Cas9 orthologue nuclease and uses thereof

Abstract

Compositions and methods are provided for novel Cas9 orthologues, including, but not limited to, systems comprising novel Cas9 nucleases and at least one guide RNA molecule. The present disclosure also describes methods for cleaving or editing a target polynucleotide and methods for genome modification of a target sequence.

Description

CAS9 ORTHOLOGUE NUCLEASE AND USES THEREOF 1. CROSS-REFERENCE TO RELATED

[0001] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 515,022, filed July 21, 2023, the disclosure of which is incorporated herein by reference in its entirety. 2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on July 18, 2024, is named 56083WO_CRF_sequencelisting.xml, and is 2,313,354 bytes in size. 3. BACKGROUND

[0003] The ability to make precise and deliberate changes at targeted genomic locations and / or modify (e.g., cleaving or editing) specific endogenous chromosomal sequences, thus altering the genomic complement of living cells has been a long-standing goal in biomedical research and development. Recently, RNA-mediated adaptive immune systems in bacteria and archaea that rely on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genomic loci and CRISPR-associated (Cas) proteins functioning together to provide protection from invading viruses and plasmids has been repositioned from prokaryotic cells to mammalian cells given a major acceleration (boost) to genome editing applications in the clinic (Doudna and Charpentier, Science.28;346(6213):11223096 (2014)).

[0004] In CRISPR-Cas systems, Cas9 functions as an RNA-guided endonuclease that uses a dual-guide RNA consisting of crRNA and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate double-stranded DNA breaks (DSBs) (Barrangou and May, Expert Opin Biol Ther 15, 311-314 (2015); Jinek et al., Science 337, 816-821(2012)). Cas9, a family of nucleases enables efficient, customizable genome editing in a wide variety of organisms and cell types. The Cas9 system derived from Streptococcus pyogenes (Barrangou and Doudna. Nat Biotechnol. 2016;34(9):933-941 (2016)), which leaves a blunt-end overhang and effects gene editing via the recognition of a Protospacer Adjacent Motif (PAM) sequence of “NGG” on the target polynucleotide. The range of target sequences that nucleic acid-guided nucleases (CRISPR-Cas9 complex) can recognize, however, is constrained by the need for a specific PAM to be located near the desired target sequence. (Doudna. Nature 578: 229–236 (2020)).

[0005] PAMs are short nucleotide sequences recognized by the CRISPR-Cas9 complex where this complex directs editing of the target sequence. The precise PAM sequence and PAM lengthrequirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-8 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5’ or 3’ to the target sequence. Yet, this technology is still limited by major constraints, mainly related to the reduced number of CRISPR-Cas systems active in mammalian cells which hardly respond to the complexity of gene therapy applications. PAM sequences, necessary for nuclease recognition and activity, are key in this context, as they dictate the compatibility of each Cas tool towards specific genomic target sites. Molecular engineering significantly improved Cas9 properties for genome editing including relaxation of PAM requirements, but this approach impacts the activity of the nucleases and still does not respond to all PAM sequences needed to tackle disease causing mutations.

[0006] Thus, there is a need for new Cas genetic tools to expand the scope and types of edits needed for therapeutic applications and related methods. 4. SUMMARY

[0007] The present disclosure provides the identification of a vast number of unreported CRISPR-Cas enzymes (including e.g. Cas endonucleases or Cas polypeptides) and their respective Protospacer Adjacent Motif (PAM) requirements, site-directed modifying polypeptides, and ribonucleoproteins comprising the polypeptides. The site-directed modifying polypeptides are useful in a variety of methods for target nucleic acid modification. In certain embodiments, the site-directed modifying polypeptides are modified for passive entry into target cells.

[0008] The identification of the PAM sequences of Cas9 nucleases is important for their exploitation as genome editing tools. A massively expanded dataset of metagenome and virome assemblies was interrogated for accurate and comprehensive PAM predictions, in order to identify novel Cas9s selected for their PAM requirements as described in Ciciani et al., Nat Commun 13, 6474 (2022), which is incorporated herein by reference in its entirety. As a test, a Cas9 was selectively isolated which uses a PAM sequence corresponding to a disease-causing mutation, P23H in the RHO gene (see Ciciani et al., 2022). This PAM prediction pipeline is useful for generating a Cas9 nuclease repertoire responding to any PAM requirement leading towards a natural PAM-free genome editing toolbox.

[0009] In one aspect, described are polypeptides, the polypeptide comprising: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas orthologue nuclease having an amino acid sequence with at least 80% identity to any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0010] In one aspect, described are polypeptides, the polypeptide comprising: a CRISPR-Cas orthologue nuclease having an amino acid sequence with at least 80% identity to any nuclease capable of complexing with a guide RNA as shown in Table 34. In some embodiments, wherein the nuclease comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO.: 1, SEQ ID NO.: 28, SEQ ID NO.: 55, SEQ ID NO.: 106, SEQ ID NO.: 129, SEQ ID NO.: 163, SEQ ID NO.: 217, SEQ ID NO.: 242, SEQ ID NO.: 254, SEQ ID NO.: 270, SEQ ID NO.: 297, SEQ ID NO.: 325, SEQ ID NO.: 341, SEQ ID NO.: 373, SEQ ID NO.: 391, SEQ ID NO.: 409, SEQ ID NO.: 426, SEQ ID NO.: 462, SEQ ID NO.: 481, SEQ ID NO.: 503, SEQ ID NO.: 523, SEQ ID NO.: 552, SEQ ID NO.: 593, SEQ ID NO.: 646, SEQ ID NO.: 686, SEQ ID NO.: 735, SEQ ID NO.: 833, SEQ ID NO.: 847, SEQ ID NO.: 886, SEQ ID NO.: 940, and SEQ ID NO: 1826.

[0011] In accordance with any of the embodiments, the polypeptide is a fusion protein. In some embodiments, the polypeptide comprises a nuclear localization signal peptide, a tag peptide, or both fused to the CRISPR-Cas orthologue nuclease. In some embodiments, the polypeptide comprises two or more nuclear localization signals fused to the CRISPR-Cas orthologue nuclease. In some embodiments, the nuclear localization signal comprises an N-terminal nuclear localization signal, a C-terminal nuclear localization signal, or both.

[0012] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 1800-1822.

[0013] In accordance with any of the embodiments, the tag peptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1823 or SEQ ID NO: 1824.

[0014] In accordance with any of the embodiments, the CRISPR-Cas orthologue nuclease has at least 90% identity with any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0015] In accordance with any of the embodiments, the CRISPR-Cas orthologue nuclease has at least 95% identity with any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0016] In accordance with any of the embodiments, the CRISPR-Cas orthologue nuclease is any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387. In some embodiments, the CRISPR-Cas orthologue nuclease is selected from any one of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0017] In some embodiments, the CRISPR-Cas orthologue nuclease is SEQ ID NO: 76 or SEQ ID NO: 1387.

[0018] In some embodiments, the CRISPR-Cas orthologue nuclease is SEQ ID NO: 362 or SEQ ID NO: 1384.

[0019] In some embodiments, the CRISPR-Cas orthologue nuclease is SEQ ID NO: 444 or SEQ ID NO: 1385.

