Type ii cas proteins and applications thereof

Abstract

Type II Cas proteins, for example Type II Cas proteins referred to as AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET 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 THEREOF1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application nos. 63 / 479,461 , filed January 11 , 2023, 63 / 488,260, filed March 3, 2023, and 63 / 601 ,409, filed November 21 , 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 January 9, 2024, is named ALA-011 WO_SL.xml and is 944,048 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). However, packaging a large Cas protein such as SpCas9 together with a guide RNA into a single AAV vector can be challenging due to the limited packaging capacity of AAVs. Thus, there is a need for Type II Cas nucleases with smaller sizes that can be packaged together with a gRNA in a single AAV. In addition, 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 bacterium from the Acidaminococcaceae genus (referred to herein as “wild-type AEQH Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Ruminococcaceae family (referred to herein as “wild-type AAOF Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Clostridia class (referred to herein as “wild-type ACEE Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Ruminococcaceae family (referred to herein as “wild-type AQSL Type II Cas”), a Type II Cas protein from an unclassified Proteobacterium (referred to herein as “wild-type ASWC Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Clostridiaceae family (referred to herein as “wild-type AVFG Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Gemmiger genus (referred to herein as “wild-type AWIT Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Gemmiger genus (referred to herein as “wild-type AWMF Type II Cas”), a Type II Cas protein from unclassified bacterium from the Firmicutes phylum (referred to herein as “wild-type BUMO Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Gemmiger genus (referred to herein as “wild-type COIA Type II Cas”), a Type II Cas protein from an unclassified bacterium from the Gemmiger genus (referred to herein as “wild-type DJQA Type II Cas”), and a Type II Cas protein from an unclassified bacterium from the Ruminococcaceae family (referred to herein as “wild-type DWET Type II Cas”). Wild-type AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT,AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins are each approximately 1000 amino acids in length, significantly shorter than SpCas9.

[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 NO:1 (such proteins referred to herein as “AEQH Type II Cas proteins”). Exemplary AEQH 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 least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95% identical, or more) to SEQ ID NO:7 (such proteins referred to herein as “AAOF Type II Cas proteins”). Exemplary AAOF 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 (such proteins referred to herein as “ACEE Type II Cas proteins”). Exemplary ACEE 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 (such proteins referred to herein as “AQSL Type II Cas proteins”). Exemplary AQSL Type II Cas protein sequences are set forth in SEQ ID NO:19, SEQ ID NQ: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 (such proteins referred to herein as “ASWC Type II Cas proteins”). Exemplary ASWC 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 to herein as “AVFG Type II Cas proteins”). Exemplary AVFG 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 (such proteins referred to herein as “AWIT Type II Cas proteins”). Exemplary AWIT 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 (such proteins referred to herein as “AWMF Type II Cas proteins”). Exemplary AWMF 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 (such proteins referred to herein as “BUMO Type II Cas proteins”). Exemplary BUMO Type II Cas protein sequences are set forth in SEQ ID NO:49, SEQ ID NQ: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 (such proteins referred to herein as “COIA Type II Cas proteins”). Exemplary COIA Type II Cas protein sequences are set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.

[0015] 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:61 (such proteins referred to herein as “DJQA Type II Cas proteins”). Exemplary DJQA Type II Cas protein sequences are set forth in SEQ ID NO:61 , SEQ ID NO:62, and SEQ ID NO:63.

[0016] 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:68 (such proteins referred to herein as “DWET Type II Cas proteins”). Exemplary DWET Type II Cas protein sequences are set forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69.

[0017] 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-l domain, RuvC-ll domain, RuvC-lll domain, BH domain, REC domain, HNH domain, WED domain, or PID domain of an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET 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 an AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and / or DWET Type II Cas protein(s) and one or more domains from a different Type II Cas protein such as SpCas9.

[0018] In some embodiments, the Type II Cas proteins of the disclosure are in the form of a fusion protein, for example, comprising an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type IICas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET 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).

[0019] Exemplary features of Type II Cas proteins of the disclosure are described in Section 6.2 and specific embodiments 1 to 295 and 1132 to 1138, infra.

[0020] In further aspects, the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs) and combinations of two or more gRNA molecules (e.g., combinations of sgRNA molecules). In various embodiments, the disclosure provides gRNAs that can be used with the AEQH Type II Cas proteins of the disclosure, gRNAs that can be used with the AAOF Type II Cas proteins of the disclosure, gRNAs that can be used with the ACEE Type II Cas proteins of the disclosure, gRNAs that can be used with the AQSL Type II Cas proteins of the disclosure, gRNAs that can be used with the ASWC Type II Cas proteins of the disclosure, gRNAs that can be used with the AVFG Type II Cas proteins of the disclosure, gRNAs that can be used with the AWIT Type II Cas proteins of the disclosure, gRNAs that can be used with the AWMF Type II Cas proteins of the disclosure, gRNAs that can be used with the BUMO Type II Cas proteins of the disclosure, gRNAs that can be used with the COIA Type II Cas proteins of the disclosure, gRNAs that can be used with the DJQA Type II Cas proteins of the disclosure, and gRNAs that can be used with the DWET Type II Cas proteins of the disclosure. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure of the disclosure are described in Section 6.3 and specific embodiments 296 to 1007, infra.

[0021] 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 a ribonucleoprotein (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 1008 to 1072, infra.

[0022] 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.

[0023] 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 an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET Type II Cas protein.

[0024] In another aspect, the disclosure provides nucleic acids encoding combinations of gRNAs of the disclosure, for example a combination of two gRNAs, and, optionally, a Type II Cas protein.

[0025] Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5 and specific embodiments 1073 to 1131 , infra.

[0026] 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 1139 to 1154, infra.

[0027] 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 1156 to 1165 and 1205, infra.

[0028] 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 1155, infra.

[0029] 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, for example retinitis pigmentosa caused by a RHO mutation. Features of exemplary methods of altering cells are described in Section 6.8 and specific embodiments 1166 to 1204, infra.5. BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1 shows PAM logos for exemplary Type II Cas proteins of the disclosure.

[0031] FIGS. 2A-2D schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for AAOF Type II Cas protein (FIG. 2A), ACEE Type II Cas protein (FIG. 2B), AEQH Type II Cas protein (FIG. 2C), and AQSL Type II Cas protein (FIG. 2D). Figures 2A-2D disclose SEQ ID NOS 124, 126, 122, and 128, respectively, in order of appearance.

[0032] FIGS. 3A-3D schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for ASWC Type II Cas protein (FIG. 3A), for AVFG Type II Cas protein (FIG. 3B), AWIT, AWMF and COIA Type II Cas proteins (FIG. 3C), and BUMO Type II Cas protein (FIG. 3D). Figures 3A-3D disclose SEQ ID NOS 130, 132, 134, and 138, respectively, in order of appearance.

[0033] FIGS. 4A-4B schematically show the hairpin structure generated for visualization after in silico folding using RNA folding form v2.3 (www.unafold.org) of the sgRNA scaffolds (not including the spacer sequence) designed from crRNAs and tracrRNAs identified for DJQA Type II Cas protein (FIG. 4A) and DWET Type II Cas protein (FIG. 4B). Figures 4A-4B disclose SEQ ID NOS 142 and 144, respectively, in order of appearance.

[0034] FIGS. 5A-5F show PAM sequence logos for AAOF Type II Cas (FIG. 5A), ACEE Type II Cas (FIG. 5C), and AEQH Type II Cas (FIG. 5E) Type II Cas proteins from 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 AAOF Type II Cas (positions 5,6 and 7,8) (FIG. 5B), ACEE Type II Cas (positions 5,6 and 7,8) (FIG. 5D), and AEQH Type II Cas (positions 3,4 and 5,6) (FIG. 5F).

[0035] FIGS. 6A-6F show PAM sequence logos for AQSL Type II Cas (FIG. 6A), ASWC Type II Cas (FIG. 6C), and AVFG Type II Cas (FIG. 6E) from 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 AQSL Type II Cas (positions 5,6 and 7,8) (FIG. 6B), ASWC Type II Cas (positions 2,3 and 5,6) (FIG 6D), and AVFG Type II Cas (positions 5,6 and 7,8) (FIG. 6F).

[0036] FIGS. 7A-7F show PAM sequence logos for AWIT Type II Cas (FIG. 7A), AWMF Type II Cas (FIG. 7C), and COIA Type II Cas (FIG. 7E) from 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 AWIT Type II Cas (positions 5,6 and 7,8) (FIG. 7B), AWMF Type II Cas (positions 5,6 and 7,8) (FIG. 7D), and COIA Type II Cas (positions 5,6 and 7,8) (FIG. 7F).

[0037] FIGS. 8A-8F show PAM sequence logos for BUMO Type II Cas (FIG. 8A), DJQA Type II Cas (FIG. 8C) and DWET Type II Cas (FIG. 8E) from 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 BUMO Type II Cas (positions 5,6 and 7,8) (FIG. 8B), DJQA Type II Cas (positions 5,6 and 7,8) (FIG. 8D), and DWET Type II Cas (positions 5,6 and 7,8) (FIG. 8F).

[0038] FIG. 9 shows the activity of selected Type II Cas proteins evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells stably expressing EGFP.

[0039] FIG. 10 (including subpart FIG. 10-1) shows a schematic representation of the rs7984 SNP locus with the position of exemplary sgRNAs for AAOF, ACEE, AEQH, AVFG, BUMO, DJQA and DWET Type II Cas proteins (Example 3). Figure 10 discloses SEQ ID NOS 494, 491 , 491 , 485, 493, 493, 487, 487, 490, 490, 492, 495, 495, 859, 486, 486, 484, 496, 497, 488, and 488, respectively, in order of appearance.

[0040] FIG. 11 shows the editing activity towards the rs7984A RHO SNP allele of AAOF, ACEE, AEQH, AVFG, BUMO, DJQA and DWET Type II Cas proteins in combination with selected sgRNAs reported after transient plasmid transfection in HEK293T cells (Example 3). Data reported as mean ± SEM for n=2 independent runs.

[0041] FIGS. 12A-C shows editing activity towards the rs7984A RHO SNP allele using guides having different spacer length (20-24 nucleotides) for ACEE (FIG. 12A), DJQA (FIG. 12B), and AVFG (FIG. 12C) Type II Cas proteins (Example 3). Data reported as mean ± SEM for n=2 independent runs.

[0042] FIGS. 13A-13B shows the editing activity and allele specificity of selected gRNAs towards theRHO SNP locus (Example 3). FIG. 13A shows the activity and allele specificity against the rs7984A SNPallele after transient transfection of HEK293T cells with ACEE, DJQA or AVFG Type II Cas in combination with the indicated guides either perfectly matching the rs7984A locus (on-target activity) or the rs7984G locus (off-target activity - allele specificity). FIG. 13B shows the same study performed in HEK293T-rs7984G which are homozygous for the rs7984G allele, with the guides targeting the rs7984G locus used to measure the on-target activity while those targeting the rs7984A locus were used to evaluate the allele-specificity. Data reported as mean ± SEM for n=2 independent runs.

[0043] FIGS. 14A-14B (including subparts FIGS. 14A-1 to 14A-7 and FIGS. 14B-1 to 14B-7) show a schematic representation of the RHO intron 1 sequence reporting the position of the evaluated guide RNAs for ACEE Type II Cas (FIG. 14A) and DJQA Type II Cas (FIG. 14B) (Example 3). Figures 14A-14B disclose SEQ ID NOS 521 , 860, 518, 520, 517, 519, 862-863, 865, 866, 868, 532, 528, 533, 869-873, 535-536, and 875, respectively, in order of appearance.

[0044] FIGS. 15A-15B show the evaluation of the editing activity of guide RNAs for ACEE (FIG. 15A) and DJQA (FIG. 15B) Type II Cas targeting RHO intron 1 after transient transfection in HEK293T cells (Example 3).

[0045] FIGS. 16A-16B show the evaluation of large editing events at the target RHO locus (Example 3). FIG. 16A shows a representative image of an agarose gel electrophoresis of endpoint PCR products generated with primers spanning the deleted RHO fragment using genomic DNA extracted from HEK293T cells transfected with the indicated Type II Cas protein and relative guides. A high molecular weight band corresponds to undeleted / inverted products while low molecular weight bands correspond to deleted RHO alleles. FIG. 16B reports the results of a qPCR assay exploiting specifically designed primers to assess the relative amount of RHO alleles which do not contain large edits (deletions / inversions) in a population of HEK293T cells transiently transfected with expression plasmids for ACEE or DJQA Type II Cas together with the selected sgRNAs, as indicated on the graph. NT : non treated cells included as unmodified controls. Data reported as mean ± SEM for n=2 independent runs.

[0046] FIG. 17 shows the levels of deletions and inversions, as measured by a specifically designed ddPCR assay, which were generated in HEK293T cells after transient transfection of either ACEE or DJQA Type II Cas in combination with the indicated sgRNAs targeting the rs7984A RHO SNP allele and RHO intron 1 (Example 3). NT: non treated cells included as unmodified controls. Data reported as mean ± SEM for n=2 independent runs, except samples including ACEE SNP g1 for which n=1 .

[0047] FIGS. 18A-18C show the editing activity of AVFG, ACEE, AEQH, DJQA and DWET Type II Cas in combination with panels of sgRNAs targeting TRAC (FIG. 18A), B2M (FIG. 18B), PD1 (FIG. 18C) after transient plasmid transfection in HEK293T cells (Example 4). Data presented as mean ± SEM for n=2 independent runs.6. DETAILED DESCRIPTION

[0048] In one aspect, the disclosure provides Type II Cas proteins (e.g., AEQH Type II Cas proteins, AAOF Type II Cas proteins, ACEE Type II Cas proteins, AQSL Type II Cas proteins, ASWC Type II Cas proteins, AVFG Type II Cas proteins, AWIT Type II Cas proteins, AWMF Type II Cas proteins, BUMO Type II Cas proteins, COIA Type II Cas proteins, DJQA Type II Cas proteins, and DWET Type II Casproteins). 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).

[0049] 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-l domain, RuvC-ll domain, RuvC-lll domain, BH domain, REC domain, HNH domain, WED domain, or PID domain of an AEQH Type II Cas protein, AAOF Type II Cas protein, ACEE Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, or DWET 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 an AEQH Type II Cas protein and / or AAOF Type II Cas protein and / or ACEE Type II Cas protein and / or AQSL Type II Cas protein and / or ASWC Type II Cas protein and / or AVFG Type II Cas protein and / or AWIT Type II Cas protein and / or AWMF Type II Cas protein and / or BUMO Type II Cas protein and / or COIA Type II Cas protein and / or DJQA Type II Cas protein and / or or DWET Type II Cas protein, and one or more domains from a different Type II Cas protein such as SpCas9.

[0050] Exemplary features of Type II Cas proteins of the disclosure are described in Section 6.2.

[0051] In further aspects, the disclosure provides guide (gRNA) molecules, for example single guide RNAs (sgRNAs) and combinations of guide RNA molecules, for example combinations of two or more sgRNAs. Combinations of gRNAs can include, for example, a gRNA targeting the RHO rs7984 SNP and a second gRNA targeting RHO intron 1. Combinations of gRNAs targeting the RHO rs7984 SNP and RHO intron 1 can be used to selectively edit RHO alleles having pathogenic mutations. This dual targeting approach is further described Section 6.8 and Example 3. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure are further described in Section 6.3.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 be obtained 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

[0059] 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.

[0060] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0061] Unless indicated otherwise, an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term “and / or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.

[0062] 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 disclosure pertaining to a “Type II Cas” or “Type II Cas protein” pertains to wild-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).

[0063] 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 alignedsequences 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.

[0064] 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 comprise a spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold. Various non-limiting examples of 3’ sgRNA scaffolds are described in Section 6.3.

[0065] An sgRNA can in some embodiments comprise no uracil base at the 3’ end of the sgRNA sequence. Alternatively, a sgRNA can comprise one or more uracil bases at the 3’ end of the sgRNA sequence. For example, a sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence, 2 uracil (UU) at the 3’ end of the sgRNA sequence, 3 uracil (UUU) at the 3’ end of the sgRNA sequence, 4 uracil (UUUU) at the 3’ end of the sgRNA sequence, 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence, 6 uracil (UUUUUU) at the 3’ end of the sgRNA sequence, 7 uracil (UUUUUUU) at the 3’ end of the sgRNA sequence, or 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence. Different length stretches of uracil can be appended at the 3’ end of a sgRNA as terminators. Thus, for example, the 3’ 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.

[0066] 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 (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (lie, 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 (Vai, V). The terms “polypeptide sequence” or “amino acid sequence” are an alphabetical representation of a polypeptide molecule.

[0067] Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, the term “nucleotide sequence” is the alphabetical representation of a polynucleotide molecule. The letters used in polynucleotide sequences described herein correspond to IUPAC notation. For example, the letter “N” in a nucleotide sequence represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA sequence; the letter “R” in a nucleotide sequence represents a nucleotide which can be A or G; and the letter “V” in a nucleotide sequence represents a nucleotide which can be “A, C, or G.

[0068] 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 sequence is located 3’ of the target sequence on the non-target strand.

[0069] 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 Proteins6.2.1. Type HA Cas Proteins6.2.1.1. AEQH Type II Cas Proteins

[0070] In one aspect, the disclosure provides AEQH Type II Cas proteins. AEQH Type II Cas proteins can be further classified as Type IIA Cas proteins. The AEQH 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 AEQH Type II Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO:1 . In some embodiments, an AEQH Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:1 .

[0071] Exemplary AEQH Type II Cas protein sequences and nucleotide sequences encoding exemplary AEQH Type II Cas proteins are set forth in Table 1A.

