAGGCAATTTCCTCCACCAAGACAATCCCAGCTTG AGCGCATCTGCTGCCAACATGGAAGATTCTGTTGAGCAGCTGTGTCT TGCGCTGGAGGATCGATGCATCACCCTGGACATTCCTTGCTCTTGCG ATGCGGCTTCTATCCAGCGGGTGCTCGAGGATTCGTCTCTCTCCCGC GCGGAGAAGTCGGAGTCGGTTGGCAAGCTGTTCGGCCTTGATAAGG ACGCCCAGAAGACGATGGGGAGGGGCATCTCTCGCGCGATTGTGGG CTATAAGGTTGATTTTGCAGCCGTGCTCGGATGCGAGTTCGAGGACT CGTCTTTCACCCTTTCCGACGATGAGAAGGTAGATGCTGCCCTTGCG GCAATTCCCGACGTTGCCATGAGCTTGTTCGATGCGATTCGAGAGGT ATATTCTGCATATGTGCTTTTGGGCATCTTGAGTTCCGGAAATGGTGC CCCTATCTCCTCGGGTTCTCTTTCATCTGTTTCTGGTAGAACGATTTC CTTTTGCAAGGTTCGGGAGTATGAGGTGTATAGAGCCGACCTCGCCC TGCTGAAGTCTCTTGTGCGTACGTATGCGCCCAGCCAGTATGACGCC TTCTTCCGTGGTGAGCTGATTCCGGGCACTTGCAACTATGACCCGAC AAAGGCCAAGGGCTATACGCGTTACGACTTGGTTCATAAGGTTGCATTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO ATGCTGACTTCTTTAAAGAGGTCAAGAGCCTTCTCGATAAGACCGATG CCGTAACGGACGAACGTTATAAGGACATGCTGGGCCGCTTTGAGGA GGAGCGCTTCCTGCGGAGGCTCAAAACGAGCGATAACGGCAGCATC CCGTATCAACTCCATCTGGAGGAGATGGACGCGATTCTCAAGAATCA AGGCAAGTATTATCCGTTCCTGCTCGAGAACCTAGACAAAATCGAGT CGCTTGTCAGCTTCAGGATTCCATATTACTTGGGGCCTTTGACACAGA AAAACGCTGCTCTCGATCACCATGGCAATCCACGTTTTGCCTGGGCA ACTCGTAAGCCCGGTATGGAGGACGAGCCTGTGTATCCCTGGAATTG GGAGGAGGTTATCGACAAGGGTCATGCGGCGCATACGTTTATCCAGC GCATGACGAGCGATTGCACCTACTTGATAGGGGAGGGCGTTCTTCCT CGCAATTCTCTTTTGTATGAGGAGTTCTGTGTCCTCAATGAGCTTAAC GGCGCTCGGTATTCCGTTGATGGCGATGATTGGCGGCGCTTCGACTA TGCCGACCGTATGGGTATCATGGATGACCTGTTCAGGCAGCGTCGAA GTGTTACGTACAAAATGGTCGAAGATTGGATGCGCGCCAATCGCGGA TGGGCCCGTGTTCATGTCCGCGGTGGTCAGGGAGAGTGTAAATTCG AATCCTCCCTTTCGGCGTACCGCTTCTTTTGCAAAGACGTTTTCAAGA CCGACGAACTGTCGCCTTCGATAATTCCTATGGTTGAGACCATAATCC TGTGGAGTACCTTATTCGAGGATCGTTCGATTTTGAAAGAGCAGCTC GTCCGCAATTTCTCAGATCGATTGAGCTCCGAACAGATCAAGATTATC TGCAAAAAGCGTCTGACGGGCTGGGGCAACCTATCGGAGAAGTTCCT GGCTGAGGTCGAGGCTGAGACGGATTGCGGACCCCGCTCCATCATG GATATCCTTCGCGAAGGCAGCCCTGTAGGCGGTGAACAGGGTAGGA CCATGGTCCTTATGGAGGTGTTGCATGATGAACGCCTGGGCTTTGAA GATAAAATCGAAGAAATAAACGCTGAAAGAATCGCTGATGCCGGTCG CCTTGAGGTGGTTGACCTGCCTGGATCTCCTGCGCTGCGCAGGACG GTCAATCAGGCCGTCCGTGTTGTCGAGGAGATTGTCCATATAGCGGG CAAGCCTCCTGTGAACATCTTTATCGAGAACACGCGTGATGAAGATG CGTCGAAAAAGGGCAAGAGGACGAAGAGGCGCTACGATACGATAAA AGAGGCTGTTAATGCGTTTAAGAGGGAAAACGCTGATTTGGCGAGAG AATTGAAGGAGTTCAAGCCTGCCGATTTCGACGATGAGAGGCTGGCC TTGTATTTCATTCAGGGAGGTAAGAGCCTGTACTCCAAGGCTCCTCTT GACGTGACGAGGCTGTCTGAATATGAAGTCGATCACATCATTCCACA GTCCTACATCAAAGATGATAGCTTTGAAAACAAAGCCCTCGTTCTAAA GAGTGAAAATCAGACCAAGACGAATCGGCTATTGTTGCCTCAAGGGG TTCGCGTAAAGATGGCGAGCTATTGGCAAGAGCTGCACCGTTGCGG CCTTATAGGTGATAAGAAACTGAGAAACCTCATGTGCTCAGATGTCAG CGAGCGTCGGGTTAAGGGCTTTATCGCACGTCAGCTTGTTGAAACCT CGCAGATCGTCAAGTTGACGAAGATGATGCTCGAGAATCGCCTGCCT GAGTCGAGGCTGGTTCCCATCAAGGCGAGCCTGTCGCATGAGCTGC GCGAGGCCAAGCATTATTACAAGTGCCGTGAGATCAACGATTTTCAC CATGCGCACGATGCACTTCTCGCTGCGGAGATCGGTAGGTTTTTGCT GCTGAGGCATGCTGGAATGTATGACAATCCCATCGGCTATGCGCATG TTGTTAAGGATTTTGTGCGCGTTCAAGCAGACGAGGCAAAGAGGACG GGCCGCCTGCCTGGATCGGCTGGCTTCATCGTGTCGAGCTTCCTGC ATTCGGGTTTCGACAAAGATACGGGCGAGATCTTCAAGGATACGTGG GACGCCGAGTTCGAATGTGAGCGCATTCGCAAGTATCTCAACTATCG GCAGGTGTACCTGAGCCGTATGCCGGAGGAGACATCTGGTGCGTTTT GGGATGCGACTATCTATTCGCCGCGTGGAAAGATGAAACTCTCCCTC CCGCTTAAAGAAGGGCTCGATCCGTCTAAATACGGCGGCTATTCATC GGAGAAATATGCATATTTCTTCTGCTATTACGCGAAGGATAAGAAGGG CAAGCGACTTATTGACTTTGCTCCAGTTCCGGTGTCTCGCGCTGCTG GGGGGCAAGTCGATATCGAGGCTTTCGGGCGTGAGGTTGCCGAAGA GAGGGGATATGCGTTCGAGTCTATCGCTCGTGCGAAGATAGCTATCA AGCAGCTCATTGAAGTTGACGGATGCAGGTTGTTTATCACCGGTGCC GACCAGGTCCGGAGTGCCGTTCCATTGGCCTTTGGCCAGGAAGAGA CGCATTTGGTTGAGAGGCTGCTTTCGGGCGCTCCGGTTGGTCCTCG CGTACGTGATTCCCTGTTCAATCAAATGATGGTCGGTATTAGGAGGTTTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO TGACAGGCGTCTGTATGACAACTTGAAGTTGGATGACCGATCGGCTG CGTTTGAGCGGTTGGGGGAAGAAGATAAGGACCTCATCCTGAAAGG GCTTCTTGCGTTGTCCTCTGCTTCAAACAACATGGCAGACATGCGGC CGATTGGAGGCGCGAAGACTGCAGGCCAGTTGAAAATCGTCTTTAGA AACGTACTGAGCAATCAGGGCATTACGTTTATCGATCAGTCTGTGACC GGCATGTTTGAGAGGAAAACTTACATTGGGCTTTAG CBGI Type II AAGCTGAGAAACATCCAGGGCGACTACAACATTGGCCTGGACCTGG 23 Cas coding GCACAGGCAGCGTGGGCTGGGCCGTGACCGGAGTGGATGGCGAAC sequence (nt) TGCTGACACAGAACGGCAGACCTACCCTGGGCTCAAGGGTCTTTCCT (human AGCGGCGAGACAGCCGCTGATGCCAGACTGAAGCGGGGACAGCGC codon- AGGCGGTATGAAAGAAGGCGCTGGCGGCTGGACCTGCTTCAGAGAT optimized; TCTTCGAGAACGACATGGCCGACGTGGACCGGACCTTCTTCATTAGA lacking N- CTGAAGCAGGCCAGACTGCTCCGGGAGGATAGAGATGAGAGCTGCA terminal GAGACTACCACAGTCCCCTGTTTCTCACCGTTGATGCAGAAAGAGAC methionine; TACTACAAGAGATTCCCTACAATCTACCACCTGAGAGTGTGGCTGATG no stop ACGACCGAGAAGAAGGCTGATCTGAGAGAGATCTACCTGGCCTTGCA codon) CAACATCGTGAAGCACAGAGGCAATTTCCTGCACCAGGACAATCCCA GCCTGAGCGCCTCAGCAGCTAATATGGAAGATTCTGTGGAGCAGCTC TGTCTGGCCTTGGAGGACAGATGCATCACCCTGGATATCCCTTGTTC TTGTGACGCCGCCAGCATCCAGAGAGTCCTGGAGGACAGCAGCCTG AGCCGGGCTGAGAAGAGCGAGTCTGTGGGCAAGCTGTTCGGCCTGG ACAAAGACGCCCAGAAAACAATGGGCAGAGGAATCTCTCGGGCGAT CGTGGGCTACAAGGTTGACTTCGCCGCTGTGCTGGGATGTGAGTTTG AGGACAGCTCTTTTACCCTGTCTGACGACGAAAAGGTGGACGCCGCC CTGGCCGCCATCCCGGACGTTGCAATGAGCTTGTTCGACGCCATCAG AGAGGTGTACAGCGCTTACGTGCTGCTGGGCATCCTGAGCAGCGGC AACGGCGCTCCAATCAGCTCCGGCTCTTTGTCCTCTGTGAGTGGCAG AACCATCAGCTTCTGCAAGGTGAGAGAATATGAGGTGTACCGGGCCG ACCTGGCCCTATTGAAATCACTGGTGCGCACCTACGCTCCTAGTCAG TACGACGCCTTCTTTAGAGGCGAGTTAATTCCTGGAACATGCAACTAC GACCCCACCAAAGCCAAGGGCTACACCAGATACGACCTGGTCCATAA GGTGGCCTACGCTGATTTCTTCAAGGAAGTGAAGTCCCTGCTCGATA AGACCGACGCCGTGACCGATGAGAGATACAAGGACATGCTGGGTAG ATTCGAAGAGGAGCGCTTCCTGCGGAGACTGAAAACCAGCGATAATG GCAGCATCCCTTATCAGCTGCACCTGGAAGAGATGGATGCCATCCTG AAGAATCAGGGCAAGTACTACCCTTTCCTGCTGGAAAACCTGGACAA GATCGAGTCCCTGGTGAGCTTTAGAATCCCTTACTACCTGGGCCCTC TGACCCAGAAGAACGCCGCTCTGGATCACCACGGAAACCCTAGATTC GCCTGGGCGACCCGGAAGCCCGGGATGGAGGACGAGCCTGTGTAC CCCTGGAACTGGGAGGAAGTGATCGACAAAGGCCACGCCGCCCATA CATTTATCCAGCGGATGACCAGCGACTGCACCTACTTGATCGGCGAG GGTGTGCTGCCCAGAAACAGCCTGCTGTACGAGGAATTTTGTGTGCT GAACGAGCTGAACGGCGCCCGGTACAGCGTGGATGGCGATGACTGG AGAAGATTCGATTACGCTGATAGGATGGGCATCATGGACGACCTGTT TCGGCAAAGAAGAAGTGTAACCTACAAGATGGTCGAGGATTGGATGC GGGCCAATAGAGGCTGGGCTAGAGTGCACGTGAGAGGCGGCCAGG GCGAGTGCAAGTTCGAGAGCAGCCTGTCCGCCTATAGATTCTTTTGT AAGGACGTGTTCAAGACAGACGAGCTGTCCCCTTCTATCATCCCTAT GGTGGAAACCATCATCCTGTGGTCGACCCTGTTCGAGGACAGATCTA TCCTCAAAGAGCAGCTGGTTAGAAATTTCAGCGACCGGCTGAGCTCC GAGCAAATCAAGATCATCTGCAAGAAGAGACTGACCGGCTGGGGCAA CCTTAGTGAAAAATTCCTGGCCGAAGTGGAAGCCGAAACCGACTGCG GCCCCCGGAGCATCATGGACATCCTGAGAGAGGGAAGCCCTGTGGG AGGAGAGCAGGGCAGAACAATGGTGCTGATGGAAGTGCTCCACGAT GAGAGACTGGGTTTCGAGGATAAAATCGAGGAGATCAACGCCGAGC GGATCGCTGACGCCGGCAGACTGGAAGTGGTGGATCTGCCCGGATC TCCCGCCCTGCGGAGAACCGTGAACCAGGCTGTGCGAGTCGTAGAGTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO GAAATCGTGCACATCGCCGGCAAACCACCTGTGAACATCTTTATTGA GAACACCCGGGACGAGGACGCCAGCAAGAAGGGCAAGCGGACCAA GCGGCGATACGATACCATCAAGGAAGCCGTGAACGCCTTCAAGCGG GAGAACGCCGACCTGGCCAGAGAACTGAAAGAGTTCAAGCCTGCGG ATTTCGATGATGAGCGGCTCGCTCTGTATTTCATTCAGGGCGGCAAG TCTCTGTACAGCAAGGCCCCTCTGGATGTGACAAGACTGAGCGAGTA CGAGGTGGACCACATCATCCCCCAAAGCTACATCAAAGACGACTCTT TCGAAAACAAGGCTCTGGTGCTCAAGTCCGAGAACCAGACCAAGACA AACAGACTGCTGCTGCCACAAGGAGTGAGAGTGAAGATGGCCTCCTA CTGGCAGGAACTGCATAGATGCGGCCTGATCGGCGACAAGAAGCTG AGAAACCTGATGTGCAGCGATGTGTCCGAACGGAGAGTGAAGGGCT TCATCGCCAGACAGCTGGTGGAAACAAGCCAGATCGTGAAGCTGACT AAGATGATGCTGGAGAATAGACTGCCTGAGTCTAGGCTGGTGCCCAT CAAGGCCAGCCTGAGCCACGAGCTGAGGGAGGCCAAGCACTATTAC AAGTGCCGGGAAATCAACGACTTCCACCACGCCCACGACGCCCTGC TGGCCGCCGAGATCGGACGGTTTCTGCTGCTGCGGCACGCCGGCAT GTACGATAACCCCATCGGCTACGCCCACGTGGTCAAAGACTTTGTGC GCGTGCAGGCCGACGAAGCCAAGAGAACCGGCCGACTGCCTGGCTC TGCCGGTTTCATCGTTTCCAGCTTCCTGCACAGCGGCTTCGACAAGG ATACGGGCGAGATCTTCAAGGATACATGGGACGCCGAGTTCGAATGC GAGAGAATCAGAAAGTACCTGAATTACAGACAGGTGTACCTGAGTAG AATGCCTGAGGAAACAAGCGGCGCCTTTTGGGACGCCACAATCTACA GCCCTCGGGGAAAAATGAAGCTGAGCCTACCTCTGAAGGAGGGCCT GGACCCTAGCAAGTACGGCGGATACAGCAGCGAGAAGTATGCTTACT TCTTTTGCTACTATGCTAAAGACAAGAAAGGCAAACGGCTCATCGACT TCGCCCCTGTGCCAGTGAGCCGGGCCGCCGGAGGACAGGTGGACA TCGAGGCCTTCGGCAGAGAAGTGGCCGAAGAGCGGGGCTATGCCTT CGAAAGCATCGCCCGAGCTAAGATCGCCATCAAGCAGCTGATCGAG GTCGACGGCTGCAGACTGTTCATCACCGGCGCCGACCAGGTTAGAA GCGCTGTGCCTCTGGCCTTCGGCCAAGAAGAAACCCACCTGGTGGA ACGGCTGCTGAGCGGCGCCCCCGTGGGACCTAGAGTGAGAGATAGC CTGTTCAACCAGATGATGGTCGGCATCAGACGGTTTGACAGGCGGCT GTACGACAACCTGAAGCTGGATGACAGAAGCGCCGCCTTTGAACGG CTGGGCGAGGAGGATAAGGATCTCATCCTCAAAGGCCTGCTGGCCC TGTCGAGCGCCTCCAACAACATGGCTGACATGCGGCCTATCGGCGG CGCCAAAACGGCCGGTCAACTGAAGATCGTGTTCCGGAATGTGCTGT CCAACCAGGGGATCACCTTCATCGATCAGAGCGTGACCGGCATGTTC GAACGTAAAACATACATTGGGTTA CBGI Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 24 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCAAGCTGAGAAACATCCAGGGCGACTACAACATTGG expression CCTGGACCTGGGCACAGGCAGCGTGGGCTGGGCCGTGACCGGAGT construct GGATGGCGAACTGCTGACACAGAACGGCAGACCTACCCTGGGCTCA (with N- AGGGTCTTTCCTAGCGGCGAGACAGCCGCTGATGCCAGACTGAAGC terminal GGGGACAGCGCAGGCGGTATGAAAGAAGGCGCTGGCGGCTGGACC methionine, TGCTTCAGAGATTCTTCGAGAACGACATGGCCGACGTGGACCGGACC stop codon, TTCTTCATTAGACTGAAGCAGGCCAGACTGCTCCGGGAGGATAGAGA V5 tag, N- TGAGAGCTGCAGAGACTACCACAGTCCCCTGTTTCTCACCGTTGATG terminal NLS CAGAAAGAGACTACTACAAGAGATTCCCTACAATCTACCACCTGAGA and C- GTGTGGCTGATGACGACCGAGAAGAAGGCTGATCTGAGAGAGATCTA terminal NLS) CCTGGCCTTGCACAACATCGTGAAGCACAGAGGCAATTTCCTGCACC (nt) AGGACAATCCCAGCCTGAGCGCCTCAGCAGCTAATATGGAAGATTCT GTGGAGCAGCTCTGTCTGGCCTTGGAGGACAGATGCATCACCCTGG ATATCCCTTGTTCTTGTGACGCCGCCAGCATCCAGAGAGTCCTGGAG GACAGCAGCCTGAGCCGGGCTGAGAAGAGCGAGTCTGTGGGCAAGC TGTTCGGCCTGGACAAAGACGCCCAGAAAACAATGGGCAGAGGAAT CTCTCGGGCGATCGTGGGCTACAAGGTTGACTTCGCCGCTGTGCTGTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO GGATGTGAGTTTGAGGACAGCTCTTTTACCCTGTCTGACGACGAAAA GGTGGACGCCGCCCTGGCCGCCATCCCGGACGTTGCAATGAGCTTG TTCGACGCCATCAGAGAGGTGTACAGCGCTTACGTGCTGCTGGGCAT CCTGAGCAGCGGCAACGGCGCTCCAATCAGCTCCGGCTCTTTGTCCT CTGTGAGTGGCAGAACCATCAGCTTCTGCAAGGTGAGAGAATATGAG GTGTACCGGGCCGACCTGGCCCTATTGAAATCACTGGTGCGCACCTA CGCTCCTAGTCAGTACGACGCCTTCTTTAGAGGCGAGTTAATTCCTG GAACATGCAACTACGACCCCACCAAAGCCAAGGGCTACACCAGATAC GACCTGGTCCATAAGGTGGCCTACGCTGATTTCTTCAAGGAAGTGAA GTCCCTGCTCGATAAGACCGACGCCGTGACCGATGAGAGATACAAG GACATGCTGGGTAGATTCGAAGAGGAGCGCTTCCTGCGGAGACTGA AAACCAGCGATAATGGCAGCATCCCTTATCAGCTGCACCTGGAAGAG ATGGATGCCATCCTGAAGAATCAGGGCAAGTACTACCCTTTCCTGCT GGAAAACCTGGACAAGATCGAGTCCCTGGTGAGCTTTAGAATCCCTT ACTACCTGGGCCCTCTGACCCAGAAGAACGCCGCTCTGGATCACCAC GGAAACCCTAGATTCGCCTGGGCGACCCGGAAGCCCGGGATGGAGG ACGAGCCTGTGTACCCCTGGAACTGGGAGGAAGTGATCGACAAAGG CCACGCCGCCCATACATTTATCCAGCGGATGACCAGCGACTGCACCT ACTTGATCGGCGAGGGTGTGCTGCCCAGAAACAGCCTGCTGTACGA GGAATTTTGTGTGCTGAACGAGCTGAACGGCGCCCGGTACAGCGTG GATGGCGATGACTGGAGAAGATTCGATTACGCTGATAGGATGGGCAT CATGGACGACCTGTTTCGGCAAAGAAGAAGTGTAACCTACAAGATGG TCGAGGATTGGATGCGGGCCAATAGAGGCTGGGCTAGAGTGCACGT GAGAGGCGGCCAGGGCGAGTGCAAGTTCGAGAGCAGCCTGTCCGC CTATAGATTCTTTTGTAAGGACGTGTTCAAGACAGACGAGCTGTCCCC TTCTATCATCCCTATGGTGGAAACCATCATCCTGTGGTCGACCCTGTT CGAGGACAGATCTATCCTCAAAGAGCAGCTGGTTAGAAATTTCAGCG ACCGGCTGAGCTCCGAGCAAATCAAGATCATCTGCAAGAAGAGACTG ACCGGCTGGGGCAACCTTAGTGAAAAATTCCTGGCCGAAGTGGAAG CCGAAACCGACTGCGGCCCCCGGAGCATCATGGACATCCTGAGAGA GGGAAGCCCTGTGGGAGGAGAGCAGGGCAGAACAATGGTGCTGATG GAAGTGCTCCACGATGAGAGACTGGGTTTCGAGGATAAAATCGAGGA GATCAACGCCGAGCGGATCGCTGACGCCGGCAGACTGGAAGTGGTG GATCTGCCCGGATCTCCCGCCCTGCGGAGAACCGTGAACCAGGCTG TGCGAGTCGTAGAGGAAATCGTGCACATCGCCGGCAAACCACCTGT GAACATCTTTATTGAGAACACCCGGGACGAGGACGCCAGCAAGAAG GGCAAGCGGACCAAGCGGCGATACGATACCATCAAGGAAGCCGTGA ACGCCTTCAAGCGGGAGAACGCCGACCTGGCCAGAGAACTGAAAGA GTTCAAGCCTGCGGATTTCGATGATGAGCGGCTCGCTCTGTATTTCA TTCAGGGCGGCAAGTCTCTGTACAGCAAGGCCCCTCTGGATGTGACA AGACTGAGCGAGTACGAGGTGGACCACATCATCCCCCAAAGCTACAT CAAAGACGACTCTTTCGAAAACAAGGCTCTGGTGCTCAAGTCCGAGA ACCAGACCAAGACAAACAGACTGCTGCTGCCACAAGGAGTGAGAGT GAAGATGGCCTCCTACTGGCAGGAACTGCATAGATGCGGCCTGATC GGCGACAAGAAGCTGAGAAACCTGATGTGCAGCGATGTGTCCGAAC GGAGAGTGAAGGGCTTCATCGCCAGACAGCTGGTGGAAACAAGCCA GATCGTGAAGCTGACTAAGATGATGCTGGAGAATAGACTGCCTGAGT CTAGGCTGGTGCCCATCAAGGCCAGCCTGAGCCACGAGCTGAGGGA GGCCAAGCACTATTACAAGTGCCGGGAAATCAACGACTTCCACCACG CCCACGACGCCCTGCTGGCCGCCGAGATCGGACGGTTTCTGCTGCT GCGGCACGCCGGCATGTACGATAACCCCATCGGCTACGCCCACGTG GTCAAAGACTTTGTGCGCGTGCAGGCCGACGAAGCCAAGAGAACCG GCCGACTGCCTGGCTCTGCCGGTTTCATCGTTTCCAGCTTCCTGCAC AGCGGCTTCGACAAGGATACGGGCGAGATCTTCAAGGATACATGGG ACGCCGAGTTCGAATGCGAGAGAATCAGAAAGTACCTGAATTACAGA CAGGTGTACCTGAGTAGAATGCCTGAGGAAACAAGCGGCGCCTTTTG GGACGCCACAATCTACAGCCCTCGGGGAAAAATGAAGCTGAGCCTACTABLE 1D CBGI Type II Cas Sequences Name Sequence SEQ ID NO CTCTGAAGGAGGGCCTGGACCCTAGCAAGTACGGCGGATACAGCAG CGAGAAGTATGCTTACTTCTTTTGCTACTATGCTAAAGACAAGAAAGG CAAACGGCTCATCGACTTCGCCCCTGTGCCAGTGAGCCGGGCCGCC GGAGGACAGGTGGACATCGAGGCCTTCGGCAGAGAAGTGGCCGAAG AGCGGGGCTATGCCTTCGAAAGCATCGCCCGAGCTAAGATCGCCAT CAAGCAGCTGATCGAGGTCGACGGCTGCAGACTGTTCATCACCGGC GCCGACCAGGTTAGAAGCGCTGTGCCTCTGGCCTTCGGCCAAGAAG AAACCCACCTGGTGGAACGGCTGCTGAGCGGCGCCCCCGTGGGACC TAGAGTGAGAGATAGCCTGTTCAACCAGATGATGGTCGGCATCAGAC GGTTTGACAGGCGGCTGTACGACAACCTGAAGCTGGATGACAGAAG CGCCGCCTTTGAACGGCTGGGCGAGGAGGATAAGGATCTCATCCTC AAAGGCCTGCTGGCCCTGTCGAGCGCCTCCAACAACATGGCTGACAT GCGGCCTATCGGCGGCGCCAAAACGGCCGGTCAACTGAAGATCGTG TTCCGGAATGTGCTGTCCAACCAGGGGATCACCTTCATCGATCAGAG CGTGACCGGCATGTTCGAACGTAAAACATACATTGGGTTATCTAGAAA GCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAA AGGTGTGA

[0085] In some embodiments a CBGI Type II 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 CBGI Type II 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 D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:20. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H890A substitution, wherein the position of the H890A substitution is defined with respect to the amino acid numbering of SEQ ID NO:20. In some embodiments, a CBGI Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with an H890A substitution. 6.2.1.5. ASDR Type II Cas Proteins

[0086] In one aspect, the disclosure provides ASDR Type II Cas proteins. ASDR Type II Cas proteins can be further classified as Type IIA Cas proteins. The ASDR Type II 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 ASDR Type II 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, an ASDR Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:25.