[0020] In some embodiments, the CRISPR-Cas orthologue nuclease is SEQ ID NO: 721 or SEQ ID NO: 1383.

[0021] In some embodiments, the CRISPR-Cas orthologue nuclease is SEQ ID NO: 768 or SEQ ID NO: 1386.

[0022] In some embodiments, the CRISPR-Cas orthologue nuclease is capable of complexing with a guide RNA.

[0023] In some embodiments, the guide RNA comprises: a crRNA comprising a spacer and a crRNA scaffold wherein the spacer is positioned 5’ to the crRNA scaffold; and a tracrRNA.

[0024] In some embodiments, the guide RNA is a single guide RNA (sgRNA) comprising a spacer and a sgRNA scaffold, wherein the spacer is positioned 5’ to the sgRNA scaffold.

[0025] In one aspect, described are guide RNAs capable of complexing with the polypeptide in accordance with any of the embodiments.

[0026] In some embodiments, the guide RNA comprises a spacer.

[0027] In some embodiments, the spacer comprises a nucleic acid sequence that is partially or fully complementary to a target mammalian genomic sequence.

[0028] In some embodiments, the target mammalian genomic sequence is upstream of a Protospacer Adjacent Motif (PAM) sequence in the non-target strand recognized by the polypeptide.

[0029] In some embodiments, the PAM polynucleotide comprises a nucleic acid sequence as set forth in Table 32.

[0030] In some embodiments, the guide RNA comprises separate crRNA and tracrRNA molecules.

[0031] In some embodiments, the crRNA comprises, in 5’ to 3’ order, the spacer and a crRNA scaffold.

[0032] In various embodiments, the guide RNA comprises, in 5’ to 3’ order, the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1362, and the tracrRNAmolecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1367; the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1363, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1368; the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1364, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1369; the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1365, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1370; or the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1366, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1371.

[0033] In some embodiments, the guide RNA comprises , in 5’ to 3’ order, the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1362, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1367; the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1363, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1368;the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1364, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1369; the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1365, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1370; or the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1366, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1371.

[0034] In various embodiments, the crRNA molecule has at least 90% identity with SEQ ID NO: 1362, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1367; the crRNA molecule has at least 90% identity with SEQ ID NO: 1363, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1368; the crRNA molecule has at least 90% identity with SEQ ID NO: 1364, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1369; the crRNA molecule has at least 90% identity with SEQ ID NO: 1365, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1370; or the crRNA molecule has at least 90% identity with SEQ ID NO: 1366, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1371.

[0035] In some embodiments, the crRNA molecule has at least 95% identity with SEQ ID NO: 1362, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1367; the crRNA molecule has at least 95% identity with SEQ ID NO: 1363, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1368; the crRNA molecule has at least 95% identity with SEQ ID NO: 1364, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1369; the crRNA molecule has at least 95% identity with SEQ ID NO: 1365, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1370; or the crRNA molecule has at least95% identity with SEQ ID NO: 1366, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1371.

[0036] In various embodiments, the guide RNA comprises a crRNA molecule comprising at least 20, at least 30, at least 40, at least 50, or at least 60 consecutive nucleotides of any one of SEQ ID NOs: 1388-1418 as shown in Table 34, and the crRNA is capable of complexing with a tracrRNA molecule comprising at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 consecutive nucleotides of any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

[0037] In various embodiments, the guide RNA comprises a crRNA molecule comprising a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs: 1388-1418 as shown in Table 34, and the crRNA is capable of complexing with a tracrRNA molecule comprising a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any one of any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

[0038] In various embodiments, the guide RNA is a sgRNA comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs: 1450- 1483 and SEQ ID NO: 1825.

[0039] In some embodiments, the guide RNA is a sgRNA.

[0040] In some embodiments, the sgRNA comprises, in 5’ to 3’ order, a spacer and a sgRNA scaffold.

[0041] In some embodiments, the sgRNA comprises at least one modified nucleotide or modified internucleoside linkage.

[0042] In some embodiments, the sgRNA comprises at least one modified nucleotide selected from 2'-fluoro, 2'-amino or 2'-O-methyl modification on the ribose of pyrimidines, abasic residues, and an inverted base at the 3' end of the RNA.

[0043] In some embodiments, the sgRNA comprises at least one modified internucleoside linkage selected from phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl inter-sugar linkages, and short chain heteroatomic or heterocyclic inter-sugar linkages.

[0044] In various embodiments, the sgRNA comprises at least 50 consecutive nucleotides of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0045] In some embodiments, the sgRNA comprises at least 80 consecutive nucleotides of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0046] In various embodiments, the sgRNA has at least 90% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0047] In some embodiments, the sgRNA has at least 95% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0048] In various embodiments, the guide RNA is a sgRNA comprising, in 5’ to 3’ order, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 consecutive nucleotides of any one of SEQ ID NOs: 1450-1483 and SEQ ID NO: 1825 as shown in Table 34.

[0049] In one aspect, described are guide RNAs, the guide RNA is capable of complexing with a polypeptide described here.

[0050] In one aspect, described are systems, the system comprising: a polypeptide comprising the CRISPR-Cas9 orthologue nuclease in accordance with any one of the embodiments disclosed herein, and a guide RNA in accordance with any one of the embodiments disclosed herein, wherein the CRISPR-Cas9 orthologue nuclease is capable of complexing with the guide RNA.

[0051] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 76 or SEQ ID NO: 1387, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1366, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1371.

[0052] In some embodiments, the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1366, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1371.

[0053] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 76 or SEQ ID NO: 1387, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1377.

[0054] In some embodiments, the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1377.

[0055] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 362 or SEQ ID NO: 1384, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO:1363, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1368.

[0056] In some embodiments, the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1363; and the tracrRNA polynucleotide has at least 90% identity to SEQ ID NO: 1368.

[0057] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 362 or SEQ ID NO: 1384, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1373.

[0058] In some embodiments, the sgRNA comprises a polynucleotide having at least 90% identity to SEQ ID NO: 1373.

[0059] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 444 or SEQ ID NO: 1385, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1364, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1369.

[0060] In some embodiments, the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1364; and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1369.

[0061] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 444 or SEQ ID NO: 1385, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1374.

[0062] In some embodiments, the sgRNA comprises a polynucleotide having at least 90% identity to SEQ ID NO: 1374.

[0063] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 721 or SEQ ID NO: 1383, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1362, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1367.

[0064] In some embodiments, the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1362, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1367.

[0065] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 721 or SEQ ID NO: 1383, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1372.

[0066] In some embodiments, the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1372.

[0067] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 768 or SEQ ID NO: 1386, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1365, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1370.

[0068] In some embodiments, the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1365, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1370.

[0069] In some embodiments, the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 765 or SEQ ID NO: 1386, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1375 or SEQ ID NO: 1376.

[0070] In some embodiments, the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1375 or SEQ ID NO: 1376.

[0071] In one aspect, described are nucleic acids, the nucleic acid comprising a polynucleotide encoding the polypeptide as disclosed herein, or the polypeptide in the system in accordance with any of the embodiments as disclosed herein.

[0072] In some embodiments, the polynucleotide has at least 80% identity with any one of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, and SEQ ID NO: 1382.