[0072] In some embodiments an AEQH Type II Cas protein comprises an amino acid sequence of SEQID NO:1 , SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, an AEQH Type II Cas protein hasnickase 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 D10A substitution, wherein the position of the D10A 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 a N627A substitution, wherein the position of the N627A 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 H604A substitution, wherein the position of the H604A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2. In some embodiments, an AEQH Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N627A substitution or an H604A substitution.6.2.2. Type IIC Cas Proteins6.2.2.1. AAOF Type II Cas Proteins

[0073] In one aspect, the disclosure provides AAOF Type II Cas proteins. AAOF Type II Cas proteins can be further classified as Type IIC Cas proteins. The AAOF 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 AAOF 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 NOT. In some embodiments, an AAOF Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NOT.

[0074] Exemplary AAOF Type II Cas protein sequences and nucleotide sequences encoding exemplary AAOF Type II Cas proteins are set forth in Table 2A.

[0075] In some embodiments an AAOF 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, an AAOF 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 D9A substitution, wherein the position of the D9A 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 a N610A substitution, wherein the position of the N610A 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 a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8. In some embodiments, an AAOF Type II Cas protein iscatalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.6.2.2.2. ACEE Type II Cas Proteins

[0076] In one aspect, the disclosure provides ACEE Type II Cas proteins. ACEE Type II Cas proteins can be further classified as Type IIC Cas proteins. The ACEE 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:13. In some embodiments, the ACEE 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 ACEE Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:13.

[0077] Exemplary ACEE Type II Cas protein sequences and nucleotide sequences encoding exemplary ACEE Type II Cas proteins are set forth in Table 2B.

[0078] In some embodiments an ACEE Type II Cas protein comprises an amino acid sequence of SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, an ACEE 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 D8A substitution, wherein the position of the D8A 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 a N613A substitution, wherein the position of the N613A 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 a H590A substitution, wherein the position of the H590A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14. In some embodiments, an ACEE Type II Cas protein is catalytically inactive, for example due a D8A substitution in combination with a N613A substitution or a H590A subsitution.6.2.2.3. AQSL Type II Cas Proteins

[0079] In one aspect, the disclosure provides AQSL Type II Cas proteins. AQSL Type II Cas proteins can be further classified as Type IIC Cas proteins. The AQSL 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 AQSL 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, an AQSL Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:19.

[0080] Exemplary AQSL Type II Cas protein sequences and nucleotide sequences encoding exemplary AQSL Type II Cas proteins are set forth in Table 2C.

[0081] In some embodiments an AQSL Type II Cas protein comprises an amino acid sequence of SEQID NO:19, SEQ ID NO:20, or SEQ ID NO:21 . In some embodiments, an AQSL 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 NQ:20, or SEQ ID NO:21 . In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20. In some embodiments, an AQSL Type II Cas protein is catalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.6.2.2.4. ASWC Type II Cas Proteins

[0082] In one aspect, the disclosure provides ASWC Type II Cas proteins. ASWC Type II Cas proteins can be further classified as Type IIC Cas proteins. The ASWC 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 ASWC 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 ASWC Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:25.

[0083] Exemplary ASWC Type II Cas protein sequences and nucleotide sequences encoding exemplary ASWC Type II Cas proteins are set forth in Table 2D.

[0084] In some embodiments an ASWC Type II Cas protein comprises an amino acid sequence of SEQID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, an ASWC 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 a N633A substitution, wherein the position of the N633A 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 H610A substitution, wherein the position of the H610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26. In some embodiments, an ASWC Type II Cas protein is catalytically inactive, for example due to a D15A substitution in combination with a N633A substitution or an H610A substitution.6.2.2.5. AVFG Type II Cas Proteins

[0085] In one aspect, the disclosure provides AVFG Type II Cas proteins. AVFG Type II Cas proteins can be further classified as Type IIC Cas proteins. The AVFG 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 AVFG 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, an AVFG Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:31 .

[0086] Exemplary AVFG Type II Cas protein sequences and nucleotide sequences encoding exemplary AVFG Type II Cas proteins are set forth in Table 2E.

[0087] In some embodiments an AVFG Type II Cas protein comprises an amino acid sequence of SEQID NO:31 , SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, an AVFG Type II Cas protein hasnickase 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 D12A substitution, wherein the position of the D12A 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 a N607A substitution, wherein the position of the N607A 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 H584A substitution, wherein the position of the H584A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32. In some embodiments, an AVFG Type II Cas protein is catalytically inactive, for example due to a D12A substitution in combination with a N607A substitution or an H584A substitution.6.2.2.6. AWIT Type II Cas Proteins

[0088] In one aspect, the disclosure provides AWIT Type II Cas proteins. AWIT Type II Cas proteins can be further classified as Type IIC Cas proteins. The AWIT 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 AWIT 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, an AWIT Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:37.

[0089] Exemplary AWIT Type II Cas protein sequences and nucleotide sequences encoding exemplary AWIT Type I Oas proteins are set forth in Table 2F.

[0090] In some embodiments an AWIT Type II Cas protein comprises an amino acid sequence of SEQID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, an AWIT 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 D10A substitution, wherein the position of the D10A 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 a N615A substitution, wherein the position of the N615A 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 H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38. In some embodiments, an AWIT Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.6.2.2.7. AWMF Type II Cas Proteins

[0091] In one aspect, the disclosure provides AWMF Type II Cas proteins. AWMF Type II Cas proteins can be further classified as Type IIC Cas proteins. The AWMF 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:43. In some embodiments, the AWMF 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, an AWMF Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:43.

[0092] Exemplary AWMF Type II Cas protein sequences and nucleotide sequences encoding exemplary AWMF Type II Cas proteins are set forth in Table 2G.

[0093] In some embodiments an AWMF Type II Cas protein comprises an amino acid sequence of SEQID NO:43, SEQ ID NO:44, or SEQ ID NO:45. In some embodiments, an AWMF 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 D10A substitution, wherein the position of the D10A 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 a N615A substitution, wherein the position of the N615A 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 H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44. In some embodiments, an AWMF Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.6.2.2.8. BUMO Type II Cas Proteins

[0094] In one aspect, the disclosure provides BUMO Type II Cas proteins. BUMO Type II Cas proteins can be further classified as Type IIC Cas proteins. The BUMO 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 BUMO 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, a BUMO Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:49.

[0095] Exemplary BUMO Type II Cas protein sequences and nucleotide sequences encoding exemplary BUMO Type II Cas proteins are set forth in Table 2H.

[0096] In some embodiments an BUMO 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, a BUMO 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 D8A substitution, wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50. In some embodiments, the one or more amino acid substitutions providing nickase activity comrpise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50. In some embodiments, the one or more amino acid substitutions providing nickase activity comrpise an H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50. In some embodiments, a BUMO Type II Cas protein is catalytically inactive, for example due to a D8A substitution in combination with a N610A substitution or an H587A substitution.6.2.2.9. COIA Type II Cas Proteins

[0097] In one aspect, the disclosure provides COIA Type II Cas proteins. COIA Type II Cas proteins can be further classified as Type IIC Cas proteins. The COIA 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 COIA 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 COIA Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:55.

[0098] Exemplary COIA Type II Cas protein sequences and nucleotide sequences encoding exemplary COIA Type II Cas proteins are set forth in Table 2I.

[0099] In some embodiments an COIA Type II Cas protein comprises an amino acid sequence of SEQID NO:55, SEQ ID NO:56, or SEQ ID NO:57. In some embodiments, a COIA 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 D10A substitution, wherein the position of the D10A 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 a N615A substitution, wherein the position of the N615A 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 a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56. In some embodiments, a COIA Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.6.2.2.10. DJQA Type II Cas Proteins

[0100] In one aspect, the disclosure provides DJQA Type II Cas proteins. DJQA Type II Cas proteins can be further classified as Type IIC Cas proteins. The DJQA 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:61 . In some embodiments, the DJQA 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:61 . In some embodiments, a DJQA Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:61 .

[0101] Exemplary DJQA Type II Cas protein sequences and nucleotide sequences encoding exemplary DJQA Type II Cas proteins are set forth in Table 2J.

[0102] In some embodiments an DJQA Type II Cas protein comprises an amino acid sequence of SEQID NO:61 , SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, a DJQA 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:61 , SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62. In some embodiments, a DJQA Type II Cas protein is catalytically inactive, for example due to a D10A substitution in combination with a N615A substitution or an H592A substitution.6.2.2.11 . DWET Type II Cas Proteins

[0103] In one aspect, the disclosure provides DWET Type II Cas proteins. DWET Type II Cas proteins can be further classified as Type IIC Cas proteins. The DWET 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:67. In some embodiments, the DWET 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:67. In some embodiments, a DWET Type II Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:67.

[0104] Exemplary DWET Type II Cas protein sequences and nucleotide sequences encoding exemplary DWET Type I Cas proteins are set forth in Table 2K.

[0105] In some embodiments an DWET Type II Cas protein comprises an amino acid sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, a DWET 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:67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68. In some embodiments, a DWET Type II Cas protein is catalytically inactive, for example due to a D9A substitution in combination with a N610A substitution or an H587A substitution.6.2.3. Fusion and Chimeric Proteins

[0106] The disclosure provides Type II Cas proteins e.g., an AEQH Type II Cas protein as described in Section 6.2.1 .1 , an AAOF Type II Cas protein as described in Section 6.2.2.1 , an ACEE Type II Cas protein as described in Section 6.2.2.2, an AQSL Type II Cas protein as described in Section 6.2.2.3, an ASWC Type II Cas protein as described in Section 6.2.2.4, an AVFG Type II Cas protein as described in Section 6.2.2.5, an AWIT Type II Cas protein as described in Section 6.2.2.6, an AWMF Type II Cas protein as described in Section 6.2.2.7, a BUMO Type II Cas protein as described in Section 6.2.2.8, a COIA Type II Cas protein as described in Section 6.2.2.9, a DJQA Type II Cas protein as described in Section 6.2.2.10, or a DWET Type II Cas protein as described in Section 6.2.2.11 ,) 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., Kruppel associated box (KRAB)), a histone-modifying protein, an integrase, or a recombinase.

[0107] 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.

[0108] Non-limiting examples of nuclear localization signals include KRTADGSEFESPKKKRKV (SEQ ID NO:145), PKKKRKV (SEQ ID NO:146), PKKKRRV (SEQ ID NO:147), KRPAATKKAGQAKKKK (SEQ ID NO:148), YGRKKRRQRRR (SEQ ID NO:149), RKKRRQRRR (SEQ ID NQ:150), PAAKRVKLD (SEQ ID NO:151), RQRRNELKRSP (SEQ ID NO:152), VSRKRPRP (SEQ ID NO:153), PPKKARED (SEQ ID NO:154), PQPKKKPL (SEQ ID NO:155), SALIKKKKKMAP (SEQ ID NO:156), PKQKKRK (SEQ ID NO:157), RKLKKKIKKL (SEQ ID NO:158), REKKKFLKRR (SEQ ID NO:159),KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160), RKCLQAGMNLEARKTKK (SEQ ID NO:161), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:162), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:163).

[0109] Exemplary fusion partners include protein tags (e.g., V5-tag e.g., having the sequence GKPIPNPLLGLDST (SEQ ID NO:164) or IPNPLLGLD (SEQ ID NO:165)), 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:166)

[0110] 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:166.

[0111] Type II Cas proteins of the disclosure in the form of a fusion protein comprising an adenosine deaminase can be used as an adenine base editor to change an “A” to a “G” in DNA. Type II Cas proteins of the disclosure in the form of a fusion protein comprising a cytidine deaminase can be used as a cytosine base editor to change a “C” to a “T” in DNA.

[0112] 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).

[0113] 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 the disclosure in the form of a fusion protein comprising a reverse transcriptase (RT) can be used as a prime editor to carry out precise base editing without double-stranded DNA breaks.

[0114] 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).

[0115] 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., an AEQH 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:145).

[0116] The disclosure provides chimeric Type II Cas proteins comprising one or more domains of an AEQH 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 AAOF 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 ACEE 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 AQSL 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 ASWC 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 AVFG 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 AWIT 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 AWMF 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 BUMO 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 COIA 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 DJQA 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 DWET 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).

[0117] The domain structures of wild-type AIK, BNK, HPLH, and ANAB Type II Cas proteins were inferred 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. The domains identified in Type II Cas proteins are: the RuvC catalytic domain (discontinuous, represented by RuvC-l, RuvC-ll, and RuvC-lll domains), bridge helix (BH), recognition (REC) domain, HNH catalytic domain, wedge (WED) domain, and PAM-interacting domain (PID).

[0118] Tables 3A-3B below report the amino acid positions corresponding to the boundaries between different functional domains in wild-type AEQH (SEQ ID NO:2), AAOF (SEQ ID NO:8), ACEE (SEQ ID NO:14), AQSL (SEQ ID NO:20), ASWC (SEQ ID NO:26), AVFG (SEQ ID NO:32), AWIT (SEQ ID NO:38), AWMF (SEQ ID NO:44), BUMO (SEQ ID NQ:50), COIA (SEQ ID NO:56), DJQA (SEQ ID NO:62), and DWET (SEQ ID NO:68) Type II Cas proteins.

[0119] 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 an AAOF Type II Cas protein, ACEE Type II Cas protein, AEQH Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, and / or DWET 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-l, BH, REC, RuvC-ll, HNH, RuvC-lll, WED, PID. 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).

[0120] In some embodiments, a Type II Cas protein of the disclosure comprises one, two, three, four, five, six, seven, or eight of a RuvC-l domain, a BH domain, a REC domain, a RuvC-ll domain, a HNH domain, a RuvC-lll domain, a WED domain, and a PID domain arranged in the N-terminal to C-terminal direction. In some embodiments, all domains are from an AEQH Type II Cas protein (e.g., an AEQH Type II Cas protein whose amino acid sequence comprises SEQ ID NO:1 , 2, or 3). In some embodiments, all domains are from an AAOF Type II Cas protein (e.g., an AAOF Type II Cas protein whose amino acid sequence comprises SEQ ID NO:7, 8, or 9). In some embodiments, all domains are from an ACEE Type II Cas protein (e.g., an ACEE Type II Cas protein whose amino acid sequence comprises SEQ ID NO:13, 14, or 15). In some embodiments, all domains are from an AQSL Type II Cas protein (e.g., an AQSL Type II Cas protein whose amino acid sequence comprises SEQ ID NO:19, 20, or 21). In some embodiments, all domains are from an ASWC Type II Cas protein (e.g., an ASWC Type II Cas protein whose amino acid sequence comprises SEQ ID NO:25, 26, or 27). In some embodiments, all domains are from an AVFG Type II Cas protein (e.g., an AVFG Type II Cas protein whose amino acid sequence comprises SEQ ID NO:31 , 32, or 33). In some embodiments, all domains are from an AWIT Type II Cas protein (e.g., an AWIT Type II Cas protein whose amino acid sequence comprises SEQ ID NO:37, 38, or 39). In some embodiments, all domains are from an AWMF Type II Cas protein (e.g., an AWMF Type II Cas protein whose amino acid sequence comprises SEQ ID NO:43, 44, or 45). In some embodiments, all domains are from an BUMO Type II Cas protein (e.g., an BUMO Type II Cas protein whose amino acid sequence comprises SEQ ID NO:49, 50, or 51). In some embodiments, all domains are from an COIA Type II Cas protein (e.g., an COIA Type II Cas protein whose amino acid sequence comprises SEQ ID NO:55, 56, or 57). In some embodiments, all domains are from an DJQA Type II Cas protein (e.g., an DJQA Type II Cas protein whose amino acid sequence comprises SEQ ID NO:61 , 62, or 63). In some embodiments, all domains are from an DWET Type II Cas protein (e.g., an DWET Type II Cas protein whose amino acid sequence comprises SEQ ID NO:67, 68, or 69). In other embodiments, one or more domains (e.g., one domain), e.g., a PID domain, is from another Type II Cas protein.

[0121] 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 SaCas9 providing nickase activity are the D10A substitution in the RuvC domain and the N580A substitution in the HNH domain. Combining both the D10A and N580A substitutions in SaCas9 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, an AEQH Type II Cas protein can include a D10A substitution (corresponding to D10A in SaCas9) or a N627A substitution (corresponding to N580A in SaCas9) to provide a nickase, or D10A and N627A substitutions to provide a catalytically inactive Casprotein, where the positions of the D13A and N589A substitutions are defined with respect to amino acid numbering of SEQ ID NO:2. Positions corresponding to D10 and N580 of SaCas9 for Type II Cas proteins of the disclosure as shown in Table 4. An exemplary amino acid substitution in CjCas9 providing nickase activity is the H559A substitution. Corresponding substitutions can be introduced into the Type II Cas nucleases of the disclosure to provide nickases. Substitutions at the position corresponding to H559A of CjCas9 can be combined with substitutions at positions corresponding to D10 of SaCas9 to make catalytically inactive Type II Cas proteins. 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.6.3. Guide RNAs

[0122] 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 disclosure typically comprise a spacer of 15 to 30 nucleotides in length. The spacer can be positioned 5’ 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.

[0123] An exemplary crRNA scaffold sequence that can be used for AEQH Type II Cas gRNAs comprises GUUUUAGUACUCUGUUGGAUAUUGAUAAACUUACAC (SEQ ID NO:73) and an exemplary tracrRNA sequence that can be used for AEQH Type II Cas gRNAs comprises UGUGAGUUUAUCAAUAUCCAACAAUAGUUCUAAGAUAAGGCUAUUUAUGCCGUAGGGUAUGGCG GUAUCCCGUUAAUCCGCCUUUAAGCCAUUGCUUUGCAAUGGCUUA (SEQ ID NO:74).

[0124] An exemplary crRNA scaffold sequence that can be used for AAOF Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:75) and anexemplary tracrRNA sequence that can be used for AAOF Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUGCAGGUUACUAUGAUAAGGUAGUACACCGCAGAGCUCUAACGCC UCGCGUAAGCGGGGCGUUAUCUCU (SEQ ID NO:76).