[0087] Exemplary ASDR Type II Cas protein sequences and nucleotide sequences encoding exemplary ASDR Type II Cas proteins are set forth in Table 1E. TABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO ASDR Type II NLRNYPGSFNIGLDLGPASAGWSVVDSEGKLFHFKKKPTWGSRLFDSA 25 Cas amino QTASEARMHRGQRRRYVRRRWRLNLLQALFTEEMEKVDPGFFMRLNH acid SRTVEGDPIFTKDFTKKDYYKRFPTIYHLRAHLMETDDPADLRLVYLAIHN sequence IVKHRGNFLRQEEKLTAKIANTSEALGKLSVSLKEWCDSRGYSVGKVDETABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO (without N- EAIAAVLADKRISRSQKAKNVTLLISVDTGDSKANAKLAKALANAIVGLQA terminal EFKDLFGDFECEATKLNLSEEEKLEALQAACPDDSAELLEAVCGAYSAY methionine) VLQGLLSYAEGQTISHNMIKKYDCYAEDLECLKALVRDYLPKTKGDDGLK PFDRFFRGVTYHDLDKSDPSRDYDASKAKGYTAYNLHKLSYDEFRKEVE KLLKGTGAQDDERYITMMDAFEKQRFLRRLKTSDNGAIYYQLHLEELQAI LENQGRFYPFLKQEASKIESLVTFRIPYYVGPLTSKNAAKDAHNKNRFQW SERKPGMEDAIITPWNWESVIDKNRSAENFIMRMTGDCTYLAGEKVLPR HSLLYEEFCVLNELNGVRFTNDGDNWHRLDSEQRAGIIRDLFHKKRSVK YADIAAWLVRMGYLERTPQVRGGQGESGLESKMSSYLLFAKDVFGVDE LDEALFPAIEQIILWNTLFEDRSIFEEKLRDEFGPNGNGMLNNEQIKKCKK RLSGWGRLSEKFLTGIRVVTQGGRQMSIMNVLREGSPNPDRRQGEALV MMEVLHDNTLGFQKKIDEVNRAYYAQNAKAMGVNELPGSPGIRRSINQA IRIVDEIVKIAGHAPDNIFIEVTRDEDEKKKGKRTVKRWDRIEAALNAFKAE GGDLAVMSDFKRLKNTEADLDERLTLYLLQNGKCLYSGRAIDLDALMTG SGEYEVDHIIPRAYIKDDSLDNKALVYRSENQRKTDQLLIDENVRQKMGE TWKQLHDAKMISDKKYNNLMRLTVTEGAMKGFIARQLVETSQIVKMTKA LLEARYGDAGTRIVPVKASMSHNLREVAGLVKCREANDFHHAHDAYLAC RMGLFIQKRYPGIYENPIGYAHAMKKYVLEQSELFKKTHRMPGSAGLIVN SFMTAGFDPETGEMFEDAWAGNDEIKHCKKKHYDDAWNAAAEVEGIRR ALNYRQCYVSRMPYEDSGKFWDANPISKRSNPKLALKNTLDPACFGGY NTKTAAYFFIYEVTDKKGKTGLRIAQMPIFMKAKCESDNAAFEKYCQDLE SESRVSFTRIVIPRLLKWQLVEIDGNLLRINAEEEMKSASQLAFSQEELER ILLVCTDGYLDPDVNRSVYMAIAKALDRAKCPMLTNDLDLISRFDAFCGLA VADQKDLLKKILLLCKGNKQSSPINLSSVGGKKKAGYLRPARSKVPDGM VLIDQSVTGMFERRTRVGV ASDR Type II MNLRNYPGSFNIGLDLGPASAGWSVVDSEGKLFHFKKKPTWGSRLFDS 26 Cas amino AQTASEARMHRGQRRRYVRRRWRLNLLQALFTEEMEKVDPGFFMRLN acid HSRTVEGDPIFTKDFTKKDYYKRFPTIYHLRAHLMETDDPADLRLVYLAIH sequence NIVKHRGNFLRQEEKLTAKIANTSEALGKLSVSLKEWCDSRGYSVGKVD EEAIAAVLADKRISRSQKAKNVTLLISVDTGDSKANAKLAKALANAIVGLQ AEFKDLFGDFECEATKLNLSEEEKLEALQAACPDDSAELLEAVCGAYSA YVLQGLLSYAEGQTISHNMIKKYDCYAEDLECLKALVRDYLPKTKGDDGL KPFDRFFRGVTYHDLDKSDPSRDYDASKAKGYTAYNLHKLSYDEFRKEV EKLLKGTGAQDDERYITMMDAFEKQRFLRRLKTSDNGAIYYQLHLEELQ AILENQGRFYPFLKQEASKIESLVTFRIPYYVGPLTSKNAAKDAHNKNRFQ WSERKPGMEDAIITPWNWESVIDKNRSAENFIMRMTGDCTYLAGEKVLP RHSLLYEEFCVLNELNGVRFTNDGDNWHRLDSEQRAGIIRDLFHKKRSV KYADIAAWLVRMGYLERTPQVRGGQGESGLESKMSSYLLFAKDVFGVD ELDEALFPAIEQIILWNTLFEDRSIFEEKLRDEFGPNGNGMLNNEQIKKCK KRLSGWGRLSEKFLTGIRVVTQGGRQMSIMNVLREGSPNPDRRQGEAL VMMEVLHDNTLGFQKKIDEVNRAYYAQNAKAMGVNELPGSPGIRRSINQ AIRIVDEIVKIAGHAPDNIFIEVTRDEDEKKKGKRTVKRWDRIEAALNAFKA EGGDLAVMSDFKRLKNTEADLDERLTLYLLQNGKCLYSGRAIDLDALMT GSGEYEVDHIIPRAYIKDDSLDNKALVYRSENQRKTDQLLIDENVRQKMG ETWKQLHDAKMISDKKYNNLMRLTVTEGAMKGFIARQLVETSQIVKMTK ALLEARYGDAGTRIVPVKASMSHNLREVAGLVKCREANDFHHAHDAYLA CRMGLFIQKRYPGIYENPIGYAHAMKKYVLEQSELFKKTHRMPGSAGLIV NSFMTAGFDPETGEMFEDAWAGNDEIKHCKKKHYDDAWNAAAEVEGIR RALNYRQCYVSRMPYEDSGKFWDANPISKRSNPKLALKNTLDPACFGG YNTKTAAYFFIYEVTDKKGKTGLRIAQMPIFMKAKCESDNAAFEKYCQDL ESESRVSFTRIVIPRLLKWQLVEIDGNLLRINAEEEMKSASQLAFSQEELE RILLVCTDGYLDPDVNRSVYMAIAKALDRAKCPMLTNDLDLISRFDAFCG LAVADQKDLLKKILLLCKGNKQSSPINLSSVGGKKKAGYLRPARSKVPDG MVLIDQSVTGMFERRTRVGV ASDR Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSNLRNYPGSFNIGLDL 27 Cas GPASAGWSVVDSEGKLFHFKKKPTWGSRLFDSAQTASEARMHRGQRR mammalian RYVRRRWRLNLLQALFTEEMEKVDPGFFMRLNHSRTVEGDPIFTKDFTKTABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO expression KDYYKRFPTIYHLRAHLMETDDPADLRLVYLAIHNIVKHRGNFLRQEEKLT construct AKIANTSEALGKLSVSLKEWCDSRGYSVGKVDEEAIAAVLADKRISRSQK (with N- AKNVTLLISVDTGDSKANAKLAKALANAIVGLQAEFKDLFGDFECEATKLN terminal LSEEEKLEALQAACPDDSAELLEAVCGAYSAYVLQGLLSYAEGQTISHN methionine, MIKKYDCYAEDLECLKALVRDYLPKTKGDDGLKPFDRFFRGVTYHDLDK stop codon, SDPSRDYDASKAKGYTAYNLHKLSYDEFRKEVEKLLKGTGAQDDERYIT V5 tag, N- MMDAFEKQRFLRRLKTSDNGAIYYQLHLEELQAILENQGRFYPFLKQEAS terminal NLS KIESLVTFRIPYYVGPLTSKNAAKDAHNKNRFQWSERKPGMEDAIITPWN and C- WESVIDKNRSAENFIMRMTGDCTYLAGEKVLPRHSLLYEEFCVLNELNG terminal NLS) VRFTNDGDNWHRLDSEQRAGIIRDLFHKKRSVKYADIAAWLVRMGYLER (aa) TPQVRGGQGESGLESKMSSYLLFAKDVFGVDELDEALFPAIEQIILWNTL FEDRSIFEEKLRDEFGPNGNGMLNNEQIKKCKKRLSGWGRLSEKFLTGI RVVTQGGRQMSIMNVLREGSPNPDRRQGEALVMMEVLHDNTLGFQKKI DEVNRAYYAQNAKAMGVNELPGSPGIRRSINQAIRIVDEIVKIAGHAPDNI FIEVTRDEDEKKKGKRTVKRWDRIEAALNAFKAEGGDLAVMSDFKRLKN TEADLDERLTLYLLQNGKCLYSGRAIDLDALMTGSGEYEVDHIIPRAYIKD DSLDNKALVYRSENQRKTDQLLIDENVRQKMGETWKQLHDAKMISDKKY NNLMRLTVTEGAMKGFIARQLVETSQIVKMTKALLEARYGDAGTRIVPVK ASMSHNLREVAGLVKCREANDFHHAHDAYLACRMGLFIQKRYPGIYENP IGYAHAMKKYVLEQSELFKKTHRMPGSAGLIVNSFMTAGFDPETGEMFE DAWAGNDEIKHCKKKHYDDAWNAAAEVEGIRRALNYRQCYVSRMPYED SGKFWDANPISKRSNPKLALKNTLDPACFGGYNTKTAAYFFIYEVTDKKG KTGLRIAQMPIFMKAKCESDNAAFEKYCQDLESESRVSFTRIVIPRLLKW QLVEIDGNLLRINAEEEMKSASQLAFSQEELERILLVCTDGYLDPDVNRS VYMAIAKALDRAKCPMLTNDLDLISRFDAFCGLAVADQKDLLKKILLLCKG NKQSSPINLSSVGGKKKAGYLRPARSKVPDGMVLIDQSVTGMFERRTRV GVSRKRTADGSEFESPKKKRKV ASDR Type II ATGAACCTGAGAAATTACCCTGGGTCGTTCAATATTGGTCTTGACCTT 28 Cas coding GGTCCCGCGTCGGCTGGCTGGTCGGTAGTGGACAGTGAGGGAAAAC sequence (nt) TCTTTCATTTCAAGAAAAAACCAACATGGGGAAGTCGTTTGTTTGATA (not codon GTGCGCAGACTGCCTCGGAGGCGCGTATGCATCGCGGTCAGCGTCG optimized) ACGTTATGTGCGTCGCCGTTGGCGCCTCAACTTGTTGCAGGCACTGT TTACTGAGGAGATGGAAAAGGTTGACCCTGGGTTCTTTATGAGGCTC AACCATTCCCGAACCGTTGAGGGCGATCCTATCTTCACCAAGGACTT TACCAAGAAGGACTATTACAAGCGTTTTCCTACCATCTATCACTTGCG TGCGCACCTTATGGAAACCGACGATCCGGCCGATCTCCGTTTGGTTT ACTTGGCTATTCATAACATCGTTAAGCACCGTGGCAACTTCCTGCGTC AGGAGGAAAAGCTTACGGCAAAAATCGCAAACACCTCGGAAGCACTT GGGAAGCTTTCTGTCTCGCTAAAAGAATGGTGCGATTCGAGGGGATA TAGTGTCGGAAAGGTTGATGAGGAAGCAATCGCGGCAGTTTTAGCAG ACAAACGCATATCGCGTTCTCAAAAAGCAAAGAATGTGACATTGCTTA TCTCCGTTGATACAGGCGACAGTAAGGCAAACGCGAAGCTCGCTAAG GCGTTGGCGAATGCTATCGTGGGTCTCCAAGCTGAGTTCAAAGACCT ATTTGGCGATTTTGAGTGTGAAGCAACCAAGCTCAATCTTTCTGAAGA GGAGAAGCTCGAAGCGCTTCAGGCGGCGTGTCCCGATGATAGCGCG GAGCTGCTTGAAGCTGTATGTGGTGCGTATTCTGCGTATGTGCTCCA GGGTTTGCTTTCATATGCGGAGGGGCAGACCATTTCGCATAATATGA TAAAAAAGTACGATTGCTACGCGGAAGATCTGGAGTGCCTTAAAGCTT TGGTTCGCGACTATTTGCCGAAGACGAAGGGCGATGACGGACTCAA GCCATTTGACCGGTTTTTTCGTGGCGTCACTTATCATGATTTGGATAA GAGTGATCCCTCGCGCGACTACGATGCGTCAAAAGCCAAAGGCTATA CCGCCTATAACTTGCATAAACTTTCGTACGACGAATTCAGAAAAGAAG TTGAAAAGCTGCTAAAGGGCACAGGTGCTCAGGACGATGAGCGCTAT ATCACCATGATGGATGCGTTTGAGAAGCAGCGTTTCTTGCGTCGCTT GAAGACGAGTGATAACGGGGCAATCTACTATCAGCTGCACTTGGAAG AGTTGCAGGCGATTCTTGAAAATCAAGGTCGTTTCTATCCCTTCCTTA AACAAGAGGCAAGCAAGATAGAAAGCCTGGTTACCTTCCGGATCCCCTABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO TACTACGTGGGTCCGCTTACGTCTAAAAACGCTGCAAAGGATGCTCA TAACAAGAACCGTTTTCAGTGGTCGGAGCGCAAGCCGGGTATGGAG GATGCGATTATCACGCCGTGGAACTGGGAAAGCGTTATCGATAAGAA TCGGAGCGCGGAGAACTTTATTATGCGCATGACGGGGGATTGCACAT ACTTGGCGGGCGAGAAAGTACTGCCCAGGCATTCCTTGCTCTACGAG GAGTTCTGCGTACTCAATGAGCTAAACGGCGTTCGTTTCACTAATGAC GGCGATAATTGGCACCGCCTGGATTCCGAGCAGCGTGCAGGCATTAT TCGCGATTTGTTCCATAAGAAGCGCAGTGTGAAGTATGCAGACATTG CCGCCTGGCTTGTGCGGATGGGCTATCTTGAGAGAACTCCTCAGGTG CGCGGCGGACAGGGTGAAAGCGGCTTGGAATCAAAGATGAGCTCGT ACCTCTTGTTTGCTAAGGATGTTTTCGGCGTTGACGAGCTTGATGAAG CGCTATTCCCTGCCATTGAGCAGATCATTTTGTGGAACACGCTGTTTG AGGATCGCTCGATTTTTGAAGAAAAGCTTCGCGATGAATTCGGGCCT AACGGCAACGGCATGCTCAATAACGAGCAGATAAAGAAATGCAAAAA GCGCCTTTCGGGGTGGGGTAGGCTTTCTGAAAAGTTCCTTACTGGAA TCAGGGTCGTCACGCAGGGTGGTCGCCAGATGAGCATCATGAATGT GCTTCGCGAGGGTAGTCCGAATCCCGATCGCCGCCAGGGTGAAGCT TTGGTGATGATGGAGGTTCTTCATGACAACACCCTTGGCTTCCAGAAA AAGATCGACGAGGTAAACCGTGCGTACTATGCGCAAAATGCCAAAGC GATGGGCGTAAACGAACTTCCCGGCTCGCCTGGTATTCGCCGTAGCA TTAACCAAGCTATTCGCATTGTTGACGAAATTGTAAAGATTGCTGGTC ATGCGCCCGACAACATCTTTATTGAGGTCACGCGTGACGAAGATGAA AAGAAGAAGGGGAAGCGTACCGTCAAGCGCTGGGACCGCATAGAAG CGGCGCTCAATGCATTCAAGGCTGAGGGCGGCGACCTTGCGGTTAT GAGCGATTTCAAGCGGCTTAAGAACACAGAAGCCGATTTGGACGAAC GACTCACTTTGTATTTGCTGCAAAATGGCAAATGCCTGTATTCAGGCC GCGCTATCGACCTCGATGCGCTTATGACGGGATCGGGCGAGTATGA AGTCGACCACATTATTCCGCGTGCATATATTAAGGACGATAGCCTCGA CAACAAGGCGTTGGTGTATCGTTCCGAAAATCAACGAAAAACCGATC AACTTTTGATTGACGAGAACGTTCGCCAAAAGATGGGCGAAACATGG AAGCAGCTTCATGACGCGAAAATGATTAGCGACAAGAAGTATAACAA CCTGATGCGACTCACCGTTACCGAAGGAGCAATGAAGGGCTTTATTG CTCGCCAGCTGGTTGAGACAAGTCAGATAGTGAAGATGACAAAGGCG TTGCTTGAAGCGCGTTATGGCGATGCGGGGACGAGGATCGTCCCTG TTAAGGCAAGCATGTCCCACAACCTTCGTGAAGTGGCGGGCTTGGTT AAATGCCGTGAAGCCAATGATTTCCATCATGCTCATGACGCGTATCTG GCGTGCCGCATGGGTTTGTTTATCCAGAAGCGTTACCCAGGGATTTA CGAGAACCCCATTGGTTATGCGCATGCCATGAAAAAGTATGTTCTTGA ACAGTCGGAGCTGTTCAAGAAGACTCACCGGATGCCTGGATCTGCTG GCTTGATTGTAAACAGCTTTATGACGGCGGGGTTTGATCCCGAAACT GGCGAGATGTTCGAAGATGCCTGGGCTGGCAACGATGAAATCAAGC ATTGTAAGAAAAAGCACTATGACGATGCATGGAATGCAGCTGCGGAG GTGGAGGGTATTCGTCGTGCACTCAACTACCGACAGTGTTATGTCTC GAGGATGCCTTACGAAGATTCGGGCAAGTTCTGGGACGCGAACCCG ATTTCAAAGAGAAGCAATCCCAAGCTTGCGCTTAAAAACACCCTCGAC CCTGCGTGCTTTGGCGGCTACAACACAAAGACAGCGGCGTATTTCTT TATATATGAGGTTACTGATAAAAAAGGTAAGACCGGGTTACGAATAGC TCAGATGCCCATTTTCATGAAAGCTAAATGCGAGAGCGATAATGCCG CTTTTGAGAAATATTGCCAAGATCTGGAAAGCGAAAGTAGGGTTTCCT TTACTCGAATCGTTATCCCAAGGCTGTTGAAATGGCAACTCGTCGAAA TCGATGGAAACCTTCTGCGCATTAATGCGGAAGAGGAGATGAAATCT GCGTCTCAGTTGGCATTTTCGCAAGAAGAGCTTGAAAGAATCCTACTT GTCTGTACAGACGGCTATCTTGACCCTGACGTTAATCGATCTGTCTAT ATGGCAATTGCGAAAGCGCTCGATAGGGCAAAATGCCCCATGTTGAC GAATGATCTGGATCTTATAAGCAGATTCGATGCATTTTGCGGTCTTGC CGTGGCTGATCAGAAGGATTTATTAAAAAAGATTCTTCTTCTCTGCAA GGGAAATAAACAGTCCTCTCCAATTAATCTTTCGTCTGTTGGTGGAAATABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO GAAGAAGGCAGGGTATCTCCGCCCAGCTCGTAGTAAAGTGCCCGAT GGGATGGTGCTCATCGACCAATCCGTAACGGGTATGTTCGAGAGGA GAACCCGTGTCGGGGTTTAG ASDR Type II AACCTGAGAAACTACCCCGGCTCCTTCAACATCGGACTGGACTTGGG 29 Cas coding CCCTGCCTCTGCCGGCTGGAGCGTGGTCGATAGCGAGGGAAAGCTG sequence (nt) TTTCATTTCAAAAAGAAGCCTACATGGGGCAGCAGACTCTTTGACAGC (human GCTCAGACCGCCTCCGAGGCCAGAATGCACAGAGGACAGCGGCGG codon- CGGTATGTGCGCAGGAGATGGCGGCTCAACCTGCTGCAGGCGCTGT optimized; TCACCGAAGAGATGGAAAAAGTGGACCCTGGTTTTTTTATGAGACTGA lacking N- ACCACAGCCGGACAGTAGAGGGCGATCCCATCTTCACCAAGGACTTC terminal ACCAAGAAGGATTACTACAAGCGGTTCCCCACCATCTACCACCTGAG methionine; GGCTCACCTGATGGAAACCGACGATCCCGCCGACCTGAGACTGGTC no stop TACCTGGCCATCCACAACATCGTGAAACACAGAGGCAACTTCCTGAG codon) GCAGGAGGAGAAGCTGACCGCTAAAATCGCTAACACCAGCGAGGCA CTGGGCAAGCTGTCTGTCTCCCTGAAAGAGTGGTGCGACTCCAGAG GATATTCTGTGGGCAAGGTGGATGAAGAAGCCATTGCTGCCGTGCTG GCTGACAAGCGAATAAGCAGATCTCAAAAGGCCAAGAACGTGACCCT GCTGATCAGCGTGGACACCGGAGATAGCAAGGCCAACGCCAAGCTG GCCAAGGCCCTGGCCAATGCCATCGTGGGACTGCAAGCTGAATTTAA GGATCTGTTCGGAGATTTCGAATGCGAGGCTACAAAGCTGAACCTGA GCGAGGAAGAGAAGCTGGAAGCCCTGCAAGCTGCCTGTCCTGATGA CAGCGCCGAGCTGCTGGAGGCCGTGTGTGGCGCCTATAGCGCCTAC GTGCTGCAGGGCCTGCTGAGCTACGCCGAGGGCCAGACAATCAGCC ACAATATGATCAAGAAGTACGACTGCTACGCCGAGGACCTGGAGTGT CTGAAGGCCCTGGTGAGAGACTACCTGCCAAAGACCAAGGGCGACG ACGGCCTCAAGCCTTTCGACAGATTCTTCCGTGGCGTGACATACCAC GACCTGGACAAGAGCGACCCTAGCAGAGATTACGATGCAAGCAAGG CAAAGGGCTACACCGCCTACAACCTGCACAAGCTGTCTTATGACGAG TTCAGAAAGGAAGTGGAAAAGCTGCTGAAGGGCACCGGCGCCCAGG ACGACGAAAGATACATCACAATGATGGACGCTTTCGAAAAGCAGAGA TTCCTACGGCGGCTTAAGACATCTGACAACGGCGCCATCTACTACCA GCTGCACCTGGAGGAGCTGCAGGCCATCCTGGAAAACCAGGGCAGA TTTTACCCCTTCCTGAAACAGGAGGCCAGTAAAATCGAGTCTCTGGT GACCTTCAGAATCCCTTACTACGTGGGCCCACTGACGTCCAAGAACG CCGCCAAGGACGCCCACAACAAGAATAGATTTCAATGGTCCGAGAGA AAGCCTGGCATGGAAGATGCCATTATCACCCCTTGGAACTGGGAGTC TGTGATCGATAAGAATAGAAGCGCCGAGAACTTCATCATGCGGATGA CCGGCGATTGCACCTACCTGGCCGGCGAGAAGGTGCTGCCTAGACA CAGTCTGCTGTACGAGGAGTTCTGCGTGCTGAACGAGCTGAACGGC GTGCGGTTCACTAACGACGGCGACAACTGGCACCGGCTGGACTCAG AGCAGCGGGCTGGAATTATCAGAGATCTGTTTCACAAAAAGAGATCT GTGAAGTACGCCGACATCGCCGCCTGGCTGGTACGGATGGGCTATC TGGAGCGAACACCTCAGGTGCGCGGTGGACAGGGCGAAAGCGGCC TGGAATCTAAGATGAGCAGCTACCTGCTGTTCGCCAAAGACGTGTTC GGCGTGGACGAGCTGGACGAGGCCTTATTTCCTGCCATCGAACAGAT CATTCTGTGGAACACCCTGTTTGAGGATAGATCCATCTTCGAAGAGAA GCTGCGGGACGAGTTCGGCCCTAATGGCAATGGCATGCTGAACAAT GAACAGATCAAGAAATGCAAAAAAAGACTCAGCGGATGGGGCAGACT GAGCGAAAAGTTCCTGACAGGCATAAGGGTTGTGACACAGGGCGGC AGACAGATGAGCATCATGAACGTGCTGCGGGAAGGCAGCCCTAACC CTGACAGACGGCAGGGCGAGGCCCTCGTAATGATGGAAGTGCTGCA TGACAACACCCTGGGCTTCCAGAAAAAGATCGACGAAGTGAACAGAG CCTACTACGCCCAGAACGCTAAGGCAATGGGCGTGAATGAGCTGCC CGGGAGCCCTGGCATCCGGCGAAGCATCAATCAAGCCATTAGAATC GTTGATGAGATCGTGAAGATCGCTGGCCACGCTCCTGACAACATCTT TATCGAGGTTACCCGGGATGAGGACGAAAAGAAGAAGGGCAAGCGG ACCGTGAAAAGATGGGACCGGATCGAAGCCGCTCTCAACGCCTTTAATABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO AGCCGAGGGTGGAGATCTGGCAGTGATGAGCGATTTTAAGCGCCTG AAGAATACAGAAGCTGATCTGGATGAGCGCCTCACACTGTACTTACT GCAGAACGGCAAGTGCCTGTACAGCGGCAGAGCGATCGACTTAGAT GCCCTGATGACCGGCAGCGGCGAGTACGAAGTGGATCACATCATCC CTAGAGCCTACATCAAGGACGATTCCCTGGACAACAAGGCCCTTGTC TACCGGAGCGAGAACCAGAGAAAGACCGACCAACTGCTGATCGACG AGAACGTGCGGCAGAAAATGGGCGAAACCTGGAAGCAGCTTCATGAT GCCAAGATGATCAGCGACAAGAAGTACAACAACCTGATGAGACTGAC CGTGACAGAGGGAGCCATGAAAGGCTTCATCGCCAGACAGTTGGTC GAGACATCTCAGATCGTGAAGATGACCAAGGCCTTGCTGGAGGCCC GGTACGGCGATGCTGGCACAAGAATCGTGCCTGTGAAGGCCAGCAT GAGCCACAACCTGAGAGAGGTTGCGGGCTTGGTCAAGTGTAGAGAG GCCAACGACTTCCACCACGCCCATGACGCTTACCTGGCCTGCAGAAT GGGCCTGTTTATCCAGAAGAGATACCCAGGAATCTACGAAAACCCCA TCGGCTACGCTCACGCCATGAAGAAATACGTGCTGGAACAGAGCGAA CTATTCAAGAAAACCCACCGGATGCCTGGCTCCGCTGGCCTGATCGT GAACAGCTTCATGACAGCTGGATTCGACCCCGAGACAGGCGAGATGT TCGAGGACGCCTGGGCCGGAAACGATGAGATCAAGCACTGCAAGAA GAAGCACTACGACGACGCCTGGAACGCCGCCGCCGAGGTGGAAGG AATCAGACGGGCCCTGAATTACAGACAATGTTACGTGAGCCGGATGC CATACGAGGACTCCGGCAAGTTCTGGGATGCTAACCCTATCAGCAAG AGAAGCAACCCCAAGTTGGCTCTAAAGAACACCCTGGACCCTGCTTG CTTCGGAGGCTACAATACCAAGACCGCCGCCTACTTCTTCATCTACG AGGTGACCGACAAAAAAGGCAAGACCGGCCTGCGAATCGCCCAGAT GCCAATCTTCATGAAGGCTAAGTGCGAGAGCGATAACGCCGCCTTCG AAAAGTACTGCCAGGACCTGGAAAGCGAATCCAGAGTGAGCTTCACC CGGATCGTGATCCCCAGACTCCTCAAGTGGCAGCTCGTGGAAATCGA CGGCAACCTGCTGCGGATCAACGCCGAAGAGGAGATGAAATCCGCC TCTCAGCTGGCCTTCAGCCAGGAGGAGCTGGAGAGAATCCTGTTGGT CTGTACAGATGGCTATCTGGACCCCGACGTGAACAGAAGCGTGTACA TGGCCATCGCCAAAGCCCTCGACAGAGCGAAGTGCCCTATGCTGAC CAACGACCTCGATCTGATCAGCAGATTCGATGCCTTCTGCGGCCTCG CCGTGGCCGATCAGAAGGACCTTCTCAAGAAGATCCTGCTGCTGTGC AAGGGCAACAAACAGAGCAGTCCCATCAACCTGAGCAGCGTGGGCG GCAAGAAGAAGGCAGGCTACCTGCGGCCTGCCCGGAGCAAAGTTCC TGATGGAATGGTGCTGATCGACCAGTCCGTGACAGGGATGTTCGAGC GGAGGACAAGAGTGGGCGTG ASDR Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 30 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCAACCTGAGAAACTACCCCGGCTCCTTCAACATCGG expression ACTGGACTTGGGCCCTGCCTCTGCCGGCTGGAGCGTGGTCGATAGC construct GAGGGAAAGCTGTTTCATTTCAAAAAGAAGCCTACATGGGGCAGCAG (with N- ACTCTTTGACAGCGCTCAGACCGCCTCCGAGGCCAGAATGCACAGA terminal GGACAGCGGCGGCGGTATGTGCGCAGGAGATGGCGGCTCAACCTG methionine, CTGCAGGCGCTGTTCACCGAAGAGATGGAAAAAGTGGACCCTGGTTT stop codon, TTTTATGAGACTGAACCACAGCCGGACAGTAGAGGGCGATCCCATCT V5 tag, N- TCACCAAGGACTTCACCAAGAAGGATTACTACAAGCGGTTCCCCACC terminal NLS ATCTACCACCTGAGGGCTCACCTGATGGAAACCGACGATCCCGCCGA and C- CCTGAGACTGGTCTACCTGGCCATCCACAACATCGTGAAACACAGAG terminal NLS) GCAACTTCCTGAGGCAGGAGGAGAAGCTGACCGCTAAAATCGCTAAC (nt) ACCAGCGAGGCACTGGGCAAGCTGTCTGTCTCCCTGAAAGAGTGGT GCGACTCCAGAGGATATTCTGTGGGCAAGGTGGATGAAGAAGCCATT GCTGCCGTGCTGGCTGACAAGCGAATAAGCAGATCTCAAAAGGCCAA GAACGTGACCCTGCTGATCAGCGTGGACACCGGAGATAGCAAGGCC AACGCCAAGCTGGCCAAGGCCCTGGCCAATGCCATCGTGGGACTGC AAGCTGAATTTAAGGATCTGTTCGGAGATTTCGAATGCGAGGCTACAA AGCTGAACCTGAGCGAGGAAGAGAAGCTGGAAGCCCTGCAAGCTGCTABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO CTGTCCTGATGACAGCGCCGAGCTGCTGGAGGCCGTGTGTGGCGCC TATAGCGCCTACGTGCTGCAGGGCCTGCTGAGCTACGCCGAGGGCC AGACAATCAGCCACAATATGATCAAGAAGTACGACTGCTACGCCGAG GACCTGGAGTGTCTGAAGGCCCTGGTGAGAGACTACCTGCCAAAGA CCAAGGGCGACGACGGCCTCAAGCCTTTCGACAGATTCTTCCGTGG CGTGACATACCACGACCTGGACAAGAGCGACCCTAGCAGAGATTACG ATGCAAGCAAGGCAAAGGGCTACACCGCCTACAACCTGCACAAGCTG TCTTATGACGAGTTCAGAAAGGAAGTGGAAAAGCTGCTGAAGGGCAC CGGCGCCCAGGACGACGAAAGATACATCACAATGATGGACGCTTTCG AAAAGCAGAGATTCCTACGGCGGCTTAAGACATCTGACAACGGCGCC ATCTACTACCAGCTGCACCTGGAGGAGCTGCAGGCCATCCTGGAAAA CCAGGGCAGATTTTACCCCTTCCTGAAACAGGAGGCCAGTAAAATCG AGTCTCTGGTGACCTTCAGAATCCCTTACTACGTGGGCCCACTGACG TCCAAGAACGCCGCCAAGGACGCCCACAACAAGAATAGATTTCAATG GTCCGAGAGAAAGCCTGGCATGGAAGATGCCATTATCACCCCTTGGA ACTGGGAGTCTGTGATCGATAAGAATAGAAGCGCCGAGAACTTCATC ATGCGGATGACCGGCGATTGCACCTACCTGGCCGGCGAGAAGGTGC TGCCTAGACACAGTCTGCTGTACGAGGAGTTCTGCGTGCTGAACGAG CTGAACGGCGTGCGGTTCACTAACGACGGCGACAACTGGCACCGGC TGGACTCAGAGCAGCGGGCTGGAATTATCAGAGATCTGTTTCACAAA AAGAGATCTGTGAAGTACGCCGACATCGCCGCCTGGCTGGTACGGA TGGGCTATCTGGAGCGAACACCTCAGGTGCGCGGTGGACAGGGCGA AAGCGGCCTGGAATCTAAGATGAGCAGCTACCTGCTGTTCGCCAAAG ACGTGTTCGGCGTGGACGAGCTGGACGAGGCCTTATTTCCTGCCATC GAACAGATCATTCTGTGGAACACCCTGTTTGAGGATAGATCCATCTTC GAAGAGAAGCTGCGGGACGAGTTCGGCCCTAATGGCAATGGCATGC TGAACAATGAACAGATCAAGAAATGCAAAAAAAGACTCAGCGGATGG GGCAGACTGAGCGAAAAGTTCCTGACAGGCATAAGGGTTGTGACACA GGGCGGCAGACAGATGAGCATCATGAACGTGCTGCGGGAAGGCAGC CCTAACCCTGACAGACGGCAGGGCGAGGCCCTCGTAATGATGGAAG TGCTGCATGACAACACCCTGGGCTTCCAGAAAAAGATCGACGAAGTG AACAGAGCCTACTACGCCCAGAACGCTAAGGCAATGGGCGTGAATGA GCTGCCCGGGAGCCCTGGCATCCGGCGAAGCATCAATCAAGCCATT AGAATCGTTGATGAGATCGTGAAGATCGCTGGCCACGCTCCTGACAA CATCTTTATCGAGGTTACCCGGGATGAGGACGAAAAGAAGAAGGGCA AGCGGACCGTGAAAAGATGGGACCGGATCGAAGCCGCTCTCAACGC CTTTAAAGCCGAGGGTGGAGATCTGGCAGTGATGAGCGATTTTAAGC GCCTGAAGAATACAGAAGCTGATCTGGATGAGCGCCTCACACTGTAC TTACTGCAGAACGGCAAGTGCCTGTACAGCGGCAGAGCGATCGACTT AGATGCCCTGATGACCGGCAGCGGCGAGTACGAAGTGGATCACATC ATCCCTAGAGCCTACATCAAGGACGATTCCCTGGACAACAAGGCCCT TGTCTACCGGAGCGAGAACCAGAGAAAGACCGACCAACTGCTGATC GACGAGAACGTGCGGCAGAAAATGGGCGAAACCTGGAAGCAGCTTC ATGATGCCAAGATGATCAGCGACAAGAAGTACAACAACCTGATGAGA CTGACCGTGACAGAGGGAGCCATGAAAGGCTTCATCGCCAGACAGTT GGTCGAGACATCTCAGATCGTGAAGATGACCAAGGCCTTGCTGGAG GCCCGGTACGGCGATGCTGGCACAAGAATCGTGCCTGTGAAGGCCA GCATGAGCCACAACCTGAGAGAGGTTGCGGGCTTGGTCAAGTGTAG AGAGGCCAACGACTTCCACCACGCCCATGACGCTTACCTGGCCTGCA GAATGGGCCTGTTTATCCAGAAGAGATACCCAGGAATCTACGAAAAC CCCATCGGCTACGCTCACGCCATGAAGAAATACGTGCTGGAACAGAG CGAACTATTCAAGAAAACCCACCGGATGCCTGGCTCCGCTGGCCTGA TCGTGAACAGCTTCATGACAGCTGGATTCGACCCCGAGACAGGCGA GATGTTCGAGGACGCCTGGGCCGGAAACGATGAGATCAAGCACTGC AAGAAGAAGCACTACGACGACGCCTGGAACGCCGCCGCCGAGGTGG AAGGAATCAGACGGGCCCTGAATTACAGACAATGTTACGTGAGCCGG ATGCCATACGAGGACTCCGGCAAGTTCTGGGATGCTAACCCTATCAGTABLE 1E ASDR Type II Cas Sequences Name Sequence SEQ ID NO CAAGAGAAGCAACCCCAAGTTGGCTCTAAAGAACACCCTGGACCCTG CTTGCTTCGGAGGCTACAATACCAAGACCGCCGCCTACTTCTTCATCT ACGAGGTGACCGACAAAAAAGGCAAGACCGGCCTGCGAATCGCCCA GATGCCAATCTTCATGAAGGCTAAGTGCGAGAGCGATAACGCCGCCT TCGAAAAGTACTGCCAGGACCTGGAAAGCGAATCCAGAGTGAGCTTC ACCCGGATCGTGATCCCCAGACTCCTCAAGTGGCAGCTCGTGGAAAT CGACGGCAACCTGCTGCGGATCAACGCCGAAGAGGAGATGAAATCC GCCTCTCAGCTGGCCTTCAGCCAGGAGGAGCTGGAGAGAATCCTGT TGGTCTGTACAGATGGCTATCTGGACCCCGACGTGAACAGAAGCGTG TACATGGCCATCGCCAAAGCCCTCGACAGAGCGAAGTGCCCTATGCT GACCAACGACCTCGATCTGATCAGCAGATTCGATGCCTTCTGCGGCC TCGCCGTGGCCGATCAGAAGGACCTTCTCAAGAAGATCCTGCTGCTG TGCAAGGGCAACAAACAGAGCAGTCCCATCAACCTGAGCAGCGTGG GCGGCAAGAAGAAGGCAGGCTACCTGCGGCCTGCCCGGAGCAAAGT TCCTGATGGAATGGTGCTGATCGACCAGTCCGTGACAGGGATGTTCG AGCGGAGGACAAGAGTGGGCGTGTCTAGAAAGCGGACAGCAGACGG CTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGTGA

[0088] In some embodiments an ASDR Type II Cas protein comprises an amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, an ASDR Type II 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 comrpise a D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H898A substitution, wherein the position of the H890A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26. In some embodiments, an ASDR Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with an H898A substitution. 6.2.1.6. BCZZ Type II Cas Proteins

[0089] In one aspect, the disclosure provides BCZZ Type II Cas proteins. BCZZ Type II Cas proteins can be further classified as Type IIA Cas proteins. The BCZZ Type II 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 BCZZ Type II 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 BCZZ Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:31.