[0073] In one aspect, described are nucleic acids, the nucleic acid comprising a polynucleotide encoding the guide RNA in accordance with any of the embodiments, wherein the guide RNA comprises: a polynucleotide encoding the crRNA comprising at least 90% identity with SEQ ID NO: 1362, and a polynucleotide encoding the tracrRNA comprising at least 90% identity with SEQ ID NO: 1367; a polynucleotide encoding the crRNA comprising at least 90% identity with SEQ ID NO: 1363, and a polynucleotide encoding the tracrRNA comprising at least 90% identity with SEQ ID NO: 1368; a polynucleotide encoding the crRNA comprising at least 90% identity with SEQ ID NO: 1364, and a polynucleotide encoding the tracrRNA comprising at least 90% identity with SEQ ID NO: 1369; a polynucleotide encoding the crRNA comprising at least 90% identity with SEQ ID NO: 1365, and a polynucleotide encoding the tracrRNA comprising at least 90% identity with SEQ ID NO: 1370; or a polynucleotide encoding the crRNA comprising at least 90% identity with SEQ ID NO: 1366, and a polynucleotide encoding the tracrRNA comprising at least 90% identity with SEQ ID NO: 1371.

[0074] In one aspect, described are nucleic acids, the nucleic acid comprising a polynucleotide encoding the guide RNA in accordance with any of the embodiments, wherein the guide RNA isa sgRNA comprising a polynucleotide having at least 90% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0075] In various embodiments, the nucleic acid comprising the polynucleotide encoding the polypeptide in accordance with any of the embodiments, or the nucleic acids encoding the crRNA in accordance with any of the embodiments; or the nucleic acids encoding the tracrRNA in accordance with any of the embodiments; or the nucleic acids encoding the sgRNA in accordance with any of the embodiments, wherein the nucleic acid is obtained by in vitro transcription.

[0076] In various embodiments, the nucleic acid comprising the polynucleotide encoding the polypeptide in accordance with any of the embodiments, or the nucleic acids encoding the crRNA in accordance with any of the embodiments; or the nucleic acids encoding the tracrRNA in accordance with any of the embodiments; or the nucleic acids encoding the sgRNA in accordance with any of the embodiments, wherein the nucleic acid is operably linked to a promoter.

[0077] In various embodiments, the nucleic acid comprising the polynucleotide encoding the polypeptide in accordance with any of the embodiments, or the nucleic acids encoding the crRNA in accordance with any of the embodiments; or the nucleic acids encoding the tracrRNA in accordance with any of the embodiments; or the nucleic acids encoding the sgRNA in accordance with any of the embodiments, wherein the nucleic acid is synthetic.

[0078] In various embodiments, the nucleic acid comprising the polynucleotide encoding the polypeptide in accordance with any of the embodiments, or the nucleic acids encoding the crRNA in accordance with any of the embodiments; or the nucleic acids encoding the tracrRNA in accordance with any of the embodiments; or the nucleic acids encoding the sgRNA in accordance with any of the embodiments, wherein the nucleic acid encoding the polypeptide is human codon optimized, and the polynucleotide has a sequence of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, or SEQ ID NO: 1382. In some embodiments, the human codon optimized nucleic acid encodes a Cas orthologue polypeptide having a sequence of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, or SEQ ID NO: 1387 as shown in Table 33.

[0079] In one aspect, described are expression vectors, the expression vector comprising the nucleic acids encoding the polypeptide in accordance with any of the embodiments.

[0080] In one aspect, described are expression vectors, the expression vector comprising the nucleic acids encoding the crRNA in accordance with any of the embodiments.

[0081] In one aspect, described are expression vectors, the expression vector comprising the nucleic acids encoding the tracrRNA in accordance with any of the embodiments.

[0082] In one aspect, described are expression vectors, the expression vector comprising the nucleic acids encoding the sgRNA in accordance with any of the embodiments.

[0083] In one aspect, described are cells, the cell comprising the system in accordance with any of the embodiments.

[0084] In some embodiments, the cell is a prokaryotic cell or eukaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0085] In one aspect, described are pharmaceutical compositions, the pharmaceutical composition comprising: the polypeptide in accordance with any of the embodiments, or the polypeptide in the system in accordance with any of the embodiments; and / or the guide RNA in accordance with any of the embodiments, wherein the guide RNA comprises separate crRNA and tracrRNA, or wherein the guide RNA comprises a sgRNA; or the system in accordance with any of the embodiments; or the nucleic acid in accordance with any of the embodiments; or the expression vector in accordance with any of the embodiments; and a pharmaceutically acceptable excipient.

[0086] In one aspect, described are methods of site-specifically cleaving a DNA target in a cell, the method comprising contacting the cell with: the polypeptide in accordance with any of the embodiments or the polypeptide in the system in accordance with any of the embodiments, and the guide RNA in the system in accordance with any of the embodiments; or the system in accordance with any of the embodiments; or the nucleic acid in accordance with any of the embodiments; the expression vector in accordance with any of the embodiments; and / or the pharmaceutical composition in accordance with any of the embodiments.

[0087] In one aspect, described are systems, the system comprising the polypeptide in accordance with any one of the embodiments described herein, and their corresponding guide RNA as shown in Table 34. 5. BRIEF DESCRIPTION OF THE FIGURES

[0088] FIG.1 shows a schematic of the pipeline used to generate PAM predictions.

[0089] FIG.2 illustrates an exemplary PAM prediction method.

[0090] FIGs.3A-3E illustrate exemplary predicted PAM logos or motifs as described in Tables 1A (FIG.3A), Table 1B (FIG.3B), Table 1C (FIG.3C), Table 1D (FIG.3D), and Table 1E (FIG.3E).

[0091] FIGs.4A-4C illustrate exemplary predicted PAM logos or motifs as described in Tables 2A (FIG.4A), Table 2B (FIG.4B), and Table 2C (FIG.4C).

[0092] FIGs.5A-5B illustrate exemplary predicted PAM logos or motifs as described in Table 3A (FIG.5A), and Table 3B (FIG.5B).

[0093] FIGs.6A-6D illustrate exemplary predicted PAM logos or motifs as described in Tables 4A (FIG.6A), Table 4B (FIG.6B), Table 4C (FIG.6C), and Table 4D (FIG.6D).

[0094] FIGs.7A-7C illustrate exemplary predicted PAM logos or motifs as described in Tables 5A (FIG.7A), Table 5B (FIG.7B), and Table 5C (FIG.7C).

[0095] FIGs.8A-8B illustrate exemplary predicted PAM logos or motifs as described in Tables 6A (FIG.8A), and Table 6B (FIG.8B).

[0096] FIGs.9A-9D illustrate exemplary predicted PAM logos or motifs as described in Tables 7A (FIG.9A), Table 7B (FIG.9B), Table 7C (FIG.9C), and Table 7D (FIG.9D).

[0097] FIG.10 illustrates exemplary predicted PAM logos or motifs as described in Tables 8.

[0098] FIGs.11A-11B illustrate exemplary predicted PAM logos or motifs as described in Tables 9A (FIG.11A), and Table 9B (FIG.11B).

[0099] FIGs.12A-12D illustrate exemplary predicted PAM logos or motifs as described in Tables 10A (FIG.12A), Table 10B (FIG.12B), Table 10C (FIG.12C), and Table 10D (FIG. 12D).