[0125] An exemplary crRNA scaffold sequence that can be used for ACEE Type II Cas gRNAs comprises AUUGUAGUUCCCUAAUUUUUCUUGGUAUGUUAUAAU (SEQ ID NO:77) and an exemplary tracrRNA sequence that can be used for ACEE Type II Cas gRNAs comprisesUUAUAACAUACCAAGAACAAUUAGGUUACUACAAAAAGGUAGAAAACCGAAAAGCUCUAACGGCUC CUUUUUGGAGCCGUUAUCUUUUU (SEQ ID NO:78).

[0126] An exemplary crRNA scaffold sequence that can be used for AQSL Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:79) and an exemplary tracrRNA sequence that can be used for AQSL Type II Cas gRNAs comprisesUUAUACCAUACCAAGGACUAUGCAGGUUACUAUGAUAAGGUAGUACACCGCAGAGCACUGACGCC CCGCUUUUGCGGGGCGUUAUCUCU (SEQ ID NQ:80).

[0127] An exemplary crRNA scaffold sequence that can be used for ASWC Type II Cas gRNAs comprises GUUCUGGCCUAAGCUCAUUUCCUAACUGAUACAAUC (SEQ ID NO:81) and an exemplary tracrRNA sequence that can be used for ASWC Type II Cas gRNAs comprisesUCAGUUAGGAAAUGGGCUUUCUCCACUAACAAGCUGAGAGAUGCACAAGAUGCGGGGUCGCUAU AUGCGACCCUUUUUCGUAUC (SEQ ID NO:82).

[0128] An exemplary crRNA scaffold sequence that can be used for AVFG Type II Cas gRNAs comprises GUUAUAGUUCCUAGUAAAUUCUCGAUAUGCUAUAAU (SEQ ID NO:83) and an exemplary tracrRNA sequence that can be used for AVFG Type II Cas gRNAs comprisesUAUAGCAUAUCGAGAGUUUAACUAGUUGCUAUAACAAGGCAAUAAGCCGUAAAGUAUCCCCUGUA CUCAUUUCUUGAGUGUAGGGGUAUCUUU (SEQ ID NO:84).

[0129] An exemplary crRNA scaffold sequence that can be used for AWIT Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:85) and an exemplary tracrRNA sequence that can be used for AWIT Type II Cas gRNAs comprisesUUAUACCAUACCAAGAACUAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUU (SEQ ID NO:86).

[0130] An exemplary crRNA scaffold sequence that can be used for AWMF Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:87) and an exemplary tracrRNA sequence that can be used for AWMF Type II Cas gRNAs comprisesUUAUACCAUACCAAGAACUAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUU (SEQ ID NO:88).

[0131] An exemplary crRNA scaffold sequence that can be used for BUMO Type II Cas gRNAs comprises GUUGUAGUUCCCUGAUGAUUCUUGGUAUGGUAUAAU (SEQ ID NO:89) and an exemplary tracrRNA sequence that can be used for BUMO Type II Cas gRNAs comprisesUUAUACCAUACCAAGGAUUAUCGGGUUACUAUGAUAAGGUAGUACACCGAAAAGCUCUAACGCUC UGUCGCUUUUGACAGAGCGUUAUCUUUU (SEQ ID NO:90).

[0132] An exemplary crRNA scaffold sequence that can be used for COIA Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:91) and an exemplary tracrRNA sequence that can be used for COIA Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUU (SEQ ID NO:92).

[0133] An exemplary crRNA scaffold sequence that can be used for DJQA Type II Cas gRNAs comprises GUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAU (SEQ ID NO:93) and an exemplary tracrRNA sequence that can be used for DJQA Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUUAUGGUUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGCCGUUUGGCAGGGCGUCAUCUUU (SEQ ID NO:94).

[0134] An exemplary crRNA scaffold sequence that can be used for DWET Type II Cas gRNAs comprises GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAU (SEQ ID NO:95) and an exemplary tracrRNA sequence that can be used for DWET Type II Cas gRNAs comprises UUAUACCAUACCAAGAACUAUGCAGGUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCUAACGCC CCGCGUAAGCGGGGCGUUAUCUCU (SEQ ID NO:96).

[0135] gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise the spacer at the 5’ end of the molecule and a 3’ sgRNA scaffold. Alternatively, gRNAs can comprise separate crRNA and tracrRNA molecules.

[0136] Further features of exemplary gRNA spacer sequences are described in Section 6.3.1 and further features of exemplary 3’ sgRNA scaffolds are described in Section 6.3.2.6.3.1. Spacers

[0137] The spacer sequence is partially or fully complementary to a target sequence found in a genomic DNA sequence, for example a human genomic DNA sequence. For example, a spacer sequence can be partially or fully complementary to a nucleotide sequence in a gene having a disease causing mutation. A spacer that is partially complementary to a target sequence can have, for example, one, two, or three mismatches with the target sequence.

[0138] 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.

[0139] 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 5A and Table 5B.

[0140] Example 3 describes exemplary sequences that can be used to target RHO genomic sequences. Example 4 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 RHO. 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.

[0141] Additional exemplary spacer sequences that can be used in gRNAs of the disclosure are set forth in Table 6A, Table 6B, and Table 6C.

[0142] The RHO spacer sequences in Table 6A are useful for targeting a RHO gene in the vicinity of the rs7984 SNP, located in the 5’ untranslated region (UTR) of the RHO gene. Allele specific targeting can be achieved by using a gRNA targeting the SNP variant found in a cell or subject. For example, guides in Table 6 having “7984A” in their name can be used when the cell or subject has an “A” at the position of the rs7984 SNP, while guides having “7984G” in their name can be used when the cell or subject has a “G” at the position of the rs7984 SNP. Such guides can be used, for example, with a guide RNA targeting RHO intron 1 (for example, having a spacer as shown in Table 6B) to knock-out expression of the mutated protein. Allele-specific targeting of RHO is described further in Example 3. Exemplary combinations of guides include a first guide RNA having a spacer whose sequence is selected from SEQ ID NOS:326-334 and 336-346 and a second guide RNA having a spacer whose sequence is selected from SEQ ID NOs:402, 403, and 406.

[0143] 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 6A. 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 6A. 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 6A. 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 6A. 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 6A. 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 6A. 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 6A. 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 6A. 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 6A. In some embodiments, a gRNA of thedisclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 6A.

[0144] 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 6B. 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 6B. 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 6B. 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 6B. 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 6B. 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 6B. 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 6B. 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 6B. 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 6B. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 or more consecutive nucleotides from a sequence shown in Table 6B.

[0145] 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 6C. 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 6C. 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 6C. 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 6C. 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 6C. 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 6C. 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 6C. 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 6C. 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 6C. In some embodiments, a gRNA of the disclosure has a spacer whose nucleotide sequence comprises 24 consecutive nucleotides from a sequence shown in Table 6C.6.3.2. sgRNA Molecules

[0146] 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-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA 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-molecule guide 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.

[0147] The sgRNA can comprise a variable length spacer sequence (e.g., 15 to 30 nucleotides) at the 5’ end of the sgRNA sequence and a 3’ sgRNA segment.

[0148] Type II Cas gRNAs typically comprise a repeat-antirepeat duplex and / or one or more stem-loops generated by the gRNA’s secondary 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).

[0149] 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.

[0150] 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.

[0151] 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.

[0152] Exemplary 3’ sgRNA scaffold sequences for Type 11 A Cas sgRNAs are shown in Table 7A. Exemplary 3’ sgRNA scaffold sequences for Type 11 C Cas sgRNAs are shown in Table 7B.

[0153] The sgRNA (e.g., for use with AAOF Type II Cas protein, ACEE Type II Cas protein, AEQH Type II Cas protein, AQSL Type II Cas protein, ASWC Type II Cas protein, AVFG Type II Cas protein, AWIT Type II Cas protein, AWMF Type II Cas protein, BUMO Type II Cas protein, COIA Type II Cas protein, DJQA Type II Cas protein, and / or DWET Type II Cas proteins) can comprise no uracil base at the 3’ end of the sgRNA sequence. Typically, however, the sgRNA comprises one or more uracil bases at the 3’ end of the sgRNA sequence, for example to promote correct sgRNA folding. For example, the sgRNA can comprise 1 uracil (U) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 2 uracil (UU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 3 uracil (UUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 4 uracil (UUUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 6 uracil (UUUUUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 7 uracil (UUUUUUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the 3’ end of the sgRNA sequence. Different length stretches of uracil can be appended at the 3’end of a sgRNA as terminators. Thus, for example, the 3’ sgRNA sequences set forth in Table 7A and Table 7B can be modified by adding (or removing) one or more uracils at the end of the sequence.

[0154] In some embodiments, a sgRNA scaffold for use with an AEQH Type II Cas protein comprises the sequence GUUUUAGUACUCUGUUGGAUAUUGAUAAACUUACAGAAAUGUGAGUUUAUCAAUAUCCAACAAUA GUUCUAAGAUAAGGCUAUUUAUGCCGUAGGGUAUGGCGGUAUCCCGUUAAUCCGCCUUUAAGCCAUUGCUUUGCAAUGGCUUAUUUUUU (SEQ ID N0:121). In some embodiments, a sgRNA scaffold for use with an AEQH Type II Cas protein comprises the sequenceGUUUUAGUACUCUGUGAAAACAAUAGUUCUAAGAUAAGGCUAUUUAUGCCGUAGGGUAUGGCGG UAUCCCGUUAAUCCGCCUUUAAGCCAUUGCUUUGCAAUGGCUUAUUUUUU (SEQ ID NO:122).

[0155] In some embodiments, a sgRNA scaffold for use with an AAOF Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAgaaaUUAUACCAUACCAAGAACUAUGCAGG UUACUAUGAUAAGGUAGUACACCGCAGAGCUCUAACGCCUCGCGUAAGCGGGGCGUUAUCUCUU UUUU (SEQ ID NO:123). In some embodiments, a sgRNA scaffold for use with an AAOF Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGGUAGGAAACUAUGCAGGUUACUAUGAUAAGGUAGUACACCGCAGAGCUCU AACGCCUCGCGUAAGCGGGGCGUUAUCUCUUUUUU (SEQ ID NO:124).

[0156] In some embodiments, a sgRNA scaffold for use with an ACEE Type II Cas protein comprises the sequenceAUUGUAGUUCCCUAAUUUUUCUUGGUAUGUUAUAAGAAAUUAUAACAUACCAAGAACAAUUAGGU UACUACAAAAAGGUAGAAAACCGAAAAGCUCUAACGGCUCCUUUUUGGAGCCGUUAUCUUUUUU (SEQ ID NO:125). In some embodiments, a sgRNA scaffold for use with an ACEE Type II Cas protein comprises the sequenceAUUGUAGUUCCCUGAAAAGGUUACUACAAAAAGGUAGAAAACCGAAAAGCUCUAACGGCUCCGAA AGGAGCCGUUAUCUUUUUU (SEQ ID NO:126).

[0157] In some embodiments, a sgRNA scaffold for use with an AQSL Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGGACUAUGCAG GUUACUAUGAUAAGGUAGUACACCGCAGAGCACUGACGCCCCGCUUUUGCGGGGCGUUAUCUCU UUUUU (SEQ ID NO:127). In some embodiments, a sgRNA scaffold for use with an AQSL Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGGUAGGAAACUAUGCAGGUUACUAUGAUAAGGUAGUACACCGCAGAGCACU GACGCCCCGCUUUUGCGGGGCGUUAUCUCUUUUUU (SEQ ID NO:128).

[0158] In some embodiments, a sgRNA scaffold for use with an ASWC Type II Cas protein comprises the sequenceGUUCUGGCCUAAGCUCAUUUCCUAACUGAUGAAAUCAGUUAGGAAAUGGGCUUUCUCCACUAAC AAGCUGAGAGAUGCACAAGAUGCGGGGUCGCUAUAUGCGACCCUUUUUCGUAUCUUUUUU (SEQ ID NO:129). In some embodiments, a sgRNA scaffold for use with an ASWC Type II Cas protein comprises the sequenceGUUCUGGCCUAAGGAAACUUUCUCCACUAACAAGCUGAGAGAUGCACAAGAUGCGGGGUCGCUA UAUGCGACCCUUAUUCGUAUCCAAAUUUUUU (SEQ ID NQ:130).

[0159] In some embodiments, a sgRNA scaffold for use with an AVFG Type II Cas protein comprises the sequenceGUUAUAGUUCCUAGUAAAUUCUCGAUAUGCUAUAGAAAUAUAGCAUAUCGAGAGUUUAACUAGUUGCUAUAACAAGGCAAUAAGCCGUAAAGUAUCCCCUGUACUCAUUUCUUGAGUGUAGGGGUAUCU UUUUU (SEQ ID N0:131). In some embodiments, a sgRNA scaffold for use with an AVFG Type II Cas protein comprises the sequenceGUUAUAGUUCCUAGUAAGAAAUUAACUAGUUGCUAUAACAAGGCAAUAAGCCGUAAAGUAUCCCC UGUACUCAUUUCUUGAGUGUAGGGGUAUCUUUUUU (SEQ ID NO:132).

[0160] In some embodiments, a sgRNA scaffold for use with an AWIT Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUUAUGG UUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGCCCUGUCUUAUGACAGGGCGUCAUCU UUUUU (SEQ ID NO:133). In some embodiments, a sgRNA scaffold for use with an AWIT Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAGAAAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUUUUU (SEQ ID NO:134).

[0161] In some embodiments, a sgRNA scaffold for use with an AWMF Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUUAUGG UUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGCCCUGUCUUAUGACAGGGCGUCAUCU UUUUU (SEQ ID NO:135). In some embodiments, a sgRNA scaffold for use with an AWMF Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAGAAAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUUUUU (SEQ ID NO:136).

[0162] In some embodiments, a sgRNA scaffold for use with an BUMO Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGAUGAUUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGGAUUAUCGGG UUACUAUGAUAAGGUAGUACACCGAAAAGCUCUAACGCUCUGUCGCUUUUGACAGAGCGUUAUC UUUUUU (SEQ ID NO:137). In some embodiments, a sgRNA scaffold for use with an BUMO Type II Cas protein comprises the sequenceGUUGUAGUUCCCUGGAAACGGGUUACUAUGAUAAGGUAGUACACCGAAAAGCUCUAACGCUCUG UCGCUUUUGACAGAGCGUUAUCUUUUUU (SEQ ID NO:138).

[0163] In some embodiments, a sgRNA scaffold for use with an COIA Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUUAUGG UUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGCCCUGUCUUAUGACAGGGCGUCAUCU UUUUU (SEQ ID NO:139). In some embodiments, a sgRNA scaffold for use with an COIA Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAGAAAUUAUGGUUGCUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGUCUUAUGACAGGGCGUCAUCUUUUUU (SEQ ID NO:140).

[0164] In some embodiments, a sgRNA scaffold for use with an DJQA Type II Cas protein comprises the sequenceGUCAUAGUUCCCUAAUAGCUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUUAUGG UUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGCCCUGCCGUUUGGCAGGGCGUCAUCU UUUUU (SEQ ID N0:141). In some embodiments, a sgRNA scaffold for use with an DJQA Type II Cas protein comprises the sequence GUCAUAGUUCCCUAAGAAAUUAUGGUUACUAUGAUAAGGUCAUAGGACCGUAAAGCUCUGACGC CCUGCCGUUUGGCAGGGCGUCAUCUUUUUU (SEQ ID NO:142).

[0165] In some embodiments, a sgRNA scaffold for use with an DWET Type II Cas protein comprises the sequence GUUGUAGUUCCCUGGUAGUUCUUGGUAUGGUAUAAGAAAUUAUACCAUACCAAGAACUAUGCAG GUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCUAACGCCCCGCGUAAGCGGGGCGUUAUCUCU UUUUU (SEQ ID NO:143). In some embodiments, a sgRNA scaffold for use with an DWET Type II Cas protein comprises the sequence GUUGUAGUUCCCUGGUAGGAAACUAUGCAGGUUACUAUGAUAAGGUAGUAUACCGCAGAGCUCU AACGCCCCGCGUAAGCGGGGCGUUAUCUCUUUUUU (SEQ ID NO:144).6.3.3. Modified gRNA Molecules

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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 cycloalkylintersugar 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)-CH2and O-N (CH3)- CH2-CH2backbones, 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 other alkyl 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.

[0170] 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.

[0171] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., 2000, J. Am. Chem. Soc., 122: 8595-8602.

[0172] 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.

[0173] 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 isfrom 1 to about 10; Ci 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; SO2CH3; 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.

[0174] 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.

[0175] 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. Kornberg, 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.

[0176] 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 andguanine, 2-thiouraci I, 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-thiouraci I, 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.

[0177] 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. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by about 0.6-1 .2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, 276-278) and are aspects of base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. 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.

[0178] 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.

[0179] 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 etal, 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.

[0180] 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.

[0181] 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.

[0182] 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 theintroduction 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

[0183] 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).

[0184] 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 a sgRNA 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.

[0185] 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

[0186] The disclosure provides nucleic acids (e.g., DNA or RNA) encoding Type II Cas proteins (e.g., AAOF Type II Cas proteins, ACEE Type II Cas proteins, AEQH Type II Cas proteins, AQSL Type II Cas proteins, ASWC Type II Cas proteins, AVFG Type II Cas proteins, AWIT Type II Cas proteins, AWMF Type II Cas proteins, BUMO Type II Cas proteins, COIA Type II Cas proteins, DJQA Type II Cas proteins, and DWET Type II Cas proteins), nucleic acids encoding gRNAs of the disclosure (e.g., a single gRNA or combination of gRNAs), 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.

[0187] 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.

[0188] 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).

[0189] 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-1G and Tables 2A-2C.