[0090] Exemplary BCZZ Type II Cas protein sequences and nucleotide sequences encoding exemplary BCZZ Type II Cas proteins are set forth in Table 1F. TABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO BCZZ Type II EQKNCKKVDAYNIGLDIGTNSVGWAVTDLDNNILKRGNKNMWGARIFDE 31 Cas amino GSTAAQTRMFRGTRRRIERRKERINILQSLLLDDMEKEYPNFFPMLRETS acid KTKEDKNSDSINGKKYNLFSELELSDPIYYQKYPTIYHLRRELMESKEKH sequence DIRLVYLAIHHIIKYRGNFLYEGDLKNNNNEIIESINELLDFIQNTVEISFISEATABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO (without N- EKIQEILKDKTKSKGEKKDLIIGLFDYDKDEKSVITGIVNAILGYKFDVNKIF terminal DIAVENSNISFSNEIANEEEIKENLQDQAYVYDVLLKIYNWFILQDILQGNN methionine) SISEAFINKYDKYKKDLSNIKSLYRNYLKDEYNSMFRREKDDSYSCFEKHI SACPIENLYKRIKKDLADVPDCKEKEEILKDIEDNAFLVKINTTANSAIPYQL HYQELEKILENQAQYYQTIQENKQHILELMKFRIPYYVGPLAQEGRSRFA WIVRNSNEKILPWTFDDVVNVDETAEKFIRRMTNKCTYLINEDVIPKQSIL YSEFCVLNELANIRVNNHKLTPKTKQLIIDKLFKVKKTIKQRDLKQVLTDFQ VFPEVTSITGLAEVDKFMSNMSSYIDIENILGCINDENIEMVENIIEWITIFED KKILKRKIKNTYNLEDKVVSQLIKKNYSGWSRLSKELLVGLKAADDGKSIM EKLRTTKDNFMQIINNDMYGFNKQIESRMKLQEKQITYKDVDEIPTSPAN KRAIWQSLKIVEEIKKVMKKDPKNIYIEFAREEQEKNKRKDNRAKALLKIY EKYEEEIKMLKDYNPNVYKELKKKQNEKDFNERLYLYFIQNGRCMYSSR PLNIDTLYLYEVDHILPQSYVKDDSLSNKALVYKEENQRKSGSLLLDEKIIN RQETWWKQLRKNGLIDEKKYYNLTRRKMFETDKEKVKFVSRQLIETRQS TKYITNLLVNQYSNTDVFAIRSNLTHNFRKFFEVYKNRNVNDYHHAQDAY IISVIGNVIDQKLQYKDEYKYTEYVKKYIKKSEEEANKVWIIMGMVSNNINK EKVKKALEYKDCFITHKLEEQTGAFYNQTLYSPKDKNIEIPLKEGLDVKKY GGYSGEIKAYFTIYSYIAQKGKMQIEMIGIPVKTKYDIENGKTTLIKYIQSKN SEASKIEIIRPKILKYQEYIDENNEPMMLLSDSEIRTNRQLIINNEMSKLIYIM NQEKIADDEKEKVQEKMEEIYNYLLGKLKNEYKTFNSIYQKLSNEETKEIF QKLEYKDKVSAINGIIDLMHRGQGNLTKLKQGDRAGRKSGKSFKTEALN KMIFIDKSVTGMYERRYKVNGMENCSSK BCZZ Type II MEQKNCKKVDAYNIGLDIGTNSVGWAVTDLDNNILKRGNKNMWGARIFD 32 Cas amino EGSTAAQTRMFRGTRRRIERRKERINILQSLLLDDMEKEYPNFFPMLRET acid SKTKEDKNSDSINGKKYNLFSELELSDPIYYQKYPTIYHLRRELMESKEKH sequence DIRLVYLAIHHIIKYRGNFLYEGDLKNNNNEIIESINELLDFIQNTVEISFISEA EKIQEILKDKTKSKGEKKDLIIGLFDYDKDEKSVITGIVNAILGYKFDVNKIF DIAVENSNISFSNEIANEEEIKENLQDQAYVYDVLLKIYNWFILQDILQGNN SISEAFINKYDKYKKDLSNIKSLYRNYLKDEYNSMFRREKDDSYSCFEKHI SACPIENLYKRIKKDLADVPDCKEKEEILKDIEDNAFLVKINTTANSAIPYQL HYQELEKILENQAQYYQTIQENKQHILELMKFRIPYYVGPLAQEGRSRFA WIVRNSNEKILPWTFDDVVNVDETAEKFIRRMTNKCTYLINEDVIPKQSIL YSEFCVLNELANIRVNNHKLTPKTKQLIIDKLFKVKKTIKQRDLKQVLTDFQ VFPEVTSITGLAEVDKFMSNMSSYIDIENILGCINDENIEMVENIIEWITIFED KKILKRKIKNTYNLEDKVVSQLIKKNYSGWSRLSKELLVGLKAADDGKSIM EKLRTTKDNFMQIINNDMYGFNKQIESRMKLQEKQITYKDVDEIPTSPAN KRAIWQSLKIVEEIKKVMKKDPKNIYIEFAREEQEKNKRKDNRAKALLKIY EKYEEEIKMLKDYNPNVYKELKKKQNEKDFNERLYLYFIQNGRCMYSSR PLNIDTLYLYEVDHILPQSYVKDDSLSNKALVYKEENQRKSGSLLLDEKIIN RQETWWKQLRKNGLIDEKKYYNLTRRKMFETDKEKVKFVSRQLIETRQS TKYITNLLVNQYSNTDVFAIRSNLTHNFRKFFEVYKNRNVNDYHHAQDAY IISVIGNVIDQKLQYKDEYKYTEYVKKYIKKSEEEANKVWIIMGMVSNNINK EKVKKALEYKDCFITHKLEEQTGAFYNQTLYSPKDKNIEIPLKEGLDVKKY GGYSGEIKAYFTIYSYIAQKGKMQIEMIGIPVKTKYDIENGKTTLIKYIQSKN SEASKIEIIRPKILKYQEYIDENNEPMMLLSDSEIRTNRQLIINNEMSKLIYIM NQEKIADDEKEKVQEKMEEIYNYLLGKLKNEYKTFNSIYQKLSNEETKEIF QKLEYKDKVSAINGIIDLMHRGQGNLTKLKQGDRAGRKSGKSFKTEALN KMIFIDKSVTGMYERRYKVNGMENCSSK BCZZ Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSEQKNCKKVDAYNIG 33 Cas LDIGTNSVGWAVTDLDNNILKRGNKNMWGARIFDEGSTAAQTRMFRGT mammalian RRRIERRKERINILQSLLLDDMEKEYPNFFPMLRETSKTKEDKNSDSINGK expression KYNLFSELELSDPIYYQKYPTIYHLRRELMESKEKHDIRLVYLAIHHIIKYR construct GNFLYEGDLKNNNNEIIESINELLDFIQNTVEISFISEAEKIQEILKDKTKSK (with N- GEKKDLIIGLFDYDKDEKSVITGIVNAILGYKFDVNKIFDIAVENSNISFSNEI terminal ANEEEIKENLQDQAYVYDVLLKIYNWFILQDILQGNNSISEAFINKYDKYKK methionine, DLSNIKSLYRNYLKDEYNSMFRREKDDSYSCFEKHISACPIENLYKRIKKD stop codon, LADVPDCKEKEEILKDIEDNAFLVKINTTANSAIPYQLHYQELEKILENQAQTABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO V5 tag, N- YYQTIQENKQHILELMKFRIPYYVGPLAQEGRSRFAWIVRNSNEKILPWT terminal NLS FDDVVNVDETAEKFIRRMTNKCTYLINEDVIPKQSILYSEFCVLNELANIRV and C- NNHKLTPKTKQLIIDKLFKVKKTIKQRDLKQVLTDFQVFPEVTSITGLAEVD terminal NLS) KFMSNMSSYIDIENILGCINDENIEMVENIIEWITIFEDKKILKRKIKNTYNLE (aa) DKVVSQLIKKNYSGWSRLSKELLVGLKAADDGKSIMEKLRTTKDNFMQII NNDMYGFNKQIESRMKLQEKQITYKDVDEIPTSPANKRAIWQSLKIVEEIK KVMKKDPKNIYIEFAREEQEKNKRKDNRAKALLKIYEKYEEEIKMLKDYNP NVYKELKKKQNEKDFNERLYLYFIQNGRCMYSSRPLNIDTLYLYEVDHIL PQSYVKDDSLSNKALVYKEENQRKSGSLLLDEKIINRQETWWKQLRKNG LIDEKKYYNLTRRKMFETDKEKVKFVSRQLIETRQSTKYITNLLVNQYSNT DVFAIRSNLTHNFRKFFEVYKNRNVNDYHHAQDAYIISVIGNVIDQKLQYK DEYKYTEYVKKYIKKSEEEANKVWIIMGMVSNNINKEKVKKALEYKDCFIT HKLEEQTGAFYNQTLYSPKDKNIEIPLKEGLDVKKYGGYSGEIKAYFTIYS YIAQKGKMQIEMIGIPVKTKYDIENGKTTLIKYIQSKNSEASKIEIIRPKILKY QEYIDENNEPMMLLSDSEIRTNRQLIINNEMSKLIYIMNQEKIADDEKEKV QEKMEEIYNYLLGKLKNEYKTFNSIYQKLSNEETKEIFQKLEYKDKVSAIN GIIDLMHRGQGNLTKLKQGDRAGRKSGKSFKTEALNKMIFIDKSVTGMYE RRYKVNGMENCSSKSRKRTADGSEFESPKKKRKV BCZZ Type II ATGGAACAAAAAAATTGCAAAAAAGTTGACGCTTATAATATAGGATTG 34 Cas coding GACATTGGAACAAATTCAGTTGGATGGGCTGTAACAGATTTGGATAAT sequence (nt) AATATTTTAAAAAGAGGCAACAAGAACATGTGGGGTGCACGCATATTT (not codon GATGAAGGAAGTACTGCAGCACAAACAAGAATGTTTAGAGGAACAAG optimized) AAGAAGGATTGAAAGAAGAAAAGAGAGAATAAATATACTTCAAAGTTT ACTATTAGATGACATGGAAAAGGAATATCCAAATTTCTTTCCAATGTTA AGGGAAACATCTAAGACTAAAGAAGATAAAAATAGTGACTCAATTAAT GGAAAAAAATATAATTTATTTTCAGAATTGGAATTATCAGATCCAATAT ATTATCAAAAATATCCAACAATATATCATTTAAGACGAGAGCTAATGGA AAGTAAAGAAAAGCATGACATTAGGCTTGTTTATCTTGCAATTCATCAT ATAATAAAATATCGTGGAAACTTCCTATATGAAGGAGACTTAAAAAATA ATAACAATGAAATAATTGAATCTATAAATGAGCTATTAGATTTTATACA AAATACAGTGGAAATAAGCTTTATTTCAGAAGCGGAAAAAATTCAAGA AATATTAAAAGATAAAACGAAATCAAAGGGAGAAAAAAAGGATTTAAT AATTGGACTTTTTGATTATGATAAGGATGAGAAGTCTGTAATTACTGG AATTGTTAATGCAATTTTGGGGTATAAATTTGACGTTAATAAGATATTT GATATTGCAGTAGAAAATTCAAATATTTCTTTTAGTAATGAAATAGCAA ATGAAGAAGAAATAAAAGAAAACTTGCAAGACCAAGCTTATGTGTATG ATGTATTACTAAAAATATATAATTGGTTTATATTACAAGATATTCTACAA GGTAATAATAGTATTTCAGAAGCTTTCATAAATAAGTATGACAAGTATA AAAAGGATTTAAGCAATATAAAAAGCTTATATAGAAATTATTTAAAAGA TGAATATAATAGTATGTTCAGAAGAGAAAAAGATGATAGCTATTCATGT TTCGAAAAGCATATATCAGCGTGTCCAATAGAAAACTTGTATAAAAGA ATAAAAAAAGATTTAGCAGATGTTCCAGATTGTAAGGAAAAAGAAGAA ATATTAAAAGATATAGAAGATAACGCTTTTTTAGTAAAAATAAATACAA CAGCAAATTCGGCAATTCCATATCAATTACATTATCAAGAATTGGAAAA AATACTTGAAAATCAAGCACAATATTATCAAACAATACAAGAAAATAAA CAACATATATTAGAACTAATGAAATTCAGAATTCCATATTATGTTGGAC CTTTGGCACAAGAGGGAAGAAGTAGATTTGCTTGGATTGTTAGAAATT CAAACGAAAAAATATTGCCATGGACATTTGATGACGTGGTCAATGTAG ATGAAACGGCAGAAAAGTTTATAAGAAGGATGACCAATAAATGTACAT ACCTAATAAATGAAGATGTAATTCCAAAACAATCAATTTTATACTCTGA GTTTTGTGTATTAAATGAACTTGCAAATATAAGAGTAAATAATCATAAA CTAACTCCAAAGACTAAACAACTTATAATAGACAAGTTATTTAAAGTCA AAAAAACAATTAAGCAAAGAGATTTAAAACAAGTATTAACAGATTTTCA AGTTTTTCCTGAAGTCACATCAATTACTGGATTAGCAGAAGTAGATAA ATTTATGTCAAACATGTCATCATATATTGATATTGAAAATATACTTGGC TGTATAAATGATGAAAACATTGAAATGGTTGAAAACATAATTGAATGGA TTACAATATTTGAAGATAAAAAAATATTAAAAAGAAAAATAAAAAATACATABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO TACAATTTAGAAGATAAAGTAGTAAGTCAACTGATAAAGAAAAATTATA GTGGATGGTCGAGATTGTCAAAAGAATTACTTGTTGGATTAAAGGCTG CTGATGATGGAAAAAGCATAATGGAAAAGCTAAGGACAACAAAAGAC AATTTTATGCAAATTATAAATAATGATATGTATGGTTTTAATAAACAAAT TGAAAGTAGAATGAAACTACAAGAAAAACAAATAACATATAAAGATGT AGATGAAATTCCAACATCTCCAGCTAATAAAAGGGCCATATGGCAAAG CCTAAAAATAGTTGAAGAAATTAAAAAAGTAATGAAAAAAGATCCTAAA AACATTTATATTGAATTTGCAAGAGAAGAGCAGGAAAAAAATAAGAGA AAAGATAATAGAGCAAAAGCACTTCTTAAAATATACGAAAAATATGAA GAAGAAATCAAAATGCTAAAAGACTATAATCCAAATGTATATAAAGAG CTAAAGAAGAAACAAAATGAAAAAGATTTTAATGAAAGATTGTACTTAT ACTTTATTCAAAATGGAAGATGTATGTATAGTTCAAGACCATTAAATAT TGATACGCTTTACTTATATGAGGTGGATCATATATTGCCTCAAAGTTAT GTTAAAGATGATAGTTTATCAAACAAAGCGTTGGTATATAAAGAAGAA AATCAAAGAAAAAGTGGAAGTTTATTGTTAGATGAAAAAATAATAAATA GACAAGAAACATGGTGGAAGCAATTACGAAAAAATGGTTTGATTGATG AGAAAAAATATTATAACTTGACAAGAAGAAAAATGTTTGAAACTGATAA AGAAAAAGTGAAATTTGTGTCTAGACAATTGATAGAAACAAGGCAGAG CACAAAATATATAACAAATTTATTAGTTAATCAATATTCAAACACAGAT GTATTTGCAATAAGGTCAAATCTTACTCATAATTTTAGAAAATTCTTTG AAGTATACAAAAACAGAAATGTTAATGATTATCATCATGCACAGGATG CATATATTATAAGTGTAATAGGCAATGTAATAGATCAGAAATTACAATA CAAAGATGAATACAAATATACAGAGTATGTAAAAAAATATATAAAAAAG AGTGAAGAAGAAGCAAATAAAGTTTGGATAATAATGGGAATGGTTTCT AACAATATAAACAAAGAAAAAGTTAAAAAGGCTCTTGAATATAAAGATT GTTTTATAACACATAAATTAGAAGAACAAACAGGTGCATTTTATAACCA AACACTATATAGTCCAAAAGATAAAAACATAGAAATACCTTTAAAAGAG GGATTAGATGTTAAAAAATATGGAGGCTATTCTGGAGAAATTAAGGCA TATTTTACAATTTACAGTTATATTGCTCAAAAGGGAAAAATGCAAATAG AAATGATAGGAATTCCTGTAAAAACTAAATATGATATAGAAAATGGAAA AACTACACTAATTAAATATATACAAAGCAAAAATAGTGAAGCTTCTAAA ATAGAGATTATTAGACCTAAAATTTTAAAATATCAAGAATATATTGATG AAAATAACGAACCAATGATGCTATTAAGTGATAGTGAGATTAGAACTA ATAGGCAACTAATTATAAATAACGAAATGTCTAAACTAATATATATAAT GAATCAAGAAAAAATAGCAGATGATGAAAAAGAAAAAGTGCAAGAAAA AATGGAAGAAATTTATAATTATTTATTAGGTAAATTAAAAAATGAATATA AAACATTTAACTCTATATATCAAAAATTAAGTAATGAAGAGACAAAAGA AATTTTTCAAAAATTAGAATATAAAGACAAGGTGTCTGCTATAAATGGA ATTATTGATCTTATGCACAGAGGACAAGGCAATTTAACAAAATTAAAAC AAGGTGATAGAGCTGGAAGAAAAAGTGGAAAAAGTTTTAAAACAGAG GCATTAAATAAAATGATATTTATAGATAAATCTGTAACAGGAATGTACG AAAGAAGGTATAAAGTAAATGGGATGGAGAACTGTAGTAGTAAATAA BCZZ Type II GAGCAGAAGAACTGCAAGAAAGTGGATGCGTATAACATCGGCCTGGA 35 Cas coding CATAGGCACCAATAGCGTGGGCTGGGCCGTGACCGACTTGGACAAC sequence (nt) AACATCCTGAAGCGGGGCAACAAGAACATGTGGGGCGCCAGAATCTT (human TGACGAGGGAAGCACCGCAGCCCAGACAAGAATGTTCAGAGGGACC codon- CGGCGGAGAATCGAAAGACGGAAGGAGAGAATCAACATCCTGCAGT optimized; CCCTGCTGCTGGACGACATGGAAAAGGAATACCCTAACTTCTTCCCT lacking N- ATGCTGCGGGAAACCAGCAAGACAAAAGAAGATAAGAACAGCGATTC terminal TATCAACGGCAAAAAATACAATCTGTTTTCTGAACTAGAACTCAGCGA methionine; CCCTATCTACTACCAGAAGTATCCTACCATCTATCACCTGAGAAGAGA no stop GCTGATGGAAAGCAAGGAGAAGCACGACATCAGACTGGTCTACCTAG codon) CCATTCACCATATCATCAAGTATAGAGGCAATTTCCTGTACGAGGGCG ACCTGAAAAACAACAACAACGAAATCATCGAGAGCATCAACGAGCTG CTGGATTTCATCCAGAACACCGTGGAAATCAGCTTCATAAGCGAGGC CGAGAAGATCCAGGAGATACTGAAGGACAAAACCAAGAGCAAAGGC GAGAAGAAAGACCTGATCATCGGCCTGTTTGACTATGATAAGGACGATABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO GAAAAGCGTGATCACAGGCATCGTGAACGCCATCCTGGGCTACAAGT TTGACGTGAACAAGATTTTCGATATCGCTGTTGAAAACAGCAATATCA GCTTCAGCAATGAGATCGCCAACGAGGAAGAAATCAAGGAAAACCTG CAGGACCAGGCCTACGTGTACGATGTGCTGCTGAAAATTTACAACTG GTTCATACTGCAGGATATCCTGCAGGGAAACAACTCCATCAGCGAGG CCTTTATTAACAAGTACGACAAGTACAAAAAGGATCTGAGCAACATCA AAAGCCTGTACAGAAATTACCTGAAGGATGAGTACAACAGCATGTTTC GAAGAGAGAAGGACGACTCCTACTCATGCTTCGAGAAGCACATTAGC GCTTGTCCAATCGAGAACTTATACAAGCGTATCAAGAAGGACCTGGC TGACGTTCCCGACTGCAAGGAGAAGGAAGAAATCCTGAAAGACATCG AAGATAATGCCTTTCTGGTGAAGATCAATACCACAGCTAACAGTGCCA TCCCCTACCAACTGCACTACCAGGAACTGGAAAAGATCCTTGAAAAC CAGGCCCAGTACTACCAGACCATCCAGGAAAACAAACAACACATCCT GGAGCTGATGAAGTTCAGAATCCCTTACTACGTCGGACCTCTGGCTC AGGAGGGCAGAAGCAGATTCGCCTGGATCGTGCGGAACAGCAACGA GAAGATACTGCCTTGGACCTTCGATGATGTGGTGAATGTGGACGAGA CAGCCGAGAAATTTATCCGGAGAATGACCAACAAATGTACCTACCTG ATTAACGAGGACGTTATCCCCAAACAGAGCATCCTGTACAGCGAGTT CTGCGTGCTGAACGAGCTGGCCAACATTCGGGTGAACAATCACAAGC TGACTCCTAAGACCAAGCAACTGATCATCGACAAACTGTTCAAGGTGA AGAAAACCATTAAGCAGAGAGATCTAAAGCAGGTGCTGACAGATTTC CAAGTGTTCCCAGAGGTCACCAGCATCACCGGCCTCGCTGAAGTGG ATAAATTCATGAGCAACATGAGCAGCTACATTGACATCGAGAACATCC TGGGCTGCATCAACGACGAGAATATCGAGATGGTGGAGAACATCATT GAGTGGATCACTATCTTTGAGGACAAGAAGATCCTGAAGCGGAAAAT CAAGAACACCTATAACCTGGAAGATAAGGTGGTGAGCCAGTTGATCA AGAAGAACTACTCTGGCTGGAGCCGGCTGTCTAAGGAGCTGCTCGT GGGCCTCAAGGCCGCCGATGACGGCAAGAGCATCATGGAGAAGCTG AGAACCACCAAGGATAATTTCATGCAGATCATCAACAATGACATGTAC GGCTTCAACAAGCAGATCGAGAGCAGGATGAAACTGCAGGAGAAGC AGATCACCTACAAGGACGTGGATGAGATCCCCACAAGCCCCGCTAAC AAGCGGGCCATCTGGCAGAGCCTGAAGATCGTCGAAGAGATCAAGA AAGTCATGAAAAAAGACCCCAAGAACATTTATATCGAGTTCGCCAGG GAAGAACAGGAGAAGAACAAGAGGAAGGACAACAGAGCCAAGGCTC TGTTGAAGATCTACGAGAAGTACGAAGAAGAGATCAAGATGCTGAAG GACTACAACCCCAACGTGTACAAGGAGCTTAAGAAGAAGCAAAATGA GAAAGACTTCAATGAGCGGCTGTACCTGTACTTCATCCAAAACGGCC GGTGCATGTACTCCAGCCGGCCTCTGAATATCGACACCCTGTACTTA TACGAAGTGGACCACATCCTGCCTCAGTCCTACGTGAAGGACGACAG CCTGAGCAACAAGGCCCTGGTGTACAAGGAAGAAAATCAAAGAAAGA GCGGCTCTCTGCTGCTGGACGAGAAGATCATCAACCGGCAGGAAAC CTGGTGGAAGCAGCTGAGAAAAAATGGACTGATCGACGAGAAGAAGT ACTACAACCTGACAAGACGCAAAATGTTCGAAACCGACAAAGAAAAG GTGAAGTTCGTGAGCAGACAGCTGATCGAGACAAGACAGTCTACCAA ATACATCACCAACCTCCTCGTGAACCAGTACAGCAACACCGATGTGTT CGCCATCCGGAGCAACCTGACCCATAACTTCAGAAAGTTCTTCGAGG TCTACAAGAATAGAAACGTGAACGATTACCACCACGCCCAGGACGCC TACATCATCAGCGTGATCGGCAATGTGATCGATCAGAAGCTGCAGTA CAAGGACGAATACAAGTACACAGAGTACGTGAAGAAGTACATCAAGA AGTCTGAAGAGGAAGCCAACAAAGTGTGGATCATCATGGGCATGGTG AGCAATAACATCAATAAAGAAAAGGTTAAGAAGGCCCTGGAATACAAG GATTGTTTTATCACCCACAAGCTGGAGGAACAGACCGGGGCCTTCTA CAACCAGACACTGTACAGTCCTAAGGATAAGAACATCGAAATCCCACT GAAGGAAGGCCTGGATGTGAAGAAGTACGGCGGATACAGCGGAGAG ATCAAGGCTTATTTCACCATCTACTCTTATATCGCCCAGAAGGGCAAG ATGCAGATCGAGATGATCGGAATCCCTGTGAAAACCAAATATGACATC GAGAACGGCAAGACCACACTCATCAAGTACATCCAGAGCAAGAACTCTABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO CGAAGCCAGCAAGATCGAGATCATTAGACCTAAGATCCTGAAGTACC AGGAGTACATAGACGAGAACAACGAGCCTATGATGCTGCTGTCTGAC TCCGAAATCAGAACCAATAGACAGCTGATCATCAACAATGAGATGAG CAAGCTCATCTACATCATGAACCAGGAGAAGATCGCCGACGACGAGA AAGAGAAAGTGCAGGAGAAGATGGAGGAGATCTACAACTACCTTCTG GGCAAGCTGAAGAATGAGTACAAAACCTTCAACTCCATCTACCAGAAA CTGTCCAACGAGGAAACAAAGGAGATCTTCCAGAAGCTGGAGTATAA GGACAAGGTGTCAGCTATCAACGGCATCATCGATCTGATGCACCGAG GACAGGGCAACCTGACCAAGCTGAAGCAGGGCGATCGGGCCGGCA GAAAATCAGGCAAGAGTTTTAAGACAGAGGCTCTGAACAAAATGATCT TCATAGACAAAAGCGTGACAGGAATGTACGAGAGAAGATACAAGGTG AACGGCATGGAGAACTGCAGCTCCAAG BCZZ Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 36 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCGAGCAGAAGAACTGCAAGAAAGTGGATGCGTATAA expression CATCGGCCTGGACATAGGCACCAATAGCGTGGGCTGGGCCGTGACC construct GACTTGGACAACAACATCCTGAAGCGGGGCAACAAGAACATGTGGG (with N- GCGCCAGAATCTTTGACGAGGGAAGCACCGCAGCCCAGACAAGAAT terminal GTTCAGAGGGACCCGGCGGAGAATCGAAAGACGGAAGGAGAGAATC methionine, AACATCCTGCAGTCCCTGCTGCTGGACGACATGGAAAAGGAATACCC stop codon, TAACTTCTTCCCTATGCTGCGGGAAACCAGCAAGACAAAAGAAGATAA V5 tag, N- GAACAGCGATTCTATCAACGGCAAAAAATACAATCTGTTTTCTGAACT terminal NLS AGAACTCAGCGACCCTATCTACTACCAGAAGTATCCTACCATCTATCA and C- CCTGAGAAGAGAGCTGATGGAAAGCAAGGAGAAGCACGACATCAGA terminal NLS) CTGGTCTACCTAGCCATTCACCATATCATCAAGTATAGAGGCAATTTC (nt) CTGTACGAGGGCGACCTGAAAAACAACAACAACGAAATCATCGAGAG CATCAACGAGCTGCTGGATTTCATCCAGAACACCGTGGAAATCAGCT TCATAAGCGAGGCCGAGAAGATCCAGGAGATACTGAAGGACAAAACC AAGAGCAAAGGCGAGAAGAAAGACCTGATCATCGGCCTGTTTGACTA TGATAAGGACGAGAAAAGCGTGATCACAGGCATCGTGAACGCCATCC TGGGCTACAAGTTTGACGTGAACAAGATTTTCGATATCGCTGTTGAAA ACAGCAATATCAGCTTCAGCAATGAGATCGCCAACGAGGAAGAAATC AAGGAAAACCTGCAGGACCAGGCCTACGTGTACGATGTGCTGCTGAA AATTTACAACTGGTTCATACTGCAGGATATCCTGCAGGGAAACAACTC CATCAGCGAGGCCTTTATTAACAAGTACGACAAGTACAAAAAGGATCT GAGCAACATCAAAAGCCTGTACAGAAATTACCTGAAGGATGAGTACA ACAGCATGTTTCGAAGAGAGAAGGACGACTCCTACTCATGCTTCGAG AAGCACATTAGCGCTTGTCCAATCGAGAACTTATACAAGCGTATCAAG AAGGACCTGGCTGACGTTCCCGACTGCAAGGAGAAGGAAGAAATCCT GAAAGACATCGAAGATAATGCCTTTCTGGTGAAGATCAATACCACAGC TAACAGTGCCATCCCCTACCAACTGCACTACCAGGAACTGGAAAAGA TCCTTGAAAACCAGGCCCAGTACTACCAGACCATCCAGGAAAACAAA CAACACATCCTGGAGCTGATGAAGTTCAGAATCCCTTACTACGTCGG ACCTCTGGCTCAGGAGGGCAGAAGCAGATTCGCCTGGATCGTGCGG AACAGCAACGAGAAGATACTGCCTTGGACCTTCGATGATGTGGTGAA TGTGGACGAGACAGCCGAGAAATTTATCCGGAGAATGACCAACAAAT GTACCTACCTGATTAACGAGGACGTTATCCCCAAACAGAGCATCCTG TACAGCGAGTTCTGCGTGCTGAACGAGCTGGCCAACATTCGGGTGAA CAATCACAAGCTGACTCCTAAGACCAAGCAACTGATCATCGACAAACT GTTCAAGGTGAAGAAAACCATTAAGCAGAGAGATCTAAAGCAGGTGC TGACAGATTTCCAAGTGTTCCCAGAGGTCACCAGCATCACCGGCCTC GCTGAAGTGGATAAATTCATGAGCAACATGAGCAGCTACATTGACATC GAGAACATCCTGGGCTGCATCAACGACGAGAATATCGAGATGGTGGA GAACATCATTGAGTGGATCACTATCTTTGAGGACAAGAAGATCCTGAA GCGGAAAATCAAGAACACCTATAACCTGGAAGATAAGGTGGTGAGCC AGTTGATCAAGAAGAACTACTCTGGCTGGAGCCGGCTGTCTAAGGAG CTGCTCGTGGGCCTCAAGGCCGCCGATGACGGCAAGAGCATCATGGTABLE 1F BCZZ Type II Cas Sequences Name Sequence SEQ ID NO AGAAGCTGAGAACCACCAAGGATAATTTCATGCAGATCATCAACAATG ACATGTACGGCTTCAACAAGCAGATCGAGAGCAGGATGAAACTGCAG GAGAAGCAGATCACCTACAAGGACGTGGATGAGATCCCCACAAGCC CCGCTAACAAGCGGGCCATCTGGCAGAGCCTGAAGATCGTCGAAGA GATCAAGAAAGTCATGAAAAAAGACCCCAAGAACATTTATATCGAGTT CGCCAGGGAAGAACAGGAGAAGAACAAGAGGAAGGACAACAGAGCC AAGGCTCTGTTGAAGATCTACGAGAAGTACGAAGAAGAGATCAAGAT GCTGAAGGACTACAACCCCAACGTGTACAAGGAGCTTAAGAAGAAGC AAAATGAGAAAGACTTCAATGAGCGGCTGTACCTGTACTTCATCCAAA ACGGCCGGTGCATGTACTCCAGCCGGCCTCTGAATATCGACACCCTG TACTTATACGAAGTGGACCACATCCTGCCTCAGTCCTACGTGAAGGA CGACAGCCTGAGCAACAAGGCCCTGGTGTACAAGGAAGAAAATCAAA GAAAGAGCGGCTCTCTGCTGCTGGACGAGAAGATCATCAACCGGCA GGAAACCTGGTGGAAGCAGCTGAGAAAAAATGGACTGATCGACGAG AAGAAGTACTACAACCTGACAAGACGCAAAATGTTCGAAACCGACAA AGAAAAGGTGAAGTTCGTGAGCAGACAGCTGATCGAGACAAGACAGT CTACCAAATACATCACCAACCTCCTCGTGAACCAGTACAGCAACACC GATGTGTTCGCCATCCGGAGCAACCTGACCCATAACTTCAGAAAGTT CTTCGAGGTCTACAAGAATAGAAACGTGAACGATTACCACCACGCCC AGGACGCCTACATCATCAGCGTGATCGGCAATGTGATCGATCAGAAG CTGCAGTACAAGGACGAATACAAGTACACAGAGTACGTGAAGAAGTA CATCAAGAAGTCTGAAGAGGAAGCCAACAAAGTGTGGATCATCATGG GCATGGTGAGCAATAACATCAATAAAGAAAAGGTTAAGAAGGCCCTG GAATACAAGGATTGTTTTATCACCCACAAGCTGGAGGAACAGACCGG GGCCTTCTACAACCAGACACTGTACAGTCCTAAGGATAAGAACATCG AAATCCCACTGAAGGAAGGCCTGGATGTGAAGAAGTACGGCGGATAC AGCGGAGAGATCAAGGCTTATTTCACCATCTACTCTTATATCGCCCAG AAGGGCAAGATGCAGATCGAGATGATCGGAATCCCTGTGAAAACCAA ATATGACATCGAGAACGGCAAGACCACACTCATCAAGTACATCCAGA GCAAGAACTCCGAAGCCAGCAAGATCGAGATCATTAGACCTAAGATC CTGAAGTACCAGGAGTACATAGACGAGAACAACGAGCCTATGATGCT GCTGTCTGACTCCGAAATCAGAACCAATAGACAGCTGATCATCAACAA TGAGATGAGCAAGCTCATCTACATCATGAACCAGGAGAAGATCGCCG ACGACGAGAAAGAGAAAGTGCAGGAGAAGATGGAGGAGATCTACAA CTACCTTCTGGGCAAGCTGAAGAATGAGTACAAAACCTTCAACTCCAT CTACCAGAAACTGTCCAACGAGGAAACAAAGGAGATCTTCCAGAAGC TGGAGTATAAGGACAAGGTGTCAGCTATCAACGGCATCATCGATCTG ATGCACCGAGGACAGGGCAACCTGACCAAGCTGAAGCAGGGCGATC GGGCCGGCAGAAAATCAGGCAAGAGTTTTAAGACAGAGGCTCTGAAC AAAATGATCTTCATAGACAAAAGCGTGACAGGAATGTACGAGAGAAG ATACAAGGTGAACGGCATGGAGAACTGCAGCTCCAAGTCTAGAAAGC GGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAG GTGTGA

[0091] In some embodiments a BCZZ Type II 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 BCZZ Type II 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 D17A substitution, wherein the position of the D17A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H836A substitution, wherein the position of the H836A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32. In some embodiments, a BCZZ Type II Cas protein is catalytically inactive, for example due to a D17A substitution in combination with a H836A substitution.6.2.1.7. DRCY Type II Cas Proteins

[0092] In one aspect, the disclosure provides DRCY Type II Cas proteins. DRCY Type II Cas proteins can be further classified as Type IIA Cas proteins. The DRCY Type II 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 DRCY Type II 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 DRCY Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:37.