[0100] FIG.13 illustrates exemplary predicted PAM logos or motifs as described in Tables 11.

[0101] FIGs.14A-14B illustrate exemplary predicted PAM logos or motifs as described in Tables 12A (FIG.14A), and Table 12B (FIG.14B).

[0102] FIG.15 illustrates exemplary predicted PAM logos or motifs as described in Tables 13.

[0103] FIG.16 illustrates exemplary predicted PAM logos or motifs as described in Tables 14.

[0104] FIG.17 illustrates exemplary predicted PAM logos or motifs as described in Table 15.

[0105] FIGs.18A-18B illustrate exemplary predicted PAM logos or motifs as described in Tables 16A (FIG.18A), and Table 16B (FIG.18B).

[0106] FIG.19 illustrates exemplary predicted PAM logos or motifs as described in Tables 17.

[0107] FIG.20 illustrates exemplary predicted PAM logos or motifs as described in Tables 18.

[0108] FIG.21 illustrates exemplary predicted PAM logos or motifs as described in Tables 19.

[0109] FIGs.22A-22B illustrate exemplary predicted PAM logos or motifs as described in Tables 20A (FIG.22A), and Table 20B (FIG.22B).

[0110] FIGs.23A-23B illustrate exemplary predicted PAM logos or motifs as described in Tables 21A (FIG.23A), and Table 21B (FIG.23B).

[0111] FIGs.24A-24B illustrate exemplary predicted PAM logos or motifs as described in Tables 22.

[0112] FIGs.25A-25C show predicted structures of sgRNAs designed for FRDB, DNDB, and GMIE CRISPR-Cas polypeptides. The capitalized four-letter identifier assigned to each CRISPR-Cas polypeptide is arbitrary and unique. Shown are schematic representation of the hairpin structure designed from crRNAs and tracrRNAs identified for FRDB CRISPR-Cas polypeptide protein (FIG.25A), DNDB CRISPR-Cas polypeptide protein (FIG.25B), and GMIE CRISPR-Cas polypeptide protein (FIG.25C). These sgRNAs have been trimmed by the insertion of a GAAA tetraloop at the level of the repeat:anti-repeat loop to reduce their size. FIGs.25A-25C disclose sgRNA scaffolds, corresponding to SEQ ID NOS 1372-1374, respectively, in order of appearance. The sgRNAs are capable of complexing with FRDB, DNDB, and GMIE CRISPR-Cas polypeptides, respectively.

[0113] FIGs.26A-26B show predicted structures of sgRNAs designed for ARHP and DUUZ CRISPR-Cas polypeptides. Shown are schematic representation of the hairpin structure designed from crRNAs and tracrRNAs identified for ARHP CRISPR-Cas polypeptide protein (FIG.26A), DUUZ CRISPR-Cas polypeptide protein (FIG.26B). These sgRNAs have been trimmed by the insertion of a GAAA tetraloop at the level of the repeat:anti-repeat loop to reduce their size. FIGs.26A-26B disclose sgRNA scaffolds, corresponding to SEQ ID NOS 1375 and 1377, respectively, in order of appearance. The sgRNAs are capable of complexing with ARHP and DUUZ, respectively.

[0114] FIGs.27A-27F illustrate experimentally determined PAM specificities of the FRDB, DNDB and GMIE CRISPR-Cas polypeptides. FIGs.27A, 27C and 27E show PAM sequence logos for FRDB CRISPR-Cas polypeptide (FIG.27A), DNDB CRISPR-Cas polypeptide (FIG. 27C), and GMIE CRISPR-Cas polypeptide (FIG.27E) as determined using an in vitro PAM discovery assay. FIGs.27B, 27D and 27F show PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for FRDB CRISPR-Cas polypeptide (FIG.27B, positions 5,6 and 7,8), DNDB CRISPR-Cas polypeptide (FIG.27D, positions 3,4 and 5,6), GMIE CRISPR-Cas polypeptide (FIG.27F, positions 5,6 and 7,8).

[0115] FIGs.28A-28D illustrate experimentally determined PAM specificities for ARHP CRISPR-Cas polypeptide protein and DUUZ CRISPR-Cas polypeptide protein. FIGs.28A and 28C show PAM sequence logos for ARHP CRISPR-Cas polypeptide (FIG.28A) and DUUZ CRISPR-Cas polypeptide (FIG.28C) as determined using the in vitro PAM discovery assay.FIGs.28B and 4D show PAM enrichment heatmaps calculated from the same in vitro PAM discovery assay showing the nucleotide preferences at different positions along the PAM for ARHP CRISPR-Cas polypeptide (FIG.28B, positions 2,3 and 4,5) and DUUZ CRISPR-Cas polypeptide (FIG.28D, positions 4,5 and 6,7).

[0116] FIG.29 shows activity of CRISPR-Cas polypeptide proteins against an EGFP reporter in mammalian cells. Data presented as mean ± SEM of n≥3 biologically independent experiments. 6. DETAILED DESCRIPTION

[0117] The present disclosure addresses the need for systems and methods for editing (e.g., cleaving, deleting, inserting, substituting) polynucleotides (e.g., DNA) using novel Cas9 orthologue nucleases. 6.1 Definitions

[0118] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0119] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. As used herein, the term “about” in reference to a value, encompasses from 90% to 110% of that value, unless the context clearly dictates otherwise.

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein canalso be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0121] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the content and context clearly dictates otherwise. Thus, for example, reference to “a CRISPR-Cas protein cluster” includes a combination of two CRISPR-Cas protein clusters, a combination of three CRISPR-Cas protein clusters, and the like.

[0122] 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.

[0123] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0124] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0125] The terms “Cas9 orthologue nuclease,” “Cas9 orthologous nuclease,” “Cas9 nuclease,” “Cas9 orthologue protein,” “Cas9 polypeptide,” “CRISPR-Cas9 polypeptide,” “CRISPR-Cas9 orthologue nuclease,” and “CRISPR-Cas enzymes” are used interchangeably herein, and they refer to a protein, polypeptide, or peptide that is capable of interacting with a nucleic acid, such as a guide nucleic acid, to form a complex (as used herein, “capable of complexing”; i.e. ribonucleotide-protein (RNP) complex) that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. The Cas9 orthologue nuclease pertains to wild-type Cas9 orthologue nuclease or Cas9 orthologuenuclease variants. The Cas9 orthologue nuclease can have nuclease activity or be catalytically inactive (e.g., as in a dCas).

[0126] The terms “hybridizable” or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine (G) of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, when a G / U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. As used herein, the terms “hybridize” or “complementary” refer to a first nucleotide sequence capable of forming non-covalently bind (hydrogen bond) with at least a portion of a specified second nucleotide sequence.

[0127] It is understood in the art that the sequence of polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). A polynucleotide can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0128] “Binding” as used herein (e.g., with reference to an RNA-binding domain of a polypeptide) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10−6M, less than 10−7M, less than 10−8M, less than 10−9M, less than 10−10M, less than 10−11M, less than 10−12M, less than 10−13M, less than 10−14M, or less than 10−15M. “Affinity” refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0129] By “binding domain” it is meant a protein domain that is able to bind non-covalently to another molecule. A binding domain can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and / or a protein molecule (a protein- binding protein). In the case of a protein domain-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of a different protein or proteins.