[0190] Nucleic acids of the disclosure, e.g., plasmids and viral vectors, can comprise one or more regulatory elements such as promoters, enhancers, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, 1990, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest or in particular cell types. Regulatory elements may also direct expression in a temporaldependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a nucleic acid of the disclosure comprises one or more pol III promoter (e.g., 1 , 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1 , 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1 , 2, 3, 4, 5, or more pol I promoters), or combinations thereof, e.g., to express a Type II Cas protein and a gRNA separately. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous Sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, 1985, Cell 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and EF1a promoters (for example, full length EF1a promoter and the EFS promoter, which is a short, intron-less form of the full EF1a promoter). Exemplary enhancer elements include WPRE; CMV enhancers; the R- U5' segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit p-globin. It will be appreciated by those skilled in the art that the design of an expression vector can depend on such factors as the choice of the host cell, the level of expression desired, etc.

[0191] The term "vector" refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked. One type of polynucleotide vector includes a "plasmid", which refers to a circular double-stranded DNA loop into which additional nucleic acid segments are or can be ligated. Another type of polynucleotide vector is a viral vector; wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0192] In some examples, vectors can be capable of directing the expression of nucleic acids to which they are operably linked. Such vectors can be referred to herein as "recombinant expression vectors", or more simply "expression vectors", which serve equivalent functions.

[0193] The term "operably linked" means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0194] Vectors can include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus (e.g., AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, AAVrhIO), SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-l, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.

[0195] In some examples, a vector can comprise one or more transcription and / or translation control elements. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. The vector can be a selfinactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.

[0196] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-l promoters (for example, the full EF1a promoter and the EFS promoter), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-l.

[0197] An expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also comprise appropriate sequences for amplifying expression. The expression vector can also include nucleotide sequences encoding non-native tags(e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.

[0198] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted and / or temporally restricted promoter (e.g., a tissue specific promoter, for example a human RHO promoter or human rhodopsin kinase promoter (hGRK), a cell type specific promoter, etc.).6.6. Particles and Cells

[0199] The disclosure further provides particles comprising a Type II Cas protein of the disclosure (e.g., an AAOF Type II Cas protein, an ACEE Type II Cas protein, an AEQH Type II Cas protein, an AQSL Type II Cas protein, an ASWC Type II Cas protein, an AVFG Type II Cas protein, an AWIT Type II Cas protein, an AWMF Type II Cas protein, a BUMO Type II Cas protein, a COIA Type II Cas protein, a DJQA Type II Cas protein, or a DWET Type II Cas protein), particles comprising a gRNA of the disclosure, particles comprising a system of the disclosure, and particles comprising a nucleic acid or plurality of nucleic acids of the disclosure. The particles can in some embodiments comprise or further comprise a gRNA, or a nucleic acid encoding the gRNA (e.g., DNA or mRNA). For example, the particles can comprise a RNP of the disclosure. Exemplary particles include lipid nanoparticles, vesicles, viral-like particles (VLPs) and gold nanoparticles. See, e.g., WO 2020 / 012335, the contents of which are incorporated herein by reference in their entireties, which describes vesicles that can be used to deliver gRNA molecules and Type II Cas proteins to cells (e.g., complexed together as a RNP).

[0200] The disclosure provides particles (e.g., virus particles) comprising a nucleic acid encoding a Type II Cas protein of the disclosure. The particles can further comprise a nucleic acid encoding a gRNA. Alternatively, a nucleic acid encoding a Type II Cas protein can further encode a gRNA.

[0201] The disclosure further provides pluralities of particles (e.g., pluralities of virus particles). Such pluralities can include a particle encoding a Type II Cas protein and a different particle encoding a gRNA. For example, a plurality of particles can comprise a virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 virus particle) encoding a Type II Cas protein and a second virus particle (e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhl 0 virus particle) encoding a gRNA. Alternatively, a plurality of particles can comprise a plurality of virus particles where each particle encodes a Type II Cas protein and a gRNA.

[0202] The disclosure further provides cells and populations of cells (e.g., ex vivo cells and populations of cells) that can comprise a Type II Cas protein (e.g., introduced to the cell as a RNP) or a nucleic acid encoding the Type II Cas protein (e.g., DNA or mRNA) (optionally also encoding a gRNA). The disclosure further provides cells and populations of cells comprising a gRNA of the disclosure (optionally complexed with a Type II Cas protein) or a nucleic acid encoding the gRNA (e.g., DNA or mRNA) (optionally also encoding a Type II Cas protein). The cells and populations of cells can be, for example, human cells such as a stem cell, e.g., a hematopoietic stem cell (HSC), a pluripotent stem cell, an induced pluripotent stem cell (iPS), or an embryonic stem cell. In some embodiments, the cells and populations of cells are T cells.Methods for introducing proteins and nucleic acids to cells are known in the art. For example, a RNP can be produced by mixing a Type II Cas protein and one or more guide RNAs in an appropriate buffer. An RNP can be introduced to a cell, for example, via electroporation and other methods known in the art.

[0203] The cell populations of the disclosure can be cells in which gene editing by the systems of the disclosure has taken place, or cells in which the components of a system of the disclosure have been introduced or expressed but gene editing has not taken place, or a combination thereof. A cell population can comprise, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have undergone gene editing by a system of the disclosure.6.7. Pharmaceutical Compositions

[0204] Also disclosed herein are pharmaceutical formulations and medicaments comprising a Type II Cas protein, gRNA, nucleic acid or plurality of nucleic acids, system, particle, or plurality of particles of the disclosure together with a pharmaceutically acceptable excipient.

[0205] Suitable excipients include, but are not limited to, salts, diluents, (e.g., Tris-HCI, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, antinfective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof. Suitable pharmaceutically acceptable excipients can be selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions.

[0206] The components of the pharmaceutical formulation can be dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3-butanediol.

[0207] In some cases, formulations can include one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. In some cases, the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

[0208] In some embodiments, the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which canbe reconstituted with an appropriate carrier or diluent prior to administration. In some embodiments, the formulations can comprise a guide RNA and a Type II Cas protein in a pharmaceutically effective amount sufficient to edit a gene in a cell. The pharmaceutical compositions can be formulated for medical and / or veterinary use.6.8. Methods of Altering a Cell

[0209] The disclosure further provides methods of using the Type II Cas proteins, gRNAs, nucleic acids (including pluralities of nucleic acids), systems, and particles (including pluralities of particles) of the disclosure for altering cells.

[0210] In one aspect, a method of altering a cell comprises contacting a eukaryotic cell (e.g., a human cell) with a nucleic acid, particle, system or pharmaceutical composition described herein.

[0211] Contacting a cell with a disclosed nucleic acid, particle, system or pharmaceutical composition can be achieved by any method known in the art and can be performed in vivo, ex vivo, or in vitro. In some embodiments, the methods can include obtaining one or more cells from a subject prior to contacting the cell(s) with a herein disclosed nucleic acid, particle, system or pharmaceutical composition. In some embodiments, the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.

[0212] Type II Cas and gRNA, as well as nucleic acids encoding Type II Cas and gRNAs can be delivered to a cell by any means known in the art, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.

[0213] A polynucleotide encoding Type II Cas and a gRNA, can be delivered to a cell (ex vivo or in vivo) by a lipid nanoparticle (LNP). LNPs can have, for example, a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm. LNPs can be made from cationic, anionic, neutral lipids, and combinations thereof. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability.

[0214] LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Lipids and combinations of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC- cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE- polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 , and 7C1 . Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG- modified lipids are: PEG-DMG, PEG- CerCI4, and PEG-CerC20. Lipids can be combined in any number of molar ratios to produce a LNP. In addition, the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.

[0215] Type II Cas and / or gRNAs can be delivered to a cell via an adeno-associated viral vector (e.g., of an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype), or by another viral vector.Other viral vectors include, but are not limited to lentivirus, adenovirus, alphavirus, enterovirus, pestivirus,baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus. In some embodiments, a Type II Cas mRNA is formulated in a lipid nanoparticle, while a sgRNA is delivered to a cell in an AAV or other viral vector. In some embodiments, one or more AAV vectors (e.g., one or more AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO serotype) are used to deliver both a sgRNA and a Type II Cas. In some embodiments, a Type II Cas and a sgRNA are delivered using separate vectors. In other embodiments, a Type II Cas and a sgRNA are delivered using a single vector. BNK Type II Cas and AIK Type II Cas, with their relatively small size, can be delivered with a gRNA (e.g., sgRNA) using a single AAV vector.

[0216] Compositions and methods for delivering Type II Cas and gRNAs to a cell and / or subject are further described in PCT Patent Application Publications WO 2019 / 102381 , WO 2020 / 012335, and WO 2020 / 053224, each of which is incorporated by reference herein in its entirety.

[0217] DNA cleavage can result in a single-strand break (SSB) or double-strand break (DSB) at particular locations within the DNA molecule. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-dependent repair (HDR) and non-homologous endjoining (NHEJ). These repair processes can edit the targeted polynucleotide by introducing a mutation, thereby resulting in a polynucleotide having a sequence which differs from the polynucleotide’s sequence prior to cleavage by a Type II Cas.

[0218] NHEJ and HDR DNA repair processes consist of a family of alternative pathways. Non- homologous end-joining (NHEJ) refers to the natural, cellular process in which a double-stranded DNA- break is repaired by the direct joining of two non-homologous DNA segments. See, e.g. Cahill et al., 2006, Front. Biosci. 11 :1958-1976. DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. Thus, NHEJ repair mechanisms can introduce mutations into the coding sequence which can disrupt gene function. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with a modification of the polynucleotide sequence such as a loss of or addition of nucleotides in the polynucleotide sequence. The modification of the polynucleotide sequence can disrupt (or perhaps enhance) gene expression.

[0219] Homology-dependent repair (HDR) utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double- stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus.

[0220] A third repair mechanism includes microhomology-mediated end joining (MMEJ), also referred to as “Alternative NHEJ (ANHEJ)”, in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome. In someinstances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0221] Modifications of a cleaved polynucleotide by HDR, NHEJ, and / or ANHEJ can result in, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and / or gene mutation. The aforementioned process outcomes are examples of editing a polynucleotide.

[0222] Advantages of ex vivo cell therapy approaches include the ability to conduct a comprehensive analysis of the therapeutic prior to administration. Nuclease-based therapeutics can have some level of off-target effects. Performing gene correction ex vivo allows a method user to characterize the corrected cell population prior to implantation, including identifying any undesirable off-target effects. Where undesirable effects are observed, a method user may opt not to implant the cells or cell progeny, may further edit the cells, or may select new cells for editing and analysis. Other advantages include ease of genetic correction in iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells that will be required for a cell-based therapy. Furthermore, iPSCs are an ideal cell type for performing clonal isolations. This allows screening for the correct genomic correction, without risking a decrease in viability.

[0223] Although certain cells present an attractive target for ex vivo treatment and therapy, increased efficacy in delivery may permit direct in vivo delivery to such cells. Ideally the targeting and editing is directed to the relevant cells. Cleavage in other cells can also be prevented by the use of promoters only active in certain cell types and / or developmental stages.

[0224] Additional promoters are inducible, and therefore can be temporally controlled if the nuclease is delivered as a plasmid. The amount of time that delivered protein and RNA remain in the cell can also be adjusted using treatments or domains added to change the half-life. In vivo treatment would eliminate a number of treatment steps, but a lower rate of delivery can require higher rates of editing. In vivo treatment can eliminate problems and losses from ex vivo treatment and engraftment.

[0225] An advantage of in vivo gene therapy can be the ease of therapeutic production and administration. The same therapeutic approach and therapy has the potential to be used to treat more than one patient, for example a number of patients who share the same or similar genotype or allele. In contrast, ex vivo cell therapy typically requires using a subject’s own cells, which are isolated, manipulated and returned to the same patient.

[0226] Progenitor cells (also referred to as stem cells herein) are capable of both proliferation and giving rise to more progenitor cells, which in turn have the ability to generate a large number of cells that can in turn give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers then to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one aspect, the term progenitor orstem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell can derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the range of cell types that each can give rise to can vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors. In many biological instances, stem cells can also be "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required.

[0227] Human cells described herein can be induced pluripotent stem cells (iPSCs). An advantage of using iPSCs in the methods of the disclosure is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then differentiated into a progenitor cell to be administered to the subject (e.g., an autologous cell). Because progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells from another subject or group of subjects. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.

[0228] Methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Pluripotent stem cells generated by such methods can be used in the method of the disclosure.

[0229] Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described. Mouse somatic cells can be converted to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and c-Myc; see, e.g., Takahashi and Yamanaka, 2006, Cell 126(4): 663-76. iPSCs resemble ES cells, as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape. In addition, mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission (see, e.g., Maherali and Hochedlinger, 2008, Cell Stem Cell. 3(6):595-605), and tetrapioid complementation.

[0230] Human iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency; see, e.g., 2014, Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57; Barrett et al, 2014, Stem Cells Trans Med 3: 1-6 sctm.2014-0121 ; Focosi et al, 2014, Blood Cancer Journal 4: e211 . The production of iPSCs can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, historically using viral vectors.

[0231] iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell. Further,reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., 2010, Cell Stem Cell, 7(5):6I8- 30. Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3 / 4 or Pouf5l), Soxl, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2, Klf4, Klf5, NR5A2, c- Myc, 1- Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming using the methods and compositions described herein can further comprise introducing one or more of Oct-3 / 4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell. The methods and compositions described herein can further comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from the reprogrammed cells are to be used in, e.g., human therapy, in one aspect the reprogramming is not affected by a method that alters the genome. Thus, in such examples, reprogramming can be achieved, e.g., without the use of viral or plasmid vectors.

[0232] Efficiency of reprogramming (the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents, e.g., small molecules, as shown by Shi et al., 2008, Cell-Stem Cell 2:525-528; Huangfu et al., 2008, Nature Biotechnology 26(7):795-797; and Marson et al., 2008, Cell-Stem Cell 3: 132-135. Thus, an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patientspecific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HD AC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others. Other non-limiting examples of reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA ( e.g ., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(l,3-Dioxo-IH,3H- benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pi valoyloxy methyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N- acetyl dinaline) and MS-27- 275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g., 6-(3- chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9, 10-epoxy decanoic acid), CHAP31 and CHAP 50. Other reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g, catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs. Such inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.

[0233] To confirm the induction of pluripotent stem cells, isolated clones can be tested for the expression of a stem cell marker. Such expression in a cell derived from a somatic cell identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdfi, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, a cell that expresses Oct4 or Nanog is identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. Detection can involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be best identified via RT-PCR, or protein detection methods such as immunocytochemistry, while cell surface markers are readily identified, e.g., by immunocytochemistry.

[0234] Pluripotency of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate into cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells can be introduced into nude mice and histology and / or immunohistochemistry can be performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0235] Patient-specific iPS cells or cell line can be created. There are many established methods in the art for creating patient specific iPS cells, e.g., as described in Takahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example, the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell. The set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1 , SOX2, SOX3, SOX15, SOX18, NANOG, KLF1 , KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

[0236] In some aspects, a biopsy or aspirate of a subject’s bone marrow can be performed. A biopsy or aspirate is a sample of tissue or fluid taken from the body. There are many different kinds of biopsies or aspirates. Nearly all of them involve using a sharp tool to remove a small amount of tissue. If the biopsy will be on the skin or other sensitive area, numbing medicine can be applied first. A biopsy or aspirate can be performed according to any of the known methods in the art. For example, in a bone marrow aspirate, a large needle is used to enter the pelvis bone to collect bone marrow.

[0237] In some aspects, a mesenchymal stem cell can be isolated from a subject. Mesenchymal stem cells can be isolated according to any method known in the art, such as from a subject’s bone marrow or peripheral blood. For example, marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on a Percoll™ density gradient. Cells, such as blood cells, liver cells, interstitial cells, macrophages, mast cells, and thymocytes, can be separated using density gradient centrifugation media, Percoll™. The cells can then be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger et. al., 1999, Science 284: 143-147).6.8.1. Exemplary Genomic Targets

[0238] The Type II Cas proteins and gRNAs of the disclosure can be used to alter various genomic targets. In some aspects, the methods of altering a cell are methods for altering a CCR5, EMX1, Fas, FANCF, HBB, ZSCAN2, Chr6, ADAMTSL1, B2M, CXCR4, PD1, DNMT1, Match8, TRAC, TRBC, VEGFAsite2, VEGFAsite3, CACNA, HEKsite3, HEKsite4, Chr8, BCR, ATM, HBG1, HPRT, IL2RG, NF1, USH2A, RHO, BcLenh, or CTFR genomic sequence. In some aspects, the methods of altering a cell are methods of altering a TRAC, B2M, PD1, or LAG3 genomic sequence. Reference sequences of RHO, TRAC, B2M, PD1, and LAG3 are available in public databases, for example those maintained by NCBI. For example, RHO has the NCBI gene ID: 6010; TRAC has the NCBI gene ID:28755; B2M has the NCBI gene ID: 567; PD1 has the NCBI gene ID:5133; and LAG3 has the NCBI gene ID: 3902.

[0239] In some embodiments, the methods of altering a cell are methods for altering a hemoglobin subunit beta (HBB) gene. HBB mutations are associated with p-thalassemia and SCD. Dever et al., 2016 Nature 539(7629):384-389.

[0240] In some embodiments, the methods of altering a cell are methods for altering a CCR5 gene. CCR5 has demonstrated involvement in several different disease states including, but not limited to, human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS). WO2018 / 119359 describes CCR5 editing by CRISPR-Cas to make loss of function CCR5 in order to provide protection against HIV infection, decrease one or more symptoms of HIV infection, halt or delay progression of HIV to AIDS, and / or decrease one or more symptoms of AIDS.

[0241] In some embodiments, the methods of altering a cell are methods for altering a PD1 , B2M gene, TRAC gene, or a combination thereof. CAR-T cells having PD1 , B2M and TRAC genes disrupted by CRISPR-Type II Cas have demonstrated enhanced activity in preclinical glioma models. Choi et al., 2019, Journal for ImmunoTherapy of Cancer 7:309.