[0093] Exemplary DRCY Type II Cas protein sequences and nucleotide sequences encoding exemplary DRCY Type II Cas proteins are set forth in Table 1G. TABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO DRCY Type II NLRNFKGDYSVGLDMGTGSVGWSVTDEDGNLLHFKKQPTWGSRLFDS 37 Cas amino AQTAAEARTPRGQRRRYVRRRWRLDLLQSLLQEEIERVDPDFFLRLRQS acid RLVNTDPNKTTSDYRWPLFNGSDFTEVDYYNKFPTIYHLRKWLMETDEQ sequence ADIRLVYLALHNIVKHRGNFLREGQKLSSENANPDDAVSEFCTALKEWCE (without N- GQEGDCGALKEADIVAVLTARGMRPSDCAADLLELLPVSFGDGDKQAD terminal KKLKKALASAMVGLKAEFKDIFGDFATEGTKISLSKDEESEALLEVCPDE methionine) GKVLFESLQRVHSAYVLQGLLSYAPGESISANMIAKYDQYAKDLRMLKLL VREYAPTSYGDFFRGPLCPNKGGYDKFDASVKGYTRYNLGVTSYDDFA KEVKKLFVGTGAEDDERYESMMDAFDDQRFLRRLKTSDNGSICYQLHLE EMETILDNQGRFYPTLANEKDKLLSLVTFRIPYYVGPLTQRNAREDAHGK LRFAWSERKPGTEGVTITPWNWEDVIDKGASAEKFITRMTGICTYLQGE DVLPKSSLLYEEFCVLNELNGMRWTSDGDEERRFDAEQREGMMRDLFR KTRTVSNKRIEDWLVQNGYAHSPKVSGGQNETGLESKLGSYIFFAKDIFG TDEIAKVDYPMIEEIILWNTLFEDRSILKEKLREKYGDAGEGRLSAEQIKKI CKKRFTGWGRLSKKFLAGIKVDTQLGEKSIMDVLREGDPDADIRRGRTM VLMEVIRDDNLGFQKKIDSFNRAYFAEAGSALGVNDLPGSPAIRRSLNQA IRIVDEIAHIAGHAPTNVFIEVTRDDDMGKKGHRTARRYDQLKEALAAFKK DDPQIWKEIQAASPTDLDERLTLYFVQRGKCMYSGKPIDINQLSNAGLYE VDHIIPRSYIKDDSFENKALVLREFNQRKTDSMLLDDGIRRNMAGYWRSL HDAKLIGDKKFNNLMRTHVGENAMRGFIARQLVETSQMVKLAQSLLSAK YPDTKITPVKASMSHDLREAAGFVKCREANDFHHAHDAFLACRVGLFIRK WYPDMYDKPILYTRAMRKYVREQAEEFKKTRRAPGSSGFVVGRFISSFV DADTGELWEGAEEVESIRRVLNYRQCHITRMPMEDSGAFWNATIYSPRD PKMGSKLSLRLKGDLDPQIYGGYSSQQFAYFFIYEARDKKGRPAFRFSQ VPVWLASRIASDEWALEEYAREQAASEQLEFVRIQRSKILKKQLVEIDGE RLIITGAEEVRNARQFAFSLDELSLLGRASARGDGEAIDEETISNGYNVLL KVMIERGELVGKGLMKKLSLGDRYEQFMQLDCAAKKSLILQLAAIVNASN RQIDLSKIGGAKTAGQYKVTYNKLLNDPNTDFYIVDQSVTGMFERRTRVG L DRCY Type II MNLRNFKGDYSVGLDMGTGSVGWSVTDEDGNLLHFKKQPTWGSRLFD 38 Cas amino SAQTAAEARTPRGQRRRYVRRRWRLDLLQSLLQEEIERVDPDFFLRLRQ acid SRLVNTDPNKTTSDYRWPLFNGSDFTEVDYYNKFPTIYHLRKWLMETDE sequence QADIRLVYLALHNIVKHRGNFLREGQKLSSENANPDDAVSEFCTALKEW CEGQEGDCGALKEADIVAVLTARGMRPSDCAADLLELLPVSFGDGDKQ ADKKLKKALASAMVGLKAEFKDIFGDFATEGTKISLSKDEESEALLEVCP DEGKVLFESLQRVHSAYVLQGLLSYAPGESISANMIAKYDQYAKDLRML KLLVREYAPTSYGDFFRGPLCPNKGGYDKFDASVKGYTRYNLGVTSYD DFAKEVKKLFVGTGAEDDERYESMMDAFDDQRFLRRLKTSDNGSICYQL HLEEMETILDNQGRFYPTLANEKDKLLSLVTFRIPYYVGPLTQRNAREDA HGKLRFAWSERKPGTEGVTITPWNWEDVIDKGASAEKFITRMTGICTYL QGEDVLPKSSLLYEEFCVLNELNGMRWTSDGDEERRFDAEQREGMMRTABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO DLFRKTRTVSNKRIEDWLVQNGYAHSPKVSGGQNETGLESKLGSYIFFA KDIFGTDEIAKVDYPMIEEIILWNTLFEDRSILKEKLREKYGDAGEGRLSAE QIKKICKKRFTGWGRLSKKFLAGIKVDTQLGEKSIMDVLREGDPDADIRR GRTMVLMEVIRDDNLGFQKKIDSFNRAYFAEAGSALGVNDLPGSPAIRR SLNQAIRIVDEIAHIAGHAPTNVFIEVTRDDDMGKKGHRTARRYDQLKEAL AAFKKDDPQIWKEIQAASPTDLDERLTLYFVQRGKCMYSGKPIDINQLSN AGLYEVDHIIPRSYIKDDSFENKALVLREFNQRKTDSMLLDDGIRRNMAG YWRSLHDAKLIGDKKFNNLMRTHVGENAMRGFIARQLVETSQMVKLAQ SLLSAKYPDTKITPVKASMSHDLREAAGFVKCREANDFHHAHDAFLACR VGLFIRKWYPDMYDKPILYTRAMRKYVREQAEEFKKTRRAPGSSGFVVG RFISSFVDADTGELWEGAEEVESIRRVLNYRQCHITRMPMEDSGAFWNA TIYSPRDPKMGSKLSLRLKGDLDPQIYGGYSSQQFAYFFIYEARDKKGRP AFRFSQVPVWLASRIASDEWALEEYAREQAASEQLEFVRIQRSKILKKQL VEIDGERLIITGAEEVRNARQFAFSLDELSLLGRASARGDGEAIDEETISN GYNVLLKVMIERGELVGKGLMKKLSLGDRYEQFMQLDCAAKKSLILQLA AIVNASNRQIDLSKIGGAKTAGQYKVTYNKLLNDPNTDFYIVDQSVTGMF ERRTRVGL DRCY Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSNLRNFKGDYSVGLD 39 Cas MGTGSVGWSVTDEDGNLLHFKKQPTWGSRLFDSAQTAAEARTPRGQR mammalian RRYVRRRWRLDLLQSLLQEEIERVDPDFFLRLRQSRLVNTDPNKTTSDY expression RWPLFNGSDFTEVDYYNKFPTIYHLRKWLETDEQADIRLVYLALHNIVKH construct RGNFLREGQKLSSENANPDDAVSEFCTALKEWCEGQEGDCGALKEADI (with N- VAVLTARGMRPSDCAADLLELLPVSFGDGDKQADKKLKKALASAMVGLK terminal AEFKDIFGDFATEGTKISLSKDEESEALLEVCPDEGKVLFESLQRVHSAY methionine, VLQGLLSYAPGESISANMIAKYDQYAKDLRMLKLLVREYAPTSYGDFFRG stop codon, PLCPNKGGYDKFDASVKGYTRYNLGVTSYDDFAKEVKKLFVGTGAEDD V5 tag, N- ERYESMMDAFDDQRFLRRLKTSDNGSICYQLHLEEMETILDNQGRFYPT terminal NLS LANEKDKLLSLVTFRIPYYVGPLTQRNAREDAHGKLRFAWSERKPGTEG and C- VTITPWNWEDVIDKGASAEKFITRMTGICTYLQGEDVLPKSSLLYEEFCVL terminal NLS) NELNGMRWTSDGDEERRFDAEQREGMMRDLFRKTRTVSNKRIEDWLV (aa) QNGYAHSPKVSGGQNETGLESKLGSYIFFAKDIFGTDEIAKVDYPMIEEIIL WNTLFEDRSILKEKLREKYGDAGEGRLSAEQIKKICKKRFTGWGRLSKKF LAGIKVDTQLGEKSIMDVLREGDPDADIRRGRTMVLMEVIRDDNLGFQKK IDSFNRAYFAEAGSALGVNDLPGSPAIRRSLNQAIRIVDEIAHIAGHAPTNV FIEVTRDDDMGKKGHRTARRYDQLKEALAAFKKDDPQIWKEIQAASPTD LDERLTLYFVQRGKCMYSGKPIDINQLSNAGLYEVDHIIPRSYIKDDSFEN KALVLREFNQRKTDSMLLDDGIRRNMAGYWRSLHDAKLIGDKKFNNLMR THVGENAMRGFIARQLVETSQMVKLAQSLLSAKYPDTKITPVKASMSHD LREAAGFVKCREANDFHHAHDAFLACRVGLFIRKWYPDMYDKPILYTRA MRKYVREQAEEFKKTRRAPGSSGFVVGRFISSFVDADTGELWEGAEEV ESIRRVLNYRQCHITRMPMEDSGAFWNATIYSPRDPKMGSKLSLRLKGD LDPQIYGGYSSQQFAYFFIYEARDKKGRPAFRFSQVPVWLASRIASDEW ALEEYAREQAASEQLEFVRIQRSKILKKQLVEIDGERLIITGAEEVRNARQ FAFSLDELSLLGRASARGDGEAIDEETISNGYNVLLKVMIERGELVGKGL MKKLSLGDRYEQFMQLDCAAKKSLILQLAAIVNASNRQIDLSKIGGAKTA GQYKVTYNKLLNDPNTDFYIVDQSVTGMFERRTRVGLSRKRTADGSEFE SPKKKRKV DRCY Type II GTTAATTTGCGGAATTTCAAGGGCGACTACAGCGTTGGTCTCGATAT 40 Cas coding GGGAACAGGCTCCGTTGGCTGGTCGGTGACGGACGAAGACGGCAAC sequence (nt) CTCCTTCACTTTAAAAAGCAACCAACATGGGGAAGCCGTTTGTTCGAT (not codon AGTGCTCAAACGGCAGCCGAGGCACGCACCCCTCGTGGGCAACGAA optimized) GGCGTTATGTTCGACGTCGTTGGCGACTCGACCTGCTGCAGAGTCTA CTCCAGGAAGAAATTGAACGTGTCGACCCAGATTTCTTCCTGCGTCTT CGCCAATCGCGTCTCGTAAATACGGACCCCAATAAGACGACCTCGGA TTATCGCTGGCCACTATTTAACGGAAGCGATTTTACCGAGGTCGATTA CTATAACAAGTTTCCAACTATCTATCATCTGCGTAAATGGCTGATGGA AACCGATGAGCAAGCTGATATCCGTCTTGTTTATTTGGCGCTTCACAATABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO CATAGTGAAGCATCGTGGTAACTTCTTGCGCGAAGGGCAGAAACTTT CTTCGGAAAATGCCAACCCTGATGACGCGGTTTCGGAATTTTGCACT GCGCTGAAAGAGTGGTGCGAAGGTCAAGAAGGTGACTGTGGGGCAC TCAAAGAGGCAGATATCGTTGCGGTTCTTACGGCGAGAGGCATGCGT CCGTCTGATTGCGCCGCTGATCTTTTGGAGCTACTCCCAGTTTCTTTT GGCGATGGTGACAAGCAGGCTGATAAGAAGCTTAAGAAAGCGCTCG CTAGCGCTATGGTTGGCTTGAAAGCTGAGTTCAAAGACATATTCGGT GATTTCGCTACTGAAGGAACGAAGATATCGCTCTCGAAGGACGAAGA ATCAGAAGCGCTGCTTGAAGTGTGCCCTGATGAGGGTAAGGTGCTTT TCGAGTCGCTGCAACGAGTTCATTCCGCATATGTACTCCAAGGGTTG CTATCTTATGCTCCTGGTGAATCAATTTCGGCAAATATGATTGCGAAG TACGATCAGTATGCTAAAGACTTGAGGATGCTGAAGTTACTGGTTCGC GAATATGCGCCAACGTCGTATGGCGACTTCTTCCGTGGTCCGCTTTG TCCGAATAAGGGCGGCTATGACAAGTTTGATGCTAGTGTTAAGGGCT ACACGCGCTACAACCTTGGAGTCACTTCATACGATGACTTTGCCAAA GAAGTAAAAAAGCTATTCGTAGGCACGGGTGCCGAGGACGATGAGC GCTACGAGTCTATGATGGACGCCTTCGATGACCAGCGATTCCTTCGT CGATTGAAAACGAGCGACAATGGAAGCATCTGTTATCAGCTGCACCT CGAAGAGATGGAGACGATCCTCGATAATCAGGGACGCTTCTATCCGA CCCTCGCGAATGAGAAGGATAAGCTATTGTCGCTGGTCACCTTCCGC ATTCCGTATTATGTGGGGCCGCTTACGCAGCGCAATGCGCGTGAGGA CGCACATGGAAAGCTTCGCTTCGCGTGGTCCGAACGCAAGCCGGGG ACCGAAGGTGTGACGATTACGCCTTGGAACTGGGAAGATGTTATCGA CAAGGGCGCGAGTGCCGAGAAATTCATCACGCGGATGACGGGTATA TGTACTTACTTGCAGGGCGAGGACGTGCTGCCGAAGTCGTCTCTTCT CTATGAGGAATTTTGCGTTCTGAATGAACTGAACGGTATGCGCTGGA CGTCCGATGGCGACGAAGAACGGCGATTCGATGCCGAGCAGCGTGA AGGGATGATGCGCGACTTGTTCCGCAAGACGCGCACGGTTTCGAATA AGAGGATTGAAGATTGGCTCGTTCAGAACGGGTACGCGCATTCTCCG AAGGTGTCGGGCGGGCAGAACGAAACAGGTTTGGAATCTAAACTCG GATCCTACATTTTCTTCGCTAAGGATATCTTCGGAACAGATGAAATTG CCAAAGTCGATTATCCGATGATTGAAGAGATCATTCTATGGAACACCT TGTTCGAAGATCGTTCGATCCTGAAAGAGAAGCTGCGAGAAAAGTAT GGCGATGCAGGTGAAGGCCGTTTGAGCGCTGAACAAATCAAGAAGAT TTGCAAAAAACGCTTCACGGGGTGGGGTCGCTTGTCGAAGAAGTTCT TGGCGGGGATTAAAGTCGATACGCAGCTTGGTGAGAAGTCGATTATG GACGTGCTGCGTGAAGGCGATCCGGATGCTGATATTCGACGTGGTC GCACGATGGTACTTATGGAAGTAATTCGCGACGATAACCTGGGTTTC CAAAAGAAGATCGACTCATTTAACCGTGCGTACTTTGCCGAGGCTGG CAGTGCGCTTGGCGTGAACGATCTTCCAGGGTCGCCCGCAATTCGTC GCAGTCTCAACCAGGCGATTCGCATTGTCGACGAGATTGCTCATATC GCAGGTCATGCTCCGACAAATGTCTTCATCGAGGTCACGCGTGATGA CGACATGGGCAAGAAGGGCCATCGCACTGCCCGTCGCTACGATCAG TTGAAAGAAGCGCTTGCTGCCTTTAAGAAGGACGATCCTCAAATATG GAAGGAGATACAGGCGGCCAGTCCAACCGATTTGGACGAACGTCTC ACACTGTATTTTGTGCAGCGAGGCAAATGCATGTATTCGGGTAAGCC CATCGATATCAATCAGCTTTCGAACGCTGGACTGTACGAGGTTGACC ATATCATCCCCCGATCCTATATTAAGGACGATAGTTTCGAAAATAAGG CGCTGGTGCTTCGCGAATTCAACCAGCGTAAAACGGATTCGATGTTG CTTGACGACGGCATCCGCCGCAACATGGCTGGCTATTGGCGCAGTTT GCATGATGCGAAACTTATTGGCGACAAGAAGTTCAATAATCTCATGCG CACGCACGTCGGCGAAAACGCTATGCGCGGATTTATCGCTCGTCAGT TGGTGGAGACAAGCCAAATGGTGAAGCTTGCCCAGTCGCTTCTTTCC GCCAAATATCCTGACACGAAGATCACTCCAGTGAAGGCGAGCATGTC TCATGATTTGCGCGAAGCGGCAGGGTTTGTGAAATGCCGCGAGGCA AATGATTTTCATCATGCACACGATGCCTTTTTGGCGTGCAGAGTGGGT CTTTTTATTCGGAAGTGGTACCCCGACATGTATGACAAGCCCATTCTCTABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO TATACGCGTGCGATGAGAAAATACGTACGAGAGCAGGCGGAGGAGT TCAAAAAGACTCGTCGGGCACCAGGCTCGAGTGGATTCGTGGTTGG GCGCTTTATAAGTTCGTTCGTTGACGCCGATACTGGTGAACTGTGGG AGGGCGCCGAAGAGGTTGAGAGTATTCGTCGTGTGCTGAATTACCGC CAGTGCCACATCACGCGCATGCCGATGGAAGACTCAGGTGCGTTTTG GAACGCAACTATTTATTCGCCGCGCGACCCGAAGATGGGGAGTAAGC TTTCGTTGCGCTTGAAGGGCGATCTGGACCCACAGATTTATGGTGGC TATTCAAGCCAGCAATTTGCATATTTCTTCATTTACGAGGCGCGCGAT AAGAAGGGGCGCCCTGCGTTCCGTTTCTCCCAGGTACCCGTGTGGC TGGCGTCGCGGATTGCGAGCGACGAATGGGCACTCGAAGAATACGC GCGTGAGCAGGCCGCAAGCGAGCAGCTCGAGTTTGTGCGTATTCAG CGTTCGAAGATCCTGAAGAAGCAGCTGGTAGAGATCGACGGAGAGC GGTTGATTATTACAGGAGCTGAAGAGGTTCGGAATGCTAGGCAGTTT GCGTTTAGTCTGGATGAACTCTCGCTTCTTGGAAGGGCGTCAGCGCG AGGCGATGGCGAGGCAATTGACGAGGAAACTATATCAAATGGATATA ACGTGCTATTGAAAGTAATGATTGAACGTGGCGAGCTGGTTGGAAAG GGCCTTATGAAGAAGCTGTCTCTCGGTGATCGCTACGAGCAATTTAT GCAATTGGACTGCGCGGCAAAGAAATCGCTAATCCTTCAGCTCGCCG CTATTGTTAATGCTTCTAATAGGCAGATTGACTTGTCGAAGATTGGAG GGGCTAAAACGGCCGGTCAGTACAAGGTAACTTATAACAAGCTTCTC AATGACCCTAACACCGATTTCTACATTGTCGATCAGTCGGTGACGGG GATGTTCGAGCGGAGGACGCGCGTTGGCCTTTAG DRCY Type II AACCTGCGCAACTTCAAGGGCGACTACAGCGTCGGACTGGACATGG 41 Cas coding GCACAGGCTCTGTGGGCTGGTCCGTGACCGATGAGGACGGCAATCT sequence (nt) GCTGCACTTCAAAAAGCAGCCTACATGGGGAAGCAGACTGTTCGACA (human GCGCCCAGACCGCCGCTGAAGCCAGAACCCCTAGAGGCCAGCGGC codon- GGCGGTACGTGCGGAGAAGGTGGCGGCTCGACCTGCTGCAAAGCCT optimized; GCTGCAGGAGGAAATCGAGAGAGTGGACCCTGATTTCTTCCTGCGG lacking N- CTGAGACAGAGCAGACTGGTGAATACCGACCCCAACAAGACCACCA terminal GCGATTATAGATGGCCTCTGTTTAACGGCAGCGACTTTACCGAGGTG methionine; GACTACTACAACAAGTTCCCCACAATCTACCACCTGAGAAAGTGGCT no stop GGAAACCGACGAGCAGGCTGACATCCGGCTGGTGTACCTGGCCCTG codon) CACAACATCGTGAAGCACAGAGGCAACTTCCTGCGCGAGGGCCAGA AGCTCAGCTCAGAGAACGCCAATCCTGACGATGCCGTGTCCGAGTTC TGCACCGCCCTGAAGGAGTGGTGTGAAGGCCAGGAGGGAGATTGCG GCGCCCTGAAAGAGGCCGACATCGTGGCCGTGCTTACAGCCCGGGG AATGCGACCCAGCGACTGCGCCGCTGACCTGCTGGAATTGCTGCCT GTGAGCTTCGGCGACGGCGATAAGCAGGCAGACAAGAAACTTAAGA AGGCCCTGGCCAGCGCTATGGTGGGCCTGAAAGCCGAGTTCAAGGA CATCTTCGGCGATTTCGCCACAGAGGGCACTAAGATCAGCCTGTCTA AGGACGAGGAAAGCGAGGCTCTGCTGGAAGTTTGTCCTGACGAGGG CAAGGTCCTGTTCGAGAGCCTGCAGAGAGTCCACTCTGCCTACGTGC TGCAAGGCCTGCTGTCTTATGCCCCTGGCGAGTCTATCTCCGCCAAT ATGATCGCCAAGTACGATCAGTACGCCAAAGATCTGCGGATGCTGAA GCTGCTGGTGCGGGAGTACGCCCCTACCAGCTACGGCGACTTCTTC AGGGGACCTCTGTGCCCCAACAAGGGCGGATACGACAAGTTCGACG CCAGCGTGAAAGGCTACACCAGATACAACCTGGGCGTGACCTCCTAC GACGACTTCGCCAAGGAGGTGAAGAAGCTGTTCGTGGGCACCGGAG CTGAGGACGACGAGAGATACGAGAGCATGATGGATGCGTTCGACGA CCAACGGTTCCTGCGGAGACTGAAAACAAGTGATAACGGATCTATCT GCTACCAGCTCCATCTGGAAGAGATGGAGACAATCCTGGACAACCAG GGCAGATTCTACCCTACGCTGGCCAACGAGAAGGACAAGCTGCTGA GCCTCGTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGACCCAG AGAAACGCTAGAGAGGATGCCCACGGCAAGCTGCGGTTCGCTTGGA GCGAGAGAAAACCTGGCACCGAGGGCGTGACAATTACACCCTGGAA CTGGGAGGATGTGATCGACAAGGGCGCCAGCGCCGAGAAGTTCATC ACCAGAATGACCGGAATCTGCACCTACCTGCAGGGAGAAGATGTTCTTABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO GCCTAAGAGCTCTCTGCTGTATGAGGAGTTCTGCGTGCTGAACGAGC TGAATGGTATGAGATGGACCAGCGACGGCGACGAGGAGAGAAGATT TGATGCCGAGCAACGGGAGGGCATGATGCGGGACCTGTTCAGAAAG ACCAGAACTGTGTCCAACAAGAGAATTGAGGACTGGCTGGTGCAGAA CGGCTACGCCCACAGCCCTAAGGTGAGCGGCGGACAAAACGAGACA GGCCTAGAAAGCAAGCTCGGATCTTACATCTTCTTTGCCAAGGACAT CTTTGGCACTGACGAGATTGCCAAGGTGGATTACCCTATGATCGAGG AGATCATCCTGTGGAACACCCTGTTTGAGGATCGGAGCATCCTCAAA GAGAAGCTAAGAGAGAAGTACGGCGACGCCGGCGAGGGCAGACTGT CAGCCGAGCAGATCAAGAAGATCTGTAAAAAAAGATTCACCGGCTGG GGCAGACTGAGCAAGAAGTTCCTCGCCGGCATCAAGGTGGATACCC AGCTGGGCGAGAAAAGCATCATGGACGTGCTGCGGGAAGGCGATCC CGACGCGGATATCAGACGGGGCCGGACAATGGTGCTGATGGAAGTG ATCCGGGACGACAATCTGGGATTTCAGAAAAAAATCGACAGCTTCAAT AGAGCATATTTCGCCGAGGCCGGTAGCGCTCTGGGCGTTAACGACC TGCCAGGCAGCCCTGCTATCCGGAGAAGCCTGAACCAGGCCATCCG GATCGTGGACGAAATCGCCCATATCGCAGGCCACGCCCCTACAAAC GTGTTCATCGAGGTGACACGGGATGACGACATGGGCAAGAAAGGCC ACAGAACTGCCCGGAGATACGACCAGCTGAAGGAGGCCCTGGCCGC CTTTAAAAAGGACGATCCCCAGATTTGGAAGGAAATCCAGGCCGCCA GTCCTACCGACCTGGACGAGCGGCTGACACTGTACTTCGTCCAGAGA GGAAAATGCATGTACAGCGGAAAGCCTATCGATATTAACCAGCTGAG CAACGCCGGCCTGTATGAAGTGGACCACATCATCCCTAGATCTTACA TCAAGGACGATTCTTTTGAAAATAAGGCCCTGGTGCTGCGGGAGTTC AACCAGAGAAAGACCGATAGCATGCTACTGGATGACGGCATCAGAAG AAACATGGCCGGCTACTGGAGAAGCCTGCACGACGCTAAGCTGATC GGCGATAAGAAGTTCAACAACCTGATGAGAACACACGTGGGTGAGAA CGCCATGAGAGGCTTTATCGCTAGGCAGCTGGTCGAAACCAGCCAG ATGGTCAAGCTGGCCCAGAGCCTGCTGTCCGCTAAGTACCCCGACA CCAAGATCACACCTGTGAAGGCCTCTATGAGCCACGACCTGAGAGAG GCCGCCGGCTTCGTGAAGTGCCGGGAAGCTAACGACTTCCACCACG CCCACGATGCCTTTCTGGCCTGCAGAGTGGGACTGTTTATTAGAAAG TGGTATCCTGACATGTACGACAAGCCCATCCTGTACACCAGGGCCAT GAGAAAGTATGTGCGAGAGCAGGCCGAAGAGTTCAAGAAAACCCGG AGAGCTCCAGGCTCCTCCGGATTCGTGGTGGGCCGGTTTATCAGCA GCTTCGTGGATGCCGACACCGGCGAGCTGTGGGAAGGCGCCGAAGA GGTGGAAAGTATCAGAAGAGTGCTGAACTACAGACAGTGCCACATCA CCCGGATGCCTATGGAGGATAGCGGCGCCTTCTGGAATGCCACAAT CTACAGCCCCAGAGATCCGAAAATGGGCAGCAAATTATCACTGCGCC TGAAGGGCGATCTGGACCCACAGATCTACGGGGGCTATAGCTCTCA GCAGTTTGCCTACTTCTTCATCTACGAGGCCCGGGATAAAAAGGGCA GACCTGCCTTCAGATTCAGCCAAGTGCCAGTGTGGCTGGCAAGCAGA ATCGCCTCCGACGAATGGGCGCTGGAGGAATATGCCAGAGAGCAGG CCGCCAGCGAACAACTGGAGTTCGTGAGAATCCAGCGGTCCAAGAT CCTGAAGAAGCAGCTGGTCGAGATCGATGGTGAGCGGCTCATCATCA CAGGCGCCGAGGAAGTGCGCAATGCCCGGCAGTTCGCTTTTAGCTT GGATGAACTGAGCCTCCTCGGCAGGGCCAGCGCTAGAGGCGACGG CGAAGCAATTGACGAAGAAACCATCTCTAATGGCTACAACGTGCTGC TGAAGGTGATGATCGAACGGGGCGAGCTGGTGGGCAAAGGCCTGAT GAAGAAACTGTCTCTGGGAGACAGATACGAGCAGTTCATGCAGCTGG ATTGTGCCGCCAAGAAGTCCCTGATCCTCCAGCTCGCAGCCATCGTG AACGCCAGCAACAGACAGATCGATCTGAGCAAAATTGGCGGCGCCAA GACCGCCGGCCAGTACAAGGTAACCTACAACAAGCTGCTGAACGAC CCTAATACCGATTTCTACATCGTGGACCAAAGCGTGACCGGCATGTT CGAGCGGAGAACCAGAGTCGGACTG DRCY Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 42 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGGTABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO mammalian AAAGTGGGATCCAACCTGCGCAACTTCAAGGGCGACTACAGCGTCG expression GACTGGACATGGGCACAGGCTCTGTGGGCTGGTCCGTGACCGATGA construct GGACGGCAATCTGCTGCACTTCAAAAAGCAGCCTACATGGGGAAGCA (with N- GACTGTTCGACAGCGCCCAGACCGCCGCTGAAGCCAGAACCCCTAG terminal AGGCCAGCGGCGGCGGTACGTGCGGAGAAGGTGGCGGCTCGACCT methionine, GCTGCAAAGCCTGCTGCAGGAGGAAATCGAGAGAGTGGACCCTGAT stop codon, TTCTTCCTGCGGCTGAGACAGAGCAGACTGGTGAATACCGACCCCAA V5 tag, N- CAAGACCACCAGCGATTATAGATGGCCTCTGTTTAACGGCAGCGACT terminal NLS TTACCGAGGTGGACTACTACAACAAGTTCCCCACAATCTACCACCTGA and C- GAAAGTGGCTGGAAACCGACGAGCAGGCTGACATCCGGCTGGTGTA terminal NLS) CCTGGCCCTGCACAACATCGTGAAGCACAGAGGCAACTTCCTGCGC (nt) GAGGGCCAGAAGCTCAGCTCAGAGAACGCCAATCCTGACGATGCCG TGTCCGAGTTCTGCACCGCCCTGAAGGAGTGGTGTGAAGGCCAGGA GGGAGATTGCGGCGCCCTGAAAGAGGCCGACATCGTGGCCGTGCTT ACAGCCCGGGGAATGCGACCCAGCGACTGCGCCGCTGACCTGCTGG AATTGCTGCCTGTGAGCTTCGGCGACGGCGATAAGCAGGCAGACAA GAAACTTAAGAAGGCCCTGGCCAGCGCTATGGTGGGCCTGAAAGCC GAGTTCAAGGACATCTTCGGCGATTTCGCCACAGAGGGCACTAAGAT CAGCCTGTCTAAGGACGAGGAAAGCGAGGCTCTGCTGGAAGTTTGTC CTGACGAGGGCAAGGTCCTGTTCGAGAGCCTGCAGAGAGTCCACTC TGCCTACGTGCTGCAAGGCCTGCTGTCTTATGCCCCTGGCGAGTCTA TCTCCGCCAATATGATCGCCAAGTACGATCAGTACGCCAAAGATCTG CGGATGCTGAAGCTGCTGGTGCGGGAGTACGCCCCTACCAGCTACG GCGACTTCTTCAGGGGACCTCTGTGCCCCAACAAGGGCGGATACGA CAAGTTCGACGCCAGCGTGAAAGGCTACACCAGATACAACCTGGGC GTGACCTCCTACGACGACTTCGCCAAGGAGGTGAAGAAGCTGTTCGT GGGCACCGGAGCTGAGGACGACGAGAGATACGAGAGCATGATGGAT GCGTTCGACGACCAACGGTTCCTGCGGAGACTGAAAACAAGTGATAA CGGATCTATCTGCTACCAGCTCCATCTGGAAGAGATGGAGACAATCC TGGACAACCAGGGCAGATTCTACCCTACGCTGGCCAACGAGAAGGA CAAGCTGCTGAGCCTCGTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGACCCAGAGAAACGCTAGAGAGGATGCCCACGGCAAGCTGCG GTTCGCTTGGAGCGAGAGAAAACCTGGCACCGAGGGCGTGACAATT ACACCCTGGAACTGGGAGGATGTGATCGACAAGGGCGCCAGCGCCG AGAAGTTCATCACCAGAATGACCGGAATCTGCACCTACCTGCAGGGA GAAGATGTTCTGCCTAAGAGCTCTCTGCTGTATGAGGAGTTCTGCGT GCTGAACGAGCTGAATGGTATGAGATGGACCAGCGACGGCGACGAG GAGAGAAGATTTGATGCCGAGCAACGGGAGGGCATGATGCGGGACC TGTTCAGAAAGACCAGAACTGTGTCCAACAAGAGAATTGAGGACTGG CTGGTGCAGAACGGCTACGCCCACAGCCCTAAGGTGAGCGGCGGAC AAAACGAGACAGGCCTAGAAAGCAAGCTCGGATCTTACATCTTCTTTG CCAAGGACATCTTTGGCACTGACGAGATTGCCAAGGTGGATTACCCT ATGATCGAGGAGATCATCCTGTGGAACACCCTGTTTGAGGATCGGAG CATCCTCAAAGAGAAGCTAAGAGAGAAGTACGGCGACGCCGGCGAG GGCAGACTGTCAGCCGAGCAGATCAAGAAGATCTGTAAAAAAAGATT CACCGGCTGGGGCAGACTGAGCAAGAAGTTCCTCGCCGGCATCAAG GTGGATACCCAGCTGGGCGAGAAAAGCATCATGGACGTGCTGCGGG AAGGCGATCCCGACGCGGATATCAGACGGGGCCGGACAATGGTGCT GATGGAAGTGATCCGGGACGACAATCTGGGATTTCAGAAAAAAATCG ACAGCTTCAATAGAGCATATTTCGCCGAGGCCGGTAGCGCTCTGGGC GTTAACGACCTGCCAGGCAGCCCTGCTATCCGGAGAAGCCTGAACC AGGCCATCCGGATCGTGGACGAAATCGCCCATATCGCAGGCCACGC CCCTACAAACGTGTTCATCGAGGTGACACGGGATGACGACATGGGCA AGAAAGGCCACAGAACTGCCCGGAGATACGACCAGCTGAAGGAGGC CCTGGCCGCCTTTAAAAAGGACGATCCCCAGATTTGGAAGGAAATCC AGGCCGCCAGTCCTACCGACCTGGACGAGCGGCTGACACTGTACTT CGTCCAGAGAGGAAAATGCATGTACAGCGGAAAGCCTATCGATATTATABLE 1G DRCY Type II Cas Sequences Name Sequence SEQ ID NO ACCAGCTGAGCAACGCCGGCCTGTATGAAGTGGACCACATCATCCCT AGATCTTACATCAAGGACGATTCTTTTGAAAATAAGGCCCTGGTGCTG CGGGAGTTCAACCAGAGAAAGACCGATAGCATGCTACTGGATGACG GCATCAGAAGAAACATGGCCGGCTACTGGAGAAGCCTGCACGACGC TAAGCTGATCGGCGATAAGAAGTTCAACAACCTGATGAGAACACACG TGGGTGAGAACGCCATGAGAGGCTTTATCGCTAGGCAGCTGGTCGA AACCAGCCAGATGGTCAAGCTGGCCCAGAGCCTGCTGTCCGCTAAG TACCCCGACACCAAGATCACACCTGTGAAGGCCTCTATGAGCCACGA CCTGAGAGAGGCCGCCGGCTTCGTGAAGTGCCGGGAAGCTAACGAC TTCCACCACGCCCACGATGCCTTTCTGGCCTGCAGAGTGGGACTGTT TATTAGAAAGTGGTATCCTGACATGTACGACAAGCCCATCCTGTACAC CAGGGCCATGAGAAAGTATGTGCGAGAGCAGGCCGAAGAGTTCAAG AAAACCCGGAGAGCTCCAGGCTCCTCCGGATTCGTGGTGGGCCGGT TTATCAGCAGCTTCGTGGATGCCGACACCGGCGAGCTGTGGGAAGG CGCCGAAGAGGTGGAAAGTATCAGAAGAGTGCTGAACTACAGACAGT GCCACATCACCCGGATGCCTATGGAGGATAGCGGCGCCTTCTGGAA TGCCACAATCTACAGCCCCAGAGATCCGAAAATGGGCAGCAAATTAT CACTGCGCCTGAAGGGCGATCTGGACCCACAGATCTACGGGGGCTA TAGCTCTCAGCAGTTTGCCTACTTCTTCATCTACGAGGCCCGGGATAA AAAGGGCAGACCTGCCTTCAGATTCAGCCAAGTGCCAGTGTGGCTG GCAAGCAGAATCGCCTCCGACGAATGGGCGCTGGAGGAATATGCCA GAGAGCAGGCCGCCAGCGAACAACTGGAGTTCGTGAGAATCCAGCG GTCCAAGATCCTGAAGAAGCAGCTGGTCGAGATCGATGGTGAGCGG CTCATCATCACAGGCGCCGAGGAAGTGCGCAATGCCCGGCAGTTCG CTTTTAGCTTGGATGAACTGAGCCTCCTCGGCAGGGCCAGCGCTAGA GGCGACGGCGAAGCAATTGACGAAGAAACCATCTCTAATGGCTACAA CGTGCTGCTGAAGGTGATGATCGAACGGGGCGAGCTGGTGGGCAAA GGCCTGATGAAGAAACTGTCTCTGGGAGACAGATACGAGCAGTTCAT GCAGCTGGATTGTGCCGCCAAGAAGTCCCTGATCCTCCAGCTCGCA GCCATCGTGAACGCCAGCAACAGACAGATCGATCTGAGCAAAATTGG CGGCGCCAAGACCGCCGGCCAGTACAAGGTAACCTACAACAAGCTG CTGAACGACCCTAATACCGATTTCTACATCGTGGACCAAAGCGTGAC CGGCATGTTCGAGCGGAGAACCAGAGTCGGACTGTCTAGAAAGCGG ACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGT GTGA

[0094] In some embodiments a DRCY Type II 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 DRCY Type II 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 D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H893A substitution, wherein the position of the H893A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38. In some embodiments, a DRCY Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with a H893A substitution. 6.2.1.8. DRPY Type II Cas Proteins

[0095] In one aspect, the disclosure provides DRPY Type II Cas proteins. DRPY Type II Cas proteins can be further classified as Type IIA Cas proteins. The DRPY Type II 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 least75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:43. In some embodiments, the DRPY Type II 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 DRPY Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:43.