[0130] “CRISPR-Cas protein cluster” refers to a set of CRISPR-Cas protein sequences grouped (or clustered) according to sequence identity level (e.g., a user-defined sequence identity level). Algorithms for clustering protein sequences are known in the art and include, for example, UCLUST, e.g., version 11.0.667, (Edgar, 2010, Bioinformatics 26:2460–2461) and CD-HIT (Weizhong & Godzik, 2006, Bioinformatics 22(13):1658-1659). Some clustering algorithms, such as UCLUST, define a cluster by a sequence known as the centroid or representative sequence, and each sequence in the cluster has a percent identity to the centroid sequence above a user- defined percent identity threshold. For example, in some embodiments the user-defined percent identity threshold can be set at 90%. Exemplary methods for generating CRISPR-Cas protein clusters are described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety.

[0131] A CRISPR-Cas locus refers to a prokaryotic genomic region having (i) a sequence encoding CRISPR-Cas proteins and (ii) a CRISPR array having spacer sequences, which are short sequences that originate from and correspond to viral DNA sequences called protospacers.CRISPR arrays are composed of repeat sequences flanking spacer sequences. A CRISPR array can have at least one spacer, each flanked by two repeats. CRISPR arrays are typically located in the vicinity of cas genes but are sometimes located distant to cas genes (Butiuc-Keul et al., 2022, Microb Physiol 32:2-17; Shmakov et al., 2020, CRISPR J.3(6):535-549. Exemplary tools for identifying and annotating CRISPR-Cas loci and CRISPR arrays include CRISPRCasTyper (Russel et al., 2020, CRISPR J.3:462–469) and CRISPRDetect (Biswas et al., 2016, BMC Genomics 17:356). A CRISPR array “corresponds to” a CRISPR-Cas protein when the sequence encoding the CRISPR-Cas protein and the CRISPR array are from the same genome and are found in close proximity.

[0132] “Protospacer Adjacent Motif (PAM)” herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system described herein. As used herein, the PAM may refer to a DNA sequence downstream (e.g., in the case of Type II Cas proteins such as Cas9) of a target sequence recognized by a CRISPR-Cas protein. The PAM may refer to a DNA sequence upstream (e.g., in the case of Type V Cas proteins such as Cas12a) of the target sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

[0133] “Putative protospacer” refers to a nucleotide sequence in a viral genome that aligns with a spacer sequence with no or only a small number of mismatches or gaps. For example, a nucleotide sequence in a viral genome is identified as a putative protospacer if the spacer aligns with the nucleotide sequence in the viral genome with no more than four, three, two, one, or zero nucleotide mismatches or gaps.

[0134] The terms “guide nucleic acid” or “guide RNA” or “guide RNA molecule” or “single guide RNA” or “sgRNA” or “guide polynucleotide” or “gRNA” are used interchangeably and refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid- guided nuclease. gRNAs of the disclosure are in some embodiments single guide RNAs (sgRNAs), which typically comprise a crRNA sequence fused to a tracrRNA sequence. As used herein, the “guide sequence” or “spacer” or “spacer sequence” refers to the polynucleotide on a gRNA that is capable of hybridizing to a genomic target locus (e.g., a target polynucleotide, a protospacer sequence). Thus, in some embodiments, the guide RNA is a sgRNA. In some embodiments, the guide RNA comprises separate crRNA and tracRNA.

[0135] The term “trans-activating CRISPR (cr) RNA (tracrRNA)” or “trans-activating crRNA (tracrRNA)” are used interchangeably and refer to a distinct RNA species that interacts with theCRISPR RNA (crRNA) to form the dual guide RNA (gRNA) in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage.

[0136] The terms “target genomic DNA sequence” or “target DNA” or “target sequence”, or “genomic target locus” or “target site” are used interchangeably and refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system. The target sequence can be a genomic locus or extrachromosomal locus. As used herein, a target sequence may be referred to “protospacer DNA” or “protospacer sequence” or “protospacer polynucleotides” presented on any locus in a genomic polynucleotide (e.g., genomic locus) of a cell or population of cells.

[0137] By “site-directed modifying polypeptide” or “RNA-binding site-directed polypeptide” or “RNA-binding site-directed modifying polypeptide” or “site-directed polypeptide” it is meant a polypeptide that binds RNA and is targeted to a specific DNA sequence. A site-directed modifying polypeptide as described herein is targeted to a specific DNA sequence by the RNA molecule to which it is bound. The RNA molecule comprises a sequence that is complementary to a target sequence within the target DNA, thus targeting the bound polypeptide to a specific location within the target DNA (the target sequence).

[0138] By “cleavage” or “cleaving” or “cleave” it is meant the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, a complex comprising a DNA- targeting RNA and a site-directed modifying polypeptide is used for targeted double-stranded DNA cleavage.

[0139] “Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for DNA cleavage.

[0140] By “cleavage domain” or “active domain” or “nuclease domain” of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for DNA cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.

[0141] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in aconstruct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences”, below).

[0142] Thus, e.g., the term “recombinant” polynucleotide or “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such can be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. It can also be performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0143] Similarly, the term “recombinant” polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a polypeptide that comprises a heterologous amino acid sequence is recombinant.

[0144] The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate expression of a coding sequence and / or production of an encoded polypeptide in a host cell.

[0145] The term “transformation” is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic change (“modification”) can beaccomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.

[0146] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms “heterologous promoter” and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.

[0147] A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible and, in some embodiments—particularly many embodiments in which selection is employed—the transcription of at least one component of the nucleic acid-guided nuclease editing system is under the control of an inducible promoter. Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res.2003 Sep.1; 31(17)), a human H1 promoter (H1), and the like.

[0148] A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding one or more biosynthetic pathway gene products such as mevalonate pathway gene products), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural,accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a subject prokaryotic host cell is a genetically modified prokaryotic host cell (e.g., a bacterium), by virtue of introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell; and a subject eukaryotic host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell. As described herein, a host cell refers to a mammalian cell, including but not limited to a human cell, mouse cell, rat cell, rabbit cell, dog cell, or non-human primate cell; bacterial cell, yeast cell, or plant cell. In some embodiments, the invention does not comprise a process for modifying the germ line genetic identity of human beings, and / or the use of human embryo for industrial or commercial purposes.

[0149] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by routine screening assays.

[0150] “Identical or percent identity”, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same as measured using a sequence comparison algorithm. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0151] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol.215:403-410. Software for performing BLAST analyses is publicly available, for example, through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0152] Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol.70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970).