[0242] In some embodiments, the methods of altering a cell are methods for altering an USH2A gene. Mutations in the USH2A gene can cause Usher syndrome type 2A, which is characterized by progressive hearing and vision loss.

[0243] In some embodiments, the methods of altering a cell are methods for altering a RHO gene. Mutations in the RHO gene can cause retinitis pigmentosa (RP).

[0244] Allele specific editing of human RHO alleles having pathogenic mutations (e.g., a P23 mutation such as P23H or a P347 mutation such as P347L, P347S, P347R, P347Q, P347T, or P347A) can be achieved using guide RNA (gRNA) molecules targeting the rs7984 SNP (for example having spacers as shown in Table 6A) located in the 5’ untranslated region (UTR) of the RHO gene. SNPs are very common in the human population, and a significant proportion of subjects are heterozygous for the rs7984 SNP. For a subject heterozygous for the rs7984 SNP and heterozygous for a pathogenic RHO gene mutation, allele specific editing of the RHO allele having the pathogenic mutation can be achieved through the use of a gRNA targeting the SNP variant found in the subject’s RHO allele having the pathogenic mutation. This allele-specific editing strategy, which does not directly target a specific pathogenic RHO gene mutation, advantageously allows editing of RHO genes having a variety of different pathogenicmutations. A rs7984 SNP targeting gRNA of the disclosure can be used in combination with a second gRNA targeting a second site in the RHO gene, for example a site in intron 1 (e.g., a gRNA having a spacer as shown in Table 6B), to promote two cuts in the RHO gene having the pathogenic mutation. Cleaving the RHO gene having the pathogenic mutation at two sites can promote a deletion in the RHO gene having the pathogenic mutation, which can result in reduced mutant RHO protein expression.

[0245] Editing a subject’s RHO allele can comprise editing a RHO allele in one or more cells from the subject (e.g., photoreceptor cells or retinal progenitor cells) or one or more cells derived from a cell of the subject (e.g., an induced pluripotent stem cell (iPSC)). For example, one or more cells from the subject or one or more cells derived from a cell of the subject can be contacted with a nucleic acid, system, or particle of the disclosure ex vivo, and cells having an edited RHO gene or progeny thereof can subsequently be implanted into the subject. Edited iPSCs can be differentiated, for instance into photoreceptor cells or retinal progenitor cells. In some embodiments, resultant differentiated cells can be implanted into the subject. When differentiated cells from the subject are edited, implantation of edited cells can proceed without an intervening differentiation step.

[0246] An in vivo method of RHO allele editing can comprise editing a RHO allele having a pathogenic mutation in a cell of a subject, such as photoreceptor cells or retinal progenitor cells. In some embodiments, the in vivo methods comprise administering one or more pharmaceutical compositions of the disclosure to or near the eye of a subject, e.g., by sub-retinal injection or intravitreal injection. For example, a single pharmaceutical composition comprising one or more AAV particles encoding one or more gRNAs (e.g., a gRNA targeting the rs7984 SNP and a gRNA targeting RHO intron 1) and a Type II Cas protein of the disclosure can be used; or alternatively, multiple pharmaceutical compositions can be used, for example a first pharmaceutical composition comprising an AAV particle encoding the gRNA(s) and a second, separate pharmaceutical composition comprising a second AAV particle encoding the Type II Cas protein. When multiple pharmaceutical compositions are used, they are preferably administered sufficiently close in time so that the gRNA(s) and Type II Cas protein provided by the pharmaceutical compositions are present together in vivo.

[0247] Targeting of (one or more of) human TRAC, human B2M, human PD1, and human LAG3 genes can be used, for example, in the engineering of chimeric antigen receptor (CAR) T cells. For example, CRISPR / Cas technology has been used to deliver CAR-encoding DNA sequences to loci such as TRAC and PD1 (see, e.g., Eyquem et al., 2017, Nature 543(7643):113-117; Hu et al., 2023, eClinicalMedicine 60:102010), while TRAC, B2M, PD1, and LAG3 knockout CAR T-cells have been reported (see, e.g., Dimitri et al., 2022, Molecular Cancer 21 :78; Liu et al., 2016, Cell Research 27:154-157; Ren et al., 2017, Clin Cancer Res. 23(9):2255-2266; Zhang et al., 2017, Front Med. 11 (4): 554-562). Thus, the Type II Cas proteins and TRAC, B2M, PD1, and LAG3 guides of the disclosure can be used for targeted knock-in of an exogenous DNA sequence to a desired genomic site in a human cell and / or knock-out of TRAC, B2M, PD1, or LAG3 in a human cell, for example a human T cell. In some embodiments, T cells are edited ex vivo to produce CAR-T cells and subsequently administered to a subject in need of CAR-T cell therapy.

[0248] In some embodiments, the methods of altering a cell are methods for altering a DNMT1 gene.Mutations in the DNMT1 gene can cause DNMT1 -related disorder, which is a degenerative disorder ofthe central and peripheral nervous systems. DNMT1 -related disorder is characterized by sensory impairment, loss of sweating, dementia, and hearing loss.7. EXAMPLES7.1. Example 1 : Identification and Characterization of Type II Cas Proteins

[0249] This Example describes studies performed to identify and characterize AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins.7.1.1. Materials and Methods7.1.1.1. Identification of Type II Cas Proteins From Metagenomic Data

[0250] 154,723 bacterial and archaeal metagenome-assembled genomes (MAGs) reconstructed from the human microbiome (Pasolli, et al., 2019, Cell 176(3):649-662.e20) were screened in order to find new Type II Cas proteins. cas1, cas2 and cas9 genes were identified from the protein annotation, performed with Prokka version 1.12 (Seemann, 2014, Bioinformatics 30(14):2068-2069). CRISPR arrays were identified using MinCED version 0.4.2 (with default parameters) (Bland, et al., 2007, BMC bioinformatics 8:209). Only loci having a CRISPR array and cas1-2-9 genes at a maximum distance of 10 kbp from each other were considered. Loci containing Type II Cas proteins shorter than 950 aa were discarded. The resulting 17173 CRISPR-Type II Cas loci were filtered by selecting short proteins (less than 1100 aa) from putative unknown species. Type II Cas proteins from the same species, having similar length but slightly different sequence, were compared by multiple sequence alignment. Proteins presenting deletions in nucleasic domains were discarded. The remaining proteins were compared for sequencing coverage and the ortholog with the highest coverage was selected for each species.7.1.1.2. tracrRNA Identification

[0251] Identification of tracrRNAs for CRISPR-Type II Cas loci of interest was performed with a method based on a work by Chyou and Brown (Chyou and Brown, 2019, RNA biology 16(4):423-434). Starting from unique direct repeats in the CRISPR array, BLAST® (National Library of Medicine) version 2.2.31 (with parameters -task blastn-short -gapopen 2 -gapextend 1 -penalty -1 -reward 1 -evalue 1 -word_size 8) (Altschul, et al., 1990, Journal of Molecular Biology 215(3):403-410) was used to identify anti-repeats within a 3000 bp window flanking the CRISPR-Type II Cas locus. A custom version of RNIE (Gardner, et al., 2011 , Nucleic Acids Research 39(14):5845-5852) was used to predict Rho-independent transcription terminators (RITs) near anti-repeats. Putative tracrRNA sequences, starting with an anti-repeat and ending with either a RIT (when found) or a poly-T, were combined with directed repeats to form sgRNA scaffolds. The secondary structure of sgRNA scaffolds was predicted using RNAsubopt version 2.4.14 (with parameters -noLP -e 5) (Lorenz, et al., 2011 , Algorithms for Molecular Biology 6(1):26). sgRNAs lacking the functional modules identified by (Briner, et al., 2014 Molecular Cell 56(2):333-339), namely the repeatanti-repeat duplex, nexus and 3’ hairpin-like folds, were discarded.7.1.2. Results

[0252] AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins were identified. Amino acid sequences of AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins and nucleotide sequences encoding exemplary AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA,and DWET Type II Cas proteins are shown in Tables 1 A-2K. Exemplary PAM sequences are shown in Table 5A. PAM logos are shown in FIG. 1 . crRNA and tracrRNA for the nucleases are described in Section 6.3. Exemplary sgRNA scaffolds are shown in Tables 7A-7B.7.2. Example 2: Additional Characterization of Type II Cas Proteins

[0253] This Example describes studies performed to further characterize AEQH, AAOF, ACEE, AQSL, ASWC, AVFG, AWIT, AWMF, BUMO, COIA, DJQA, and DWET Type II Cas proteins.7.2.1. Materials and Methods7.2.1.1. Plasmids

[0254] Type II Cas proteins were expressed in mammalian cells from a plasmid vector characterized by an EF1 alpha-driven cassette. Each Type II Cas protein coding sequence was human codon-optimized and modified by the addition of an SV5 tag at the N-terminus and two bipartite nuclear localization signals (1 at the N-term and 1 at the C-term). The sgRNA were expressed from a U6-driven cassette located on an independent plasmid construct. The human codon-optimized coding sequence of the Type II Cas proteins, as well as the sgRNA scaffolds, were obtained by synthesis from Twist Bioscience. Spacer sequences were cloned into the sgRNA plasmid as annealed DNA oligonucleotides (Eurofins Genomics) using a double Bsal site present in the plasmid. The list of spacer sequences and relative cloning oligonucleotides used in the present example is reported in Table 8. In all cases in which a spacer did not contain a matching native 5’-G, this nucleotide was appended upstream the targeting sequence in order to allow efficient transcription from a U6 promoter.7.2.1.2. Cell Lines

[0255] U2OS-EGFP cells, harboring a single integrated copy of an EGFP reporter gene, were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 2 mM L-Glutamine (Life Technologies) and penicillin / streptomycin (Life Technologies). All cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. All cells tested mycoplasma negative (PlasmoTest, Invivogen).7.2.1.3. In vitro Cas PAM identification assay

[0256] The in vitro PAM evaluation of the novel Type II Cas proteins was performed according to the protocol from Karvelis, et al., 2019, Methods in Enzymology, 616, pp. 219-240. In brief: the human codon optimized version of the Type II Cas protein gene obtained as a synthetic construct (Twist Bioscience) was cloned into an expression vector for in vitro transcription and translation (IVT) (pT7-N-His-GST, Thermo Fisher Scientific). The sgRNAs to perform the assay were obtained by in vitro transcription of the guide using the High Yield T7 RNA Synthesis Kit (Jena Bioscience) starting from a PCR template generated by amplification from each sgRNA expression construct, as commonly done in the field. The primers used to generate the IVT templates are reported in Table 9. In vitro transcribed gRNAs were subsequently purified using the MEGACIear Transcription Clean-up kit (Thermo Fisher Scientific). The in vitro transcription and translation reaction for Cas expression was performed according to the manufacturer’s protocol (1-Step Human High-Yield Mini IVT Kit, Thermo Fisher Scientific). The nuclease- guide RNA RNP complex was assembled by combining 20 pL of the supernatant containing the soluble Type II Cas protein with 1 pL of RiboLock RNase Inhibitor (Thermo Fisher Scientific) and 2pg of guide RNA (previously transcribed in vitro). The RNP complex was used to digest 1 ug of a PAM plasmid DNA library (containing a defined target sequence flanked at the 3’-end by a randomized 8 nucleotide PAM sequence) for 1 hour at 37°C.

[0257] A double stranded DNA adapter (Table 10) was ligated to the DNA ends generated by the targeted Cas cleavage and the final ligation product was purified using a GeneJet PCR Purification Kit (Thermo Fisher Scientific).

[0258] One round of a two-step PCR (Phusion HF DNA polymerase, Thermo Fisher Scientific) was performed to enrich the sequences that were cut using a set of forward primers annealing on the adapter and a reverse primer designed on the plasmid backbone downstream of the PAM (Table 11). A second round of PCR was performed to attach the Illumina indexes and adapters. PCR products were purified using the GeneJet PCR Purification Kit (Thermo Fisher Scientific).

[0259] The library was analysed with a 71-bp single read sequencing, using a flow cell v2 micro, on an Illumina MiSeq® sequencer.

[0260] PAM sequences were extracted from Illumina MiSeq reads and used to generate PAM sequence logos, using Logomaker version 0.8. PAM heatmaps were used to display PAM enrichment, computed dividing the frequency of PAM sequences in the cleaved library by the frequency of the same sequences in a control uncleaved library.7.2.1.4. Cell line transfections

[0261] To perform editing studies, 200,000 U2OS-EGFP cells were nucleofected with 500 ng of nuclease-expressing plasmid and 250 ng of sgRNA-expressing plasmid containing a guide designed to target EGFP using the 4D-Nucleofector™ SE Kit (Lonza), DN-100 program, according to the manufacturer’s protocol. After electroporation, cells were plated in a 24-well plate. EGFP knock-out was analysed 4 days after nucleofection using a BD FACSymphony A1 (BD) flow cytometer.7.2.2. Results

[0262] Having determined the sgRNA requirements for the selected Type II Cas proteins, it was possible to proceed with the discovery of the PAM sites recognized by each nuclease. To this aim a previously described in vitro PAM assay (see Methods) was exploited. Briefly, the assay uses in vitro translated Type II Cas proteins coupled with an in vitro synthesized sgRNA to generate a functional ribonucleoprotein complex to cleave a plasmid library characterized by a defined target sequence followed by a randomized 8 nt stretch corresponding to the putative PAMs. Cleaved PAMs can then be recovered after library preparation by next generation sequencing. Table 12 reported here below contains the PAM preferences as determined based on the assay outcome. The PAM logos and the PAM heatmaps reporting the nucleotide preferences for specific positions along the PAMs are reported in FIG. 5A-FIG. 8F.

[0263] After the discovery of the PAM sequences and the sgRNAs of the selected Type II Cas proteins and after obtaining preliminary information on the ability of these nucleases to cut a desired target in vitro (plasmid target used during the PAM assay), their ability to cleave selected targets in mammalian cells was investigated. An EGFP reporter system was used as it allowed an easier readout on the editing activity, based on the loss of fluorescence of treated cells quantitatively measured by cytofluorimetry. sgRNAs targeting the EGFP coding sequence (3 for each evaluated Type II Cas protein) were thus designed for all the Type II Cas proteins and evaluated in U2OS cells stably expressing a single copy of an EGFP reporter by transient electroporation. Loss of EGFP fluorescence, expressed as % of EGFP- negative cells, was measured by cytofluorimetry. Data presented in FIG. 9 as mean ± SEM of n=2 biologically independent studies.

[0264] Surprisingly, as reported in FIG. 9, some of the evaluated guides in combination with their respective Type II Cas protein were able to significantly downregulate EGFP expression in target cells. In particular, AVFG, ACEE AEQH DJQA and DWET Type II Cas proteins showed very high (>90% EGFP KO) with one or more of the evaluated guides; AAOF, BUMO and COIA Type II Cas proteins showed appreciable knock-out activity (>50% EGFP KO) with at least one of the evaluated sgRNAs; AWIT Type II Cas protein showed lower editing activity (>30% with one guide RNA out of three); ASWC and AQSL Type II Cas proteins showed lower, but still some activity (>10% EGFP KO) with at least one of the evaluated guide RNAs. The remaining AWMF Type II Cas protein did not show editing levels above the background of the assay against the currently evaluated targets in the EGFP coding sequence. Thesedata clearly demonstrate that some of the selected Type II Cas proteins were able to very efficiently modify genetic targets in mammalian cells and can thus be exploited to edit the mammalian genome.7.3. Example 3: Allele Specific RHO Editing with AAOF, ACEE, AEQH, AVFG, BUMO, DJQA, and DWET Type II Cas

[0265] This Example describes the design and evaluation of a mutation independent allele-specific strategy to selectively inactivate mutated RHO alleles. The RHO gene, which encodes for the photopigment rhodopsin, is one of the most frequently mutated genes in autosomal dominant retinitis pigmentosa and more than one hundred mutations have been described in the art. The great heterogeneity of mutations in affected patients and the overall low prevalence of most of these mutations makes a mutation independent approach to target the disease particularly desirable. Additionally, effective knock-out of diseased alleles can be effectively obtained using gene editing tools, such as Type II Cas enzymes. Key for the success of the approach is the ability to preferentially downregulate RHO mutated alleles while sparing the wild-type counterpart, in order to preserve photoreceptor function.

[0266] The strategy described in this Example exploits a commonly occurring non-pathogenic SNP in the RHO gene, rs7984, located in the 5 -UTR of the gene and common in the general population, to selectively target only the one RHO allele containing dominant negative mutations, independently of the exact nature of the mutation. Only patients which are heterozygous for the rs7984 SNP are potentially eligible for this targeting strategy, which is based on the exact knowledge of the phase between the SNP alleles and the mutation affecting each patient. Allele-selectivity is achieved selectively targeting the rs7984 allele which is in phase with the patient’s mutation.