[0096] Exemplary DRPY Type II Cas protein sequences and nucleotide sequences encoding exemplary DRPY Type II Cas proteins are set forth in Table 1H. TABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO DRPY Type II KKRAIEEYYLGLDIGTNSVGYAVTDPQYKILKYHGEPMWGSHVFEEGSQ 43 Cas amino CAERRGFRTARRRLNRRQQRVRLVQEIFAHEIEKVDSRFFLRLKESALYR acid EDANGNDPFILFNDNGYTDKEYYLKYPTIHHLIMDLIDDENPHDVRLVYLA sequence VAWLVAHRGHFLSDVEKTNVEKVLDFSTSYNALKEMYSGIGQDLPWIDE (without N- EETFKNILLKKCGVKDKEKAFKDKLLKNNSNKKNSDITEESEVLGISAVITL terminal LSGGTVAADKLFVQAEFQDKISISFKKNEDEFEQILTELDEYAEYLLKIRAV methionine) YDWAVLYEASKGQVYISKAKIEVYEQHKKDLAGLKAFIRKYCPDKYNEIF RDAGDNNYVAYSYNFNSLNLTDGEKRPKRKASQEDFCTYIRKIVKDIKCE MEDEEFYDDMWQRLELGTFMPKQVNTDNRVLPYQLYYFELARLLGRVS AYLPFINEKDNDGYITKEKLLSIMEFRIPYYVGPLHRENKDNNSFAWIKRK AQGRIYPWNYEEKIDLDASEQAFINKMTNQCSYLPGEDVLPKYSLLYCKY EVLNEINNIKINGQKITVECKKGIFGLFKKNKKVTVKKIKDFLISNNYMGTS DEITGLDISVKSSLKSYHDFRKMLENKIVNEEDVEEIIKRLTYTEDKKRINR YLEENYPNISEEDRRYISKLKYKDFGRLSEKFLTGIKGVVKETGEILSVIQI MLETNDNLMQIIFSDHYMIKDALEREKKDYYSEHPASVEQILSDMYISNAV KRPIYRTLDIISDVTKACQNAPRKIFVEMARGGGEKGKRTISRRDKIKELY KNMDKQEVREISKEISELLGELDKKTDNELQSEVLFLYFMQLGICMYSGD PIDITKLKSDAYVNVDHIYPQAYVKDDSLNNKVLVKSKLNGEKSDNYPINK DIRDKMSSIWKHYRKEGLISEEKYNRLTRKTPFTDEEKQGFVNRQLVETR QSTKAVAEILKMMFPETEVVYVKAGLASEFRHVFGIIKSRQINDLHHAKDA YLNVVCGNVYHSTFTKNFFLKHQTYSIKTETVYKRRIEADGKLIWNGQES LSFVKKMLGKNNIHYTRYAFCRKGGFFDQMPVTAKEGLVSRKKDLPSEK YGGYNKPTASYFIMAKYTEKVKKEKQDIMIVPVELMVGEKVLNDKNFAVG YIQQQIAQISNRKVEEIVNVSFPLGLRPLKVNTRFSFDGFEACVTGKDSG GKNISMSSLTSLKVSQANEKYIKKVERFAEKKEVNNKITLDEKYDEINENL NFEIYRMLQQKITKSCFGVAFGNLSNVFEKGENKFVALKTDMQCEVLLNII NVFKTGRSGTCNLKSIGGVEKAASYRISAKVSNWKKNYTCVKIIDESASGI FRKESENILEWL DRPY Type II MKKRAIEEYYLGLDIGTNSVGYAVTDPQYKILKYHGEPMWGSHVFEEGS 44 Cas amino QCAERRGFRTARRRLNRRQQRVRLVQEIFAHEIEKVDSRFFLRLKESAL acid YREDANGNDPFILFNDNGYTDKEYYLKYPTIHHLIMDLIDDENPHDVRLVY sequence LAVAWLVAHRGHFLSDVEKTNVEKVLDFSTSYNALKEMYSGIGQDLPWI DEEETFKNILLKKCGVKDKEKAFKDKLLKNNSNKKNSDITEESEVLGISAV ITLLSGGTVAADKLFVQAEFQDKISISFKKNEDEFEQILTELDEYAEYLLKI RAVYDWAVLYEASKGQVYISKAKIEVYEQHKKDLAGLKAFIRKYCPDKYN EIFRDAGDNNYVAYSYNFNSLNLTDGEKRPKRKASQEDFCTYIRKIVKDIK CEMEDEEFYDDMWQRLELGTFMPKQVNTDNRVLPYQLYYFELARLLGR VSAYLPFINEKDNDGYITKEKLLSIMEFRIPYYVGPLHRENKDNNSFAWIK RKAQGRIYPWNYEEKIDLDASEQAFINKMTNQCSYLPGEDVLPKYSLLYC KYEVLNEINNIKINGQKITVECKKGIFGLFKKNKKVTVKKIKDFLISNNYMG TSDEITGLDISVKSSLKSYHDFRKMLENKIVNEEDVEEIIKRLTYTEDKKRI NRYLEENYPNISEEDRRYISKLKYKDFGRLSEKFLTGIKGVVKETGEILSVI QIMLETNDNLMQIIFSDHYMIKDALEREKKDYYSEHPASVEQILSDMYISN AVKRPIYRTLDIISDVTKACQNAPRKIFVEMARGGGEKGKRTISRRDKIKE LYKNMDKQEVREISKEISELLGELDKKTDNELQSEVLFLYFMQLGICMYS GDPIDITKLKSDAYVNVDHIYPQAYVKDDSLNNKVLVKSKLNGEKSDNYPI NKDIRDKMSSIWKHYRKEGLISEEKYNRLTRKTPFTDEEKQGFVNRQLVETABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO TRQSTKAVAEILKMMFPETEVVYVKAGLASEFRHVFGIIKSRQINDLHHAK DAYLNVVCGNVYHSTFTKNFFLKHQTYSIKTETVYKRRIEADGKLIWNGQ ESLSFVKKMLGKNNIHYTRYAFCRKGGFFDQMPVTAKEGLVSRKKDLPS EKYGGYNKPTASYFIMAKYTEKVKKEKQDIMIVPVELMVGEKVLNDKNFA VGYIQQQIAQISNRKVEEIVNVSFPLGLRPLKVNTRFSFDGFEACVTGKD SGGKNISMSSLTSLKVSQANEKYIKKVERFAEKKEVNNKITLDEKYDEINE NLNFEIYRMLQQKITKSCFGVAFGNLSNVFEKGENKFVALKTDMQCEVLL NIINVFKTGRSGTCNLKSIGGVEKAASYRISAKVSNWKKNYTCVKIIDESA SGIFRKESENILEWL DRPY Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSKKRAIEEYYLGLDIG 45 Cas TNSVGYAVTDPQYKILKYHGEPMWGSHVFEEGSQCAERRGFRTARRRL mammalian NRRQQRVRLVQEIFAHEIEKVDSRFFLRLKESALYREDANGNDPFILFND expression NGYTDKEYYLKYPTIHHLIMDLIDDENPHDVRLVYLAVAWLVAHRGHFLS construct DVEKTNVEKVLDFSTSYNALKEMYSGIGQDLPWIDEEETFKNILLKKCGV (with N- KDKEKAFKDKLLKNNSNKKNSDITEESEVLGISAVITLLSGGTVAADKLFV terminal QAEFQDKISISFKKNEDEFEQILTELDEYAEYLLKIRAVYDWAVLYEASKG methionine, QVYISKAKIEVYEQHKKDLAGLKAFIRKYCPDKYNEIFRDAGDNNYVAYS stop codon, YNFNSLNLTDGEKRPKRKASQEDFCTYIRKIVKDIKCEMEDEEFYDDMW V5 tag, N- QRLELGTFMPKQVNTDNRVLPYQLYYFELARLLGRVSAYLPFINEKDND terminal NLS GYITKEKLLSIMEFRIPYYVGPLHRENKDNNSFAWIKRKAQGRIYPWNYE and C- EKIDLDASEQAFINKMTNQCSYLPGEDVLPKYSLLYCKYEVLNEINNIKIN terminal NLS) GQKITVECKKGIFGLFKKNKKVTVKKIKDFLISNNYMGTSDEITGLDISVKS (aa) SLKSYHDFRKMLENKIVNEEDVEEIIKRLTYTEDKKRINRYLEENYPNISEE DRRYISKLKYKDFGRLSEKFLTGIKGVVKETGEILSVIQIMLETNDNLMQIIF SDHYMIKDALEREKKDYYSEHPASVEQILSDMYISNAVKRPIYRTLDIISDV TKACQNAPRKIFVEMARGGGEKGKRTISRRDKIKELYKNMDKQEVREISK EISELLGELDKKTDNELQSEVLFLYFMQLGICMYSGDPIDITKLKSDAYVN VDHIYPQAYVKDDSLNNKVLVKSKLNGEKSDNYPINKDIRDKMSSIWKHY RKEGLISEEKYNRLTRKTPFTDEEKQGFVNRQLVETRQSTKAVAEILKMM FPETEVVYVKAGLASEFRHVFGIIKSRQINDLHHAKDAYLNVVCGNVYHS TFTKNFFLKHQTYSIKTETVYKRRIEADGKLIWNGQESLSFVKKMLGKNNI HYTRYAFCRKGGFFDQMPVTAKEGLVSRKKDLPSEKYGGYNKPTASYFI MAKYTEKVKKEKQDIMIVPVELMVGEKVLNDKNFAVGYIQQQIAQISNRK VEEIVNVSFPLGLRPLKVNTRFSFDGFEACVTGKDSGGKNISMSSLTSLK VSQANEKYIKKVERFAEKKEVNNKITLDEKYDEINENLNFEIYRMLQQKIT KSCFGVAFGNLSNVFEKGENKFVALKTDMQCEVLLNIINVFKTGRSGTCN LKSIGGVEKAASYRISAKVSNWKKNYTCVKIIDESASGIFRKESENILEWL SRKRTADGSEFESPKKKRKV DRPY Type II ATGAAAAAAAGAGCAATTGAAGAATATTATTTAGGACTTGATATTGGAA 46 Cas coding CAAATTCTGTGGGATATGCAGTTACTGATCCACAGTATAAAATTTTAAA sequence (nt) ATATCACGGAGAGCCGATGTGGGGATCTCATGTGTTTGAGGAGGGCA (not codon GCCAGTGTGCGGAAAGGCGTGGCTTTAGAACTGCCAGAAGGAGATT optimized) AAACAGACGTCAGCAAAGAGTACGCTTGGTTCAGGAGATATTTGCCC ACGAGATTGAAAAAGTTGATAGCAGATTTTTCCTTCGCCTAAAGGAAA GTGCTCTGTATCGTGAAGACGCAAACGGGAATGATCCATTTATATTGT TTAATGACAACGGATACACTGACAAAGAATATTATTTAAAATATCCAAC GATACATCATCTTATCATGGATCTGATTGATGATGAAAATCCGCACGA TGTCCGCCTGGTTTATCTTGCGGTGGCATGGCTTGTTGCACACAGAG GTCATTTTTTAAGTGATGTTGAAAAAACAAATGTAGAAAAGGTGTTAGA TTTTTCCACTTCATATAATGCACTTAAGGAAATGTATTCTGGTATTGGA CAGGATTTACCTTGGATTGACGAAGAGGAAACATTTAAAAACATTTTA TTGAAAAAATGTGGGGTGAAAGATAAAGAAAAAGCATTTAAGGATAAA TTATTGAAAAATAACTCTAATAAAAAGAATTCAGATATAACGGAGGAGA GCGAAGTACTGGGAATAAGTGCGGTCATAACCTTACTTTCTGGAGGA ACGGTTGCAGCAGATAAGCTTTTTGTGCAAGCCGAGTTTCAGGACAA AATTAGCATTAGTTTTAAAAAGAATGAAGATGAGTTTGAACAGATTTTA ACAGAATTAGATGAATATGCGGAATATTTATTAAAGATCAGAGCTGTATABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO TATGACTGGGCTGTATTATATGAGGCATCAAAGGGACAAGTATACATT TCAAAGGCCAAAATAGAGGTGTATGAACAGCACAAAAAAGATTTGGC AGGGCTCAAAGCCTTTATAAGAAAATATTGTCCGGATAAATACAATGA AATTTTCCGTGATGCAGGCGATAACAATTATGTGGCGTATTCGTATAA TTTTAATTCTTTGAATCTTACTGATGGGGAAAAGCGACCGAAGAGAAA AGCTTCACAGGAAGATTTCTGCACTTATATACGGAAAATTGTTAAAGA TATTAAGTGCGAAATGGAGGATGAAGAATTTTATGACGATATGTGGCA GCGGTTAGAACTTGGAACATTTATGCCGAAACAGGTTAATACGGATAA TCGTGTCCTTCCGTATCAGTTATATTATTTTGAACTTGCCCGCCTTTTA GGAAGGGTATCGGCATATCTTCCATTTATCAATGAAAAGGATAATGAT GGATACATTACGAAAGAAAAATTGCTTTCTATTATGGAATTTCGGATTC CTTATTATGTAGGTCCATTACATAGAGAGAATAAAGATAATAATTCATT TGCCTGGATTAAGAGAAAGGCTCAAGGACGTATTTATCCATGGAACTA TGAAGAAAAAATTGATCTTGATGCAAGTGAGCAGGCATTTATCAACAA AATGACAAATCAGTGTAGTTACCTGCCGGGAGAGGATGTACTGCCGA AATATTCCCTGTTGTACTGTAAATATGAAGTGTTAAATGAAATTAATAA TATTAAAATAAATGGTCAAAAAATTACAGTGGAATGTAAAAAAGGAATC TTTGGTCTTTTTAAAAAGAACAAAAAAGTTACAGTAAAGAAGATTAAAG ATTTTTTGATCAGTAATAACTATATGGGGACTTCAGATGAGATAACGG GACTTGACATTTCTGTAAAATCATCTTTGAAATCATACCATGATTTTCG AAAAATGTTGGAAAATAAAATTGTCAATGAGGAGGACGTGGAAGAAAT TATCAAGCGTCTTACCTATACGGAAGACAAAAAAAGAATTAATAGGTA TTTAGAAGAAAATTATCCTAATATTTCGGAGGAAGACAGGCGTTATAT TTCAAAATTGAAATATAAGGATTTTGGAAGATTATCGGAAAAATTTTTG ACAGGAATTAAGGGGGTTGTAAAAGAAACGGGAGAAATCTTAAGTGT TATACAGATTATGTTGGAAACAAATGACAACCTGATGCAGATTATTTTT TCTGATCATTATATGATTAAAGATGCACTTGAAAGAGAAAAGAAAGATT ATTATAGTGAACATCCGGCAAGTGTGGAACAGATATTATCGGATATGT ACATTTCGAATGCGGTGAAACGTCCGATTTATAGAACATTGGATATTA TAAGTGATGTTACAAAAGCATGTCAGAATGCACCACGCAAGATATTTG TCGAGATGGCACGCGGAGGTGGTGAAAAGGGGAAAAGAACCATTTC GCGTCGCGATAAAATTAAGGAATTGTACAAGAATATGGACAAACAGG AAGTAAGAGAAATATCAAAAGAGATCTCCGAGCTTCTTGGTGAGTTGG ATAAAAAGACAGACAATGAGCTGCAGAGTGAGGTGTTGTTCTTATATT TTATGCAGTTAGGAATATGTATGTATTCGGGAGATCCGATTGACATTA CCAAACTGAAATCGGACGCATATGTGAATGTAGATCATATTTATCCGC AGGCGTATGTGAAAGATGACAGCTTGAATAATAAAGTATTGGTAAAAT CAAAATTAAATGGAGAAAAGTCAGATAATTATCCGATAAACAAGGATA TCCGAGATAAAATGTCATCAATTTGGAAGCACTATAGAAAAGAAGGGT TAATTTCTGAGGAAAAATATAACCGCTTAACAAGAAAAACGCCATTTA CGGATGAGGAAAAACAAGGATTTGTGAATCGTCAGTTAGTAGAAACA AGACAATCCACAAAGGCTGTGGCTGAAATTTTAAAAATGATGTTTCCG GAAACGGAAGTTGTATATGTAAAAGCAGGGTTAGCGTCAGAGTTTCG ACATGTATTTGGAATTATAAAATCTCGTCAGATTAATGACCTGCATCAT GCGAAGGACGCTTATTTAAACGTAGTATGTGGAAATGTTTATCATTCT ACATTTACAAAAAACTTTTTCTTGAAGCATCAGACGTATTCGATAAAAA CAGAAACGGTATATAAACGAAGAATTGAGGCAGATGGAAAGCTGATA TGGAATGGACAGGAGTCGTTATCTTTTGTTAAAAAGATGCTTGGCAAA AATAATATACATTATACCAGATATGCATTTTGCAGGAAGGGCGGATTT TTTGATCAGATGCCGGTTACAGCAAAAGAGGGGCTGGTTTCGAGAAA GAAAGATTTACCATCAGAAAAATATGGTGGATATAATAAACCAACAGC GTCCTATTTCATAATGGCAAAATATACGGAAAAAGTGAAGAAGGAAAA ACAGGATATTATGATTGTACCTGTAGAGCTAATGGTGGGTGAAAAAGT ACTGAATGATAAAAACTTTGCGGTTGGTTATATTCAGCAACAGATTGC ACAGATTTCAAATAGGAAAGTAGAAGAAATTGTTAATGTTTCGTTTCC GTTGGGATTGAGACCACTTAAGGTAAATACGAGATTTTCTTTTGATGG GTTTGAAGCATGTGTAACGGGAAAAGATAGTGGAGGAAAAAATATAATABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO GTATGAGTTCACTTACTTCTTTAAAAGTGAGTCAAGCAAATGAAAAATA TATAAAGAAAGTAGAAAGATTTGCAGAAAAGAAGGAGGTAAACAACAA AATAACTTTAGATGAAAAATATGATGAAATAAATGAAAACTTAAATTTT GAAATATATAGAATGTTGCAACAAAAAATAACAAAAAGTTGCTTTGGG GTTGCATTTGGTAATTTATCTAATGTGTTTGAAAAAGGAGAAAACAAAT TTGTTGCTTTAAAGACAGATATGCAATGTGAGGTGTTACTAAACATAAT TAATGTTTTTAAAACAGGTAGAAGTGGTACATGTAATTTGAAAAGCATT GGTGGAGTGGAAAAGGCTGCATCATATAGAATTAGTGCAAAAGTTTC AAATTGGAAAAAGAACTATACATGTGTAAAGATAATCGACGAATCAGC ATCAGGAATTTTCCGTAAAGAATCGGAAAATATATTGGAGTGGTTATG A DRPY Type II AAGAAAAGAGCCATCGAGGAATACTACCTGGGCCTGGATATTGGCAC 47 Cas coding CAACAGCGTGGGGTACGCCGTGACCGATCCTCAGTACAAAATCCTGA sequence (nt) AGTACCACGGCGAGCCTATGTGGGGTAGCCACGTGTTTGAGGAAGG (human CAGCCAGTGCGCTGAGAGAAGGGGCTTCAGAACCGCCAGACGGAGA codon- TTGAATAGGAGGCAGCAAAGAGTCAGACTGGTCCAGGAGATCTTTGC optimized; CCACGAGATCGAGAAGGTGGACAGCAGATTCTTCCTGAGACTGAAGG lacking N- AGAGCGCCCTGTATAGAGAGGACGCCAACGGCAACGACCCTTTCATC terminal CTGTTTAACGACAACGGCTACACAGACAAGGAGTACTACCTGAAGTA methionine; CCCCACCATCCACCACCTGATCATGGACCTGATCGACGATGAAAACC no stop CCCACGACGTGAGACTGGTGTACCTCGCTGTGGCCTGGCTGGTGGC codon) TCACAGAGGCCACTTCCTGTCTGATGTGGAGAAAACCAATGTGGAAA AAGTGCTGGACTTCTCCACCTCGTACAATGCTCTGAAGGAGATGTAC TCCGGAATCGGCCAAGACCTGCCTTGGATCGACGAAGAGGAAACCTT CAAGAACATCCTCCTGAAGAAGTGCGGCGTCAAGGACAAGGAGAAG GCTTTTAAGGACAAGCTGCTGAAGAACAACAGTAACAAGAAAAACAG CGACATTACAGAAGAATCTGAAGTCCTGGGCATCTCAGCCGTGATCA CCCTGCTGTCTGGTGGCACCGTGGCTGCCGACAAGCTGTTCGTGCA GGCCGAGTTCCAGGACAAGATCAGCATCTCTTTCAAGAAGAATGAGG ATGAGTTCGAACAGATCCTGACAGAGCTGGACGAGTACGCGGAGTAC CTGCTGAAGATCAGGGCCGTTTATGACTGGGCCGTGCTGTACGAAGC CAGCAAAGGCCAGGTGTACATCAGCAAGGCTAAGATCGAGGTTTACG AGCAACACAAGAAGGACCTGGCCGGACTGAAGGCCTTTATCAGAAAG TATTGTCCTGATAAGTACAACGAAATCTTCCGCGACGCAGGCGACAA CAATTACGTGGCTTATTCCTACAACTTCAACAGCCTGAACCTGACCGA CGGCGAGAAGCGGCCCAAAAGAAAGGCCAGCCAGGAGGACTTCTGC ACCTACATCCGGAAGATTGTGAAGGATATAAAGTGTGAAATGGAAGAT GAGGAGTTCTATGACGACATGTGGCAGAGACTGGAACTCGGAACCTT CATGCCTAAGCAGGTGAACACCGATAACCGGGTGCTGCCGTACCAG CTGTATTACTTCGAGCTGGCACGGCTGCTGGGCAGAGTGAGCGCCT ATCTGCCTTTTATCAATGAAAAAGACAACGACGGCTACATCACCAAAG AGAAGCTGCTGAGCATCATGGAGTTCAGAATCCCATACTACGTTGGA CCTCTGCATAGAGAAAACAAGGATAACAACTCCTTTGCCTGGATCAAG CGGAAGGCCCAGGGAAGAATCTACCCTTGGAACTACGAGGAGAAGA TCGACCTGGACGCTTCCGAGCAGGCCTTCATCAACAAGATGACCAAC CAATGTAGCTACCTACCTGGCGAGGACGTGCTGCCTAAGTATTCTCT GCTGTACTGCAAGTACGAGGTCCTGAACGAGATCAACAATATTAAGAT CAACGGACAGAAAATAACCGTGGAGTGCAAGAAGGGCATCTTCGGC CTGTTCAAGAAGAACAAAAAGGTGACCGTGAAGAAGATCAAGGACTT CCTGATCTCTAACAACTACATGGGCACAAGCGACGAAATCACAGGCC TGGACATCAGCGTGAAGTCAAGCCTGAAATCTTACCATGATTTCCGG AAGATGCTGGAGAACAAGATCGTGAACGAGGAGGACGTGGAAGAAA TCATCAAGAGGCTGACATACACCGAGGACAAGAAGCGGATCAACAGA TACCTGGAGGAAAATTACCCTAACATTTCTGAGGAAGATAGACGGTAC ATCTCCAAGCTGAAATACAAGGACTTCGGCAGACTGAGCGAAAAGTT CCTGACCGGCATCAAGGGCGTGGTGAAGGAAACCGGCGAAATTCTG AGCGTCATTCAGATCATGCTGGAAACAAACGACAATCTGATGCAGATTABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO CATATTCAGCGATCACTACATGATCAAGGACGCCCTGGAAAGAGAAA AGAAGGACTACTACAGCGAGCACCCCGCGAGCGTGGAACAAATCCT GAGCGACATGTACATCTCCAACGCCGTGAAGCGGCCTATCTACCGCA CCCTGGATATCATCAGCGATGTGACCAAAGCCTGCCAGAACGCCCCT AGAAAGATCTTCGTCGAAATGGCCAGAGGCGGCGGCGAGAAGGGCA AGAGAACAATCAGCAGAAGAGACAAGATCAAAGAGCTGTATAAGAAT ATGGACAAACAGGAGGTGCGGGAAATCAGCAAGGAGATCAGCGAGC TGCTGGGCGAGCTGGACAAGAAAACCGATAACGAGCTGCAGAGCGA AGTACTGTTCTTGTACTTCATGCAGCTGGGAATCTGCATGTACAGCG GAGATCCTATCGACATCACTAAGCTGAAGAGCGACGCCTACGTGAAT GTCGACCACATTTACCCCCAGGCTTACGTGAAGGACGACAGCCTGAA CAACAAAGTGCTGGTGAAGTCTAAACTGAATGGGGAAAAAAGCGACA ATTACCCTATTAACAAGGACATCCGGGACAAGATGAGCAGCATCTGG AAGCACTACAGAAAGGAAGGCTTGATTTCAGAAGAAAAATATAACAGA CTGACCAGAAAGACACCTTTCACAGATGAAGAAAAGCAGGGCTTCGT GAATAGACAGCTGGTCGAAACCAGACAGAGTACCAAGGCGGTTGCT GAAATCCTGAAGATGATGTTCCCCGAAACCGAGGTTGTGTACGTGAA GGCCGGACTGGCCAGCGAGTTCAGACACGTGTTCGGTATCATCAAG AGCAGACAGATCAATGATCTGCATCACGCCAAAGACGCCTACCTGAA TGTGGTGTGCGGCAACGTGTACCACAGCACCTTCACCAAGAACTTTT TCCTGAAGCACCAGACTTATTCAATCAAGACCGAGACAGTGTACAAG CGCAGAATCGAGGCCGATGGCAAGCTGATCTGGAATGGCCAGGAGA GCCTGTCTTTCGTGAAGAAAATGCTGGGAAAGAACAACATCCACTAC ACCAGGTACGCGTTCTGCAGAAAGGGCGGCTTCTTCGACCAGATGC CTGTGACCGCCAAGGAGGGACTGGTGTCCCGGAAAAAGGACCTGCC CAGCGAGAAGTACGGCGGCTATAACAAGCCAACGGCCAGCTATTTTA TCATGGCCAAATACACCGAAAAAGTGAAAAAGGAAAAGCAGGATATC ATGATCGTGCCCGTGGAGCTGATGGTCGGCGAGAAAGTGCTGAACG ACAAAAACTTCGCCGTGGGCTACATCCAACAGCAGATCGCCCAGATC AGCAACCGGAAGGTGGAGGAGATTGTGAACGTGTCTTTCCCTCTGGG CCTGCGGCCTCTGAAGGTGAACACCCGGTTCAGCTTTGATGGCTTTG AGGCCTGTGTGACAGGCAAGGATTCTGGCGGCAAGAACATCTCCATG AGCTCTTTGACAAGCCTGAAAGTTAGCCAAGCTAATGAAAAGTACATC AAGAAGGTGGAGAGATTCGCCGAGAAGAAAGAGGTGAATAACAAGAT CACCCTGGACGAGAAATACGACGAGATCAATGAGAACCTGAACTTCG AGATCTACAGAATGCTGCAGCAGAAGATAACAAAGTCCTGTTTTGGC GTGGCCTTTGGCAACCTGAGCAACGTGTTCGAGAAGGGCGAGAACA AGTTCGTGGCCCTGAAAACCGATATGCAGTGCGAGGTGCTGCTCAAC ATCATCAACGTGTTCAAGACTGGAAGAAGCGGCACATGCAACCTGAA GAGCATCGGAGGAGTGGAAAAGGCCGCCAGCTACCGGATCTCTGCC AAAGTGTCCAACTGGAAGAAGAATTACACCTGCGTGAAAATTATCGAC GAGAGCGCCAGCGGCATCTTTAGAAAGGAGTCTGAGAACATCCTGGA ATGGCTG DRPY Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 48 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCAAGAAAAGAGCCATCGAGGAATACTACCTGGGCCT expression GGATATTGGCACCAACAGCGTGGGGTACGCCGTGACCGATCCTCAG construct TACAAAATCCTGAAGTACCACGGCGAGCCTATGTGGGGTAGCCACGT (with N- GTTTGAGGAAGGCAGCCAGTGCGCTGAGAGAAGGGGCTTCAGAACC terminal GCCAGACGGAGATTGAATAGGAGGCAGCAAAGAGTCAGACTGGTCC methionine, AGGAGATCTTTGCCCACGAGATCGAGAAGGTGGACAGCAGATTCTTC stop codon, CTGAGACTGAAGGAGAGCGCCCTGTATAGAGAGGACGCCAACGGCA V5 tag, N- ACGACCCTTTCATCCTGTTTAACGACAACGGCTACACAGACAAGGAG terminal NLS TACTACCTGAAGTACCCCACCATCCACCACCTGATCATGGACCTGAT and C- CGACGATGAAAACCCCCACGACGTGAGACTGGTGTACCTCGCTGTG terminal NLS) GCCTGGCTGGTGGCTCACAGAGGCCACTTCCTGTCTGATGTGGAGA (nt) AAACCAATGTGGAAAAAGTGCTGGACTTCTCCACCTCGTACAATGCTCTABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO TGAAGGAGATGTACTCCGGAATCGGCCAAGACCTGCCTTGGATCGAC GAAGAGGAAACCTTCAAGAACATCCTCCTGAAGAAGTGCGGCGTCAA GGACAAGGAGAAGGCTTTTAAGGACAAGCTGCTGAAGAACAACAGTA ACAAGAAAAACAGCGACATTACAGAAGAATCTGAAGTCCTGGGCATC TCAGCCGTGATCACCCTGCTGTCTGGTGGCACCGTGGCTGCCGACA AGCTGTTCGTGCAGGCCGAGTTCCAGGACAAGATCAGCATCTCTTTC AAGAAGAATGAGGATGAGTTCGAACAGATCCTGACAGAGCTGGACGA GTACGCGGAGTACCTGCTGAAGATCAGGGCCGTTTATGACTGGGCC GTGCTGTACGAAGCCAGCAAAGGCCAGGTGTACATCAGCAAGGCTAA GATCGAGGTTTACGAGCAACACAAGAAGGACCTGGCCGGACTGAAG GCCTTTATCAGAAAGTATTGTCCTGATAAGTACAACGAAATCTTCCGC GACGCAGGCGACAACAATTACGTGGCTTATTCCTACAACTTCAACAG CCTGAACCTGACCGACGGCGAGAAGCGGCCCAAAAGAAAGGCCAGC CAGGAGGACTTCTGCACCTACATCCGGAAGATTGTGAAGGATATAAA GTGTGAAATGGAAGATGAGGAGTTCTATGACGACATGTGGCAGAGAC TGGAACTCGGAACCTTCATGCCTAAGCAGGTGAACACCGATAACCGG GTGCTGCCGTACCAGCTGTATTACTTCGAGCTGGCACGGCTGCTGG GCAGAGTGAGCGCCTATCTGCCTTTTATCAATGAAAAAGACAACGAC GGCTACATCACCAAAGAGAAGCTGCTGAGCATCATGGAGTTCAGAAT CCCATACTACGTTGGACCTCTGCATAGAGAAAACAAGGATAACAACTC CTTTGCCTGGATCAAGCGGAAGGCCCAGGGAAGAATCTACCCTTGGA ACTACGAGGAGAAGATCGACCTGGACGCTTCCGAGCAGGCCTTCATC AACAAGATGACCAACCAATGTAGCTACCTACCTGGCGAGGACGTGCT GCCTAAGTATTCTCTGCTGTACTGCAAGTACGAGGTCCTGAACGAGA TCAACAATATTAAGATCAACGGACAGAAAATAACCGTGGAGTGCAAGA AGGGCATCTTCGGCCTGTTCAAGAAGAACAAAAAGGTGACCGTGAAG AAGATCAAGGACTTCCTGATCTCTAACAACTACATGGGCACAAGCGA CGAAATCACAGGCCTGGACATCAGCGTGAAGTCAAGCCTGAAATCTT ACCATGATTTCCGGAAGATGCTGGAGAACAAGATCGTGAACGAGGAG GACGTGGAAGAAATCATCAAGAGGCTGACATACACCGAGGACAAGAA GCGGATCAACAGATACCTGGAGGAAAATTACCCTAACATTTCTGAGG AAGATAGACGGTACATCTCCAAGCTGAAATACAAGGACTTCGGCAGA CTGAGCGAAAAGTTCCTGACCGGCATCAAGGGCGTGGTGAAGGAAA CCGGCGAAATTCTGAGCGTCATTCAGATCATGCTGGAAACAAACGAC AATCTGATGCAGATCATATTCAGCGATCACTACATGATCAAGGACGCC CTGGAAAGAGAAAAGAAGGACTACTACAGCGAGCACCCCGCGAGCG TGGAACAAATCCTGAGCGACATGTACATCTCCAACGCCGTGAAGCGG CCTATCTACCGCACCCTGGATATCATCAGCGATGTGACCAAAGCCTG CCAGAACGCCCCTAGAAAGATCTTCGTCGAAATGGCCAGAGGCGGC GGCGAGAAGGGCAAGAGAACAATCAGCAGAAGAGACAAGATCAAAG AGCTGTATAAGAATATGGACAAACAGGAGGTGCGGGAAATCAGCAAG GAGATCAGCGAGCTGCTGGGCGAGCTGGACAAGAAAACCGATAACG AGCTGCAGAGCGAAGTACTGTTCTTGTACTTCATGCAGCTGGGAATC TGCATGTACAGCGGAGATCCTATCGACATCACTAAGCTGAAGAGCGA CGCCTACGTGAATGTCGACCACATTTACCCCCAGGCTTACGTGAAGG ACGACAGCCTGAACAACAAAGTGCTGGTGAAGTCTAAACTGAATGGG GAAAAAAGCGACAATTACCCTATTAACAAGGACATCCGGGACAAGAT GAGCAGCATCTGGAAGCACTACAGAAAGGAAGGCTTGATTTCAGAAG AAAAATATAACAGACTGACCAGAAAGACACCTTTCACAGATGAAGAAA AGCAGGGCTTCGTGAATAGACAGCTGGTCGAAACCAGACAGAGTACC AAGGCGGTTGCTGAAATCCTGAAGATGATGTTCCCCGAAACCGAGGT TGTGTACGTGAAGGCCGGACTGGCCAGCGAGTTCAGACACGTGTTC GGTATCATCAAGAGCAGACAGATCAATGATCTGCATCACGCCAAAGA CGCCTACCTGAATGTGGTGTGCGGCAACGTGTACCACAGCACCTTCA CCAAGAACTTTTTCCTGAAGCACCAGACTTATTCAATCAAGACCGAGA CAGTGTACAAGCGCAGAATCGAGGCCGATGGCAAGCTGATCTGGAAT GGCCAGGAGAGCCTGTCTTTCGTGAAGAAAATGCTGGGAAAGAACAATABLE 1H DRPY Type II Cas Sequences Name Sequence SEQ ID NO CATCCACTACACCAGGTACGCGTTCTGCAGAAAGGGCGGCTTCTTCG ACCAGATGCCTGTGACCGCCAAGGAGGGACTGGTGTCCCGGAAAAA GGACCTGCCCAGCGAGAAGTACGGCGGCTATAACAAGCCAACGGCC AGCTATTTTATCATGGCCAAATACACCGAAAAAGTGAAAAAGGAAAAG CAGGATATCATGATCGTGCCCGTGGAGCTGATGGTCGGCGAGAAAG TGCTGAACGACAAAAACTTCGCCGTGGGCTACATCCAACAGCAGATC GCCCAGATCAGCAACCGGAAGGTGGAGGAGATTGTGAACGTGTCTTT CCCTCTGGGCCTGCGGCCTCTGAAGGTGAACACCCGGTTCAGCTTT GATGGCTTTGAGGCCTGTGTGACAGGCAAGGATTCTGGCGGCAAGA ACATCTCCATGAGCTCTTTGACAAGCCTGAAAGTTAGCCAAGCTAATG AAAAGTACATCAAGAAGGTGGAGAGATTCGCCGAGAAGAAAGAGGTG AATAACAAGATCACCCTGGACGAGAAATACGACGAGATCAATGAGAA CCTGAACTTCGAGATCTACAGAATGCTGCAGCAGAAGATAACAAAGT CCTGTTTTGGCGTGGCCTTTGGCAACCTGAGCAACGTGTTCGAGAAG GGCGAGAACAAGTTCGTGGCCCTGAAAACCGATATGCAGTGCGAGG TGCTGCTCAACATCATCAACGTGTTCAAGACTGGAAGAAGCGGCACA TGCAACCTGAAGAGCATCGGAGGAGTGGAAAAGGCCGCCAGCTACC GGATCTCTGCCAAAGTGTCCAACTGGAAGAAGAATTACACCTGCGTG AAAATTATCGACGAGAGCGCCAGCGGCATCTTTAGAAAGGAGTCTGA GAACATCCTGGAATGGCTGTCTAGAAAGCGGACAGCAGACGGCTCC GAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGTGA

[0097] In some embodiments a DRPY Type II 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 DRPY Type II 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 D14A substitution, wherein the position of the D14A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H878A substitution, wherein the position of the H878A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44. In some embodiments, a DRPY Type II Cas protein is catalytically inactive, for example due to a D14A substitution in combination with an H878A substitution. 6.2.1.9. EAJR Type II Cas Proteins

[0098] In one aspect, the disclosure provides EAJR Type II Cas proteins. EAJR Type II Cas proteins can be further classified as Type IIA Cas proteins. The EAJR Type II 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 EAJR Type II 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, an EAJR Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:49.