[0153] A “vector” or “expression vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and / or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like. As used herein, the “vector” comprises a coding sequence for a nuclease to be used in the nucleic acid- guided nuclease systems and methods of the present disclosure. The vector may also comprise, in a bacterial system, the X Red recombineering system or an equivalent thereto. Vectors also typically comprise a selectable marker. As an example, for the purpose of genome editing, the “vector” may comprise a donor nucleic acid, optionally including an alteration to the target sequence that prevents nuclease binding at a PAM or spacer in the target sequence after editing has taken place, and a coding sequence for a gRNA. The vector may also comprise a selectable marker and / or a barcode. Further, the vector may comprise control sequences operably linked to, e.g., the nuclease coding sequence, recombineering system coding sequences (if present), donor nucleic acid, guide nucleic acid, and selectable marker(s). 6.2 Compositions for Editing in Nucleic Acid-Guided Nuclease Genome Systems

[0154] Described herein are compositions for editing polynucleotides (e.g., DNA) in a cell or genome using nucleic acid-guided nuclease systems. In some embodiments, the nucleic acid- guided nuclease systems utilize an RNA-guided nuclease. In one aspect, the composition comprises a nuclease system comprising the components: a polypeptide or an amino acid sequence having at least about 90% identity to any one of the amino acid sequences set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. In some embodiments, the nucleic acid-guided nuclease systems utilize an RNA-guided nuclease. In one aspect, the composition comprises a nuclease system comprising the components: a polypeptide or an amino acid sequence having at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. In some embodiments, the polypeptide or amino acid sequence of the composition is capable of recognizing or hybridizing to at least one protospacer adjacent motif (PAM) polynucleotide sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. In some embodiments, the polypeptide or amino acid sequence of the composition and the corresponding PAM polynucleotide nucleic acid sequence are as shown in in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the composition further comprises a guide polynucleotide such as a guide RNA (gRNA). In the composition according to any of the embodiments, nucleic acid-guided nuclease systems are CRISPR-Cas (CRISPR associated) systems. In the composition according to any of the embodiments, the nuclease is a Cas9 orthologue endonuclease. In the composition according to any of the embodiments, the nucleic acid-guided nuclease systems are capable of editing such as cleaving a nucleic acid molecule (e.g., double stranded or single stranded), inserting or deleting or substituting one or more polynucleotides to a target polynucleotide sequence. As used herein, a “functional fragment” in relation to a nucleic acid sequence or amino acid sequence refers to a partial or truncated sequence relative to a full-length reference sequence, which is derived from and / or shares high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity) with the full-length reference sequence and retains the same functionality or activity (e.g., a functional fragment of a Cas endonuclease sequence may encompass a polypeptide comprising a truncated form of that reference sequence and retaining endonuclease activity). The skilled person will be able to readily determine the structure and activity of said functional fragments using methods known in the art.

[0155] In one aspect, the composition comprises a polypeptide comprising: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas orthologue nuclease having anamino acid sequence with at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387. In some embodiments, the CRISPR-Cas orthologue nuclease has an amino acid sequence of any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387. In some embodiments, the CRISPR-Cas orthologue nuclease has an amino acid sequence of any one of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0156] In various embodiments, the CRISPR-Cas orthologue nuclease is capable of complexing with a sgRNA comprising a polynucleotide sequence having at least 80% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0157] In various embodiments, the CRISPR-Cas orthologue nuclease is capable of complexing with a crRNA comprising a polynucleotide sequence having at least 80% identity with any one of SEQ ID NO: 1362, SEQ ID NO: 1363, SEQ ID NO: 1364, SEQ ID NO: 1365, and SEQ ID NO: 1366, and the crRNA is capable of complexing with a tracrRNA comprising a polynucleotide sequence having at least 80% identity with any one of SEQ ID NO: 1367, SEQ ID NO: 1368, SEQ ID NO: 1369, SEQ ID NO: 1370, and SEQ ID NO: 1371.

[0158] In one aspect, the composition comprises a polypeptide comprising: a CRISPR-Cas orthologue nuclease having an amino acid sequence with at least 80% identity to any nuclease capable of complexing with a guide RNA as shown in Table 34. In some embodiments, wherein the nuclease comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO.: 1, SEQ ID NO.: 28, SEQ ID NO.: 55, SEQ ID NO.: 106, SEQ ID NO.: 129, SEQ ID NO.: 163, SEQ ID NO.: 217, SEQ ID NO.: 242, SEQ ID NO.: 254, SEQ ID NO.: 270, SEQ ID NO.: 297, SEQ ID NO.: 325, SEQ ID NO.: 341, SEQ ID NO.: 373, SEQ ID NO.: 391, SEQ ID NO.: 409, SEQ ID NO.: 426, SEQ ID NO.: 462, SEQ ID NO.: 481, SEQ ID NO.: 503, SEQ ID NO.: 523, SEQ ID NO.: 552, SEQ ID NO.: 593, SEQ ID NO.: 646, SEQ ID NO.: 686, SEQ ID NO.: 735, SEQ ID NO.: 833, SEQ ID NO.: 847, SEQ ID NO.: 886, SEQ ID NO.: 940, and SEQ ID NO: 1826. In some embodiments, the CRISPR-Cas orthologue nuclease is capable of complexing with a sgRNA having a polypeptide sequence having at least 80% identity with any one of SEQ ID NOs: 1450-1483 and SEQ ID NO: 1825 as shown in Table 34. In some embodiments, the CRISPR-Cas orthologue nuclease is capable of complexing with a guide RNA comprising separate cRNA and tracrRNA, and the crRNA comprises a nucleic acid sequence having at least 80% identity with SEQ ID NOs: 1388-1418 as shown in Table 34, and the crRNAis capable of complexing with a tracrRNA comprising a nucleic acid sequence having at least 80% identity with any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

[0159] In accordance with any of the embodiments, the CRISPR-Cas orthologue nuclease is a fusion protein, in which the CRISPR-Cas orthologue nuclease protein sequence is fused to one more amino acid sequences, such as one or more nuclear localization signal peptides, one or more non-native tags, or both. As used herein, a nuclear localization sequence refers to an amino acid sequence that tags the polypeptide for importation into the cell nucleus by nuclear transport. In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 1800-1822. In some embodiments, the tag comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1823 or SEQ ID NO: 1824.

[0160] In one aspect, the composition comprises a guide RNA capable of complexing with a polypeptide (e.g., a CRISPR-Cas nuclease), the guide RNA comprises a crRNA capable of complexing with a tracrRNA as shown in Tables 29 and Table 30, or Table 34; or a sgRNA as shown in Table 31 or Table 34.

[0161] In one aspect, the composition comprises a system, the system comprising: a polypeptide comprising a CRISPR-Cas9 orthologue nuclease capable of complexing with a guide RNA, such as a crRNA capable of complexing with a tracrRNA as shown in Table 29 and Table 30, or Table 34; or a sgRNA as shown in Table 31 or Table 34.

[0162] In one aspect, the composition comprises a nucleic acid comprising a polynucleotide encoding a polypeptide capable of complexing with a guide RNA, optionally, the polypeptide is a Cas orthologue polypeptide.

[0163] In some embodiments, the polynucleotide encoding the Cas orthologue polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, and SEQ ID NO: 1382. In some embodiments, the polynucleotide encoding the Cas orthologue polypeptide is human codon optimized and the polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0164] In one aspect, the composition comprises a nucleic acid comprising a polynucleotide encoding a guide RNA. In some embodiments, the nucleic acid comprises a polynucleotide encoding a sgRNA having a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ IDNO: 1376, and SEQ ID NO: 1377; or any one of SEQ ID Nos: 1388-1418 as shown in Table 34, and the crRNA is capable of complexing with a tracrRNA comprising a polynucleotide having a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1419- 1449 as shown in Table 34.