[0267] Since the rs7984 SNP is located outside the RHO coding sequence, a second cut in RHO intron 1 can be introduced to remove the entire exon 1 and knock-out expression of the mutated protein. This second cut, which has to occur synchronously with the cut on the rs7984 locus to produce the desired deletion, can be bi-allelic, targeting a site present on both RHO alleles.7.3.1. Materials and Methods7.3.1.1. Plasmids

[0268] EF1 alpha-driven expression plasmids were used to express AAOF, ACEE, AEQH, AVFG, BUMO, DJQA, AND DWET Type II Cas protein in mammalian cells. Briefly, the human codon-optimized coding sequences of the different Type II Cas were cloned into the aforementioned expression plasmid. The sgRNA scaffold of each Type II Cas (trimmed scaffold reported in Table 7A or Table 7B, with added 3’ uracils) was cloned into an expression plasmid containing a human U6 promoter to drive guide RNA expression in mammalian cells. Each Type II Cas coding sequence, modified by the addition of an SV5 tag at the N-terminus and two bipartite nuclear localization signals (1 at the N-terminus and 1 at the C- terminus) and human codon-optimized, as well as the sgRNA expression cassettes (U6 promoter + sgRNA scaffolds), were obtained as synthetic constructs from Twist Bioscience. Spacer sequences were cloned into the sgRNA expression plasmids as annealed DNA oligonucleotides using a double Bsal site present in the plasmid. The list of spacer sequences and relative cloning oligonucleotides used in the present example is reported in Table 13.7.3.1.2. Cell Lines

[0269] HEK293T cells (obtained from ATCC) and HEK293-rs7984G cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 2 mM L-Glutamine (Life Technologies) and penicillin / streptomycin (Life Technologies). HEK293-rs7984G cells, which are homozygous for the rs7984G SNP allele, were obtained by base editing and single clones were subsequently isolated, expanded and characterized to select the one to be used for further studies. All cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. All cells tested mycoplasma negative (PlasmoTest™, Invivogen).7.3.1.3. Cell Line Transfections

[0270] To perform editing studies on target RHO locus 100,000 HEK293T or HEK293-rs7984G cells were seeded in a 24-well plate 24 hours before transfection. Cells were then transfected with 500 ng of nuclease-expressing plasmid together with 250 ng of sgRNA expression vector targeting the locus of interest using the TranslT®-LT1 reagent (Mirus Bio) according to the manufacturer’s protocol. Cell pellets were collected three days from transfection for analysis.7.3.1.4. Evaluation of Gene Editing

[0271] Three days after transfection cells were collected and DNA was extracted using the QuickExtract™ DNA Extraction Solution (Lucigen) according to the manufacturer’s instructions. To amplify the target loci, PCR reactions were performed using the HOT FIREPol® polymerase (Solis BioDyne) and the oligonucleotides listed in Table 14. The amplified products were purified, sent for Sanger sequencing (EasyRun service, Microsynth) and analyzed with the TIDE web tool (shinyapps.datacurators.nl / tide / ) to quantify indels. The primers used for Sanger sequencing reactions on amplicons are reported in Table 15, associated with their respective target locus.7.3.1.5. Evaluation of Large Edits at the Target Locus

[0272] The presence of large editing events at the target RHO locus (deletions and inversions produced by the double cut) was evaluated using multiple approaches. Deletion formation was detected by endpoint PCR amplification of the target locus using the primers reported in Table 16 followed by visualization of the amplified products on agarose gel. Alternatively, a qPCR assay (HOT FIREPol® Multiplex qPCR Mix) using the primers and probes reported in Table 17 was developed to detect the amount of RHO alleles which were not rearranged and by complementary those which contained large edits. Additionally, a high sensitivity ddPCR assay to specifically quantify the desired deletion and inversion events was exploited, using the primers reported in Table 17 and Biorad ddPCR Supermix for Probes (no dUTP). Both the qPCR and ddPCR assays were probe-based and shared the same probe and some of the primers (Table 17 and Table 18). An internal reference was included in both assays by positioning a specific primer-probe pair in downstream exons (RHO exon-intron 4) of the RHO gene (see Table 17 and Table 18).7.3.2. Results7.3.2.1. Type II Cas sgRNAs for the allele-specific targeting of the RHO rs7984 SNP

[0273] A set of sgRNAs associated with PAMs strongly recognized by AAOF, ACEE, AEQH, AVFG, BUMO, DJQA or DWET Type II Cas spanning the rs7984 SNP were designed (FIG. 10). The editing activity of the selected guides in combination with the respective nucleases was evaluated by transient transfection of HEK293T cells. These cells are homozygous for the rs7984A allele of the SNP and sgRNAs targeting the rs7984A allele were used. As shown in FIG. 11 , many of the evaluated guides showed appreciable generation of indels, with the ACEE, AVFG and DJQA Type II Cas proteins showing particularly high levels of modification (above 40% indel formation) with at least one of the designed sgRNAs. On the other hand, AEQH Type II Cas, for which only one guide was evaluated, showed intermediate levels of modification while DWET, AAOF and BUMO Type II Cas proteins produced only modest levels of editing at the target locus. Given these results, ACEE+g1 / g3, DJQA+g1 and AVFG+g2 nuclease-guides combinations were selected for further characterization.

[0274] Different spacer lengths of the selected guides were evaluated spanning from 20 to 24 matching nucleotides (all targeting the rs7984A allele). Where needed, a 5’-end non-matching G nucleotide was added to allow efficient transcription for the human U6 promoter, as reported in the art. As shown in FIGS. 12A-12C, after transient transfection in HEK293T cells, for all the evaluated nucleases the highest editing levels were obtained using 23 nucleotide (nt) spacers, as originally designed. This spacer length was thus used in subsequent studies.

[0275] Next, to verify the allele specificity of the candidate Type II Cas (ACEE, AVFG, DJQA) in combination with the selected guides towards the rs7984 SNP target site, two parallel approaches were undertaken. First, HEK293T cells were transiently transfected with two alternative versions of each selected sgRNA targeting either the rs7984A or rs7984G alleles together with the corresponding Type II Cas. Since HEK293T cells are homozygous for rs7984A, the on-target editing activity was measured by using rs7984A-targeting guides (FIG. 13A, left bars), while the allele specificity (G vs A direction) was evaluated by using rs7984G-targeting sgRNAs (FIG. 13A, right bars).

[0276] In parallel, an engineered HEK293T cell clone modified by base editing to be homozygous for the rs7984G SNP allele (HEK293T-rs7984G) was transfected with the same combination of constructs to evaluate the on-target cleavage activity towards the rs7984G allele (FIG. 13B, left bars) and to verify the allelic specificity in the A vs G direction (FIG. 13B, right bars). AVFG Type II Cas was not included in these evaluations.

[0277] This allowed the complete evaluation of activity and specificity of alternative versions of the candidate guide directed to both alleles of the rs7984 SNP which will be necessarily exploited in order to cover the entire putatively eligible patient population. Overall, all evaluated sgRNAs showed allelicpreference towards the intended target which is more efficiently edited compared to non-target counter allele. Notably, a very pronounced selectivity was observed for all the guides in the A vs G direction. Based on the combined data on activity and specificity towards the rs7984A / G alleles, the candidates ACEE+g1 / g3 and DJQA+g1 were selected for further studies.7.3.2.2. ACEE and DJQA Type II Cas sgRNAs to target RHO intron 1

[0278] Next, sgRNAs for ACEE and DJQA Type II Cas targeting RHO intron 1 were screened for cleavage activity with the final goal of identifying high performing guides to be used in combination with guide RNAs targeting the rs7984 SNP to generate the desired large edit (deletions or inversions) encompassing RHO exon 1 , which includes the ATG translation start site, to knock-out mutant protein expression. A schematic representation of the sgRNAs positions is reported in FIGS. 14A-14B. HEK293T cells were transfected with either ACEE or DJQA Type II Cas together with a panel of sgRNAs targeting the first half of RHO intron 1 in order to generate a deletion below 1000bp in size, when used in combination with the selected sgRNAs targeting the rs7984 SNP for each nuclease (ACEE: g1 / g3; DJQA: g1). Variable levels of indel formation were observed with the different sgRNAs, with some of the guides failing to generate appreciable editing at the target site in this study (FIGS. 15A-15B). Notably, for all evaluated nucleases, highly efficient guide RNAs were identified. Guides g481 , g485, g543, g546, g794 and g948 for ACEE Type II Cas, and g700 g825, g884 and g973 for DJQA Type II Cas performed particularly well, and were thus selected for further studies.7.3.2.3. Evaluation of the formation of large editing events in the RHO gene using best performing sgRNA couples

[0279] Next, the formation of large editing events (e.g., large deletion and inversions) at the target RHO locus was assessed after transient transfection of HEK293T cells with combinations of SNP-targeting sgRNAs (directed towards the rs7984A allele) for ACEE and DJQA Type II Cas with guides selected to target RHO intron 1 . As a control, guide RNAs which were not observed to produce high levels of indel formation in RHO intron 1 were included in the evaluated combinations to evaluate the sensitivity of the readouts.

[0280] Deletion formation was initially assessed using a specifically designed PCR assay with primers spanning the deleted region and was visualized using agarose gel electrophoresis, where a low molecular weight band should be present when the desired deletion is correctly formed. As shown in FIG. 16A, each of the tested combinations was producing the desired deletion in the RHO locus efficiently. Of note, control guides (g594 for ACEE and g703 for DJQA) showed reduced (g594 for ACEE) or abolished (g703 for DJQA) deletion formation, as expected (FIG. 16A).

[0281] Next, to measure quantitatively the extent of modification of the RHO gene and rank the same candidates were evaluated for their efficacy by using a qPCR assay specifically designed to detect unmodified RHO alleles. This, conversely, allows to estimate the amount of large edits (among which the desired deletion and inversion) produced by each nuclease-guide RNA couple. As shown in FIG. 16B, many candidates showed high levels of RHO gene modification (low levels of non-modified RHO), with ACEE Type II Cas being generally more effective than DJQA Type II Cas in these studies. Control guides(g594+ACEE and g703+DJQA) showed again the highest level of non-modified RHO alleles, in accordance with previous data (see FIG. 16B).

[0282] Among the evaluated candidates, those more effective in modifying the target locus were further investigated using a very sensitive droplet digital PCR (ddPCR) assay, which for the measurement of the absolute number of RHO alleles characterized by the desired deletion and inversion events after transient transfection of a population of cells. To this aim, HEK293T cells (homozygous rs7984A) were transiently transfected with either ACEE Type II Cas in combination with the g1 or g3 SNP-targeting guides (rs7984A version) together with intron guides g481 , g543, g546, g794 or g948 or DJQA Type II Cas in combination with the g1 SNP-targeting guide (rs7984A version) together with intron guides g825 or g884. Deletion and inversion formation was then quantified by ddPCR, showing efficient modification of the target locus for most of the evaluated candidates (FIG. 17). Interestingly, different guide combinations yielded different ratios between deletions and inversions, demonstrating specific repair preferences based on the exact pattern of DSB produced by specific guide RNAs at the two target sites (e.g., see FIG. 17 ACEE g1+g794 vs ACEE g3+g794). Among all the evaluated candidates, ACEE Type II Cas showed a generally higher efficacy in generating the desired edits at the RHO target locus, with SNP g1 / g3 in combination with intron g543, g546 or g794 being among the top performers (FIG. 17).

[0283] Overall, sgRNAs targeting the RHO gene were identified that generate high levels of desired deletions / inversions of the first exon of the gene and also have selectivity for the desired RHO copy.7.4. Example 4: Gene Editing with BDLP and EQSC Type II Cas proteins

[0284] To extensively evaluate the cleavage activity AVFG, ACEE, AEQH, DJQA and DWET Type II Cas, a panel of endogenous loci (B2M, TRAC, PD-1) which are commonly targeted to generate allogeneic CAR-T cells (Chimeric Antigen Receptor T cells) were selected for editing studies. For each target locus multiple sgRNAs were designed and evaluated in parallel by transient plasmid transfection in HEK293T cells.

[0285] Materials and methods were similar to those used in Example 3. Table 19 shows protospacer and oligo sequences used for cloning sgRNA spacers. Table 20 shows oligos used for TIDE analysis.

[0286] As shown in FIGS. 18A-18C, for each of the evaluated loci, for each of the tested loci most of the nucleases showed significant editing activity with at least one of the selected guide RNAs, demonstrating that these novel Type II Cas proteins have the ability to effectively modify genomic targets of interest.8. SPECIFIC EMBODIMENTS