[0099] Exemplary EAJR Type II Cas protein sequences and nucleotide sequences encoding exemplary EAJR Type II Cas proteins are set forth in Table 1J.TABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO EAJR Type II EEKKTIRSHSPYYIGLDIGTNSVGWAVTDPEYNVLRFKGNSMWGARLFE 49 Cas amino EAKPAADRRAARIARRRLERRKQRLLYLEEFFSAEIGKSDPLFFRRLHES acid ALFPDDKTTGTGKYTLFNDPDFTDRDYLRRYPTVYHLRSELVHSSEPHD sequence PRLVFLALHHIIKHRGHFLYETSENGDECPTVDAAMQELTEYLSAEMNVE (without N- LGLEPEYFTILTDRNMRISQKKAALRAALGRSVQTEESGADIQALSDLLA terminal GAKVKLAALYDDESLKNADPASIALSDDLDAVFDALSSALGERVELLLAA methionine) KKVFDAARLSEMRGGEAYLCDAKVRQYEKNHADLRALKDYVREAAPDK YRNIFYKNAKFPNNYAAYSRYRSESGDKTCKQEDFCAFLKSVLPKPAAE HSFAPQYVQIEEKTFLPRLTGSENGIIPYQLHRQELRKILDNASAYLPFLLE EDEDGISVREKIEAVFSFRVPYYVGPLKGRWAERTEEAKRTGEKAYPWN FEKVVDLDASAAAFLKDLIGKCTYTGAPVLPKDSLLYSEFMLLNELNPLRL NGNELPVSVKQQLIEDLFKKENRRVTKKRILDYLVMHGYAAKTDEISGIDD PVKTRLRSYHDFAPLIPVLGEEAVEKIIEGTLVYSDDKRMLRRWLKKNFP ALNEDDVRKICRLKYADWGRLSKEFLTELYSGADEQGEAQNILYFLRNTN LNLMQLLNDPEHANLPAAAEERRRARFGRDSLRSQLDEMYISPAVRRGI WQTLKISEEIAHIRGGAPAKIFVEVARGEDKEKKGTRTKSRKEQLLELYKA CERDSGELYEKLAATEESALRRDKLFLYYTQFGKCMYSGEPIDLETALRD DTTYDIDHIFPRSRIKDDSIENRVLVKSVLNREKTNTYPISQDIRSRMLPFW SMLHDKKMIGDKKYDRLKRNTELTEDELASFVQRQLTETQQSTKALAQL LKERYPESEIVYSKAGNISEFRQKFEMLKCREVNDLHHAKDAYLNIVVGN VYKTEFTDRFFLNIRKENYSLKKVFDGDTPGAWKADGTSIATVKKYMAKN NPIITRMPREEKGQLFNLNIMPAGLGQLPIKAGKPIEKYGGYNKLAGSYY CVVEHTEKKKRVRSIETVLLYARGLYESDPVKYCREILGLVDPVVVAPKIR INSLLQLDGFRLYPSGRTGNRIIYKHAYQLAIDAERERYIRNLTKYIERCAKI EYPATAFDGISAEENVALYNWFVERLESSAYGKLMQALLANLKASDETF KNMDILSQAKTLMEVLKAFKCDAQSSNLKDLCGKGSVGTILKNASLQGV QSAYLIHQSPTGLYEYRTDLLH EAJR Type II MEEKKTIRSHSPYYIGLDIGTNSVGWAVTDPEYNVLRFKGNSMWGARLF 50 Cas amino EEAKPAADRRAARIARRRLERRKQRLLYLEEFFSAEIGKSDPLFFRRLHE acid SALFPDDKTTGTGKYTLFNDPDFTDRDYLRRYPTVYHLRSELVHSSEPH sequence DPRLVFLALHHIIKHRGHFLYETSENGDECPTVDAAMQELTEYLSAEMNV ELGLEPEYFTILTDRNMRISQKKAALRAALGRSVQTEESGADIQALSDLLA GAKVKLAALYDDESLKNADPASIALSDDLDAVFDALSSALGERVELLLAA KKVFDAARLSEMRGGEAYLCDAKVRQYEKNHADLRALKDYVREAAPDK YRNIFYKNAKFPNNYAAYSRYRSESGDKTCKQEDFCAFLKSVLPKPAAE HSFAPQYVQIEEKTFLPRLTGSENGIIPYQLHRQELRKILDNASAYLPFLLE EDEDGISVREKIEAVFSFRVPYYVGPLKGRWAERTEEAKRTGEKAYPWN FEKVVDLDASAAAFLKDLIGKCTYTGAPVLPKDSLLYSEFMLLNELNPLRL NGNELPVSVKQQLIEDLFKKENRRVTKKRILDYLVMHGYAAKTDEISGIDD PVKTRLRSYHDFAPLIPVLGEEAVEKIIEGTLVYSDDKRMLRRWLKKNFP ALNEDDVRKICRLKYADWGRLSKEFLTELYSGADEQGEAQNILYFLRNTN LNLMQLLNDPEHANLPAAAEERRRARFGRDSLRSQLDEMYISPAVRRGI WQTLKISEEIAHIRGGAPAKIFVEVARGEDKEKKGTRTKSRKEQLLELYKA CERDSGELYEKLAATEESALRRDKLFLYYTQFGKCMYSGEPIDLETALRD DTTYDIDHIFPRSRIKDDSIENRVLVKSVLNREKTNTYPISQDIRSRMLPFW SMLHDKKMIGDKKYDRLKRNTELTEDELASFVQRQLTETQQSTKALAQL LKERYPESEIVYSKAGNISEFRQKFEMLKCREVNDLHHAKDAYLNIVVGN VYKTEFTDRFFLNIRKENYSLKKVFDGDTPGAWKADGTSIATVKKYMAKN NPIITRMPREEKGQLFNLNIMPAGLGQLPIKAGKPIEKYGGYNKLAGSYY CVVEHTEKKKRVRSIETVLLYARGLYESDPVKYCREILGLVDPVVVAPKIR INSLLQLDGFRLYPSGRTGNRIIYKHAYQLAIDAERERYIRNLTKYIERCAKI EYPATAFDGISAEENVALYNWFVERLESSAYGKLMQALLANLKASDETF KNMDILSQAKTLMEVLKAFKCDAQSSNLKDLCGKGSVGTILKNASLQGV QSAYLIHQSPTGLYEYRTDLLH EAJR Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSEEKKTIRSHSPYYIG 51 Cas LDIGTNSVGWAVTDPEYNVLRFKGNSMWGARLFEEAKPAADRRAARIA mammalian RRRLERRKQRLLYLEEFFSAEIGKSDPLFFRRLHESALFPDDKTTGTGKYTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO expression TLFNDPDFTDRDYLRRYPTVYHLRSELVHSSEPHDPRLVFLALHHIIKHR construct GHFLYETSENGDECPTVDAAMQELTEYLSAEMNVELGLEPEYFTILTDR (with N- NMRISQKKAALRAALGRSVQTEESGADIQALSDLLAGAKVKLAALYDDES terminal LKNADPASIALSDDLDAVFDALSSALGERVELLLAAKKVFDAARLSEMRG methionine, GEAYLCDAKVRQYEKNHADLRALKDYVREAAPDKYRNIFYKNAKFPNNY stop codon, AAYSRYRSESGDKTCKQEDFCAFLKSVLPKPAAEHSFAPQYVQIEEKTF V5 tag, N- LPRLTGSENGIIPYQLHRQELRKILDNASAYLPFLLEEDEDGISVREKIEAV terminal NLS FSFRVPYYVGPLKGRWAERTEEAKRTGEKAYPWNFEKVVDLDASAAAF and C- LKDLIGKCTYTGAPVLPKDSLLYSEFMLLNELNPLRLNGNELPVSVKQQLI terminal NLS) EDLFKKENRRVTKKRILDYLVMHGYAAKTDEISGIDDPVKTRLRSYHDFA (aa) PLIPVLGEEAVEKIIEGTLVYSDDKRMLRRWLKKNFPALNEDDVRKICRLK YADWGRLSKEFLTELYSGADEQGEAQNILYFLRNTNLNLMQLLNDPEHA NLPAAAEERRRARFGRDSLRSQLDEMYISPAVRRGIWQTLKISEEIAHIR GGAPAKIFVEVARGEDKEKKGTRTKSRKEQLLELYKACERDSGELYEKL AATEESALRRDKLFLYYTQFGKCMYSGEPIDLETALRDDTTYDIDHIFPRS RIKDDSIENRVLVKSVLNREKTNTYPISQDIRSRMLPFWSMLHDKKMIGD KKYDRLKRNTELTEDELASFVQRQLTETQQSTKALAQLLKERYPESEIVY SKAGNISEFRQKFEMLKCREVNDLHHAKDAYLNIVVGNVYKTEFTDRFFL NIRKENYSLKKVFDGDTPGAWKADGTSIATVKKYMAKNNPIITRMPREEK GQLFNLNIMPAGLGQLPIKAGKPIEKYGGYNKLAGSYYCVVEHTEKKKRV RSIETVLLYARGLYESDPVKYCREILGLVDPVVVAPKIRINSLLQLDGFRLY PSGRTGNRIIYKHAYQLAIDAERERYIRNLTKYIERCAKIEYPATAFDGISA EENVALYNWFVERLESSAYGKLMQALLANLKASDETFKNMDILSQAKTL MEVLKAFKCDAQSSNLKDLCGKGSVGTILKNASLQGVQSAYLIHQSPTG LYEYRTDLLHSRKRTADGSEFESPKKKRKV EAJR Type II ATGGAAGAGAAAAAAACGATCCGATCGCATTCTCCGTATTATATCGGC 52 Cas coding CTTGACATAGGCACAAATTCCGTCGGCTGGGCGGTGACCGATCCAGA sequence (nt) ATACAACGTTCTGCGCTTCAAAGGAAACTCCATGTGGGGCGCGAGAT (not codon TGTTTGAGGAGGCAAAACCGGCGGCGGATCGGCGCGCGGCGCGCA optimized) TTGCCCGCCGGCGTCTGGAGCGGAGAAAACAGAGACTTTTGTATCTG GAAGAATTCTTCTCTGCGGAGATCGGGAAGTCTGACCCGCTCTTTTT CCGACGGCTGCATGAAAGCGCGCTCTTTCCGGACGATAAGACAACC GGAACCGGGAAATACACGCTTTTCAATGACCCGGATTTTACGGATCG GGATTATCTGCGCCGGTACCCCACGGTGTACCACCTGCGCAGCGAA CTGGTGCATTCCTCCGAGCCGCACGACCCGCGGCTCGTATTTCTGG CGCTGCACCATATCATCAAGCACCGCGGGCATTTCCTTTATGAAACAT CGGAGAACGGCGACGAATGCCCGACGGTAGACGCTGCGATGCAGGA GCTGACAGAGTATCTCTCAGCGGAAATGAATGTGGAGCTGGGATTGG AGCCGGAGTACTTCACGATCCTTACCGACCGCAATATGCGCATATCC CAGAAGAAGGCGGCGCTGCGCGCCGCTCTGGGGAGGTCGGTGCAG ACGGAGGAGAGCGGCGCGGATATTCAGGCCCTGTCCGATCTGCTTG CGGGGGCCAAAGTCAAGCTGGCTGCGCTCTATGACGATGAGTCGCT CAAAAATGCCGACCCGGCGAGCATCGCGCTGAGCGACGATCTGGAC GCCGTGTTTGACGCTCTCTCGTCGGCGTTGGGCGAGCGGGTGGAGC TGCTTCTTGCGGCAAAAAAGGTGTTTGATGCGGCACGGCTGTCGGAA ATGCGCGGCGGCGAAGCGTATCTCTGCGATGCAAAGGTCCGTCAGT ATGAGAAAAACCATGCCGACCTTCGTGCGCTGAAAGACTATGTGAGA GAAGCGGCGCCGGACAAATACCGGAACATATTCTATAAAAACGCGAA GTTCCCGAATAACTATGCCGCCTACAGCCGCTACCGTTCGGAGAGCG GGGATAAGACCTGCAAGCAGGAAGACTTTTGCGCGTTCCTCAAGTCT GTGCTGCCGAAGCCGGCGGCGGAGCATTCGTTCGCGCCGCAGTATG TGCAGATCGAAGAGAAGACGTTTCTTCCGCGGCTGACCGGCAGCGA AAACGGCATCATCCCGTATCAGCTCCACCGGCAGGAGCTGCGGAAA ATTCTGGATAACGCATCGGCGTATCTGCCGTTCCTGCTCGAAGAGGA CGAGGACGGCATCTCCGTGCGCGAGAAGATCGAAGCGGTGTTTTCC TTCCGTGTGCCATATTATGTCGGGCCGCTGAAAGGCCGTTGGGCAGA GCGGACTGAGGAAGCAAAGAGAACCGGCGAGAAAGCCTACCCTTGGTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO AATTTTGAAAAAGTTGTGGATCTCGACGCGAGCGCGGCGGCCTTTCT GAAAGACCTGATCGGAAAATGCACCTACACCGGCGCGCCGGTGCTG CCGAAGGACTCTCTGCTGTACAGCGAATTTATGCTTCTCAACGAGCT GAACCCGCTGCGTCTCAACGGAAACGAGCTCCCGGTCAGCGTAAAG CAGCAGTTGATCGAAGACCTGTTCAAAAAAGAAAACCGCCGCGTGAC GAAAAAACGCATTCTGGACTATCTGGTCATGCACGGCTATGCCGCAA AAACCGATGAGATCAGCGGCATCGACGACCCGGTGAAGACCAGACT GCGTTCGTACCACGATTTTGCGCCGCTCATCCCCGTGCTCGGCGAG GAAGCGGTGGAAAAGATCATCGAAGGAACGCTGGTATACAGCGACG ACAAGCGTATGCTGCGCCGCTGGCTGAAAAAGAATTTCCCTGCGCTG AACGAGGACGATGTGCGGAAGATCTGCCGTCTGAAATATGCCGACTG GGGACGCCTTTCCAAAGAGTTTTTGACGGAACTCTATTCCGGCGCGG ATGAGCAGGGCGAGGCGCAGAACATTCTGTATTTTCTGCGAAACACC AATCTCAATCTCATGCAGCTTCTCAATGATCCGGAGCATGCCAATCTT CCGGCCGCGGCGGAAGAGCGGCGGCGCGCTCGTTTCGGAAGAGAC AGTCTCCGCTCACAGCTGGACGAAATGTACATTTCTCCCGCCGTGCG CCGCGGCATATGGCAGACGCTGAAGATCTCCGAAGAGATCGCCCAT ATCCGCGGCGGCGCACCGGCGAAGATCTTTGTCGAGGTGGCGCGC GGCGAGGATAAGGAGAAGAAGGGAACGCGAACCAAATCCAGAAAAG AACAGCTTCTGGAGCTCTATAAGGCCTGCGAGCGGGACAGCGGCGA GCTGTACGAAAAGCTTGCGGCGACGGAGGAGAGCGCGCTGCGGAG AGACAAGCTCTTCCTGTACTATACACAGTTCGGGAAATGCATGTATTC CGGCGAGCCCATCGATCTGGAAACGGCTCTGCGCGATGATACAACG TATGATATCGACCATATTTTCCCGCGCTCCCGCATCAAGGACGACAG CATTGAGAACCGCGTGCTCGTAAAGAGCGTTCTCAACCGCGAAAAAA CGAACACCTATCCGATCTCGCAGGATATCCGCAGCCGGATGCTGCC GTTCTGGTCGATGCTGCACGATAAGAAAATGATCGGCGATAAAAAGT ACGACCGCCTGAAACGGAATACAGAACTGACGGAGGACGAACTGGC GTCCTTTGTACAGCGCCAACTGACGGAAACGCAGCAGTCCACCAAGG CGCTTGCCCAGCTTTTAAAGGAACGGTACCCGGAGTCGGAGATCGTA TACTCCAAGGCGGGCAACATTTCGGAGTTCCGGCAGAAGTTTGAAAT GCTCAAATGCCGCGAGGTGAACGACCTGCACCACGCCAAGGACGCC TATCTGAACATCGTTGTCGGCAACGTATATAAGACAGAGTTTACCGAC CGGTTTTTCCTGAATATCCGAAAAGAGAATTACAGTCTCAAAAAAGTC TTTGACGGGGATACGCCGGGCGCGTGGAAAGCGGACGGAACGAGC ATCGCCACGGTGAAAAAGTACATGGCGAAGAATAACCCCATCATCAC CCGCATGCCGCGAGAGGAGAAGGGACAGCTCTTCAACCTGAACATC ATGCCTGCCGGACTGGGCCAGCTTCCGATCAAAGCCGGAAAACCCA TTGAGAAATACGGCGGATACAACAAGCTGGCCGGTTCGTATTACTGT GTCGTGGAGCATACGGAAAAGAAGAAACGTGTTCGTTCCATCGAAAC GGTACTGCTTTACGCACGGGGACTGTATGAGAGCGACCCGGTAAAAT ACTGCCGTGAGATACTTGGCCTTGTTGACCCGGTCGTTGTTGCACCG AAGATCCGAATAAACTCCCTTTTGCAATTGGATGGTTTCCGCTTGTAC CCGTCCGGAAGAACCGGAAACAGAATCATTTACAAACACGCCTATCA GCTTGCCATTGACGCCGAAAGAGAACGCTACATCCGTAATCTGACAA AATACATAGAGCGCTGTGCCAAAATCGAATATCCGGCAACAGCCTTT GACGGGATCAGTGCGGAAGAGAATGTGGCGCTGTACAACTGGTTTGT CGAAAGACTGGAAAGCAGCGCATATGGAAAACTCATGCAGGCTCTTC TTGCCAATCTGAAAGCAAGTGATGAGACGTTTAAAAACATGGATATCC TTTCACAGGCAAAGACGCTGATGGAAGTTTTAAAAGCATTTAAGTGCG ATGCGCAAAGCTCAAATTTAAAGGACCTCTGCGGCAAAGGGTCGGTC GGAACAATCCTGAAAAATGCTTCACTGCAAGGCGTCCAATCGGCCTA TCTTATCCACCAGTCGCCGACCGGTCTGTACGAGTATCGTACCGACC TTCTGCACTGA EAJR Type II GAAGAAAAGAAAACCATCCGGAGTCACAGTCCTTACTACATCGGCCT 53 Cas coding CGACATCGGCACCAACAGCGTGGGCTGGGCCGTGACCGATCCTGAA sequence (nt) TACAACGTGCTGAGATTCAAGGGAAACAGCATGTGGGGCGCTAGGCTTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO (human GTTCGAAGAGGCCAAACCCGCCGCTGATAGACGGGCCGCCAGGATC codon- GCCAGGCGCCGGCTGGAAAGACGCAAGCAGAGACTGCTCTACCTGG optimized; AAGAGTTTTTCAGCGCTGAGATCGGCAAGTCTGACCCTCTGTTTTTCA lacking N- GACGGCTGCACGAGTCCGCCCTGTTCCCCGACGATAAGACAACCGG terminal CACCGGCAAGTACACCCTGTTCAACGACCCTGACTTTACCGACCGGG methionine; ACTACCTTAGAAGATACCCTACCGTGTACCACCTGAGATCTGAGCTG no stop GTGCACAGCAGCGAACCCCACGATCCTAGACTGGTGTTCCTGGCCCT codon) GCACCACATCATCAAGCATAGAGGTCACTTCCTCTACGAAACCAGCG AGAACGGCGATGAGTGCCCCACCGTTGATGCCGCCATGCAGGAGCT GACAGAGTACCTATCTGCCGAGATGAACGTGGAACTGGGCCTGGAA CCTGAGTACTTCACTATCCTGACCGACCGTAACATGCGAATCTCACA GAAGAAGGCCGCCCTGAGAGCCGCTCTGGGTAGAAGCGTGCAGACC GAAGAGTCTGGAGCCGATATACAGGCCCTGAGCGATCTGCTGGCCG GAGCCAAAGTGAAGCTCGCCGCCCTTTACGACGACGAATCTCTGAAG AACGCCGACCCTGCCAGCATCGCCCTGTCCGACGACCTGGATGCTG TGTTTGACGCTCTGAGCAGCGCCTTGGGCGAGAGAGTCGAACTGCT GCTGGCTGCCAAAAAAGTGTTCGACGCCGCCAGACTGAGCGAGATG AGAGGAGGCGAGGCTTACCTGTGTGATGCCAAGGTGCGGCAGTACG AGAAGAACCACGCCGACCTGAGAGCCCTGAAAGATTACGTGAGAGA GGCCGCCCCTGATAAGTACCGGAATATCTTCTACAAGAATGCTAAGTT CCCAAACAATTACGCTGCGTATAGCAGATACAGAAGCGAGTCTGGAG ACAAAACATGTAAGCAGGAGGATTTCTGCGCCTTCCTAAAGAGCGTG CTGCCCAAGCCTGCCGCTGAGCACAGCTTCGCCCCTCAGTACGTGC AGATCGAGGAAAAGACATTTCTGCCCAGGCTCACCGGGAGCGAGAA CGGCATCATCCCTTACCAGCTGCACAGACAGGAGCTGCGGAAGATC CTGGACAACGCCAGCGCCTACCTGCCCTTCCTCCTGGAGGAGGACG AGGACGGCATCAGCGTGCGGGAAAAGATCGAGGCAGTGTTCAGCTT TCGGGTCCCCTACTACGTGGGCCCTCTGAAGGGCAGATGGGCCGAA AGAACCGAGGAAGCCAAGAGAACCGGCGAAAAGGCCTATCCTTGGA ATTTCGAGAAGGTGGTGGATCTGGATGCCAGCGCCGCCGCTTTTCTA AAGGACCTGATCGGCAAGTGTACCTACACCGGCGCCCCAGTGCTGC CTAAGGACAGCCTGCTGTACTCTGAGTTCATGCTGCTGAATGAGCTG AACCCTCTGCGACTGAACGGCAATGAGCTCCCCGTGTCCGTGAAGCA ACAACTCATCGAGGACCTGTTCAAGAAAGAAAACAGAAGGGTGACAA AGAAGAGAATCCTGGACTACCTGGTGATGCACGGCTACGCCGCCAA GACCGATGAGATCAGCGGCATCGACGACCCTGTGAAAACCAGACTG CGAAGCTACCACGACTTCGCCCCGCTGATCCCCGTGCTGGGCGAAG AAGCTGTGGAGAAAATCATTGAAGGCACCCTGGTGTACAGCGATGAC AAGCGGATGCTGAGAAGATGGCTGAAGAAGAATTTCCCTGCTTTAAA TGAGGACGACGTGCGGAAGATCTGCAGACTGAAATATGCCGACTGG GGTAGACTGAGCAAGGAGTTCCTGACAGAACTGTACAGCGGCGCAG ATGAGCAGGGCGAGGCCCAGAACATCCTGTATTTTTTGAGAAACACC AACCTGAATCTGATGCAGCTGCTGAACGACCCCGAACACGCCAACCT GCCTGCGGCTGCTGAAGAGAGAAGAAGAGCCAGATTTGGCAGAGAT TCCCTGAGATCCCAACTCGACGAGATGTACATCAGCCCCGCCGTGAG AAGAGGCATTTGGCAGACCCTGAAGATCAGCGAAGAAATCGCCCACA TCCGGGGAGGAGCCCCTGCCAAGATCTTTGTGGAAGTGGCCCGGGG AGAGGACAAAGAAAAGAAGGGCACAAGAACCAAGTCCAGAAAGGAA CAGCTGCTCGAACTGTATAAGGCCTGCGAGCGCGACAGCGGCGAGC TGTACGAGAAGCTGGCCGCTACAGAAGAATCTGCTCTGAGGCGGGA CAAGCTGTTCCTGTACTACACCCAGTTCGGCAAGTGCATGTACAGCG GCGAACCTATCGATCTGGAGACAGCCCTGCGGGACGACACCACCTA CGACATCGACCACATCTTCCCTAGAAGCAGAATTAAGGATGATTCAAT CGAAAACCGGGTGCTGGTCAAGAGCGTGCTGAACCGGGAAAAGACC AACACCTACCCTATCTCCCAGGACATCCGGAGCAGAATGCTGCCTTT CTGGAGCATGCTGCACGACAAAAAGATGATCGGCGACAAGAAGTATG ACAGACTGAAGAGAAACACCGAGCTCACCGAGGACGAGCTGGCCAGTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO CTTCGTGCAAAGACAGCTGACCGAGACACAGCAGAGCACCAAGGCC CTCGCTCAGTTACTGAAAGAAAGATACCCAGAGTCCGAGATCGTTTA CAGCAAGGCCGGAAACATCTCTGAGTTCCGGCAGAAATTCGAGATGC TGAAATGTAGAGAGGTGAACGACCTGCATCATGCCAAAGACGCCTAC CTGAATATCGTGGTAGGCAACGTGTACAAGACCGAGTTCACAGATAG ATTCTTCCTGAATATTAGGAAGGAGAACTACTCTCTGAAGAAGGTGTT CGATGGAGATACCCCTGGCGCCTGGAAGGCCGACGGAACGAGCATC GCCACCGTGAAGAAGTATATGGCCAAGAACAACCCCATCATCACAAG AATGCCTAGAGAAGAGAAAGGGCAGCTGTTTAACCTGAACATCATGC CTGCCGGCCTGGGCCAGCTGCCAATCAAGGCCGGCAAACCTATCGA GAAGTATGGCGGCTACAACAAGCTGGCAGGCTCTTACTACTGCGTGG TCGAGCACACCGAAAAAAAGAAGCGGGTGAGAAGCATCGAAACCGT GCTGCTGTACGCTAGAGGCCTGTACGAATCCGATCCAGTGAAGTACT GCAGAGAGATTCTGGGCCTGGTGGACCCTGTCGTGGTGGCCCCTAA GATCAGAATCAACAGCCTGTTGCAGCTGGACGGCTTCAGACTCTACC CCAGCGGAAGAACAGGAAACCGGATCATCTACAAGCACGCCTACCA GCTGGCTATCGACGCTGAGAGAGAGAGATACATTCGGAACCTGACAA AGTACATCGAGCGGTGCGCCAAGATCGAGTACCCAGCTACTGCCTTT GACGGCATCAGCGCCGAGGAGAACGTCGCCCTGTACAATTGGTTCG TGGAACGGCTGGAGAGCAGCGCATACGGCAAACTGATGCAAGCCCT GCTGGCCAACCTGAAGGCCAGCGACGAGACATTCAAAAATATGGACA TCTTGAGCCAGGCCAAAACCCTGATGGAAGTGCTGAAGGCCTTCAAG TGCGATGCCCAGAGCAGCAACCTGAAAGACCTGTGCGGCAAGGGCA GCGTGGGAACCATCCTGAAAAACGCCTCTCTGCAGGGCGTGCAATCT GCCTACCTGATCCACCAGAGCCCAACAGGCCTGTACGAGTACAGAAC AGATCTGCTGCAC EAJR Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 54 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCGAAGAAAAGAAAACCATCCGGAGTCACAGTCCTTA expression CTACATCGGCCTCGACATCGGCACCAACAGCGTGGGCTGGGCCGTG construct ACCGATCCTGAATACAACGTGCTGAGATTCAAGGGAAACAGCATGTG (with N- GGGCGCTAGGCTGTTCGAAGAGGCCAAACCCGCCGCTGATAGACGG terminal GCCGCCAGGATCGCCAGGCGCCGGCTGGAAAGACGCAAGCAGAGA methionine, CTGCTCTACCTGGAAGAGTTTTTCAGCGCTGAGATCGGCAAGTCTGA stop codon, CCCTCTGTTTTTCAGACGGCTGCACGAGTCCGCCCTGTTCCCCGACG V5 tag, N- ATAAGACAACCGGCACCGGCAAGTACACCCTGTTCAACGACCCTGAC terminal NLS TTTACCGACCGGGACTACCTTAGAAGATACCCTACCGTGTACCACCT and C- GAGATCTGAGCTGGTGCACAGCAGCGAACCCCACGATCCTAGACTG terminal NLS) GTGTTCCTGGCCCTGCACCACATCATCAAGCATAGAGGTCACTTCCT (nt) CTACGAAACCAGCGAGAACGGCGATGAGTGCCCCACCGTTGATGCC GCCATGCAGGAGCTGACAGAGTACCTATCTGCCGAGATGAACGTGG AACTGGGCCTGGAACCTGAGTACTTCACTATCCTGACCGACCGTAAC ATGCGAATCTCACAGAAGAAGGCCGCCCTGAGAGCCGCTCTGGGTA GAAGCGTGCAGACCGAAGAGTCTGGAGCCGATATACAGGCCCTGAG CGATCTGCTGGCCGGAGCCAAAGTGAAGCTCGCCGCCCTTTACGAC GACGAATCTCTGAAGAACGCCGACCCTGCCAGCATCGCCCTGTCCG ACGACCTGGATGCTGTGTTTGACGCTCTGAGCAGCGCCTTGGGCGA GAGAGTCGAACTGCTGCTGGCTGCCAAAAAAGTGTTCGACGCCGCC AGACTGAGCGAGATGAGAGGAGGCGAGGCTTACCTGTGTGATGCCA AGGTGCGGCAGTACGAGAAGAACCACGCCGACCTGAGAGCCCTGAA AGATTACGTGAGAGAGGCCGCCCCTGATAAGTACCGGAATATCTTCT ACAAGAATGCTAAGTTCCCAAACAATTACGCTGCGTATAGCAGATACA GAAGCGAGTCTGGAGACAAAACATGTAAGCAGGAGGATTTCTGCGCC TTCCTAAAGAGCGTGCTGCCCAAGCCTGCCGCTGAGCACAGCTTCGC CCCTCAGTACGTGCAGATCGAGGAAAAGACATTTCTGCCCAGGCTCA CCGGGAGCGAGAACGGCATCATCCCTTACCAGCTGCACAGACAGGA GCTGCGGAAGATCCTGGACAACGCCAGCGCCTACCTGCCCTTCCTCTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO CTGGAGGAGGACGAGGACGGCATCAGCGTGCGGGAAAAGATCGAG GCAGTGTTCAGCTTTCGGGTCCCCTACTACGTGGGCCCTCTGAAGGG CAGATGGGCCGAAAGAACCGAGGAAGCCAAGAGAACCGGCGAAAAG GCCTATCCTTGGAATTTCGAGAAGGTGGTGGATCTGGATGCCAGCGC CGCCGCTTTTCTAAAGGACCTGATCGGCAAGTGTACCTACACCGGCG CCCCAGTGCTGCCTAAGGACAGCCTGCTGTACTCTGAGTTCATGCTG CTGAATGAGCTGAACCCTCTGCGACTGAACGGCAATGAGCTCCCCGT GTCCGTGAAGCAACAACTCATCGAGGACCTGTTCAAGAAAGAAAACA GAAGGGTGACAAAGAAGAGAATCCTGGACTACCTGGTGATGCACGG CTACGCCGCCAAGACCGATGAGATCAGCGGCATCGACGACCCTGTG AAAACCAGACTGCGAAGCTACCACGACTTCGCCCCGCTGATCCCCGT GCTGGGCGAAGAAGCTGTGGAGAAAATCATTGAAGGCACCCTGGTG TACAGCGATGACAAGCGGATGCTGAGAAGATGGCTGAAGAAGAATTT CCCTGCTTTAAATGAGGACGACGTGCGGAAGATCTGCAGACTGAAAT ATGCCGACTGGGGTAGACTGAGCAAGGAGTTCCTGACAGAACTGTAC AGCGGCGCAGATGAGCAGGGCGAGGCCCAGAACATCCTGTATTTTTT GAGAAACACCAACCTGAATCTGATGCAGCTGCTGAACGACCCCGAAC ACGCCAACCTGCCTGCGGCTGCTGAAGAGAGAAGAAGAGCCAGATT TGGCAGAGATTCCCTGAGATCCCAACTCGACGAGATGTACATCAGCC CCGCCGTGAGAAGAGGCATTTGGCAGACCCTGAAGATCAGCGAAGA AATCGCCCACATCCGGGGAGGAGCCCCTGCCAAGATCTTTGTGGAA GTGGCCCGGGGAGAGGACAAAGAAAAGAAGGGCACAAGAACCAAGT CCAGAAAGGAACAGCTGCTCGAACTGTATAAGGCCTGCGAGCGCGA CAGCGGCGAGCTGTACGAGAAGCTGGCCGCTACAGAAGAATCTGCT CTGAGGCGGGACAAGCTGTTCCTGTACTACACCCAGTTCGGCAAGTG CATGTACAGCGGCGAACCTATCGATCTGGAGACAGCCCTGCGGGAC GACACCACCTACGACATCGACCACATCTTCCCTAGAAGCAGAATTAA GGATGATTCAATCGAAAACCGGGTGCTGGTCAAGAGCGTGCTGAACC GGGAAAAGACCAACACCTACCCTATCTCCCAGGACATCCGGAGCAGA ATGCTGCCTTTCTGGAGCATGCTGCACGACAAAAAGATGATCGGCGA CAAGAAGTATGACAGACTGAAGAGAAACACCGAGCTCACCGAGGAC GAGCTGGCCAGCTTCGTGCAAAGACAGCTGACCGAGACACAGCAGA GCACCAAGGCCCTCGCTCAGTTACTGAAAGAAAGATACCCAGAGTCC GAGATCGTTTACAGCAAGGCCGGAAACATCTCTGAGTTCCGGCAGAA ATTCGAGATGCTGAAATGTAGAGAGGTGAACGACCTGCATCATGCCA AAGACGCCTACCTGAATATCGTGGTAGGCAACGTGTACAAGACCGAG TTCACAGATAGATTCTTCCTGAATATTAGGAAGGAGAACTACTCTCTG AAGAAGGTGTTCGATGGAGATACCCCTGGCGCCTGGAAGGCCGACG GAACGAGCATCGCCACCGTGAAGAAGTATATGGCCAAGAACAACCCC ATCATCACAAGAATGCCTAGAGAAGAGAAAGGGCAGCTGTTTAACCT GAACATCATGCCTGCCGGCCTGGGCCAGCTGCCAATCAAGGCCGGC AAACCTATCGAGAAGTATGGCGGCTACAACAAGCTGGCAGGCTCTTA CTACTGCGTGGTCGAGCACACCGAAAAAAAGAAGCGGGTGAGAAGC ATCGAAACCGTGCTGCTGTACGCTAGAGGCCTGTACGAATCCGATCC AGTGAAGTACTGCAGAGAGATTCTGGGCCTGGTGGACCCTGTCGTG GTGGCCCCTAAGATCAGAATCAACAGCCTGTTGCAGCTGGACGGCTT CAGACTCTACCCCAGCGGAAGAACAGGAAACCGGATCATCTACAAGC ACGCCTACCAGCTGGCTATCGACGCTGAGAGAGAGAGATACATTCGG AACCTGACAAAGTACATCGAGCGGTGCGCCAAGATCGAGTACCCAGC TACTGCCTTTGACGGCATCAGCGCCGAGGAGAACGTCGCCCTGTACA ATTGGTTCGTGGAACGGCTGGAGAGCAGCGCATACGGCAAACTGAT GCAAGCCCTGCTGGCCAACCTGAAGGCCAGCGACGAGACATTCAAA AATATGGACATCTTGAGCCAGGCCAAAACCCTGATGGAAGTGCTGAA GGCCTTCAAGTGCGATGCCCAGAGCAGCAACCTGAAAGACCTGTGC GGCAAGGGCAGCGTGGGAACCATCCTGAAAAACGCCTCTCTGCAGG GCGTGCAATCTGCCTACCTGATCCACCAGAGCCCAACAGGCCTGTACTABLE 1J EAJR Type II Cas Sequences Name Sequence SEQ ID NO GAGTACAGAACAGATCTGCTGCACTCTAGAAAGCGGACAGCAGACG GCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGTGA

[0100] In some embodiments an EAJR Type II Cas protein comprises an amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. In some embodiments, an EAJR Type II 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 comrpise a D18A substitution, wherein the position of the D18A substitution is defined with respect to the amino acid numbering of SEQ ID NO:50. In some embodiments, the one or more amino acid substitutions providing nickase activity comrpise an H587A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:50. In some embodiments, an EAJR Type II Cas protein is catalytically inactive, for example due to a D18A substitution in combination with a H587A substitution. 6.2.1.10. CFDE Type II Cas Proteins

[0101] In one aspect, the disclosure provides CFDE Type II Cas proteins. CFDE Type II Cas proteins can be further classified as Type IIA Cas proteins. The CFDE Type II 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 CFDE Type II 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 CFDE Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:55.

[0102] Exemplary CFDE Type II Cas protein sequences and nucleotide sequences encoding exemplary CFDE Type II Cas proteins are set forth in Table 1K. TABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO CFDE Type II NLRNYSGDYNIGLDMGTGSVGWAVTDAQGKLLHFKKQPTWGSRLFDSA 55 Cas amino QPASEARVHRGQRRRYVRRRWRLDLLQKLFEPAMSEIDPGFFMRLNQS acid RTIEGDPIFTKDFTKADYYDRFPTIYHLRAYLMDTDEQADLRLVYLAIHNIV sequence KHRGNFLRQGEKLTAKTAKTSDALERLNDSLQGWCESREYEVAKINKSA (without N- VAKILADERSSRSQKAKDIQALVKVTTGDAAADKKLAKGLSNAVVGLQCE terminal FKDVFGEFECEATKLALSDDEKLETLQAACPDDCVELLESVCGVYSAYV methionine) LQGLLSYAEGQTISINMIKKYEQYAEDLALLKLLVRKYALDSYEPFFRGAT YHDSNSDDPSRDYDASKATGYTAYDLHKLSYDDFAKSVKKLFAGTGAET DERYIAMMDAFDKQRFLRRLKTSDNGAIYYQLHLEELQAILKNQGRFYPF LKDEAAKIESLVTFRIPYYVGPLTSKNAAQDKHGKPRFQWSERKPGMED ATITPWNWDAVIDKNKSAENFILRMTGDCTYLAGEKVLPKQSLLYEEFCV LNELNGVRWSEDGDNWQRLDAAQREGIIHELFHKKRRVTYKMIADWLV KEGDATNPVVRGGQRESGLESKMGSYIFFAKDIFGVDELSRAEYPVIEKII LWNTLFEDRSILKEKLQDEFGPSGNGMLDAEQIKKICKKRLTGWGRLSE KFLTGIRIDTQAGRSMSIMDVLREGNPSSEHRLGETMVMMEILHDNTLGF QEKVDEHNRKYYAEHEKSLGVNELPGSPAIRRSLNQAIRIVDEIAKIAGHA PANVFVEVTRDEDEKKKGKRTTKRWDAIEDALKAFKAEGGDLAVVDDFK ELRAANVDLDERLTLYLMQNGKCLYSGRAIDLNKLMAGSGEYEVDHIIPR AYIKDDSLENKALVYREENQHKTDQLLIDKSIRWKMGETWKQLHQAKLIGTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO DKKYRNLLRSSINENAMKGFVARQLVETSQMVKMAQALLEARYADEGTK IVPVKASVSHNLREAAGLVKCREANDFHHAHDAYLACRLGLFIQMRHPG MYENPIGYSHVIKKYVREQSELFKKTHRMPGSAGFVVNSFMTAGFDPET GEVFDDAWTEDDEVKRYRGKYLPDGWNAAAEVESIRRALNYRQCYISR MPQEDTGAFWKATIYSPRDPKMGAKLELSTKQGLDAKRYGGFSSQQFA YFFIYEAKKKGKPVFRFSQVPVWLAARIEGDSAALGEYARGLAEKEGLEY VGITRPKIYKKQLIEVDGERFFITGKKEMRNATQLAFSNEQMKLLAGDTC SEDAIKLLDMFNPAASSVSGRLLRQLKIEAIIEKCATVDAEDVVAVVKRLL ALVNGTMNMVDLSPAGGSKFSGCIQPNYSKLLSDSNVTVFLIDQSVTGM FERRTRVGL CFDE Type II MNLRNYSGDYNIGLDMGTGSVGWAVTDAQGKLLHFKKQPTWGSRLFD 56 Cas amino SAQPASEARVHRGQRRRYVRRRWRLDLLQKLFEPAMSEIDPGFFMRLN acid QSRTIEGDPIFTKDFTKADYYDRFPTIYHLRAYLMDTDEQADLRLVYLAIH sequence NIVKHRGNFLRQGEKLTAKTAKTSDALERLNDSLQGWCESREYEVAKIN KSAVAKILADERSSRSQKAKDIQALVKVTTGDAAADKKLAKGLSNAVVGL QCEFKDVFGEFECEATKLALSDDEKLETLQAACPDDCVELLESVCGVYS AYVLQGLLSYAEGQTISINMIKKYEQYAEDLALLKLLVRKYALDSYEPFFR GATYHDSNSDDPSRDYDASKATGYTAYDLHKLSYDDFAKSVKKLFAGTG AETDERYIAMMDAFDKQRFLRRLKTSDNGAIYYQLHLEELQAILKNQGRF YPFLKDEAAKIESLVTFRIPYYVGPLTSKNAAQDKHGKPRFQWSERKPG MEDATITPWNWDAVIDKNKSAENFILRMTGDCTYLAGEKVLPKQSLLYEE FCVLNELNGVRWSEDGDNWQRLDAAQREGIIHELFHKKRRVTYKMIAD WLVKEGDATNPVVRGGQRESGLESKMGSYIFFAKDIFGVDELSRAEYPV IEKIILWNTLFEDRSILKEKLQDEFGPSGNGMLDAEQIKKICKKRLTGWGR LSEKFLTGIRIDTQAGRSMSIMDVLREGNPSSEHRLGETMVMMEILHDNT LGFQEKVDEHNRKYYAEHEKSLGVNELPGSPAIRRSLNQAIRIVDEIAKIA GHAPANVFVEVTRDEDEKKKGKRTTKRWDAIEDALKAFKAEGGDLAVVD DFKELRAANVDLDERLTLYLMQNGKCLYSGRAIDLNKLMAGSGEYEVDH IIPRAYIKDDSLENKALVYREENQHKTDQLLIDKSIRWKMGETWKQLHQA KLIGDKKYRNLLRSSINENAMKGFVARQLVETSQMVKMAQALLEARYAD EGTKIVPVKASVSHNLREAAGLVKCREANDFHHAHDAYLACRLGLFIQM RHPGMYENPIGYSHVIKKYVREQSELFKKTHRMPGSAGFVVNSFMTAGF DPETGEVFDDAWTEDDEVKRYRGKYLPDGWNAAAEVESIRRALNYRQC YISRMPQEDTGAFWKATIYSPRDPKMGAKLELSTKQGLDAKRYGGFSSQ QFAYFFIYEAKKKGKPVFRFSQVPVWLAARIEGDSAALGEYARGLAEKE GLEYVGITRPKIYKKQLIEVDGERFFITGKKEMRNATQLAFSNEQMKLLAG DTCSEDAIKLLDMFNPAASSVSGRLLRQLKIEAIIEKCATVDAEDVVAVVK RLLALVNGTMNMVDLSPAGGSKFSGCIQPNYSKLLSDSNVTVFLIDQSVT GMFERRTRVGL CFDE Type II MGKPIPNPLLGLDSTKRTADGSEFESPKKKRKVGSNLRNYSGDYNIGLD 57 Cas MGTGSVGWAVTDAQGKLLHFKKQPTWGSRLFDSAQPASEARVHRGQR mammalian RRYVRRRWRLDLLQKLFEPAMSEIDPGFFMRLNQSRTIEGDPIFTKDFTK expression ADYYDRFPTIYHLRAYLMDTDEQADLRLVYLAIHNIVKHRGNFLRQGEKL construct TAKTAKTSDALERLNDSLQGWCESREYEVAKINKSAVAKILADERSSRS (with N- QKAKDIQALVKVTTGDAAADKKLAKGLSNAVVGLQCEFKDVFGEFECEA terminal TKLALSDDEKLETLQAACPDDCVELLESVCGVYSAYVLQGLLSYAEGQTI methionine, SINMIKKYEQYAEDLALLKLLVRKYALDSYEPFFRGATYHDSNSDDPSRD stop codon, YDASKATGYTAYDLHKLSYDDFAKSVKKLFAGTGAETDERYIAMMDAFD V5 tag, N- KQRFLRRLKTSDNGAIYYQLHLEELQAILKNQGRFYPFLKDEAAKIESLVT terminal NLS FRIPYYVGPLTSKNAAQDKHGKPRFQWSERKPGMEDATITPWNWDAVI and C- DKNKSAENFILRMTGDCTYLAGEKVLPKQSLLYEEFCVLNELNGVRWSE terminal NLS) DGDNWQRLDAAQREGIIHELFHKKRRVTYKMIADWLVKEGDATNPVVR (aa) GGQRESGLESKMGSYIFFAKDIFGVDELSRAEYPVIEKIILWNTLFEDRSIL KEKLQDEFGPSGNGMLDAEQIKKICKKRLTGWGRLSEKFLTGIRIDTQAG RSMSIMDVLREGNPSSEHRLGETMVMMEILHDNTLGFQEKVDEHNRKY YAEHEKSLGVNELPGSPAIRRSLNQAIRIVDEIAKIAGHAPANVFVEVTRD EDEKKKGKRTTKRWDAIEDALKAFKAEGGDLAVVDDFKELRAANVDLDETABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO RLTLYLMQNGKCLYSGRAIDLNKLMAGSGEYEVDHIIPRAYIKDDSLENK ALVYREENQHKTDQLLIDKSIRWKMGETWKQLHQAKLIGDKKYRNLLRS SINENAMKGFVARQLVETSQMVKMAQALLEARYADEGTKIVPVKASVSH NLREAAGLVKCREANDFHHAHDAYLACRLGLFIQMRHPGMYENPIGYSH VIKKYVREQSELFKKTHRMPGSAGFVVNSFMTAGFDPETGEVFDDAWT EDDEVKRYRGKYLPDGWNAAAEVESIRRALNYRQCYISRMPQEDTGAF WKATIYSPRDPKMGAKLELSTKQGLDAKRYGGFSSQQFAYFFIYEAKKK GKPVFRFSQVPVWLAARIEGDSAALGEYARGLAEKEGLEYVGITRPKIYK KQLIEVDGERFFITGKKEMRNATQLAFSNEQMKLLAGDTCSEDAIKLLDM FNPAASSVSGRLLRQLKIEAIIEKCATVDAEDVVAVVKRLLALVNGTMNM VDLSPAGGSKFSGCIQPNYSKLLSDSNVTVFLIDQSVTGMFERRTRVGLS RKRTADGSEFESPKKKRKV CFDE Type II ATGAATTTAAGAAACTACTCTGGCGACTACAATATCGGCCTCGACATG 58 Cas coding GGAACCGGCTCCGTTGGTTGGGCGGTGACTGATGCCCAGGGCAAGC sequence (nt) TTCTGCATTTTAAGAAGCAGCCGACGTGGGGTAGCCGCTTGTTCGAT (not codon AGTGCGCAGCCGGCATCCGAGGCGCGCGTTCATCGTGGCCAGCGTC optimized) GTCGTTACGTGCGTCGTCGCTGGCGTCTCGACTTGCTTCAAAAGCTG TTCGAACCCGCGATGAGCGAGATCGACCCCGGCTTTTTCATGCGACT CAATCAGTCGCGAACCATCGAAGGCGACCCGATCTTTACCAAGGACT TTACGAAGGCCGATTACTACGATCGTTTCCCGACCATCTATCATCTGC GCGCTTACCTTATGGACACCGATGAACAGGCCGATCTTCGTTTGGTG TATCTTGCGATCCATAACATCGTTAAGCATCGCGGTAACTTTTTGCGC CAGGGAGAGAAGCTGACGGCAAAAACGGCAAAGACGTCGGATGCCC TCGAGCGTCTTAATGACTCCCTGCAGGGTTGGTGCGAGTCGAGGGA ATATGAGGTTGCAAAAATCAATAAAAGCGCCGTCGCAAAGATCCTCG CTGACGAGAGGTCTTCTCGCTCGCAAAAGGCAAAAGACATTCAGGCG CTTGTAAAAGTTACCACTGGAGACGCTGCCGCAGATAAAAAGCTGGC GAAGGGCTTGTCGAATGCTGTCGTCGGCCTTCAGTGCGAGTTCAAAG ACGTGTTTGGCGAGTTTGAGTGCGAGGCAACTAAGCTTGCGCTTTCG GATGACGAGAAACTCGAGACACTGCAAGCCGCGTGTCCCGACGATT GCGTCGAGCTTCTTGAATCTGTCTGCGGGGTATATTCTGCATATGTTT TGCAGGGGCTTTTGTCATATGCAGAGGGCCAAACTATTTCGATCAAC ATGATTAAAAAATACGAGCAATACGCAGAAGACCTTGCTCTGCTTAAG CTCTTGGTCCGCAAATATGCTCTCGACTCATACGAGCCCTTCTTCCGT GGCGCGACCTATCATGATTCGAATAGCGATGACCCCTCGCGCGACTA CGATGCCTCGAAAGCAACGGGGTACACCGCGTATGACCTTCATAAGC TCTCCTACGATGACTTTGCCAAATCGGTAAAGAAGCTCTTTGCGGGAA CGGGCGCGGAGACGGATGAGCGCTATATCGCCATGATGGACGCATT CGACAAACAGCGCTTTTTGCGTCGCCTGAAGACGAGCGATAACGGC GCCATCTACTACCAGCTGCATCTTGAGGAACTTCAGGCTATTCTTAAA AACCAGGGGCGCTTTTATCCGTTCCTTAAGGATGAGGCTGCAAAAAT CGAGAGTCTGGTCACGTTCCGTATTCCGTACTATGTGGGGCCGCTGA CGTCTAAAAACGCTGCCCAGGACAAACACGGCAAGCCTCGATTTCAA TGGTCCGAGCGCAAGCCCGGCATGGAAGATGCGACGATTACGCCTT GGAACTGGGATGCCGTGATCGATAAGAATAAGAGCGCCGAGAACTTT ATCCTGCGCATGACGGGTGACTGCACGTATCTTGCTGGCGAAAAGGT GCTTCCCAAGCAGTCTTTGCTCTATGAGGAGTTTTGCGTTCTGAATGA ACTCAATGGCGTTCGCTGGTCCGAAGATGGTGACAATTGGCAACGCC TGGATGCCGCGCAGCGCGAGGGCATTATCCATGAGCTTTTCCATAAA AAGCGTCGCGTTACCTACAAGATGATTGCCGACTGGCTTGTAAAAGA GGGCGACGCGACAAATCCGGTTGTCCGTGGTGGCCAGCGTGAGAGC GGTCTTGAATCGAAGATGGGCTCGTACATCTTCTTTGCTAAGGACATC TTTGGTGTGGACGAGTTGAGCCGTGCCGAGTATCCGGTTATCGAGAA GATCATTCTTTGGAACACATTGTTTGAGGACCGTTCGATCCTTAAGGA AAAACTGCAGGATGAGTTTGGTCCAAGTGGCAATGGAATGCTCGATG CCGAGCAGATTAAGAAGATTTGCAAGAAGCGTCTAACTGGCTGGGGA CGTCTGTCGGAGAAGTTCCTGACGGGTATCAGGATTGATACCCAAGCTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO TGGACGCTCTATGAGCATCATGGACGTGCTGCGAGAGGGAAACCCC AGTTCTGAGCATCGCCTGGGCGAGACCATGGTGATGATGGAGATTCT CCACGACAACACGCTGGGCTTCCAGGAAAAAGTGGATGAACACAATC GCAAATACTACGCCGAACACGAGAAGTCTCTAGGCGTAAATGAGCTC CCTGGATCTCCGGCGATTCGTCGCAGCTTGAATCAAGCGATTCGCAT TGTTGATGAAATCGCAAAGATTGCGGGACACGCGCCGGCCAACGTTT TTGTTGAGGTGACTCGCGACGAGGACGAAAAGAAGAAGGGCAAGCG GACCACGAAGCGCTGGGACGCCATCGAGGATGCGCTCAAAGCGTTT AAGGCCGAGGGCGGCGACCTGGCTGTTGTGGATGATTTCAAGGAGC TCAGGGCAGCCAATGTAGACCTTGACGAGCGTCTGACCCTCTATTTG ATGCAGAACGGAAAATGCCTGTATTCTGGCCGCGCTATCGACCTCAA TAAGCTTATGGCGGGCTCGGGCGAGTATGAGGTCGACCATATCATTC CGCGCGCCTATATTAAGGACGACAGCCTCGAAAACAAGGCGCTGGT GTACCGCGAGGAGAATCAGCACAAGACCGACCAGCTTTTGATTGATA AGAGCATTCGCTGGAAGATGGGCGAGACGTGGAAACAGTTGCATCA GGCGAAGCTCATTGGCGATAAAAAGTATCGCAACCTGCTGAGGTCTT CCATAAACGAGAATGCCATGAAGGGCTTTGTCGCGCGCCAGCTCGTG GAGACGAGCCAGATGGTCAAGATGGCGCAGGCGTTGCTTGAGGCGC GTTATGCCGACGAGGGAACGAAGATCGTTCCCGTTAAGGCGAGCGT ATCGCACAACCTTCGTGAGGCAGCCGGCTTGGTCAAATGCCGCGAA GCCAATGATTTCCATCATGCGCACGATGCCTATCTGGCATGTCGCTT GGGCCTGTTTATTCAGATGCGCCACCCCGGAATGTATGAAAACCCCA TTGGCTACTCGCACGTGATTAAGAAGTACGTGCGCGAGCAGTCTGAG CTGTTTAAGAAAACCCATCGCATGCCTGGATCTGCCGGTTTTGTCGTC AATAGCTTTATGACTGCGGGTTTTGACCCCGAGACGGGCGAGGTCTT TGACGATGCCTGGACCGAGGACGACGAGGTCAAGCGTTATAGGGGA AAGTATCTTCCGGACGGCTGGAATGCAGCAGCGGAGGTTGAGAGCA TTCGCCGTGCGCTTAACTATCGCCAGTGCTACATCTCGAGGATGCCG CAGGAGGATACGGGAGCGTTCTGGAAGGCAACGATTTACTCGCCGC GTGATCCCAAGATGGGTGCAAAGCTTGAGTTGTCGACCAAGCAGGG GCTTGATGCTAAGCGGTACGGTGGTTTTTCGAGCCAGCAGTTTGCGT ATTTCTTTATCTATGAGGCCAAAAAGAAGGGGAAGCCGGTGTTCCGG TTCTCCCAGGTTCCCGTGTGGCTTGCGGCGCGCATCGAGGGCGACT CGGCGGCACTCGGCGAATATGCAAGGGGACTTGCGGAGAAGGAAGG TTTGGAATACGTCGGTATCACGAGGCCGAAGATCTACAAGAAACAGC TGATTGAGGTGGATGGCGAGAGATTCTTTATTACGGGGAAAAAGGAG ATGAGGAATGCTACGCAACTGGCTTTTTCAAATGAGCAAATGAAACTG CTCGCAGGAGATACGTGTAGCGAAGATGCTATCAAGCTTTTGGATAT GTTTAATCCCGCGGCTTCTTCCGTCTCTGGCAGGCTGCTGCGTCAAC TCAAAATTGAGGCGATCATTGAAAAGTGCGCAACTGTGGATGCGGAA GATGTTGTTGCAGTAGTCAAGCGCCTGCTTGCCTTGGTGAATGGAAC AATGAATATGGTTGATTTGTCACCCGCGGGCGGCTCAAAGTTTTCGG GTTGTATACAGCCTAATTATTCAAAGTTGCTGAGCGATTCAAATGTGA CGGTTTTCCTTATTGATCAGTCCGTAACAGGCATGTTCGAGAGAAGG ACCCGTGTCGGGCTTTAG CFDE Type II AACCTGAGAAACTACAGCGGCGACTACAACATCGGACTCGACATGGG 59 Cas coding CACCGGCAGCGTGGGCTGGGCCGTGACAGACGCCCAAGGTAAGTTG sequence (nt) CTGCATTTCAAGAAACAACCTACCTGGGGCTCCAGACTCTTTGACAG (human CGCCCAGCCTGCCAGCGAGGCGAGAGTGCACCGGGGACAGAGAAG codon- AAGATACGTGCGTAGAAGATGGCGGTTGGACCTGCTGCAGAAGCTCT optimized; TTGAACCTGCCATGAGCGAGATCGACCCTGGCTTCTTCATGCGGCTG lacking N- AACCAGAGCAGAACTATCGAGGGAGATCCTATCTTCACCAAGGACTT terminal TACCAAAGCCGACTACTACGACAGATTCCCTACCATCTATCACCTGCG methionine; GGCCTATCTGATGGACACCGATGAGCAGGCTGATCTGCGGCTGGTG no stop TACTTAGCCATCCACAACATCGTCAAACACCGGGGCAACTTCCTGCG codon) CCAAGGGGAGAAGTTGACCGCCAAGACCGCCAAAACCAGCGACGCC CTGGAGCGTCTGAATGATTCGCTGCAAGGATGGTGCGAAAGCAGAGTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO AGTACGAGGTTGCCAAGATCAACAAGTCGGCCGTGGCGAAGATCCT GGCGGATGAACGCAGCAGCCGGTCCCAGAAAGCCAAGGACATCCAG GCCCTGGTGAAGGTGACGACTGGCGACGCCGCCGCGGATAAGAAGC TGGCTAAGGGCCTCTCTAACGCTGTGGTGGGCCTCCAGTGTGAGTTC AAGGACGTGTTCGGGGAGTTCGAGTGCGAAGCCACCAAGCTGGCCT TGAGCGACGATGAGAAACTGGAAACACTCCAGGCTGCATGCCCTGAC GATTGCGTGGAGCTGTTGGAGAGCGTTTGTGGCGTGTACAGCGCCT ATGTCCTGCAAGGCCTGCTGAGCTACGCCGAAGGCCAGACCATCAG TATCAACATGATCAAGAAGTACGAGCAATACGCCGAAGATCTGGCCC TGCTGAAGTTGCTGGTGAGAAAGTATGCTCTGGACAGCTACGAGCCC TTCTTCCGGGGAGCCACGTACCACGATAGCAACAGCGACGACCCTTC CAGGGACTACGACGCCAGCAAGGCTACAGGCTACACCGCCTACGAC CTGCACAAGCTCAGCTATGACGACTTTGCCAAGTCCGTGAAGAAACT GTTTGCTGGAACCGGTGCCGAAACCGACGAGAGATATATCGCCATGA TGGACGCTTTCGACAAACAGAGATTCCTAAGACGGCTGAAAACCTCC GATAACGGCGCCATCTACTACCAGCTTCATCTGGAGGAACTGCAGGC CATCCTGAAAAACCAGGGCAGATTCTACCCTTTTCTGAAGGACGAGG CCGCTAAGATCGAGAGCCTGGTGACCTTCAGAATCCCTTACTACGTG GGCCCCCTGACAAGCAAGAACGCCGCCCAGGATAAGCACGGCAAGC CCAGATTTCAGTGGTCTGAGCGGAAGCCTGGCATGGAAGATGCAACA ATCACCCCTTGGAACTGGGACGCCGTTATCGACAAGAACAAGAGCGC TGAAAATTTCATCCTGCGCATGACAGGCGACTGCACCTACCTGGCTG GAGAAAAGGTGCTGCCTAAGCAGAGCCTGCTGTACGAGGAATTTTGC GTGCTGAACGAGCTGAATGGCGTGCGGTGGAGCGAGGACGGCGAC AACTGGCAGCGGCTGGACGCCGCCCAGCGGGAAGGCATTATCCATG AGCTGTTCCACAAGAAGAGAAGAGTGACCTACAAGATGATCGCCGAC TGGCTGGTGAAGGAGGGCGATGCCACCAACCCCGTGGTGCGGGGC GGCCAACGCGAGAGCGGCCTGGAAAGCAAGATGGGCTCTTACATCT TTTTCGCTAAAGATATCTTCGGCGTGGATGAGCTGAGCAGGGCCGAG TACCCCGTGATCGAGAAGATCATCCTGTGGAATACCCTGTTCGAAGA TAGATCTATCCTTAAAGAAAAGCTGCAGGACGAGTTCGGCCCTAGCG GAAATGGTATGCTGGACGCCGAGCAGATTAAGAAGATTTGTAAAAAA CGGCTGACCGGCTGGGGCAGACTGAGCGAAAAGTTCCTGACAGGTA TCAGAATCGACACCCAGGCCGGCCGGTCTATGTCTATCATGGACGTG CTGCGCGAAGGCAACCCTTCTTCCGAACACCGGCTGGGCGAGACAA TGGTGATGATGGAAATCCTGCATGACAACACACTGGGCTTTCAGGAG AAAGTCGATGAACACAACAGAAAGTACTACGCCGAGCACGAAAAGTC TCTGGGCGTCAACGAGCTGCCTGGTTCTCCTGCCATCAGGAGATCTC TGAATCAGGCGATCAGAATAGTGGACGAGATCGCCAAGATTGCCGGC CACGCTCCTGCCAATGTGTTCGTGGAAGTGACCAGAGATGAAGATGA GAAGAAGAAGGGCAAGCGGACAACAAAAAGATGGGATGCCATTGAG GATGCCCTGAAGGCATTTAAGGCCGAAGGCGGCGACCTGGCAGTGG TGGACGACTTCAAGGAACTGAGAGCCGCCAACGTGGACCTGGATGA AAGACTGACACTGTACCTGATGCAAAACGGCAAGTGTCTGTACTCCG GCAGAGCTATCGACCTGAACAAGCTGATGGCCGGCTCTGGCGAGTA CGAGGTGGACCACATCATCCCAAGAGCTTACATCAAGGACGATAGCC TGGAAAATAAAGCCCTGGTGTACCGGGAGGAGAACCAGCACAAGAC AGACCAGCTACTGATCGATAAGTCTATAAGATGGAAGATGGGAGAAA CATGGAAGCAGCTGCACCAGGCTAAACTGATCGGGGATAAGAAATAC AGAAACCTGCTGAGATCTTCCATCAACGAGAATGCAATGAAGGGCTT CGTTGCAAGACAACTCGTGGAAACCAGCCAGATGGTCAAGATGGCCC AGGCCCTGCTGGAAGCCAGATACGCCGACGAGGGCACAAAGATCGT GCCTGTGAAGGCCAGCGTGTCCCACAATCTGAGAGAAGCTGCCGGC CTGGTGAAGTGCAGAGAAGCAAACGACTTCCACCACGCCCACGACG CCTATCTGGCCTGCAGACTGGGCCTGTTCATCCAAATGAGACACCCC GGCATGTATGAGAACCCAATCGGCTACAGCCACGTGATCAAGAAGTA CGTGAGAGAGCAGTCTGAACTGTTCAAGAAAACCCACCGAATGCCCGTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO GCAGCGCCGGATTCGTGGTCAACAGCTTCATGACCGCTGGCTTCGA CCCAGAAACCGGCGAGGTGTTTGACGACGCTTGGACCGAGGATGAT GAAGTTAAACGGTATAGAGGAAAGTACCTGCCTGACGGCTGGAATGC CGCCGCAGAGGTGGAAAGCATTCGTAGAGCCCTGAACTACCGGCAG TGCTACATCAGCAGAATGCCTCAGGAGGATACCGGAGCCTTCTGGAA GGCCACAATCTACAGCCCCCGGGACCCTAAGATGGGCGCTAAGTTA GAACTGAGCACCAAGCAGGGACTGGATGCTAAAAGATACGGCGGATT CAGCAGCCAGCAGTTCGCCTACTTCTTCATCTACGAGGCCAAAAAGA AAGGCAAACCCGTGTTCAGATTCAGCCAGGTGCCAGTGTGGCTGGCT GCCAGAATCGAGGGCGATTCCGCCGCTCTGGGAGAATACGCCAGGG GCCTGGCCGAGAAGGAGGGCCTGGAGTACGTGGGCATCACCCGGC CTAAGATCTACAAGAAGCAGCTGATAGAAGTGGACGGCGAGAGATTC TTCATCACCGGCAAGAAGGAAATGAGAAACGCCACCCAGCTGGCCTT CAGCAACGAGCAGATGAAGCTGCTGGCCGGCGACACCTGCAGCGAG GATGCCATCAAGCTGTTAGACATGTTCAACCCTGCAGCCTCCAGCGT GTCTGGCAGACTGCTGAGGCAGCTCAAGATCGAGGCCATTATTGAGA AGTGCGCCACCGTGGACGCCGAGGACGTGGTCGCCGTAGTGAAGC GGCTGCTGGCTCTGGTGAACGGCACCATGAACATGGTGGACCTCAG TCCTGCAGGAGGCAGTAAGTTTTCCGGATGTATCCAGCCCAACTACT CAAAGCTGCTGAGCGACTCCAATGTCACCGTGTTTCTGATCGATCAG AGCGTGACAGGAATGTTCGAGAGAAGAACAAGAGTGGGCCTG CFDE Type II ATGGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCAA 60 Cas GCGGACTGCCGACGGCAGTGAGTTCGAGTCTCCCAAGAAAAAGAGG mammalian AAAGTGGGATCCAACCTGAGAAACTACAGCGGCGACTACAACATCGG expression ACTCGACATGGGCACCGGCAGCGTGGGCTGGGCCGTGACAGACGC construct CCAAGGTAAGTTGCTGCATTTCAAGAAACAACCTACCTGGGGCTCCA (with N- GACTCTTTGACAGCGCCCAGCCTGCCAGCGAGGCGAGAGTGCACCG terminal GGGACAGAGAAGAAGATACGTGCGTAGAAGATGGCGGTTGGACCTG methionine, CTGCAGAAGCTCTTTGAACCTGCCATGAGCGAGATCGACCCTGGCTT stop codon, CTTCATGCGGCTGAACCAGAGCAGAACTATCGAGGGAGATCCTATCT V5 tag, N- TCACCAAGGACTTTACCAAAGCCGACTACTACGACAGATTCCCTACCA terminal NLS TCTATCACCTGCGGGCCTATCTGATGGACACCGATGAGCAGGCTGAT and C- CTGCGGCTGGTGTACTTAGCCATCCACAACATCGTCAAACACCGGGG terminal NLS) CAACTTCCTGCGCCAAGGGGAGAAGTTGACCGCCAAGACCGCCAAA (nt) ACCAGCGACGCCCTGGAGCGTCTGAATGATTCGCTGCAAGGATGGT GCGAAAGCAGAGAGTACGAGGTTGCCAAGATCAACAAGTCGGCCGT GGCGAAGATCCTGGCGGATGAACGCAGCAGCCGGTCCCAGAAAGCC AAGGACATCCAGGCCCTGGTGAAGGTGACGACTGGCGACGCCGCCG CGGATAAGAAGCTGGCTAAGGGCCTCTCTAACGCTGTGGTGGGCCT CCAGTGTGAGTTCAAGGACGTGTTCGGGGAGTTCGAGTGCGAAGCC ACCAAGCTGGCCTTGAGCGACGATGAGAAACTGGAAACACTCCAGG CTGCATGCCCTGACGATTGCGTGGAGCTGTTGGAGAGCGTTTGTGG CGTGTACAGCGCCTATGTCCTGCAAGGCCTGCTGAGCTACGCCGAA GGCCAGACCATCAGTATCAACATGATCAAGAAGTACGAGCAATACGC CGAAGATCTGGCCCTGCTGAAGTTGCTGGTGAGAAAGTATGCTCTGG ACAGCTACGAGCCCTTCTTCCGGGGAGCCACGTACCACGATAGCAAC AGCGACGACCCTTCCAGGGACTACGACGCCAGCAAGGCTACAGGCT ACACCGCCTACGACCTGCACAAGCTCAGCTATGACGACTTTGCCAAG TCCGTGAAGAAACTGTTTGCTGGAACCGGTGCCGAAACCGACGAGA GATATATCGCCATGATGGACGCTTTCGACAAACAGAGATTCCTAAGAC GGCTGAAAACCTCCGATAACGGCGCCATCTACTACCAGCTTCATCTG GAGGAACTGCAGGCCATCCTGAAAAACCAGGGCAGATTCTACCCTTT TCTGAAGGACGAGGCCGCTAAGATCGAGAGCCTGGTGACCTTCAGA ATCCCTTACTACGTGGGCCCCCTGACAAGCAAGAACGCCGCCCAGG ATAAGCACGGCAAGCCCAGATTTCAGTGGTCTGAGCGGAAGCCTGG CATGGAAGATGCAACAATCACCCCTTGGAACTGGGACGCCGTTATCG ACAAGAACAAGAGCGCTGAAAATTTCATCCTGCGCATGACAGGCGACTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO TGCACCTACCTGGCTGGAGAAAAGGTGCTGCCTAAGCAGAGCCTGCT GTACGAGGAATTTTGCGTGCTGAACGAGCTGAATGGCGTGCGGTGG AGCGAGGACGGCGACAACTGGCAGCGGCTGGACGCCGCCCAGCGG GAAGGCATTATCCATGAGCTGTTCCACAAGAAGAGAAGAGTGACCTA CAAGATGATCGCCGACTGGCTGGTGAAGGAGGGCGATGCCACCAAC CCCGTGGTGCGGGGCGGCCAACGCGAGAGCGGCCTGGAAAGCAAG ATGGGCTCTTACATCTTTTTCGCTAAAGATATCTTCGGCGTGGATGAG CTGAGCAGGGCCGAGTACCCCGTGATCGAGAAGATCATCCTGTGGA ATACCCTGTTCGAAGATAGATCTATCCTTAAAGAAAAGCTGCAGGACG AGTTCGGCCCTAGCGGAAATGGTATGCTGGACGCCGAGCAGATTAA GAAGATTTGTAAAAAACGGCTGACCGGCTGGGGCAGACTGAGCGAA AAGTTCCTGACAGGTATCAGAATCGACACCCAGGCCGGCCGGTCTAT GTCTATCATGGACGTGCTGCGCGAAGGCAACCCTTCTTCCGAACACC GGCTGGGCGAGACAATGGTGATGATGGAAATCCTGCATGACAACACA CTGGGCTTTCAGGAGAAAGTCGATGAACACAACAGAAAGTACTACGC CGAGCACGAAAAGTCTCTGGGCGTCAACGAGCTGCCTGGTTCTCCTG CCATCAGGAGATCTCTGAATCAGGCGATCAGAATAGTGGACGAGATC GCCAAGATTGCCGGCCACGCTCCTGCCAATGTGTTCGTGGAAGTGAC CAGAGATGAAGATGAGAAGAAGAAGGGCAAGCGGACAACAAAAAGAT GGGATGCCATTGAGGATGCCCTGAAGGCATTTAAGGCCGAAGGCGG CGACCTGGCAGTGGTGGACGACTTCAAGGAACTGAGAGCCGCCAAC GTGGACCTGGATGAAAGACTGACACTGTACCTGATGCAAAACGGCAA GTGTCTGTACTCCGGCAGAGCTATCGACCTGAACAAGCTGATGGCCG GCTCTGGCGAGTACGAGGTGGACCACATCATCCCAAGAGCTTACATC AAGGACGATAGCCTGGAAAATAAAGCCCTGGTGTACCGGGAGGAGA ACCAGCACAAGACAGACCAGCTACTGATCGATAAGTCTATAAGATGG AAGATGGGAGAAACATGGAAGCAGCTGCACCAGGCTAAACTGATCG GGGATAAGAAATACAGAAACCTGCTGAGATCTTCCATCAACGAGAAT GCAATGAAGGGCTTCGTTGCAAGACAACTCGTGGAAACCAGCCAGAT GGTCAAGATGGCCCAGGCCCTGCTGGAAGCCAGATACGCCGACGAG GGCACAAAGATCGTGCCTGTGAAGGCCAGCGTGTCCCACAATCTGA GAGAAGCTGCCGGCCTGGTGAAGTGCAGAGAAGCAAACGACTTCCA CCACGCCCACGACGCCTATCTGGCCTGCAGACTGGGCCTGTTCATC CAAATGAGACACCCCGGCATGTATGAGAACCCAATCGGCTACAGCCA CGTGATCAAGAAGTACGTGAGAGAGCAGTCTGAACTGTTCAAGAAAA CCCACCGAATGCCCGGCAGCGCCGGATTCGTGGTCAACAGCTTCAT GACCGCTGGCTTCGACCCAGAAACCGGCGAGGTGTTTGACGACGCT TGGACCGAGGATGATGAAGTTAAACGGTATAGAGGAAAGTACCTGCC TGACGGCTGGAATGCCGCCGCAGAGGTGGAAAGCATTCGTAGAGCC CTGAACTACCGGCAGTGCTACATCAGCAGAATGCCTCAGGAGGATAC CGGAGCCTTCTGGAAGGCCACAATCTACAGCCCCCGGGACCCTAAG ATGGGCGCTAAGTTAGAACTGAGCACCAAGCAGGGACTGGATGCTAA AAGATACGGCGGATTCAGCAGCCAGCAGTTCGCCTACTTCTTCATCT ACGAGGCCAAAAAGAAAGGCAAACCCGTGTTCAGATTCAGCCAGGTG CCAGTGTGGCTGGCTGCCAGAATCGAGGGCGATTCCGCCGCTCTGG GAGAATACGCCAGGGGCCTGGCCGAGAAGGAGGGCCTGGAGTACG TGGGCATCACCCGGCCTAAGATCTACAAGAAGCAGCTGATAGAAGTG GACGGCGAGAGATTCTTCATCACCGGCAAGAAGGAAATGAGAAACGC CACCCAGCTGGCCTTCAGCAACGAGCAGATGAAGCTGCTGGCCGGC GACACCTGCAGCGAGGATGCCATCAAGCTGTTAGACATGTTCAACCC TGCAGCCTCCAGCGTGTCTGGCAGACTGCTGAGGCAGCTCAAGATC GAGGCCATTATTGAGAAGTGCGCCACCGTGGACGCCGAGGACGTGG TCGCCGTAGTGAAGCGGCTGCTGGCTCTGGTGAACGGCACCATGAA CATGGTGGACCTCAGTCCTGCAGGAGGCAGTAAGTTTTCCGGATGTA TCCAGCCCAACTACTCAAAGCTGCTGAGCGACTCCAATGTCACCGTG TTTCTGATCGATCAGAGCGTGACAGGAATGTTCGAGAGAAGAACAAGTABLE 1K CFDE Type II Cas Sequences Name Sequence SEQ ID NO AGTGGGCCTGTCTAGAAAGCGGACAGCAGACGGCTCCGAATTTGAAA GCCCTAAGAAAAAGAGAAAGGTGTGA