[0165] The nucleic acid comprises a polynucleotide encoding a CRISPR-Cas orthologue nuclease, a sgRNA, a crRNA, or a tracrRNA can be obtained by in vitro transcription. In some embodiments, the nucleic acid is operably linked to a promoter. In some embodiments, the nucleic acid is a natural molecule.

[0166] In some embodiments, the nucleic acid comprises the polynucleotide encoding a CRISPR-Cas orthologue nuclease, a sgRNA, a crRNA, or a tracrRNA that is artificially synthesized.

[0167] In one aspect, the composition comprises one or more expression vectors comprising nucleic acids encoding a CRISPR-Cas orthologue nuclease, a guide RNA (e.g., a sgRNA, a crRNA, or a tracrRNA) as described herein. In some embodiments, the CRISPR-Cas orthologue nuclease and guide RNA are expressed in a single expression vector. In some embodiments, the CRISPR-Cas orthologue nuclease and guide RNA are expressed in separate vectors.

[0168] In one aspect, the composition comprises a cell comprising the CRISPR-Cas orthologue nuclease system and the guide RNA as disclosed herein. In some embodiments, the cell is a prokaryotic cell or eukaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0169] In one aspect, the composition comprises a pharmaceutical composition comprising the CRISPR-Cas orthologue nuclease system and the guide RNA, and a pharmaceutically acceptable excipient as disclosed herein.

[0170] RNA-guided nucleases (RGNs) have rapidly become the foundational tools for genome engineering of prokaryotes and eukaryotes. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are an adaptive immunity system which protect prokaryotes against mobile genetic elements (MGEs). RGNs are a major part of this defense system because they identify and destroy MGEs. RGNs can be repurposed for genome editing in various organisms by reprogramming the CRISPR RNA (crRNA) that guides the RGN to a specific target DNA. A number of different RGNs have been identified to date for various applications; however, there are various properties that make some RGNs more desirable than others for specific applications. RGNs can be used for creating specific double strand breaks (DSBs), specific nicks of one strand of DNA, or guide another moiety to a specific DNA sequence.

[0171] The ability of an RGN to specifically target any genomic sequence is perhaps the most desirable feature of RGNs; however, RGNs can only access their desired target if the target DNA also contains a short motif called PAM (protospacer adjacent motif) that is specific for every RGN. Type V RGNs such as MAD7, AsCas12a and LbCas12a tend to access DNA targets that contain YTTN / TTTN on the 5′ end whereas type II RGNs target DNA sequences containing a specific short motif on the 3′ end. An example well known in the art for a type II RGN is SpCas9 which requires an NGG on the 3′ end of the target DNA. Type II RGNs have substantially different domain architecture relative to type V RGNs. Further, type II RGNs also require a transactivating RNA (tracrRNA) in addition to a crRNA for optimal function. Compared to type V RGNs, the type II RGNs create a double-strand break closer to the PAM sequence, which is highly desirable for precise genome editing applications.

[0172] A number of type II RGNs have been discovered so far; however, their use in widespread applications is limited by restrictive PAMs. For example, the PAM of SpCas9 occurs less frequently in AT-rich regions of the genome. New RGNs with new and less restrictive PAMs are beneficial for the field. Further, not all type II nucleases are active in multiple organisms. For example, a number of RGNs have been discussed in the scientific literature but only a few have been demonstrated to be active in vitro and fewer still are active in cells, particularly in mammalian cells. The present disclosure identifies multiple RGNs (e.g., CRISPR- Cas systems) that have novel PAMs and are active in mammalian cells. 6.2.1 Pharmaceutical compositions

[0173] Also disclosed herein are pharmaceutical compositions, formulations and medicaments comprising a CRISPR-Cas orthologue nuclease, gRNA (e.g., sgRNA, crRNA and tracrRNA complex), nucleic acid or plurality of nucleic acids, system, particles, cells, or plurality of particles of the disclosure together with a pharmaceutically acceptable excipient.

[0174] 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 intoliposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions.

[0175] The components of the pharmaceutical compositions 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.

[0176] In some cases, compositions 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.

[0177] In some embodiments, the compositions can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration. In some embodiments, the compositions can comprise a guide RNA and a CRISPR-Cas orthologue nuclease 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. 6.3 Nuclease System for Editing / Cleaving Polynucleotides

[0178] In general, “nuclease system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of Cas genes, including sequences encoding a Cas9 orthologue gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- processed partial direct repeat in the context of an endogenous nuclease system), a guide sequence (also referred to as a “spacer” in the context of an endogenous nuclease system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a nuclease system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a nuclease system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a nuclease system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous nuclease system). In the context of formation of a CRISPR complex (e.g., a protein- polypeptide complex), “target sequence” or “protospacer sequence” refers to a sequence to whicha guide sequence or “spacer sequence” is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence”. In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.

[0179] Typically, in the context of an endogenous nuclease system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more nucleases such as Cas9 orthologue proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracrRNA sequence, which may comprise or consist of all or a portion of a wild-type tracrRNA sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracrRNA sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracrRNA sequence to all or a portion of a tracrRNA mate sequence that is operably linked to the guide sequence. In some embodiments, the tracrRNA sequence has sufficient complementarity to a tracrRNA mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracrRNA sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracrRNA mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a nuclease system are introduced into a host cell such that expression of the elements of the nuclease system direct formation of a CRISPR complex at one or more target sites. For example, a Cas9 orthologue enzyme, a guide sequence linked to a tracrRNA-mate sequence, and a tracrRNA sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nuclease system not included in the first vector. Nuclease system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracrRNA mate sequence (optionally operably linked to the guide sequence), and a tracrRNA sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracrRNA mate sequence, and tracrRNA sequence are operably linked to and expressed from the same promoter. 6.3.1. CRISPR-Cas Systems

[0180] CRISPR-Cas (CRISPR associated) systems are protein-RNA complexes that use an RNA molecule (sgRNA) as a guide to localize the complex to a target DNA sequence via base- pairing. A Cas protein then acts as an endonuclease to cleave the targeted DNA sequence. The target DNA sequence typically is complementary to the sgRNA and also contains a protospacer- adjacent motif (PAM) at the 3 '-end of the complementary region in order for the system to function.

[0181] A CRISPR-Cas system comprises, at a minimum, a CRISPR RNA (crRNA) molecule and at least one CRISPR-associated (Cas) endonuclease to form crRNA ribonucleoprotein (crRNP) effector complexes. The Cas endonuclease is guided by a CRISPR RNA (crRNA) through direct RNA-DNA base-pairing to recognize a DNA target site that is in close vicinity to a protospacer adjacent motif (PAM) (Jore, M.M. et al, 2011, Nat. Struct. Mol. Biol.18:529-536, Westra, E.R. et al, 2012, Molecular Cell 46:595-605, and Sinkunas, T. et al, 2013, EMBO J. 32:385-394).