[0287] The present disclosure is exemplified by the specific embodiments below.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-l domain of a reference protein sequence;(b) the amino acid sequence of a RuvC-ll domain of a reference protein sequence;(c) the amino acid sequence of a RuvC-lll 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 REC domain of a reference protein sequence;(f) the amino acid sequence of a HNH domain of a reference protein sequence;(g) the amino acid sequence of a WED domain of a reference protein sequence;(h) the amino acid sequence of a PID domain of a reference protein sequence; or(i) 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 NQ: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 NQ:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:67, or SEQ ID NO:68.2. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.3. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.4. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.5. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.6. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.7. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.8. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.9. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.10. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.11. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.12. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.13. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.14. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.15. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.16. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-l domain of the reference protein sequence.17. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.18. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.19. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.20. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.21 . The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.22. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.23. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.24. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.25. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.26. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.27. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.28. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.29. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.30. The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.31 . The Type II Cas protein of any one of embodiments 1 to 16, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-ll domain of the reference protein sequence.32. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.33. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.34. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.35. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.36. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.37. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.38. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.39. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.40. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.41 . The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.42. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.43. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.44. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.45. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.46. The Type II Cas protein of any one of embodiments 1 to 31 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the RuvC-lll domain of the reference protein sequence.47. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the BH domain of the reference protein sequence.48. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the BH domain of the reference protein sequence.49. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the BH domain of the reference protein sequence.50. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the BH domain of the reference protein sequence.51 . The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the BH domain of the reference protein sequence.52. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the BH domain of the reference protein sequence.53. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the BH domain of the reference protein sequence.54. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the BH domain of the reference protein sequence.55. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the BH domain of the reference protein sequence.56. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the BH domain of the reference protein sequence.57. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the BH domain of the reference protein sequence.58. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the BH domain of the reference protein sequence.59. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the BH domain of the reference protein sequence.60. The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the BH domain of the reference protein sequence.61 . The Type II Cas protein of any one of embodiments 1 to 46, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the BH domain of the reference protein sequence.62. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the REC domain of the reference protein sequence.63. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the REC domain of the reference protein sequence.64. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the REC domain of the reference protein sequence.65. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the REC domain of the reference protein sequence.66. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the REC domain of the reference protein sequence.67. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the REC domain of the reference protein sequence.68. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the REC domain of the reference protein sequence.69. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the REC domain of the reference protein sequence.70. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the REC domain of the reference protein sequence.71 . The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the REC domain of the reference protein sequence.72. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the REC domain of the reference protein sequence.73. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the REC domain of the reference protein sequence.74. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the REC domain of the reference protein sequence.75. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the REC domain of the reference protein sequence.76. The Type II Cas protein of any one of embodiments 1 to 61 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the REC domain of the reference protein sequence.77. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the HNH domain of the reference protein sequence.78. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the HNH domain of the reference protein sequence.79. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the HNH domain of the reference protein sequence.80. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the HNH domain of the reference protein sequence.81 . The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the HNH domain of the reference protein sequence.82. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the HNH domain of the reference protein sequence.83. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the HNH domain of the reference protein sequence.84. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the HNH domain of the reference protein sequence.85. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the HNH domain of the reference protein sequence.86. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the HNH domain of the reference protein sequence.87. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the HNH domain of the reference protein sequence.88. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the HNH domain of the reference protein sequence.89. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the HNH domain of the reference protein sequence.90. The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the HNH domain of the reference protein sequence.91 . The Type II Cas protein of any one of embodiments 1 to 76, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the HNH domain of the reference protein sequence.92. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the WED domain of the reference protein sequence.93. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the WED domain of the reference protein sequence.94. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the WED domain of the reference protein sequence.95. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the WED domain of the reference protein sequence.96. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the WED domain of the reference protein sequence.97. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the WED domain of the reference protein sequence.98. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the WED domain of the reference protein sequence.99. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the WED domain of the reference protein sequence.100. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the WED domain of the reference protein sequence.101 . The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the WED domain of the reference protein sequence.102. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the WED domain of the reference protein sequence.103. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the WED domain of the reference protein sequence.104. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the WED domain of the reference protein sequence.105. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the WED domain of the reference protein sequence.106. The Type II Cas protein of any one of embodiments 1 to 91 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the WED domain of the reference protein sequence.107. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of the PID domain of the reference protein sequence.108. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the amino acid sequence of the PID domain of the reference protein sequence.109. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of the PID domain of the reference protein sequence.110. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence of the PID domain of the reference protein sequence.111. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the PID domain of the reference protein sequence.112. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of the PID domain of the reference protein sequence.113. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the PID domain of the reference protein sequence.114. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of the PID domain of the reference protein sequence.115. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the PID domain of the reference protein sequence.116. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of the PID domain of the reference protein sequence.117. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of the PID domain of the reference protein sequence.118. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of the PID domain of the reference protein sequence.119. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of the PID domain of the reference protein sequence.120. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of the PID domain of the reference protein sequence.121. The Type II Cas protein of any one of embodiments 1 to 106, wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is identical to the amino acid sequence of the PID domain of the reference protein sequence.122. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 55% identical to the full length of the reference protein sequence.123. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 60% identical to the full length of the reference protein sequence.124. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 65% identical to the full length of the reference protein sequence.125. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 70% identical to the full length of the reference protein sequence.126. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 75% identical to the full length of the reference protein sequence.127. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 80% identical to the full length of the reference protein sequence.128. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 85% identical to the full length of the reference protein sequence.129. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 90% identical to the full length of the reference protein sequence.130. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 95% identical to the full length of the reference protein sequence.131 . The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 96% identical to the full length of the reference protein sequence.132. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 97% identical to the full length of the reference protein sequence.133. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 98% identical to the full length of the reference protein sequence.134. The Type II Cas protein of embodiment 1 , wherein the amino acid sequence of the Type II Cas protein comprises an amino acid sequence that is at least 99% identical to the full length of the reference protein sequence.135. The Type II Cas protein of embodiment 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.136. The Type II Cas protein of any one of embodiments 1 to 135, which is a chimeric Type II Cas protein.137. The Type II Cas protein of any one of embodiments 1 to 136, which is a fusion protein.138. The Type II Cas protein of embodiment 137, which comprises one or more nuclear localization signals.139. The Type II Cas protein of embodiment 138, which comprises two or more nuclear localization signals.140. The Type II Cas protein of embodiment 138 or embodiment 139, which comprises an N- terminal nuclear localization signal.141. The Type II Cas protein of any one of embodiments 138 to 140, which comprises a C- terminal nuclear localization signal.142. The Type II Cas protein of any one of embodiments 138 to 141 , which comprises an N- terminal nuclear localization signal and a C-terminal nuclear localization signal.143. The Type II Cas protein of any one of embodiments 138 to 142, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145), PKKKRKV (SEQ ID NO:146), PKKKRRV (SEQ ID NO:147), KRPAATKKAGQAKKKK (SEQ ID NO:148), YGRKKRRQRRR (SEQ ID NO:149), RKKRRQRRR (SEQ ID NQ:150), PAAKRVKLD (SEQ ID NO:151), RQRRNELKRSP (SEQ ID NO:152), VSRKRPRP (SEQ ID NO:153), PPKKARED (SEQ ID NO:154), PQPKKKPL (SEQ ID NO:155), SALIKKKKKMAP (SEQ ID NO:156), PKQKKRK (SEQ ID NO:157), RKLKKKIKKL (SEQ ID NO:158), REKKKFLKRR (SEQ ID NO:159), KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160), RKCLQAGMNLEARKTKK (SEQ ID NO:161), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:162), or RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:163).144. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145).145. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRKV (SEQ ID NO:146).146. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKKKRRV (SEQ ID NO:147).147. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO:148).148. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO:149).149. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKKRRQRRR (SEQ ID NQ:150).150. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PAAKRVKLD (SEQ ID NO:151).151. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RQRRNELKRSP (SEQ ID NO:152).-ISO-152. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence VSRKRPRP (SEQ ID NO:153).153. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PPKKARED (SEQ ID NO:154).154. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PQPKKKPL (SEQ ID NO:155).155. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence SALIKKKKKMAP (SEQ ID NO:156).156. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence PKQKKRK (SEQ ID NO:157).157. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKLKKKIKKL (SEQ ID NO:158).158. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence REKKKFLKRR (SEQ ID NO:159).159. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160).160. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RKCLQAGMNLEARKTKK (SEQ ID NO:161).161. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:162).162. The Type II Cas protein of embodiment 143, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:163).163. The Type II Cas protein of any one of embodiments 138 to 162, wherein the amino acid sequence of each nuclear localization signal is the same.164. The Type II Cas protein of any one of embodiments 136 to 163, 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, anacetyltransferase, 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.165. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a means for deaminating adenosine, optionally wherein the means for deaminating adenosine is an adenosine deaminase.166. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a fusion partner which is an adenosine deaminase, optionally wherein the amino acid sequence of the adenosine deaminase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:166, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e.167. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a means for deaminating cytidine, optionally wherein the means for deaminating cytidine is a cytidine deaminase.168. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a fusion partner which is a cytidine deaminase.169. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a means for synthesizing DNA from a single-stranded template, optionally wherein the means for synthesizing DNA from a single-stranded template is a reverse transcriptase.170. The Type II Cas protein of any one of embodiments 136 to 164, which comprises a fusion partner which is a reverse transcriptase.171 . The Type II Cas protein of any one of embodiments 136 to 170, which comprises a tag.172. The Type II Cas protein of embodiment 171 , wherein the tag is a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:164) or IPNPLLGLD (SEQ ID NO:165).173. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:1 .174. The Type II Cas protein of embodiment 173, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1 .175. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:2.176. The Type II Cas protein of any one of embodiments 173 to 175, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:2.177. The Type II Cas protein of embodiment 173 or embodiment 174, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:3.178. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:7.179. The Type II Cas protein of embodiment 178, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:7.180. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:8.181 . The Type II Cas protein of any one of embodiments 178 to 180, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:8.182. The Type II Cas protein of embodiment 178 or embodiment 179, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:9.183. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:13.184. The Type II Cas protein of embodiment 183, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:13.185. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:14.186. The Type II Cas protein of any one of embodiments 183 to 185, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:14.187. The Type II Cas protein of embodiment 183 or embodiment 184, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:15.188. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:19.189. The Type II Cas protein of embodiment 188, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:19.190. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NQ:20.191 . The Type II Cas protein of any one of embodiments 188 to 190, whose amino acid sequence comprises the amino acid sequence of SEQ ID NQ:20.192. The Type II Cas protein of embodiment 188 or embodiment 189, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:21 .193. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:25.194. The Type II Cas protein of embodiment 193, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:25.195. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:26.196. The Type II Cas protein of any one of embodiments 193 to 195, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:26.197. The Type II Cas protein of embodiment 194 or embodiment 195, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:27.198. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:31 .199. The Type II Cas protein of embodiment 198, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:31 .200. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:32.201. The Type II Cas protein of any one of embodiments 199 to 200, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:32.202. The Type II Cas protein of embodiment 198 or embodiment 199, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:33.203. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:37.204. The Type II Cas protein of embodiment 203, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:37.205. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:38.206. The Type II Cas protein of any one of embodiments 203 to 205, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:38.207. The Type II Cas protein of embodiment 203 or embodiment 204, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:39.208. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:43.209. The Type II Cas protein of embodiment 208, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:43.210. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:44.211 . The Type II Cas protein of any one of embodiments 208 to 210, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:44.212. The Type II Cas protein of embodiment 208 or embodiment 209, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:45.213. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:49.214. The Type II Cas protein of embodiment 213, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:49.215. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NQ:50.216. The Type II Cas protein of any one of embodiments 213 to 215, whose amino acid sequence comprises the amino acid sequence of SEQ ID NQ:50.217. The Type II Cas protein of embodiment 213 or embodiment 214, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:51 .218. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:55.219. The Type II Cas protein of embodiment 218, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:55.220. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:56.221. The Type II Cas protein of any one of embodiments 218 to 220, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:56.222. The Type II Cas protein of embodiment 218 or embodiment 219, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:57.223. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:61 .224. The Type II Cas protein of embodiment 223, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:61 .225. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:62.226. The Type II Cas protein of any one of embodiments 223 to 225, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:62.227. The Type II Cas protein of embodiment 223 or embodiment 224, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:63.228. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:67.229. The Type II Cas protein of embodiment 228, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:67.230. The Type II Cas protein of any one of embodiments 1 to 172, wherein the reference protein sequence is SEQ ID NO:68.231 . The Type II Cas protein of any one of embodiments 228 to 230, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:68.232. The Type II Cas protein of embodiment 228 or embodiment 229, whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:69.233. A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of embodiments 1 to 232 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 substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4).234. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions that provide nickase activity are in a RuvC or HNH domain.235. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.236. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N627A substitution, whereinthe position of the N627A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.237. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H604A substitution, wherein the position of the H604A substitution is defined with respect to the amino acid numbering of SEQ ID NO:2.238. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.239. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.240. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:8.241 . The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D8A substitution, wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.242. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N613A substitution, wherein the position of the N613A substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.243. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H590 substitution, wherein the position of the H590 substitution is defined with respect to the amino acid numbering of SEQ ID NO:14.244. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.245. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.246. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20.247. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D15A substitution, whereinthe position of the D15A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.248. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N633A substitution, wherein the position of the N633A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.249. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H610A substitution, wherein the position of the H610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:26.250. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide 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:32.251 . The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N607A substitution, wherein the position of the N607A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.252. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H584A substitution, wherein the position of the H584A substitution is defined with respect to the amino acid numbering of SEQ ID NO:32.253. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.254. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.255. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:38.256. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.257. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, whereinthe position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.258. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:44.259. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D8A substitution, wherein the position of the D8A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.260. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.261 . The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50.262. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.263. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.264. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, wherein the position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:56.265. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D10A substitution, wherein the position of the D10A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.266. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N615A substitution, wherein the position of the N615A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.267. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H592A substitution, whereinthe position of the H592A substitution is defined with respect to the amino acid numbering of SEQ ID NO:62.268. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a D9A substitution, wherein the position of the D9A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.269. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a N610A substitution, wherein the position of the N610A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.270. The Type II Cas of embodiment 233, wherein the one or more amino acid substitutions relative to the reference sequence that provide nickase activity comprise a H587A substitution, wherein the position of the H587A substitution is defined with respect to the amino acid numbering of SEQ ID NO:68.271 . A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of embodiments 1 to 232 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 substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4), or a combination thereof.272. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N627A substitutions, wherein the positions of the D10A and N627A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:2.273. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H604A substitutions, wherein the positions of the D10A and H604A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:2.274. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N610A substitutions, wherein the positions of the D9A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:8.275. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:8.276. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and N613A substitutions, wherein the positions of the D8A and N613A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:14.277. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and H590A substitutions, wherein the positions of the D8A and H590A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:14.278. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N610A substitutions, wherein the positions of the D9A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:20.279. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NQ:20.280. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D15A and N633A substitutions, wherein the positions of the D15A and N633A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:26.281 . The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D15A and H610A substitutions, wherein the positions of the D15A and H610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:26282. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D12A and N607A substitutions, wherein the positions of the D12A and N607A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:32.283. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D12A and H584A substitutions, wherein the positions of the D12A and H584A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:32284. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:38.285. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:38.286. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:44.287. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:44.288. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and N610A substitutions, wherein the positions of the D8A and N610A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:50.289. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D8A and H587A substitutions, wherein the positions of the D8A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NQ:50.290. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:56.291 . The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:56.292. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and N615A substitutions, wherein the positions of the D10A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:62.293. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D10A and H592A substitutions, wherein the positions of the D10A and H592A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:62.294. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and N615A substitutions, wherein the positions of the D9A and N615A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:68.295. The Type II Cas protein of embodiment 271 , wherein the one or more amino acid substitutions that render the Type II Cas protein catalytically inactive comprise D9A and H587A substitutions, wherein the positions of the D9A and H587A substitutions are defined with respect to the amino acid numbering of SEQ ID NO:68.296. An AEQH Type II Cas guide RNA (gRNA) molecule.297. An AAOF Type II Cas guide RNA (gRNA) molecule.298. An ACEE Type II Cas guide RNA (gRNA) molecule.299. An AQSL Type II Cas guide RNA (gRNA) molecule.300. An ASWC Type II Cas guide RNA (gRNA) molecule.301 . An AVFG Type II Cas guide RNA (gRNA) molecule.302. An AWIT Type II Cas guide RNA (gRNA) molecule.303. An AWMF Type II Cas guide RNA (gRNA) molecule.304. A BUMO Type II Cas guide RNA (gRNA) molecule.305. A COIA Type II Cas guide RNA (gRNA) molecule.306. A DJQA Type II Cas guide RNA (gRNA) molecule.307. A DWET Type II Cas guide RNA (gRNA) molecule.308. The gRNA of any one of embodiments 296 to 307, which is a gRNA for editing a human RHO gene.309. The gRNA of any one of embodiments 296 to 307, which is a gRNA for editing a human B2 / W gene.310. The gRNA of any one of embodiments 296 to 307, which is a gRNA for editing a human TRAC gene.311 . The gRNA of any one of embodiments 296 to 307, which is a gRNA for editing a human LAG3 gene.312. The gRNA of any one of embodiments 296 to 307, which is a gRNA for editing a human PD1 gene.313. A guide RNA (gRNA) molecule for editing a human RHO 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:314-399.314. A guide RNA (gRNA) molecule for editing a human RHO 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 N0s:400-419.315. 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 NQs:420-441 .316. 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:442-461 .317. 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:462-482.318. The gRNA of any one of embodiments 313 to 317, which comprises a spacer that is 15 to 30 nucleotides in length.319. The gRNA of embodiment 318, wherein the spacer is 18 to 30 nucleotides in length.320. The gRNA of embodiment 318, wherein the spacer is 20 to 28 nucleotides in length.321 . The gRNA of embodiment 318, wherein the spacer is 22 to 26 nucleotides in length.322. The gRNA of embodiment 318, wherein the spacer is 23 to 25 nucleotides in length.323. The gRNA of embodiment 318, wherein the spacer is 22 to 25 nucleotides in length.324. The gRNA of embodiment 318, wherein the spacer is 15 to 25 nucleotides in length.325. The gRNA of embodiment 318, wherein the spacer is 16 to 24 nucleotides in length.326. The gRNA of embodiment 318, wherein the spacer is 17 to 23 nucleotides in length.327. The gRNA of embodiment 318, wherein the spacer is 18 to 22 nucleotides in length.328. The gRNA of embodiment 318, wherein the spacer is 19 to 21 nucleotides in length.329. The gRNA of embodiment 318, wherein the spacer is 25 nucleotides in length.330. The gRNA of embodiment 318, wherein the spacer is 24 nucleotides in length.331 . The gRNA of embodiment 318, wherein the spacer is 23 nucleotides in length.332. The gRNA of embodiment 318, wherein the spacer is 22 nucleotides in length.333. The gRNA of embodiment 318, wherein the spacer is 21 nucleotides in length.334. The gRNA of embodiment 318, wherein the spacer is 20 nucleotides in length.335. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises 16 or more consecutive nucleotides of the reference sequence.336. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises 17 or more consecutive nucleotides of the reference sequence.337. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises 18 or more consecutive nucleotides of the reference sequence.338. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises 19 or more consecutive nucleotides of the reference sequence.339. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises 20 consecutive nucleotides of the reference sequence.340. The gRNA of any one of embodiments 313 to 333, wherein the reference sequence is a reference sequence having at least 21 and the spacer comprises 21 consecutive nucleotides of the reference sequence.341 . The gRNA of any one of embodiments 313 to 332, wherein the reference sequence is a reference sequence having at least 22 and the spacer comprises 22 consecutive nucleotides of the reference sequence.342. The gRNA of any one of embodiments 313 to 331 , wherein the reference sequence is a reference sequence having at least 23 nucleotides and the spacer comprises 23 consecutive nucleotides of the reference sequence.343. The gRNA of any one of embodiments 313 to 330, wherein the reference sequence is a reference sequence having at least 24 nucleotides and the spacer comprises 24 consecutive nucleotides of the reference sequence.344. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises a nucleotide sequence that is at least 90% identical to the reference sequence.345. The gRNA of embodiment 344, wherein the spacer comprises a nucleotide sequence that is at least 95% identical to the reference sequence.346. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises a nucleotide sequence that has one mismatch relative to the reference sequence.347. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises a nucleotide sequence that has two mismatches relative to the reference sequence.348. The gRNA of any one of embodiments 313 to 334, wherein the spacer comprises the reference sequence.349. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCAUUCUUGGGUGGGAGCAGCCR (SEQ ID NO:314).350. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCAUUCUUGGGUGGGAGCAGCCA (SEQ ID NO:315).351 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCAUUCUUGGGUGGGAGCAGCCG (SEQ ID NO:316).352. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GUGGCUGACCCGYGGCUGCUCCCA (SEQ ID NO:317).353. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GUGGCUGACCCGUGGCUGCUCCCA (SEQ ID NO:318).354. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GUGGCUGACCCGCGGCUGCUCCCA (SEQ ID NO:319).355. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCRCGGGUC (SEQ ID NQ:320).356. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCACGGGUC (SEQ ID NO:321).357. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCGCGGGUC (SEQ ID NO:322).358. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGCUGACCCGYGGCUGCUCCCAC (SEQ ID NO:323).359. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGCUGACCCGUGGCUGCUCCCAC (SEQ ID NO:324).360. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGCUGACCCGCGGCUGCUCCCAC (SEQ ID NO:325).361 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGYGGCU (SEQ ID NO:326).362. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGUGGCU (SEQ ID NO:327).363. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGCGGCU (SEQ ID NO:328).364. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGUGGCUGACCCGUGGCU (SEQ ID NO:329).365. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CCUUGUGGCUGACCCGUGGCU (SEQ ID NQ:330).366. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGCCCUUGUGGCUGACCCGUGGCU (SEQ ID NO:331).367. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGUGGCUGACCCGCGGCU (SEQ ID NO:332).368. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CCUUGUGGCUGACCCGCGGCU (SEQ ID NO:333).369. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGCCCUUGUGGCUGACCCGCGGCU (SEQ ID NO:334).370. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUCGCAGCAUUCUUGGGUGGGA (SEQ ID NO:335).371 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCRCGGG (SEQ ID NO:336).372. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCACGGG (SEQ ID NO:337).373. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCGCGGG (SEQ ID NO:338).374. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCACGGG (SEQ ID NO:339).375. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCACGGG (SEQ ID NQ:340).376. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCACGGG (SEQ ID NO:341).377. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUCUUGGGUGGGAGCAGCCACGGG (SEQ ID NO:342).378. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCGCGGG (SEQ ID NO:343).379. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCGCGGG (SEQ ID NO:344).380. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCGCGGG (SEQ ID NO:345).381 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUCUUGGGUGGGAGCAGCCGCGGG (SEQ ID NO:346).382. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCRCGGGUCAGCCACA (SEQ ID NO:347).383. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCACGGGUCAGCCACA (SEQ ID NO:348).384. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCGCGGGUCAGCCACA (SEQ ID NO:349).385. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGUGGCUGACCCGYGGCUGCUCC (SEQ ID NQ:350).386. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGUGGCUGACCCGUGGCUGCUCC (SEQ ID NO:351).387. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGUGGCUGACCCGCGGCUGCUCC (SEQ ID NO:352).388. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCRCGGGUCA (SEQ ID NO:353).389. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCACGGGUCA (SEQ ID NO:354).390. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCGCGGGUCA (SEQ ID NO:355).391 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GUGGGAGCAGCCACGGGUCA (SEQ ID NO:356).392. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGUGGGAGCAGCCACGGGUCA (SEQ ID NO:357).393. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGGUGGGAGCAGCCACGGGUCA (SEQ ID NO:358).394. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCACGGGUCA (SEQ ID NO:359).395. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GUGGGAGCAGCCGCGGGUCA (SEQ ID NQ:360).396. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGUGGGAGCAGCCGCGGGUCA (SEQ ID NO:361).397. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGGUGGGAGCAGCCGCGGGUCA (SEQ ID NO:362).398. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCGCGGGUCA (SEQ ID NO:363).399. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGYGGCUGCUC (SEQ ID NO:364).400. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGUGGCUGCUC (SEQ ID NO:365).401 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGCGGCUGCUC (SEQ ID NO:366).402. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCRCGGGU (SEQ ID NO:367).403. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCACGGGU (SEQ ID NO:368).404. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCGCGGGU (SEQ ID NO:369).405. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGYGGCU (SEQ ID NQ:370).406. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGUGGCU (SEQ ID NO:371).407. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GCCCUUGUGGCUGACCCGCGGCU (SEQ ID NO:372).408. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUCACAGCAUUCUUGGGUGGGA (SEQ ID NO:373).409. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCRCGGG (SEQ ID NO:374).410. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCACGGG (SEQ ID NO:375).411 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UCUUGGGUGGGAGCAGCCGCGGG (SEQ ID NO:376).412. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCRCGGGUCAGCCACA (SEQ ID NO:377).413. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCACGGGUCAGCCACA (SEQ ID NO:378).414. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGAGCAGCCGCGGGUCAGCCACA (SEQ ID NO:379).415. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCRCGGGUC (SEQ ID NQ:380).416. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCACGGGUC (SEQ ID NO:381).417. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGGGUGGGAGCAGCCGCGGGUC (SEQ ID NO:382).418. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGUGGGAGCAGCCACGGGUC (SEQ ID NO:383).419. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGGUGGGAGCAGCCACGGGUC (SEQ ID NO:384).420. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCACGGGUC (SEQ ID NO:385).421 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCACGGGUC (SEQ ID NO:386).422. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGUGGGAGCAGCCGCGGGUC (SEQ ID NO:387).423. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GGGUGGGAGCAGCCGCGGGUC (SEQ ID NO:388).424. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UGGGUGGGAGCAGCCGCGGGUC (SEQ ID NO:389).425. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCGCGGGUC (SEQ ID NO:390).426. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGYGGCUGCUC (SEQ ID NO:391).427. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGUGGCUGCUC (SEQ ID NO:392).428. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is UUGUGGCUGACCCGCGGCUGCUC (SEQ ID NO:393).429. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCRCGGGU (SEQ ID NO:394).430. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCACGGGU (SEQ ID NO:395).431 . The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is CUUGGGUGGGAGCAGCCGCGGGU (SEQ ID NO:396).432. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GAGCAGCCRCGGGUCAGCCACAA (SEQ ID NO:397).433. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GAGCAGCCACGGGUCAGCCACAA (SEQ ID NO:398).434. The gRNA of any one of embodiments 313 and 318 to 348 when depending from embodiment 313, wherein the reference sequence is GAGCAGCCGCGGGUCAGCCACAA (SEQ ID NO:399).435. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AUGCUCCCGGGCUCCUGCACACC (SEQ ID NQ:400).436. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CUCCAUGCUCCCGGGCUCCUGCA (SEQ ID NQ:401).437. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AGGAGAAGGGAGAAGGCCUCUCA (SEQ ID NQ:402).438. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is GACAGGAGAAGGGAGAAGGCCUC (SEQ ID NO:403).439. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is GUUAUCCAAAGCCCUCAUAUAUU (SEQ ID NQ:404).440. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is GCCCGAGAUAGAUGCGGGCUUCC (SEQ ID NQ:405).441 . The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is ACAUGGCCCGAGAUAGAUGCGGG (SEQ ID NQ:406).442. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AUGACUGGAGAAUGGAAAAUCCA (SEQ ID NQ:407).443. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AGACCCAAUGACUGGAGAAUGGA (SEQ ID NQ:408).444. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is UGCUUCAGAGCGGCUGCUUGCGG (SEQ ID NQ:409).445. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is UGUUGACUGAAUAUAUGAGGGCU (SEQ ID NQ:410).446. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is UAUCCAAAGCCCUCAUAUAUUCA (SEQ ID NO:411).447. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CAAGGCAGUGUUCAGUGCCAGCC (SEQ ID NO:412).448. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AAAUUAGACAAGCGCAUAUUGCU (SEQ ID NO:413).449. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AGCUCAGUUUUCUUGCUGUGAAA (SEQ ID NO:414).450. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CUGAACACUGCCUUGAUCUUAUU (SEQ ID NO:415).451 . The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is AGCAAUAUGCGCUUGUCUAAUUU (SEQ ID NO:416).452. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CUCCAAGGGAAACAGAGGCUUGG (SEQ ID NO:417).453. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CCAGGCUGGGCACUGAGGGAGAG (SEQ ID NO:418).454. The gRNA of any one of embodiments 314 and 318 to 348 when depending from embodiment 314, wherein the reference sequence is CCAGUCAUUGGGUCUUCCCUGUG (SEQ ID NO:419).455. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is GAGAGAGUAGCGCGAGCACAGCU (SEQ ID NQ:420).456. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CGCUCCGUGGCCUUAGCUGUGCU (SEQ ID NO:421).457. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UUGCUAUGUGUCUGGGUUUCAUC (SEQ ID NO:422).458. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CAAUUCUCUCUCCAUUCUUCAGU (SEQ ID NO:423).459. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCAGACUUGUCUUUCAGCAAGGA (SEQ ID NO:424).460. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCUGGCCUGGAGGCUAUCCAGCG (SEQ ID NO:425).461 . The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCACGUCAUCCAGCAGAGAAUGG (SEQ ID NO:426).462. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CUUUCCAUUCUCUGCUGGAUGAC (SEQ ID NO:427).463. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is GUCGGAUGGAUGAAACCCAGACA (SEQ ID NO:428).464. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CUUUUUCAAUUCUCUCUCCAUUC (SEQ ID NO:429).465. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is AAGUGGAGCAUUCAGACUUGUCU (SEQ ID NQ:430).466. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is AGAGUAGCGCGAGCACAGCUAAG (SEQ ID NO:431).467. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCGGGCCGAGAUGUCUCGCUCCG (SEQ ID NO:432).468. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CAGAAAGAGAGAGUAGCGCGAGC (SEQ ID NO:433).469. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is GAGACUCACGCUGGAUAGCCUCC (SEQ ID NO:434).470. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CACGUCAUCCAGCAGAGAAUGGA (SEQ ID NO:435).471 . The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CAGAAAGAGAGAGUAGCGCGAGC (SEQ ID NO:436).472. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CAGGCCAGAAAGAGAGAGUAGCG (SEQ ID NO:437).473. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is GAGACUCACGCUGGAUAGCCUCC (SEQ ID NO:438).474. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCGGAUGGAUGAAACCCAGACAC (SEQ ID NO:439).475. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is UCUCUCCAUUCUUCAGUAAGUCA (SEQ ID NQ:440).476. The gRNA of any one of embodiments 315 and 318 to 348 when depending from embodiment 315, wherein the reference sequence is CAAUGUCGGAUGGAUGAAACCCA (SEQ ID NO:441).477. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is ACCAGCUGAGAGACUCUAAAUCC (SEQ ID NO:442).478. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UGCCUAUUCACCGAUUUUGAUUC (SEQ ID NO:443).479. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UUGAUUCUCAAACAAAUGUGUCA (SEQ ID NO:444).480. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UAGACAUGAGGUCUAUGGACUUC (SEQ ID NO:445).481 . The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is AUGCUGUUGUUGAAGGCGUUUGC (SEQ ID NO:446).482. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is AGACAUGAGGUCUAUGGACUUCA (SEQ ID NO:447).483. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is CAAGAGCAACAGUGCUGUGGCCU (SEQ ID NO:448).484. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is AUGCAAAGUCAGAUUUGUUGCUC (SEQ ID NO:449).485. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is AUCUGACUUUGCAUGUGCAAACG (SEQ ID NQ:450).486. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UGACUUUGCAUGUGCAAACGCCU (SEQ ID NO:451).487. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is CUCAGCUGGUACACGGCAGGGUC (SEQ ID NO:452).488. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is GAGUCUCUCAGCUGGUACACGGC (SEQ ID NO:453).489. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UCACUGGAUUUAGAGUCUCUCAG (SEQ ID NO:454).490. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is ACACAUUUGUUUGAGAAUCAAAA (SEQ ID NO:455).491 . The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is ACAGACAAAACUGUGCUAGACAU (SEQ ID NO:456).492. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is ACCCUGCCGUGUACCAGCUGAGA (SEQ ID NO:457).493. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is GAUUUUGAUUCUCAAACAAAUGU (SEQ ID NO:458).494. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is UAAGGAUUCUGAUGUGUAUAUCA (SEQ ID NO:459).495. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is AGACAUGAGGUCUAUGGACUUCA (SEQ ID NQ:460).496. The gRNA of any one of embodiments 316 and 318 to 348 when depending from embodiment 316, wherein the reference sequence is GAGCAACAGUGCUGUGGCCUGGA (SEQ ID NO:461).497. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GGUGGGGCUGCUCCAGGCAUGCA (SEQ ID NO:462).498. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCCAGCCCAGUUGUAGCACCGCC (SEQ ID NO:463).499. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCGCCCUGGCCAGUCGUCUGGGC (SEQ ID NO:464).500. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCCCUGGCCAGUCGUCUGGGCGGU (SEQ ID NO:465).501 . The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCCAGGAUGGUUCUUAGGUAGGU (SEQ ID NO:466).502. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is AGGCGCCCUGGCCAGUCGUCUGG (SEQ ID NO:467).503. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCUACAACUGGGCUGGCGGCCAGG (SEQ ID NO:468).504. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is UGGCGGCCAGGAUGGUUCUUAGG (SEQ ID NO:469).505. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is UGCAGAUCCCACAGGCGCCCUGG (SEQ ID NQ:470).506. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCCCUGGCCAGUCGUCUGGGCGG (SEQ ID NO:471).507. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is AGGCAUGCAGAUCCCACAGGCGC (SEQ ID NO:472).508. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is CCAGUUGUAGCACCGCCCAGACG (SEQ ID NO:473).509. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GGCGCCCUGGCCAGUCGUCUGGG (SEQ ID NO:474).510. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GUGGGGCUGCUCCAGGCAUGCAG (SEQ ID NO:475).511 . The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GGCCGCCAGCCCAGUUGUAGCAC (SEQ ID NO:476).512. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is ACAACUGGGCUGGCGGCCAGGAU (SEQ ID NO:477).513. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GCCCUGGCCAGUCGUCUGGGCGGU (SEQ ID NO:478).514. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is GGCAUGCAGAUCCCACAGGCGCC (SEQ ID NO:479).515. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein the reference sequence is CAGUUGUAGCACCGCCCAGACGA (SEQ ID NQ:480).516. The gRNA of any one of embodiments 317 and 318 to 348 when depending from embodiment 317, wherein t...