[0103] In some embodiments a CFDE Type II 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 CFDE Type II 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 D15A substitution, wherein the position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H890A substitution, wherein the position of the H890A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56. In some embodiments, a CFDE Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with an H890A substitution. 6.2.2. Fusion and Chimeric Proteins

[0104] The disclosure provides Type II Cas proteins (e.g., a DGHJ Type II Cas protein as described in Section 6.2.1.1, a CUAZ Type II Cas protein as described in Section 6.2.1.2, an AHWY Type II Cas protein as described in Section 6.2.1.3, a CBGI Type II Cas protein as described in Section 6.2.1.4, an ASDR Type II Cas protein as described in Section 6.2.1.5, a BCZZ Type II Cas protein as described in Section 6.2.1.6, a DRCY Type II Cas protein as described in Section 6.2.1.7, a DRPY Type II Cas protein as described in Section 6.2.1.8, an EAJR Type II Cas protein as described in Section 6.2.1.9, or a CFDE Type II Cas protein as described in Section 6.2.1.10) which are in the form of fusion proteins comprising a Type II 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., Krüppel associated box (KRAB)), a histone-modifying protein, an integrase, or a recombinase.

[0105] In some embodiments, a fusion protein of the disclosure comprises a means for localizing the Type II Cas protein to the nucleus, for example a nuclear localization signal.

[0106] Non-limiting examples of nuclear localization signals include KRTADGSEFESPKKKRKV (SEQ ID NO:116), PKKKRKV (SEQ ID NO:117), PKKKRRV (SEQ ID NO:118), KRPAATKKAGQAKKKK (SEQ ID NO:119), YGRKKRRQRRR (SEQ ID NO:120), RKKRRQRRR (SEQ ID NO:121), PAAKRVKLD (SEQ ID NO:122), RQRRNELKRSP (SEQ ID NO:123), VSRKRPRP (SEQ ID NO:124), PPKKARED (SEQ ID NO:125), PQPKKKPL (SEQ ID NO:126), SALIKKKKKMAP (SEQ ID NO:127), PKQKKRK (SEQ ID NO:128), RKLKKKIKKL (SEQ ID NO:129), REKKKFLKRR (SEQ ID NO:130), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:131), RKCLQAGMNLEARKTKK (SEQ ID NO:132), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:133), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:134) PAAKKKKLD (SEQ ID NO:582) and KRPAATKKAGQAKKKKLD (SEQ ID NO:583). In some embodiments, a nuclear localizationsignal comprises. PAAKKKKLD (SEQ ID NO 582) In some embodiments, a nuclear localization signal comprises KRPAATKKAGQAKKKKLD (SEQ ID NO:583)

[0107] Exemplary fusion partners include protein tags (e.g., V5-tag (e.g., having the sequence GKPIPNPLLGLDST (SEQ ID NO:135) or IPNPLLGLD (SEQ ID NO:136)), 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 II 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; US patent 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: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILAD ECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO:137)

[0108] 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:137.

[0109] Type II Cas proteins of the disclosure in the form of a fusion protein comprising an adenosine GHDPLQDVH^FDQ^EH^XVHG^DV^DQ^DGHQLQH^EDVH^HGLWRU^WR^FKDQJH^DQ^³$´^WR^D^³*´^LQ^'1$^^7\SH^,,^&DV^ proteins of the disclosure in the form of a fusion protein comprising a cytidine deaminase can be used as a F\WRVLQH^EDVH^HGLWRU^WR^FKDQJH^D^³&´^WR^D^³7´^LQ^'1$^

[0110] 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 (Cheng et al., 2019, Nat Commun.10(1):3612; Gehrke et al., 2018, Nat Biotechnol. 36(10):977-982).

[0111] 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. Type II Cas proteins of thedisclosure in the form of a fusion protein comprising a reverse transcriptase (RT) can be used as a prime editor to carry out precise base editing without double-stranded DNA breaks.

[0112] In some embodiments, a fusion protein of the disclosure is a prime editor, e.g., a Type II Cas protein fused to a suitable RT (e.g., Moloney murine leukemia virus (M-MLV) RT or other RT enzyme). Such fusion proteins can be used in conjunction with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit (Anzalone et al., 2019, Nature, 576(7785):149- 157).

[0113] 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 II Cas protein sequence (e.g., a DGHJ Type II Cas protein having a sequence of SEQ ID NO:1). In some embodiments, a fusion protein of the disclosure comprises an N-terminal and a C-terminal nuclear localization signal, for example each having the sequence KRTADGSEFESPKKKRKV (SEQ ID NO:116).

[0114] The disclosure provides chimeric Type II Cas proteins comprising one or more domains of a DGHJ Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of a CUAZ Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an AHWY Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of a CBGI Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an ASDR Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of a BCZZ Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of a DRCY Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of a DRPY Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), chimeric Type II Cas proteins comprising one or more domains of an EAJR Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins), and chimeric Type II Cas proteins comprising one or more domains of a CFDE Type II Cas protein and one or more domains of one or more different proteins (e.g., one or more different Type II Cas proteins).

[0115] The domain boundaries of wild-type Type II Cas proteins of the disclosure were determined by multiple alignment with the amino acid sequences of Type II Cas proteins for which the crystal structure is known and for which it is thus possible to define the boundaries of each functional domain. Domains identified in Type II Cas proteins include: the RuvC catalytic domain (discontinuous, represented by RuvC-I, RuvC-II, and RuvC-III domains), bridge helix (BH), recognition (REC) domain (which can be divided into REC1_1, REC2, and REC1_2 domains), HNH catalytic domain, and PAM-interacting domain(PID). The PID domain can be divided into a topoisomerase homology (Topo) domain and a C-terminal domain (CTD).

[0116] Table 2 below report the amino acid positions corresponding to the boundaries between different functional domains in wild-type DGHJ (SEQ ID NO:2), CUAZ (SEQ ID NO:8), AHWY (SEQ ID NO:14), CBGI (SEQ ID NO:20), ASDR (SEQ ID NO:26), BCZZ (SEQ ID NO:32), DRCY (SEQ ID NO:38), DRPY (SEQ ID NO:44), EAJR (SEQ ID NO:50), and CFDE (SEQ ID NO:56) Type II Cas proteins. Table 2 Amino Acid Positions of DGHJ, CUAZ, AHWY CBGI, ASDR, BCZZ, DRCY, DRPY, EAJR, and CFDE Domains DGHJ CUAZ AHWY CBGI ASDR BCZZ DRCY DRPY EAJR CFDERuvC-I1-49 1-50 1-51 1-51 1-52 1-54 1-52 1-51 1-55 1-52BH50-83 51-79 52-85 53-86 53-86 55-88 53-86 52-85 56-89 53-8684- 80- 86-178 87- 87- 89- 87- 86- 90- 87- REC1_1 176 153 178 167 180 178 171 179 167 177- 154- 179- 179- 168- 181- 179- 172- 180- 168- REC2 303 285 322 309 303 307 316 315 313 303 304- 286- 323- 310- 304- 308- 317- 316- 314- 304- REC1_2 738 699 769 765 765 706 765 741 727 757 739- 700- 770- 766- 766- 707- 766- 742- 728- 758- RuvC-II 794 764 825 821 822 762 822 798 784 814 795- 765- 826- 822- 823- 763- 823- 799- 785- 815- HNH 934 924 966 956 964 902 959 945 924 956 935- 925- 967- 957- 965- 903- 960- 946- 925- 957- RuvC-III 1075 1102 1130 1121 1149 1043 1120 1082 1058 1141 1076- 1103- 1131- 1122- 1150- 1044- 1121- 1083- 1059- 1142- Topo 1176 1205 1234 1221 1250 1143 1226 1191 1151 1246 1177- 1206- 1235- 1222- 1251- 1144- 1227- 1192- 1152- 1247- CTD 1344 1408 1392 1374 1404 1311 1388 1377 1324 1396

[0117] A chimeric Type II 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 CUAZ Type II Cas protein, AHWY Type II Cas protein, DGHJ Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, and / or CFDE Type II Cas protein, and one or more domains from one or more other proteins, for example SaCas9, SpCas9 or a Type II Cas protein described in US 2020 / 0332273, US 2019 / 0169648, or 2015 / 0247150 (the contents of each of which are incorporated herein by reference in their entirety): RuvC-I, BH, REC (or a subpart thereof, for example one, two, or three of REC1_1, REC2, and REC1_2), RuvC-II, HNH, RuvC-III, PID (or a subpart thereof, for example one or both of the Topo domain and CTD). For example, the PID domain can be swapped between different Type II 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).

[0118] In some embodiments, a Type II Cas protein of the disclosure comprises one, two, three, four, five, six, seven, eight, nine or ten of a RuvC-I domain, a BH domain, a REC1_1 domain, a REC2 domain, a REC1_2 domain, a RuvC-II domain, a HNH domain, a RuvC-III domain, a Topo domain and a CTDarranged in the N-terminal to C-terminal direction. In some embodiments, all domains are from a DGHJ Type II Cas protein (e.g., a DGHJ Type II Cas protein whose amino acid sequence comprises SEQ ID NO:1, 2, or 3). In some embodiments, all domains are from a CUAZ Type II Cas protein (e.g., a CUAZ Type II Cas protein whose amino acid sequence comprises SEQ ID NO:7, 8, or 9). In some embodiments, all domains are from an AHWY Type II Cas protein (e.g., an AHWY Type II Cas protein whose amino acid sequence comprises SEQ ID NO:13, 14, or 15). In some embodiments, all domains are from a CBGI Type II Cas protein (e.g., a CBGI Type II Cas protein whose amino acid sequence comprises SEQ ID NO:19, 20, or 21). In some embodiments, all domains are from an ASDR Type II Cas protein (e.g., an ASDR Type II Cas protein whose amino acid sequence comprises SEQ ID NO:25, 26, or 27). In some embodiments, all domains are from a BCZZ Type II Cas protein (e.g., a BCZZ Type II Cas protein whose amino acid sequence comprises SEQ ID NO:31, 32, or 33). In some embodiments, all domains are from a DRCY Type II Cas protein (e.g., a DRCY Type II Cas protein whose amino acid sequence comprises SEQ ID NO:37, 38, or 39). In some embodiments, all domains are from a DRPY Type II Cas protein (e.g., a DRPY Type II Cas protein whose amino acid sequence comprises SEQ ID NO:43, 44, or 45). In some embodiments, all domains are from an EAJR Type II Cas protein (e.g., an EAJR Type II Cas protein whose amino acid sequence comprises SEQ ID NO:49, 50, or 51). In some embodiments, all domains are from a CFDE Type II Cas protein (e.g., a CFDE Type II Cas protein whose amino acid sequence comprises SEQ ID NO:55, 56, or 57). In other embodiments, one or more domains (e.g., one domain), e.g., a PID domain, is from another Type II Cas protein.

[0119] 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 SpCas9 providing nickase activity are the D10A substitution in the RuvC domain and the H840A substitution in the HNH domain. Combining both the D10A and H840A substitutions in SpCas9 provides a catalytically inactive nuclease. Corresponding substitutions can be introduced into the Type II Cas nucleases of the disclosure to provide nickases and catalytically inactive Cas proteins. For example, a DGHJ Type II Cas protein can include a D12A substitution (corresponding to D10A in SpCas9) or a H868A substitution (corresponding to H840A in SpCas9) to provide a nickase, or D12A and H868A substitutions to provide a catalytically inactive Cas protein, where the positions of the D12A and H868A substitutions are defined with respect to amino acid numbering of SEQ ID NO:2. Positions corresponding to D10 and H840 of SpCas9 for Type II Cas proteins of the disclosure as shown in Table 3. Nickases and catalytically inactive Type II Cas proteins of the disclosure can be used, for example, in base editors comprising a cytosine or adenosine deaminase fusion partner. Catalytically inactive Type II Cas proteins can also be used, for example, as fusion partners for transcriptional activators or repressors. Table 3 Position Type II corresponding Position Reference SEQ ID NO Cas to D10 of corresponding to defining amino acid Protein SpCas9 H840 of SpCas9 numbering DGHJ 12 868 2Table 3 Position Type II corresponding Position Reference SEQ ID NO Cas to D10 of corresponding to defining amino acid Protein SpCas9 H840 of SpCas9 numbering CUAZ 18 845 8 AHWY 14 900 14 CBGI 15 890 20 ASDR 15 898 26 BCZZ 17 836 32 DRCY 15 893 38 DRPY 14 878 44 EAJR 18 857 50 CFDE 15 890 56 6.3. Guide RNAs

[0120] The disclosure provides gRNA molecules that can be used with Type II Cas proteins of the disclosure to edit genomic DNA, for example mammalian DNA, e.g., human DNA. gRNAs of the GLVFORVXUH^W\SLFDOO\^FRPSULVH^D^VSDFHU^RI^^^^WR^^^^QXFOHRWLGHV^LQ^OHQJWK^^7KH^VSDFHU^FDQ^EH^SRVLWLRQHG^^¶^ of a crRNA scaffold to form a full crRNA. The crRNA can be used with a tracrRNA to effect cleavage of a target genomic sequence.

[0121] An exemplary crRNA scaffold sequence that can be used for DGHJ Type II Cas gRNAs comprises GUUUUAGAAUUAUGUUAUUUUAGAUAGAAAUAAUAA (SEQ ID NO:61) and an exemplary tracrRNA sequence that can be used for DGHJ Type II Cas gRNAs comprises UAUUAAUUCUAUCUAAAAUAGCAUAAUGAGUUAAAAUAAGGUUUAACCUUUAAUGCCGAAUUGCU UCGGUUUCACUUAGGUGAAAAUUGCUCUGUUCAUCAGAGC (SEQ ID NO:62).

[0122] An exemplary crRNA scaffold sequence that can be used for CUAZ Type II Cas gRNAs comprises GAUUUUAAGUAUGGUUCUUUUUACUAAGUUACUGAC (SEQ ID NO:63) and an exemplary tracrRNA sequence that can be used for CUAZ Type II Cas gRNAs comprises GUAACUUAGUAAAAGGAUACCAUAACGAAAAAUUAUGAAUAUUCUACUACUUUUAGAGGGCAUAA UUGGUUGCCCUCA (SEQ ID NO:64).

[0123] An exemplary crRNA scaffold sequence that can be used for AHWY Type II Cas gRNAs comprises GUUUUGGAGCAGUGGCAUUCUGACUGGUAAUCAAAC (SEQ ID NO:65) and an exemplary tracrRNA sequence that can be used for AHWY Type II Cas gRNAs comprises UUGAUACCAGUCAAGAUGUCACUGCGAGUCAAAAUAAAGACUUAGUCUUAAAUGUACCUGUAAUA GGGACGCCGCGUAGGCGGCACUUUUGAAUCGCGAAAGAGCCUUCGGGCUCUUU (SEQ ID NO:66).

[0124] An exemplary crRNA scaffold sequence that can be used for CBGI Type II Cas gRNAs comprises GUUUUGGAGCAGUGUCGUUCUGCUGGUAAUCCAAC (SEQ ID NO:67) and an exemplary tracrRNA sequence that can be used for CBGI Type II Cas gRNAs comprises UGAUUACCAGUCAGGACGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUUCGGCGCC GCGUAGGCGGCAG (SEQ ID NO:68).

[0125] An exemplary crRNA scaffold sequence that can be used for ASDR Type II Cas gRNAs comprises GUUUUGGAGCAGUGUCGAUCUAACUGGUAAUCAAAC (SEQ ID NO:69) and an exemplary tracrRNA sequence that can be used for ASDR Type II Cas gRNAs comprises GAUUACCAGUUACAUCGACACUGUGAGUCAAAAUACGGCUAAGCCAAAAAUGCUGCUCUUUCGG GAGCAUCACCCCGUAGGGGGAA (SEQ ID NO:70).

[0126] An exemplary crRNA scaffold sequence that can be used for BCZZ Type II Cas gRNAs comprises GUUUUAGUAUUGUGUUAUUUUAGAUAGUAAUAAAAC (SEQ ID NO:71) and an exemplary tracrRNA sequence that can be used for BCZZ Type II Cas gRNAs comprises UAUUAAAUUACUAUCUAAAAUAGCACAAUGAGUUAAAAUAAGGUUUAAACCUUAAAUGCCAACUUG AUGUUGGUUUCACUUAGGUGAA (SEQ ID NO:72).

[0127] An exemplary crRNA scaffold sequence that can be used for DRCY Type II Cas gRNAs comprises GUUUUGGAGCAGUGUCAAUCUGACUGGUAAUCAAAC (SEQ ID NO:73) and an exemplary tracrRNA sequence that can be used for DRCY Type II Cas gRNAs comprises GAUUACCAGUCGGAUCGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUGCUUGCAGG CGCCACGUAGGUGGCAA (SEQ ID NO:74).

[0128] An exemplary crRNA scaffold sequence that can be used for DRPY Type II Cas gRNAs comprises GUUUGAGAGCAGUGUUAAUCCAUAGGGGAUUGAAAC (SEQ ID NO:75) and an exemplary tracrRNA sequence that can be used for DRPY Type II Cas gRNAs comprises GUGUCAAUCCCCUAUGGAUUAACACUGCGAGUUCAAAUAACGUUAUCGCAACGAAAUCGCCGUAU AUCGGUUGCACAGUGUGUGCA (SEQ ID NO:76).

[0129] An exemplary crRNA scaffold sequence that can be used for EAJR Type II Cas gRNAs comprises GUUUGAGAGUAGUGUAAUCCCAAGUGGUAGUAGUGU (SEQ ID NO:77) and an exemplary tracrRNA sequence that can be used for EAJR Type II Cas gRNAs comprises CUACUACCACUUGGGAUUACACUACAAGUUCAAAUAAGCGUUCUGCGCCCUUACCGCAUUAAGUU GCGGAUUCACAGUGUGUGA (SEQ ID NO:78).

[0130] An exemplary crRNA scaffold sequence that can be used for CFDE Type II Cas gRNAs comprises GUUUUGGAGCAGUGUCGAUCUAACUGGUAAUCAAAC (SEQ ID NO:79) and an exemplary tracrRNA sequence that can be used for CFDE Type II Cas gRNAs comprises GAUUACCAGUUACAUCGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUUCCUCGGAAG GCGUCCCGUAGGGGACAAA (SEQ ID NO:80).

[0131] gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically FRPSULVH^WKH^VSDFHU^DW^WKH^^¶^HQG^RI^WKH^PROHFXOH^DQG^D^^¶^VJ51$^VFDIIROd. Alternatively, gRNAs can comprise separate crRNA and tracrRNA molecules.

[0132] Further features of exemplary gRNA spacer sequences are described in Section 6.3.1 and IXUWKHU^IHDWXUHV^RI^H[HPSODU\^^¶^VJ51$^VFDIIROGV^DUH^GHVFULEHG^LQ^6HFWLRQ^6.3.2. 6.3.1. Spacers

[0133] 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 bepartially 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.

[0134] 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.

[0135] Type II Cas endonucleases require a specific sequence, called a protospacer adjacent motif (PAM) that is downstream (e.g., directly downstream) 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 II Cas protein. Exemplary PAM sequences for Type II Cas proteins are shown in Table 4. Table 4 Type II Cas protein PAM(s) Most preferred PAM(s) DGHJ Type II Cas NGG NGG CUAZ Type II Cas NNGNC NNGNCNY AHWY Type II Cas NNRC NNAC, NNACNT CBGI Type II Cas NNRTA NNRTA ASDR Type II Cas NNNNCC NNANCC, NNACCC BCZZ Type II Cas NGG NGG DRCY Type II Cas In silico predicted : NNAT, In vitro determined: NRATA NNATM, NNATA, NVAT, NVATM In vitro determined: NVRTA, NRRTA, NVATA DRPY Type II Cas NNNAC NRNAC, NRHAC, NRNACD, NRHACD, NRNACA, NRHACA EAJR Type II Cas NNNRNY NRNRNY,NRNRNC, NANANC, NNNRDC CFDE Type II Cas NNART NRART N = A, T, C, or G; M = A or C; V = G, C, or A; R = A or G; Y = C or T; H = A, C or T; D = A, G or TTable 4 Type II Cas protein PAM(s) Most preferred PAM(s) All PAMs are in vitro determined PAMs, except where noted (see Example 2).

[0136] Example 3 describes exemplary sequences that can be used to target TRAC, B2M, and PD1 genomic sequences. 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.

[0137] Additional exemplary spacer sequences that can be used in gRNAs of the disclosure are set forth in Table 5A, Table 5B, and Table 5C. Table 5A Exemplary Spacer Sequences Targeting B2M Type II Cas Name Spacer Sequence SEQ ID NO: AHWY sgRNA-AHWY-B2M-g1 CAGAAAGAGAGAGUAGCGCGAGC 138 AHWY sgRNA-AHWY-B2M-g2 CCGUGGCCUUAGCUGUGCUCGCG 139 AHWY sgRNA-AHWY-B2M-g3 UCAGGUACUCCAAAGAUUCAGGU 140 AHWY sgRNA-AHWY-B2M-g4 GUACUCCAAAGAUUCAGGUUUAC 141 AHWY sgRNA-AHWY-B2M-g5 GACACAUAGCAAUUCAGGAAAUU 142 ASDR sgRNA-ASDR-B2M-g1 ACUCUCUCUUUCUGGCCUGGAGG 143 ASDR sgRNA-ASDR-B2M-g2 CCAAAGAUUCAGGUUUACUCACG 144 ASDR sgRNA-ASDR-B2M-g3 CUGAAUUGCUAUGUGUCUGGGUU 145 ASDR sgRNA-ADSR-B2M-g4 AUUGCUAUGUGUCUGGGUUUCAU 146 ASDR sgRNA-ASDR-B2M-g5 GUCAACUUCAAUGUCGGAUGGAUG 147 ASDR sgRNA-ASDR-B2M-g5_20nt ACUUCAAUGUCGGAUGGAUG 148 CBGI sgRNA-CBGI-B2M-g1 GAUAGCCUCCAGGCCAGAAAGAGA 149 CBGI sgRNA-CBGI-B2M-g2 GGAGGGUAGGAGAGACUCACGCU 150 CBGI sgRNA-CBGI-B2M-g3 GACGUGAGUAAACCUGAAUCUUUG 151 CBGI sgRNA-CBGI-B2M-g4 UUCCAUUCUCUGCUGGAUGACGU 152 CBGI sgRNA-CBGI-B2M-g5 GUCGGAUGGAUGAAACCCAGAC 153 CFDE sgRNA-CFDE-B2M-g1 GAUAGCCUCCAGGCCAGAAAGAG 154 CFDE sgRNA-CFDE-B2M-g2 UCUGGCCUGGAGGCUAUCCAGCG 155 CFDE sgRNA-CFDE-B2M-g3 GACGUGAGUAAACCUGAAUCUUU 156 CFDE sgRNA-CFDE-B2M-g4 GUUUACUCACGUCAUCCAGCAG 157 CFDE sgRNA-CFDE-B2M-g5 GAGAAUGGAAAGUCAAAUUUCC 158 DRPY sgRNA-DRPY-B2M-g1 UUCAGUGGGGGUGAAUUCAGUGU 159 DRPY sgRNA-DRPY-B2M-g2 GCCAGAAAGAGAGAGUAGCGCG 160 DRPY sgRNA-DRPY-B2M-g3 CAAUGUCGGAUGGAUGAAACCCA 161 DRPY sgRNA-DRPY-B2M-g4 GCAAGGACUGGUCUUUCUAUCUCU 162Table 5B Exemplary Spacer Sequences Targeting TRAC Type II Cas Name Spacer Sequence SEQ ID NO: AHWY sgRNA-AHWY-TRAC-g1 AUCAAAAUCGGUGAAUAGGCAGAC 163 AHWY sgRNA-AHWY-TRAC-g2 ACACAUCAGAAUCCUUACUUUGUG 164 AHWY sgRNA-AHWY-TRAC-g3 GCACUGUUGCUCUUGAAGUCCAU 165 AHWY sgRNA-AHWY-TRAC-g4 AACUGUGCUAGACAUGAGGUCUAU 166 AHWY sgRNA-AHWY-TRAC-g5 GUAUAUCACAGACAAAACUGUGCU 167 ASDR sgRNA-ASDR-TRAC-g1 UCUUGUCCCACAGAUAUCCA 168 ASDR sgRNA-ASDR-TRAC-g2 CCCACAGAUAUCCAGAACCC 169 ASDR sgRNA-ASDR-TRAC-g3 GUGUACCAGCUGAGAGACUCU 170 ASDR sgRNA-ASDR-TRAC-g4 UGUCUGUGAUAUACACAUCA 171 ASDR sgRNA-ASDR-TRAC-g5 GCCACAGCACUGUUGCUCUUG 172 CBGI sgRNA-CBGI-TRAC-g1 GAUAUCCAGAACCCUGACCCUGCC 173 CBGI sgRNA-CBGI-TRAC-g2 CACUGGAUUUAGAGUCUCUCAGC 174 CBGI sgRNA-CBGI-TRAC-g3 UUUGUUUGAGAAUCAAAAUCGGU 175 CBGI sgRNA-CBGI-TRAC-g4 GAUUCUCAAACAAAUGUGUCACA 176 CFDE sgRNA-CFDE-TRAC-g5 GUCACAAAGUAAGGAUUCUGAU 177 CFDE sgRNA-CFDE-TRAC-g1 GCCGUGUACCAGCUGAGAGACUC 178 CFDE sgRNA-CFDE-TRAC-g2 GCUGAGAGACUCUAAAUCCAGUGA 179 CFDE sgRNA-CFDE-TRAC-g3 GCAGACAGACUUGUCACUGGAUUU 180 CFDE sgRNA-CFDE-TRAC-g4 AUUCACCGAUUUUGAUUCUCAAA 181 CFDE sgRNA-CFDE-TRAC-g5 AUUUGUUUGAGAAUCAAAAUCGG 182 CFDE sgRNA-CFDE-TRAC-g6 UGAUUCUCAAACAAAUGUGUCAC 183 CFDE sgRNA-CFDE-TRAC-g7 UUACUUUGUGACACAUUUGUUUG 184 CFDE sgRNA-CFDE-TRAC-g8 GUUUUGUCUGUGAUAUACACAUC 185 CFDE sgRNA-CFDE-TRAC-g9 CAGGCCACAGCACUGUUGCUCUU 186 CFDE sgRNA-CFDE-TRAC-g10 gAACAGUGCUGUGGCCUGGAGCAA 187 DRPY sgRNA-DRPY-TRAC-g1 ACUGGAUUUAGAGUCUCUCAGCU 188 DRPY sgRNA-DRPY-TRAC-g2 GCCUAUUCACCGAUUUUGAUUCU 189 DRPY sgRNA-DRPY-TRAC-g3 ACACAUCAGAAUCCUUACUUUGU 190 DRPY sgRNA-DRPY-TRAC-g4 CUAGCACAGUUUUGUCUGUGAUA 191 DRPY sgRNA-DRPY-TRAC-g5 UGAAGUCCAUAGACCUCAUGUCU 192 DRPY sgRNA-DRPY-TRAC-g6 GUCUAGCACAGUUUUGUCUGUGA 193 DRPY sgRNA-DRPY-TRAC-g7 AUAAUGCUGUUGUUGAAGGCGUU 194 Table 5C Exemplary Spacer Sequences Targeting PD1 Type II Cas Name Spacer Sequence SEQ ID NO: AHWY sgRNA-AHWY-PD1-g1 AUCCUGGCCGCCAGCCCAGUUGUA 195 AHWY sgRNA-AHWY-PD1-g2 GGGGCUGCUCCAGGCAUGCAGAUC 196 AHWY sgRNA-AHWY-PD1-g3 GCCCAGUUGUAGCACCGCCCAGA 197 AHWY sgRNA-AHWY-PD1-g4 GGCCAGUCGUCUGGGCGGUGCUA 198Table 5C Exemplary Spacer Sequences Targeting PD1 Type II Cas Name Spacer Sequence SEQ ID NO: ASDR sgRNA-ASDR-PD1-g1 GUGGGGCUGCUCCAGGCAUGC 199 ASDR sgRNA-ASDR-PD1-g2 CGACCCCACCUACCUAAGAA 200 ASDR sgRNA-ASDR-PD1-g3 CAGGCAUGCAGAUCCCACAG 201 ASDR sgRNA-ASDR-PD1-g4 GCCGCCAGCCCAGUUGUAGCA 202 ASDR sgRNA-ASDR-PD1-g5 CUAAGAACCAUCCUGGCCGC 203 ASDR sgRNA-ASDR-PD1-g6 UCAGACUCCCCAGACAGGCCCUG 204 ASDR sgRNA-ASDR-PD1-g7 CGGGGCCUCCGAGGCCGCACCUG 205 CBGI sgRNA-CBGI-PD1-g1 GAACCAUCCUGGCCGCCAGCCCAG 206 CBGI sgRNA-CBGI-PD1-g2 GGCUGGCGGCCAGGAUGGUUCUU 207 CBGI sgRNA-CBGI-PD1-g3 CAUCGGAGAGCUUCGUGCUAAAC 208 CBGI sgRNA-CBGI-PD1-g4 CCGUCUGGUUGCUGGGGCUCAUG 209 CBGI sgRNA-CBGI-PD1-g5 GGGCCAGGGAGAUGGCCCCACAG 210 CFDE sgRNA-CFDE-PD1-g1 GGGGGGUUCCAGGGCCUGUCUGG 211 CFDE sgRNA-CFDE-PD1-g2 CGGGCCCUGACCACGCUCAUGUG 212 DRPY sgRNA-DRPY-PD1-g1 GCCACCUUCACCUGCAGCUUCUCC 213 DRPY sgRNA-DRPY-PD1-g2 GGCCAGGACUGCCGCUUCCGUGU 214 DRPY sgRNA-DRPY-PD1-g3 UGGGGCUCAUGCGGUACCAGUUU 215 DRPY sgRNA-DRPY-PD1-g4 CAACACAUCGGAGAGCUUCGUGC 216 DRPY sgRNA-DRPY-PD1-g5 GUCACGCCCGUUGGGCAGUUGUGU 217

[0138] 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 5A. 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 5A. 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 5A. 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 5A. 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 5A. 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 5A. 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 5A. 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 5A. 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 5A. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 5A.

[0139] 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 5B. In someembodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 16 or more consecutive nucleotides from a sequence shown in Table 5B. 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 5B. 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 5B. 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 5B. 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 5B. 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 5B. 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 5B. 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 5B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 5B.

[0140] 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 5C. 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 5C. 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 5C. 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 5C. 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 5C. 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 5C. 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 5C. 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 5C. 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 5C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 5C. 6.3.2. sgRNA Molecules

[0141] gRNAs of the disclosure can be single-guide RNA (sgRNA) molecules. A sgRNA can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-PROHFXOH^JXLGH^OLQNHU^^D^PLQLPXP^WUDFU51$^VHTXHQFH^^D^^¶^WUDFU51$^ sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-moleculeguide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins.

[0142] The sgRNA can comprise a variable length spacer sequence (e.g.^^^^^WR^^^^QXFOHRWLGHV^^DW^WKH^^¶^ HQG^RI^WKH^VJ51$^VHTXHQFH^DQG^D^^¶^VJ51$^VHJPHQW^^

[0143] Type II Cas gRNAs typically comprise a repeat-antirepeat duplex and / or one or more stem-loops JHQHUDWHG^E\^WKH^J51$¶V^VHFRQdary structure. The length of the repeat-antirepeat duplex and / or one or more stem-loops can be modified in order to modulate (e.g., increase) the editing efficacy of a Type II Cas nuclease, and / or to reduce the size of a guide RNA for easier vectorization in situations in which the cargo size of the vector is limiting (e.g., AAV vectors).

[0144] For example, the repeat-antirepeat duplex (which in a sgRNA is fused through a synthetic linker to become an additional stem loop in the structure) can be trimmed at different lengths without generally having detrimental effects on nuclease function and in some cases even producing increased enzymatic activity. If bulges are present within this duplex they generally should be retained in the final guide RNA sequence.

[0145] 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.

[0146] 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.

[0147] ([HPSODU\^^¶^VJ51$^VFDIIROG^VHTXHQFHV^IRU^Type II Cas sgRNAs are shown in Table 6. Table 6 Sequences of sgRNA Scaffolds for Type IIA Cas Proteins Name Sequence SEQ ID NO: DGHJ Type II GUUUUAGAAUUAUGUUAUUUUAGAUAGAAAUAAUAAGAAAUAUUAAU 81 Cas sgRNA UCUAUCUAAAAUAGCAUAAUGAGUUAAAAUAAGGUUUAACCUUUAAU scaffold GCCGAAUUGCUUCGGUUUCACUUAGGUGAAAAUUGCUCUGUUCAUC AGAGC DGHJ Type II GUUUUAGAAUUAUGGAAACAUAAUGAGUUAAAAUAAGGUUUAACCUU 82 Cas sgRNA UAAUGCCGAAUUGCUUCGGUUUCACUUAGGUGAAAAUUGCUCUGUU scaffold CAUCAGAGC (trimmed) (v1) DGHJ Type II GUUUUAGAAUUAUGGAAACAUAAUGAGUUAAAAUAAGGUUUAACCUU 83 Cas sgRNA UAAUGCCGAAUUGCUUCGGUUUCACUUAGGUGAAAA scaffold (trimmed) (alternate) (v2)Table 6 Sequences of sgRNA Scaffolds for Type IIA Cas Proteins Name Sequence SEQ ID NO: CUAZ Type II GAUUUUAAGUAUGGUUCUUUUUACUAAGUUACUGACGAAAGUAACU 84 Cas sgRNA UAGUAAAAGGAUACCAUAACGAAAAAUUAUGAAUAUUCUACUACUUU scaffold UAGAGGGCAUAAUUGGUUGCCCUCA CUAZ Type II GAUUUUAAGUAUGGGAAACCAUAACGAAAAAUUAUGAAUAUUCUACU 85 Cas sgRNA ACUUUUAGAGGGCAUAAUUGGUUGCCCUCA scaffold (trimmed) AHWY Type II GUUUUGGAGCAGUGGCAUUCUGACUGGUAAUCAAACGAAAUUGAUA 86 Cas sgRNA CCAGUCAAGAUGUCACUGCGAGUCAAAAUAAAGACUUAGUCUUAAAU scaffold GUACCUGUAAUAGGGACGCCGCGUAGGCGGCACUUUUGAAUCGCGA AAGAGCCUUCGGGCUC AHWY Type II GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUAAAGACUUAGUC 87 Cas sgRNA UUAAAUGUACCUGUAAUAGGGACGCCGCGUAGGCGGCACUUUUGAA scaffold UCGCGAAAGAGCCUUCGGGCUC (trimmed) (v1) AHWY Type II GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUAAAGACUUAGUC 88 Cas sgRNA UUAAAUGUACCUGUAAUAGGGACGCCGCGUAGGCGGCAC scaffold (trimmed) (alternate) (v2) CBGI Type II GUUUUGGAGCAGUGUCGUUCUGCUGGUAAUCCAACGAAAUGAUUAC 89 Cas sgRNA CAGUCAGGACGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUG scaffold CCUUCGGCGCCGCGUAGGCGGCAG CBGI Type II GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUACGGCUUUGCCA 90 Cas sgRNA AAAAUGCCUUCGGCGCCGCGUAGGCGGCAG scaffold (trimmed) ASDR Type II GUUUUGGAGCAGUGUCGAUCUAACUGGUAAUCAAACGAAAGAUUAC 91 Cas sgRNA CAGUUACAUCGACACUGUGAGUCAAAAUACGGCUAAGCCAAAAAUGC scaffold UGCUCUUUCGGGAGCAUCACCCCGUAGGGGGAA ASDR Type II GUUUUGGAGCAGUGGAAACACUGUGAGUCAAAAUACGGCUAAGCCA 92 Cas sgRNA AAAAUGCUGCUCUUUCGGGAGCAUCACCCCGUAGGGGGAA scaffold (trimmed) BCZZ Type II GUUUUAGUAUUGUGUUAUUUUAGAUAGUAAUAAAACGAAAUAUUAAA 93 Cas sgRNA UUACUAUCUAAAAUAGCACAAUGAGUUAAAAUAAGGUUUAAACCUUA scaffold AAUGCCAACUUGAUGUUGGUUUCACUUAGGUGAA BCZZ Type II GUUUUAGUAUUGAAAAAUGAGUUAAAAUAAGGUUUAAACCUUAAAUG 94 Cas sgRNA CCAACUUGAUGUUGGUUUCACUUAGGUGAA scaffold (trimmed) DRCY Type II GUUUUGGAGCAGUGUCAAUCUGACUGGUAAUCAAACGAAAGAUUAC 95 Cas sgRNA CAGUCGGAUCGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUG scaffold CCUGCUUGCAGGCGCCACGUAGGUGGCAA DRCY Type II GUUUUGGAGCGAAAGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCC 96 Cas sgRNA UGCUUGCAGGCGCCACGUAGGUGGCAA scaffold (trimmed) DRPY Type II GUUUGAGAGCAGUGUUAAUCCAUAGGGGAUUGAAACGAAAGUGUCA 97 Cas sgRNA AUCCCCUAUGGAUUAACACUGCGAGUUCAAAUAACGUUAUCGCAACG scaffold AAAUCGCCGUAUAUCGGUUGCACAGUGUGUGCA DRPY Type II GUUUGAGAGCGAAAGCGAGUUCAAAUAACGUUAUCGCAACGAAAUC 98 Cas sgRNA GCCGUAUAUCGGUUGCACAGUGUGUGCATable 6 Sequences of sgRNA Scaffolds for Type IIA Cas Proteins Name Sequence SEQ ID NO: scaffold (trimmed) EAJR Type II GUUUGAGAGUAGUGUAAUCCCAAGUGGUAGUAGUGUGAAACUACUA 99 Cas sgRNA CCACUUGGGAUUACACUACAAGUUCAAAUAAGCGUUCUGCGCCCUU scaffold ACCGCAUUAAGUUGCGGAUUCACAGUGUGUGA EAJR Type II GUUUGAGAGUGAAAACAAGUUCAAAUAAGCGUUCUGCGCCCUUACC 100 Cas sgRNA GCAUUAAGUUGCGGAUUCACAGUGUGUGA scaffold (trimmed) CFDE Type II GUUUUGGAGCAGUGUCGAUCUAACUGGUAAUCAAACGAAAGAUUAC 101 Cas sgRNA CAGUUACAUCGACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUGC scaffold CUUCCUCGGAAGGCGUCCCGUAGGGGACAAA CFDE Type II GUUUUGGAGCGAAAGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCC 102 Cas sgRNA UUCCUCGGAAGGCGUCCCGUAGGGGACAAA scaffold (trimmed)

[0148] The sgRNA (e.g., for use with CUAZ Type II Cas protein, AHWY Type II Cas protein, DGHJ Type II Cas protein, CBGI Type II Cas protein, ASDR Type II Cas protein, BCZZ Type II Cas protein, DRCY Type II Cas protein, DRPY Type II Cas protein, EAJR Type II Cas protein, and / or CFDE Type II Cas protein^^FDQ^FRPSULVH^QR^XUDFLO^EDVH^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^7\SLFDOO\^^KRZHYHU^^WKH^ VJ51$^FRPSULVHV^RQH^RU^PRUH^XUDFLO^EDVHV^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^IRU^H[DPple to SURPRWH^FRUUHFW^VJ51$^IROGLQJ^^)RU^H[DPSOH^^WKH^VJ51$^FDQ^FRPSULVH^^^XUDFLO^^8^^DW^WKH^^¶^HQG^RI^WKH^ VJ51$^VHTXHQFH^^7KH^VJ51$^FDQ^FRPSULVH^^^XUDFLO^^88^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^7KH^ VJ51$^FDQ^FRPSULVH^^^XUDFLO^^888^^DW^WKH^^¶^HQG^RI^WKH sgRNA sequence. The sgRNA can comprise 4 XUDFLO^^8888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^7KH^VJ51$^FDQ^FRPSULVH^^^XUDFLO^^88888^^DW^WKH^ ^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^7KH^VJ51$^FDQ^FRPSULVH^^^XUDFLO^^888888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^ sequence. The sgRN$^FDQ^FRPSULVH^^^XUDFLO^^8888888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^7KH^ VJ51$^FDQ^FRPSULVH^^^XUDFLO^^88888888^^DW^WKH^^¶^HQG^RI^WKH^VJ51$^VHTXHQFH^^'LIIHUHQW^OHQJWK^ VWUHWFKHV^RI^XUDFLO^FDQ^EH^DSSHQGHG^DW^WKH^^¶HQG^RI^D^VJ51$^DV^WHUPLQDWRUV^^7KXV^^IRU^H[DPSOH^^WKH^^¶^ sgRNA sequences set forth in Table 6 can be modified by adding (or removing) one or more uracils at the end of the sequence.

[0149] In some embodiments, a sgRNA scaffold for use with a DGHJ Type II Cas protein comprises the sequence GUUUUAGAAUUAUGGAAACAUAAUGAGUUAAAAUAAGGUUUAACCUUUAAUGCCGAAUUGCUUCG GUUUCACUUAGGUGAAAAUUGCUCUGUUCAUCAGAGCUUUUUU (SEQ ID NO:103). In some embodiments, a sgRNA scaffold for use with a DGHJ Type II Cas protein comprises the sequence GUUUUAGAAUUAUGGAAACAUAAUGAGUUAAAAUAAGGUUUAACCUUUAAUGCCGAAUUGCUUCG GUUUCACUUAGGUGAAAAUUUUUU (SEQ ID NO:104).

[0150] In some embodiments, a sgRNA scaffold for use with a CUAZ Type II Cas protein comprises the sequenceGAUUUUAAGUAUGGGAAACCAUAACGAAAAAUUAUGAAUAUUCUACUACUUUUAGAGGGCAUAAU UGGUUGCCCUCAUUUUUU (SEQ ID NO:105).

[0151] In some embodiments, a sgRNA scaffold for use with an AHWY Type II Cas protein comprises the sequence GUUUUGGAGCAGUGGCAUUCUGACUGGUAAUCAAACGAAAUUGAUACCAGUCAAGAUGUCACUG CGAGUCAAAAUAAAGACUUAGUCUUAAAUGUACCUGUAAUAGGGACGCCGCGUAGGCGGCACUU UUGAAUCGCGAAAGAGCCUUCGGGCUCUUU (SEQ ID NO:106). In some embodiments, a sgRNA scaffold for use with an AHWY Type II Cas protein comprises the sequence GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUAAAGACUUAGUCUUAAAUGUACCUGUAAUAG GGACGCCGCGUAGGCGGCACUUUUGAAUCGCGAAAGAGCCUUCGGGCUCUUUUUU (SEQ ID NO:107). In some embodiments, a sgRNA scaffold for use with an AHWY Type II Cas protein comprises the sequence GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUAAAGACUUAGUCUUAAAUGUACCUGUAAUAG GGACGCCGCGUAGGCGGCACUUUUUU (SEQ ID NO:108).

[0152] In some embodiments, a sgRNA scaffold for use with a CBGI Type II Cas protein comprises the sequence GUUUUGGAGCAGUGGAAACACUGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUUCGGCGCCG CGUAGGCGGCAGUUUUUU (SEQ ID NO:109).

[0153] In some embodiments, a sgRNA scaffold for use with an ASDR Type II Cas protein comprises the sequence GUUUUGGAGCAGUGGAAACACUGUGAGUCAAAAUACGGCUAAGCCAAAAAUGCUGCUCUUUCGG GAGCAUCACCCCGUAGGGGGAAUUUUUU (SEQ ID NO:110).

[0154] In some embodiments, a sgRNA scaffold for use with a BCZZ Type II Cas protein comprises the sequence GUUUUAGUAUUGAAAAAUGAGUUAAAAUAAGGUUUAAACCUUAAAUGCCAACUUGAUGUUGGUUU CACUUAGGUGAAUUUUUU (SEQ ID NO:111).

[0155] In some embodiments, a sgRNA scaffold for use with a DRCY Type II Cas protein comprises the sequence GUUUUGGAGCGAAAGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUGCUUGCAGGCGCCACGU AGGUGGCAAUUUUUU (SEQ ID NO:112).

[0156] In some embodiments, a sgRNA scaffold for use with a DRPY Type II Cas protein comprises the sequence GUUUGAGAGCGAAAGCGAGUUCAAAUAACGUUAUCGCAACGAAAUCGCCGUAUAUCGGUUGCAC AGUGUGUGCAUUUUUU (SEQ ID NO:113).

[0157] In some embodiments, a sgRNA scaffold for use with an EAJR Type II Cas protein comprises the sequence GUUUGAGAGUGAAAACAAGUUCAAAUAAGCGUUCUGCGCCCUUACCGCAUUAAGUUGCGGAUUC ACAGUGUGUGAUUUUUU (SEQ ID NO:114).

[0158] In some embodiments, a sgRNA scaffold for use with a CFDE Type II Cas protein comprises the sequence GUUUUGGAGCGAAAGCGAGUCAAAAUACGGCUUUGCCAAAAAUGCCUUCCUCGGAAGGCGUCCC GUAGGGGACAAAUUUUUU (SEQ ID NO:115). 6.3.3. Modified gRNA Molecules

[0159] 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.

[0160] 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 II 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.

[0161] 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.

[0162] 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 CH2-NH-O-CH2, CH,~N(CH3)-O-CH2 (known as a methylene(methylimino) or MMI backbone), CH2-O-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P- O- CH,); amide backbones (see De Mesmaeker et al.1995, Ace. Chem. Res., 28:366-374); morpholino backbone structures (see U.S. Patent 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 otheralkyl phosphonates comprising 3'alkylene 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. Patent 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.

[0163] 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. Patent No.5,034,506.

[0164] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., 2000, J. Am. Chem. Soc., 122: 8595-8602.

[0165] 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 intemucleoside 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 CH2 component parts; see U.S. Patent 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.

[0166] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or bi- alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; 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'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al., 1995, Helv. Chim. Acta, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'- OCH2CH2CH3) 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.

[0167] 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. Patent 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.

[0168] 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.

[0169] 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.

[0170] Further, nucleobases can comprise those disclosed in U.S. Patent 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. Theseinclude 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. Patent No.3,687,808, as well as 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.

[0171] 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.

[0172] 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. Patent 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.

[0173] 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.

[0174] 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. Patent 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.

[0175] 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 KA 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

[0176] The disclosure provides systems comprising a Type II Cas protein of the disclosure (e.g., as described in Section 6.2) and a means for targeting the Type II Cas protein to a target genomic sequence. The means for targeting the Type II Cas protein to a target genomic sequence can be a guide RNA (gRNA) (e.g., as described in Section 6.3).

[0177] The disclosure also provides systems comprising a Type II 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 II Cas protein is complexed with a gRNA, for example asgRNA or separate crRNA and tracrRNA. Systems of the disclosure can in some embodiments further comprise genomic DNA complexed with the Type II Cas protein and the gRNA. Accordingly, the disclosure provides systems comprising a Type II Cas protein, a genomic DNA, and gRNA, all complexed with one another.

[0178] 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

[0179] The disclosure provides nucleic acids (e.g., DNA or RNA) encoding Type II Cas proteins (e.g., CUAZ Type II Cas proteins, AHWY Type II Cas proteins, DGHJ Type II Cas proteins, CBGI Type II Cas proteins, ASDR Type II Cas proteins, BCZZ Type II Cas proteins, DRCY Type II Cas proteins, DRPY Type II Cas proteins, EAJR Type II Cas proteins, and CFDE Type II Cas proteins), nucleic acids encoding gRNAs of the disclosure, nucleic acids encoding both Type II Cas proteins and gRNAs, and pluralities of nucleic acids, for example comprising a nucleic acid encoding a Type II Cas protein and a gRNA.

[0180] A nucleic acid encoding a Type II 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 II Cas and gRNA coding sequences in bacterial (e.g., E. coli) or eukaryotic (e.g., yeast) cells.

[0181] A nucleic acid encoding a Type II 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).

[0182] Nucleic acids encoding a Type II 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 II Cas can be used for producing a Type II Cas polypeptide. Exemplary codon-optimized sequences are shown in Tables 1A-1K.

[0183] 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...

Claims

WHAT IS CLAIMED IS:

1. A Type II Cas protein comprising an amino acid sequence having at least 50% sequence identity to: (a) the amino acid sequence of a RuvC-I domain of a reference protein sequence; (b) the amino acid sequence of a RuvC-II domain of a reference protein sequence; (c) the amino acid sequence of a RuvC-III domain of a reference protein sequence; (d) the amino acid sequence of a BH domain of a reference protein sequence; (e) the amino acid sequence of a REC1_1 domain of a reference protein sequence; (f) the amino acid sequence of a REC2 domain of a reference protein sequence; (g) the amino acid sequence of a REC1_2 domain of a reference protein sequence; (h) the amino acid sequence of a HNH domain of a reference protein sequence; (i) the amino acid sequence of a Topo domain of a reference protein sequence; (j) the amino acid sequence of a CTD domain of a reference protein sequence; or (k) the amino acid sequence of the full length of a reference protein sequence; 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, or SEQ ID NO:

56.

2. The Type II Cas protein of claim 1, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or is identical to the full length of the reference protein sequence.

3. The Type II Cas protein of claim 1, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or is identical to the full length of the reference protein sequence.

4. The Type II Cas protein of claim 1, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the full length of the reference protein sequence 5. The Type II Cas protein of any one of claims 1 to 4, which is a fusion protein.

6. The Type II Cas protein of claim 5, which comprises one or more nuclear localization signals, such as two or more nuclear localization signals, and which optionally comprises an N-terminal nuclear localization signal and / or a C-terminal nuclear localization signal.

7. The Type II Cas protein of claim 6, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:116), PKKKRKV (SEQ ID NO:117), PKKKRRV (SEQ ID NO:118), KRPAATKKAGQAKKKK (SEQ ID NO:119), YGRKKRRQRRR (SEQ ID NO:120), RKKRRQRRR (SEQ ID NO:121), PAAKRVKLD (SEQ ID NO:122), RQRRNELKRSP (SEQ ID NO:123), VSRKRPRP (SEQ ID NO:124), PPKKARED (SEQ ID NO:125), PQPKKKPL (SEQ ID NO:126), SALIKKKKKMAP (SEQ ID NO:127), PKQKKRK (SEQ ID NO:128), RKLKKKIKKL (SEQ ID NO:129), REKKKFLKRR (SEQ ID NO:130),KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:131), RKCLQAGMNLEARKTKK (SEQ ID NO:132), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:133), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:134).

8. The Type II Cas protein of claim 6, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PAAKKKKLD (SEQ ID NO:582) or KRPAATKKAGQAKKKKLD (SEQ ID NO:583) 9. The Type II Cas protein of any one of claims 6 to 8, wherein the amino acid sequence of each nuclear localization signal is the same.

10. The Type II Cas protein of any one of claims 5 to 9, 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.

11. The Type II Cas protein of any one of claims 5 to 10, which comprises (a) 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:137, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e; (b) a fusion partner which is a cytidine deaminase; or (c) a fusion partner which is a reverse transcriptase.

12. The Type II Cas protein of any one of claims 5 to 11, which comprises a tag, e.g., a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:135).

13. The Type II Cas protein of any one of claims 1 to 12, wherein (a) the reference protein sequence is SEQ ID NO:1 or SEQ ID NO:2, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (b) the reference protein sequence is SEQ ID NO:7 or SEQ ID NO:8, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; (c) the reference protein sequence is SEQ ID NO:13 or SEQ ID NO:14, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (d) the reference protein sequence is SEQ ID NO:19 or SEQ ID NO:20, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21; (e) the reference protein sequence is SEQ ID NO:25 or SEQ ID NO:26, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27; (f) the reference protein sequence is SEQ ID NO:31 or SEQ ID NO:32, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33; (g) the reference protein sequence is SEQ ID NO:37 or SEQ ID NO:38,optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39; (h) the reference protein sequence is SEQ ID NO:43 or SEQ ID NO:44, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45; (i) the reference protein sequence is SEQ ID NO:49 or SEQ ID NO:50, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; (j) the reference protein sequence is SEQ ID NO:55 or SEQ ID NO:56, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:

57.

14. A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of claims 1 to 13 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 D10 of SpCas9 or H840 of SpCas9 (e.g., as shown in Table 3).

15. A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of claims 1 to 13 except for one or more amino acid substitutions relative to the reference sequence that render the Type II Cas protein catalytically inactive, optionally wherein the one or more amino acid substitutions comprise a substitution (e.g., alanine substitution) at a position corresponding to position D10 of SpCas9 or H840 of SpCas9 (e.g., as shown in Table 3), or a combination thereof.

16. 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:138-162.

17. 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:163-194.

18. 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:195-217.

19. The gRNA of any one of claims 16 to 18, which comprises a spacer that is 15 to 30 nucleotides in length, 18 to 30 nucleotides in length, 20 to 28 nucleotides in length, 22 to 26 nucleotides in length, 23 to 25 nucleotides in length, 22 to 25 nucleotides in length, 15 to 25 nucleotides in length, 16 to 24 nucleotides in length, 17 to 23 nucleotides in length, 18 to 22 nucleotides in length, 19 to 21 nucleotides in length, 25 nucleotides in length, 24 nucleotides in length, 23 nucleotides in length, 22 nucleotides in length, 21 nucleotides in length, or 20 nucleotides in length.

20. The gRNA of any one of claims 16 to 19, wherein the spacer comprises the reference sequence.

21. The gRNA of any one of claims 16 to 20, which is a single guide RNA (sgRNA).

22. A gRNA comprising a spacer and a sgRNA scaffold, wherein:(a) WKH^VSDFHU^LV^SRVLWLRQHG^^¶^WR^WKH^VJ51$^VFDIIROG^^DQG (b) the nucleotide sequence of the sgRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is any one of SEQ ID NOS:81-102.

23. The gRNA of claim 22, wherein the sgRNA scaffold comprises a nucleotide sequence that is at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the reference scaffold sequence; comprises a nucleotide sequence that has no more than 5 nucleotide mismatches, no more than 4 nucleotide mismatches, no more than 3 nucleotide mismatches, no more than 2 nucleotide mismatches, or no more than 1 nucleotide mismatch with the reference scaffold sequence; or comprises a nucleotide sequence that is 100% identical to the reference scaffold sequence.

24. The gRNA of claim 22 or claim 23, wherein the sgRNA scaffold comprises 1 to 8 uracils DW^LWV^^¶^HQG^ 25. The gRNA of any one of claims 22 to 24, wherein the nucleotide sequence of the spacer is partially or fully complementary to a target mammalian genomic sequence.

26. A gRNA comprising (i) a crRNA comprising a spacer and a crRNA scaffold, wherein the VSDFHU^LV^^¶^WR^WKH^FU51$^VFDIIROG^^DQG^^LL^^D^WracrRNA, wherein the nucleotide sequence of the spacer is partially or fully complementary to a target mammalian genomic sequence and the nucleotide sequence of the crRNA scaffold comprises the nucleotide sequence of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, or SEQ ID NO:

79.

27. The gRNA of claim 26, wherein the gRNA comprises separate crRNA and tracrRNA molecules.

28. The gRNA of claim 26, wherein the gRNA is a single guide RNA (sgRNA).

29. The gRNA of any one of claims 22 to 28, wherein the target mammalian genomic sequence is a human genomic sequence.

30. The gRNA of any one of claims 22 to 29, wherein the target mammalian genomic sequence is upstream of a protospacer adjacent motif (PAM) sequence in the non-target strand recognized by a Type II Cas protein, optionally wherein the Type II Cas protein is a Type II Cas protein according to any one of claims 1 to 15.

31. The gRNA of claim 30, wherein the PAM sequence is NGG, NNGNC, NNGNCNY, NNRC, NNAC, NNACNT, NNRTA, NNNNCC, NNANCC, NNACCC, NNAT, NNATM, NNATA, NVAT, NVATM, NNNAC, NRNAC, NRHAC, NRNACD, NRHACD, NRNACA, NRHACA, NNNRNY, NRNRNY,NRNRNC, NANANC, NNNRDC, NNART, or NRART.

32. The gRNA of claim 30, wherein the PAM sequence is NVRTA, NRRTA, NVATA, or NRATA.

33. The gRNA of any one of claims 22 to 32, wherein the spacer is 15 to 30 nucleotides in length.

34. A system comprising the Type II Cas protein of any one of claims 1 to 15 and a guide RNA (gRNA) comprising a spacer sequence, optionally wherein the gRNA is a gRNA according to any one of claims 16 to 33.

35. A nucleic acid encoding the Type II Cas protein of any one of claims 1 to 15, optionally wherein the nucleotide sequence encoding the Type II Cas protein is operably linked to a promoter that is heterologous to the Type II Cas protein.

36. A nucleic acid encoding the gRNA of any one of claims 16 to 33.

37. A nucleic acid encoding the Type II Cas protein and gRNA of the system of claim 34.

38. A plurality of nucleic acids comprising separate nucleic acids encoding the Type II Cas protein and gRNA of the system of claim 34.

39. A Type II Cas protein according to any one of claims 1 to 15, a gRNA according to any one of claims 16 to 33, a system according to claim 34, a nucleic acid according to any one of claims 35 to 37, or a plurality of nucleic acids according to claim 38 for use in a method of editing a human genomic sequence.

40. A particle comprising a Type II Cas protein according to any one of claims 1 to 15, a gRNA according to any one of claims 16 to 33, a system according to claim 34, a nucleic acid according to any one of claims 35 to 37, or a plurality of nucleic acids according to claim 38.

41. A pharmaceutical composition comprising a Type II Cas protein according to any one of claims 1 to 15, a gRNA according to any one of claims 16 to 33, a system according to claim 34, a nucleic acid according to any one of claims 35 to 37, or a plurality of nucleic acids according to claim 38, or a particle according to claim 40 and at least one pharmaceutically acceptable excipient.

42. An ex vivo cell comprising a Type II Cas protein according to any one of claims 1 to 15, a gRNA according to any one of claims 16 to 33, a system according to claim 34, a nucleic acid according to any one of claims 35 to 37, or a plurality of nucleic acids according to claim 38, or a particle according to claim 40.

43. A method for altering an ex vivo cell, the method comprising contacting the cell with a Type II Cas protein according to any one of claims 1 to 15, a gRNA according to any one of claims 16 to 33, a system according to claim 34, a nucleic acid according to any one of claims 35 to 37, a plurality of nucleic acids according to claim 38, a particle according to claim 40, or a pharmaceutical composition according to claim 41.

44. An ex vivo cell or population of cells produced by the method of claim 43.

Images