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-l domain of a reference protein sequence;(b) the amino acid sequence of a RuvC-ll domain of a reference protein sequence;(c) the amino acid sequence of a RuvC-lll 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 REC domain of a reference protein sequence;(f) the amino acid sequence of a HNH domain of a reference protein sequence;(g) the amino acid sequence of a WED domain of a reference protein sequence;(h) the amino acid sequence of a PID domain of a reference protein sequence; or(i) 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 NQ: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 NQ:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:67, or SEQ ID NO:68.

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 identical to the full length of the reference protein sequence.

4. The Type II Cas protein of any one of claims 1 to 3, which is a fusion protein.

5. The Type II Cas protein of claim 4, 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.

6. The Type II Cas protein of claim 5, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:145), PKKKRKV (SEQ ID NO:146), PKKKRRV (SEQ ID NO:147), KRPAATKKAGQAKKKK (SEQ ID NO:148), YGRKKRRQRRR (SEQ ID NO:149), RKKRRQRRR (SEQ ID NQ:150), PAAKRVKLD (SEQ ID NO:151), RQRRNELKRSP (SEQ ID NO:152), VSRKRPRP (SEQ ID NO:153), PPKKARED (SEQ ID NO:154), PQPKKKPL (SEQ ID NO:155), SALIKKKKKMAP (SEQ ID NO:156), PKQKKRK (SEQ ID NO:157), RKLKKKIKKL (SEQ ID NO:158), REKKKFLKRR (SEQ ID NO:159), KRKGDEVDGVDEVAKKKSKK (SEQ ID NQ:160), RKCLQAGMNLEARKTKK (SEQ ID NO:161), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:162), or RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:163).

7. The Type II Cas protein of claim 5 or claim 6, wherein the amino acid sequence of each nuclear localization signal is the same.

8. The Type II Cas protein of any one of claims 4 to 7, 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.

9. The Type II Cas protein of any one of claims 4 to 8, 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:166, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e; (b) a fusion partner which is a cytodine deaminase; or (c) a fusion partner which is a reverse transcriptase.

10. The Type II Cas protein of any one of claims 4 to 9, which comprises a tag, e.g., a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:164).11 . The Type II Cas protein of any one of claims 1 to 10, 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 NOT, 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 NQ: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 NQ: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 NQ:50, optionally wherein the amino acid sequence of theType 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; (k) the reference protein sequence is SEQ ID NO:61 or SEQ ID NO:62, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:61 , SEQ ID NO:62, or SEQ ID NO:63; (I) the reference protein sequence is SEQ ID NO:67 or SEQ ID NO:68, optionally wherein the amino acid sequence of the Type II Cas protein comprises the amino acid sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69.

12. A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of claims 1 to 11 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 substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4).

13. A Type II Cas protein whose amino acid sequence is identical to a Type II Cas protein of any one of claims 1 to 11 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 substiution (e.g., alanine substitution) at a position corresponding to position D10 of SaCas9, N580 of SaCas9, or H559 of CjCas9 (e.g., as shown in Table 4), or a combination thereof.

14. A guide RNA (gRNA) molecule for editing a human RHO 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:314-399.

15. A guide RNA (gRNA) molecule for editing a human RHO 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 NQs:400-419.

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 NQs:420-441.

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:442-461.

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:462-482.

19. The gRNA of any one of claims 14 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 14 to 19, wherein the spacer comprises the reference sequence.21 . The gRNA of any one of claims 14 to 20, which is a single guide RNA (sgRNA).

22. A gRNA comprising a spacer and a sgRNA scaffold, wherein:(a) the spacer is positioned 5’ to the sgRNA scaffold; and(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:97-120.

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 at its 3’ end.

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. The gRNA of any one of claims 22 to 25, wherein the nucleotide sequence of the spacer comprises a sequence selected from SEQ ID NOs:314-482.

27. A combination of gRNAs comprising (a) a first gRNA comprising a spacer whose sequence comprises the sequence of any one of SEQ ID NOs.326-348 and a second gRNA comprising a spacer whose sequence comprises the sequence of any one of SEQ ID NQs:400-409, optionally wherein the spacer of the first and / or second gRNA is positioned 5’ to a sgRNA scaffold whose sequence comprises the sequence of SEQ ID NQ:101 , 102, 125, or 126 or (b) a first gRNA comprising a spacer whose sequence comprises the sequence of any one of SEQ ID NOs.380-390 and a second gRNA comprising a spacer whose sequence comprises the sequence of any one of SEQ ID NOs:410-419, optionally wherein the spacer of the first and / or second gRNA is positioned 5’ to a sgRNA scaffold whose sequence comprises the sequence of SEQ ID NO:117, 118, 141 , or 142.

28. A system comprising the Type II Cas protein of any one of claims 1 to 13 and a guide RNA (gRNA) comprising a spacer sequence, optionally wherein the gRNA is a gRNA according to any one of claims 14 to 26.

29. A nucleic acid encoding the Type II Cas protein of any one of claims 1 to 13, 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.

30. The nucleic acid of claim 29, wherein the nucleotide sequence encoding the Type II Cas protein is codon optimized for expression in human cells.31 . The nucleic acid of any one of claims 37 to 39, which is a plasmid or a viral genome, optionally an adeno-associated virus (AAV) genome, e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrh 10 genome.

32. The nucleic acid of any one of claims 29 to 31 , further encoding a gRNA33. A nucleic acid encoding the gRNA of any one of claims 14 to 26 or the combination of gRNAs of claim 27.

34. A nucleic acid encoding the Type II Cas protein and gRNA of the system of claim 28.

35. A plurality of nucleic acids comprising separate nucleic acids encoding the Type II Cas protein and gRNA of the system of claim 28.

36. A particle comprising a Type II Cas protein according to any one of claims 1 to 13, a gRNA according to any one of claims 14 to 26, a combination of gRNAs according to claim 27, a system according to claim 28, a nucleic acid according to any one of claims 29 to 34, or a plurality of nucleic acids according to claim 35.

37. A pharmaceutical composition comprising a Type II Cas protein according to any one of claims 1 to 13, a gRNA according to any one of claims 14 to 26, a combination of gRNAs according to claim 27, a system according to claim 28, a nucleic acid according to any one of claims 29 to 34, a plurality of nucleic acids according to claim 35, or a particle according to claim 36 and at least one pharmaceutically acceptable excipient.

38. A Type II Cas protein according to any one of claims 1 to 13, a gRNA according to any one of claims 14 to 26, a combination of gRNAs according to claim 27, a system according to claim 28, a nucleic acid according to any one of claims 29 to 34, a plurality of nucleic acids according to claim 35, a particle according to claim 36, or a pharmaceutical composition according to claim 37 for use in a method of editing a human genomic sequence.

39. The Type II Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to claim 38, wherein the human genomic sequence is a RHO genomic sequence, optionally wherein the RHO genomic sequence has a pathogenic mutation.

40. The Type II Cas protein, gRNA, combination of gRNAs, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to claim 38, wherein the human genomic sequence is a TRAC, B2M, PD1, or LAG3 genomic sequence, optionally wherein the human genomic sequence is in a T cell.41 . An ex vivo human cell comprising a Type II Cas protein according to any one of claims 1 to 13, a gRNA according to any one of claims 14 to 26, a combination of gRNAs according to claim 27, a system according to claim 28, a nucleic acid according to any one of claims 29 to 34, a plurality of nucleic acids according to claim 35, or a particle according to claim 36.

Images