[0182] In some embodiments, a CRISPR-Cas system (also referred to as CRISPR system herein) refers to a polypeptide of the Cas endonuclease and a guide polynucleotide including a crRNA polynucleotide and a tracrRNA polynucleotide. The crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA (trans-activating CRISPR RNA) forming a RNA duplex that directs the Cas endonuclease to cleave the DNA target. 6.3.1.1. Cas endonucleases

[0183] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Cas endonucleases, either as single effector proteins or in an effector complex with other components, unwind the DNA duplex at the target sequence and optionally cleave at least one DNA strand, as mediated by recognition of the target sequence by apolynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas endonuclease. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Cas endonucleases that have been described include, but are not limited to, for example: Cas3, Cas9, and Cas12 (Cpfl).

[0184] Among the discovered Cas proteins, S. pyogenes Cas9 (formerly referred to as Cas5, Csnl, or Csxl2) has been mostly widely used as a tool for genome engineering. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Cas9 forms a complex with a crRNA and a tracrRNA, or with a single guide polynucleotide (sgRNA), for specifically recognizing and cleaving all or part of a DNA target sequence.

[0185] A Cas9 protein comprises a RuvC nuclease with an HNH (H-N-H) nuclease adjacent to the RuvC-II domain. The RuvC nuclease and HNH nuclease each can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, 2013, Cell 157:11228-1278). Thus, in some embodiments, the nuclease is a Cas9 nickase variant or a dCas9.

[0186] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide disclosed herein has one or more mutations in a residue homologous to D10 and H840 of SpCas9 and N580 of SaCas9, identified by multisequence alignment using Clustal Omega. The mutations can generate nickases or deactivated nucleases. For example, mutation of these residues to A (alanine) can abolish the catalytic activity of the Cas9 RuvC domain, Cas9 HNH domain, or both.

[0187] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide has one or more mutations at the position D20, H631, or N654 of SEQ ID NO: 721 or SEQ ID NO: 1383.

[0188] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide has one or more mutations at the position D10, H797, or N819 of SEQ ID NO: 362 or SEQ ID NO: 1384.

[0189] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide has one or more mutations at the position D9, H687, or N710 of SEQ ID NO: 444 or SEQ ID NO: 1385.

[0190] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide has one or more mutations at the position D9, H565, or N588 of SEQ ID NO: 768 or SEQ ID NO: 1386.

[0191] In some embodiments, the CRISPR-Cas orthologue nuclease or polypeptide has one or more mutations at the position D8, H681, N704 of SEQ ID NO: 76 or SEQ ID NO: 1387.

[0192] Cas endonucleases can be used for targeted genome editing (e.g., via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (e.g., via tethering of epigenetic effector domains to either the Cas protein or sgRNA. The most widely used Cas9 recognizes a 3’ GC-rich PAM sequence on the target dsDNA, typically comprising an NGG motif. The Cas9 orthologues described herein may recognize additional PAM sequences and used to modify target sites with different recognition sequence specificity.

[0193] In some embodiments, the Cas9 orthologues described herein are found in a bacterial or archaeal genome. In some embodiments, the Cas9 orthologues described herein are isolated from a bacterial or archaeal genome. In some embodiments, the Cas9 orthologues are found, identified, or isolated from (i) publicly available sequences on NCBI database (Benson et al., 2013, Nucleic Acids Res.41:D36–D42 (available as of January 2021), (ii) 771,529 metagenome- assembled genomes (MAGs) from an unpublished study, and (iii) 54,169 additional MAGs obtained with a validated assembly-based pipeline similarly to Pasolli et al, 2019, Cell 176:649– 662.e20.

[0194] A Cas9 orthologue protein as described herein may comprise a polypeptide having an amino acid sequence or a functional fragment thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein comprises a polypeptide having an amino acid sequence or a functional fragment thereof having at least 90% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A- 5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A- 12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein comprises a polypeptide having an amino acid sequence or a functional fragment thereof having at least 95% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein comprises a polypeptide having an amino acid sequence or a functional fragment thereof having at least 98% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0195] A Cas9 orthologue protein as described herein may comprise a polypeptide having an amino acid sequence or a functional fragment thereof having between 90% and 95%, between 95% and 96%, between 96% and 99%, between 97% and 98%, between 97% and 99%, between 98% and 99%, between 99% and 100%, or 100% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein comprises a polypeptide having an amino acid sequence or a functional fragment thereof having between 90% and 100%, or between 95% and 99% amino acid sequence identity with any of one amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0196] A Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, may recognize at least one PAM sequence categorized as any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A- 16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, recognizes at least one PAM sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, recognizes at least one PAM sequence having at least 90%, at least 95%, or at least 98% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0197] A Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, may recognize at least one PAM sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A- 5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A- 12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In someembodiments, the Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, recognizes at least one PAM sequence having between 90% and 95%, between 95% and 96%, between 96% and 99%, between 97% and 98%, between 97% and 99%, between 98% and 99%, between 99% and 100%, or 100% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the Cas9 orthologue protein or Cas9 polypeptide, or a functional fragment thereof, recognizes at least one PAM sequence having between 90% and 95%, between 90% and 99%, between 95% and 99% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0198] In various aspects, a Cas9 orthologue protein as described herein comprises a polypeptide having an amino acid sequence set forth as Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), or a functional fragment thereof, that recognizes (hybridizes to) or is capable of recognizing (or hybridizing to) a corresponding PAM polynucleotide having a nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0199] The present inventors have surprisingly identified novel CRISPR-Cas orthologue nucleases which retain strong endonuclease activity and recognize hitherto-untargeted PAM sequences. Therefore, these novel nucleases are capable of being directed towards targets which were previously inaccessible for genome editing, thus addressing a major constraint of existing CRISPR-Cas systems. Accordingly, in one embodiments, the CRISPR-Cas polypeptide encoding the Cas orthologue nuclease comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387, or any one as shown in Table 34. In some embodiments, the CRISPR-Cas orthologue nuclease has an amino acid sequence of any one of SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 721, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387, or any one as shown in Table 34.

[0200] In one embodiment, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of SEQ ID NO: 721 or SEQ ID NO: 1383.

[0201] In one embodiment, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of SEQ ID NO: 362 or SEQ ID NO: 1384.

[0202] In one embodiment, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of SEQ ID NO: 444 or SEQ ID NO: 1385.

[0203] In one embodiment, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of SEQ ID NO: 768 or SEQ ID NO: 1386.

[0204] In one embodiment, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of SEQ ID NO: 76 or SEQ ID NO: 1387.

[0205] In some embodiments, the CRISPR-Cas orthologue nuclease comprises an amino acid sequence of any one of nucleases having a SEQ ID NOs as shown in Table 34.

[0206] In some embodiments, wherein the nuclease comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO.: 1, SEQ ID NO.: 28, SEQ ID NO.: 55, SEQ ID NO.: 106, SEQ ID NO.: 129, SEQ ID NO.: 163, SEQ ID NO.: 217, SEQ ID NO.: 242, SEQ ID NO.: 254, SEQ ID NO.: 270, SEQ ID NO.: 297, SEQ ID NO.: 325, SEQ ID NO.: 341, SEQ ID NO.: 373, SEQ ID NO.: 391, SEQ ID NO.: 409, SEQ ID NO.: 426, SEQ ID NO.: 462, SEQ ID NO.: 481, SEQ ID NO.: 503, SEQ ID NO.: 523, SEQ ID NO.: 552, SEQ ID NO.: 593, SEQID NO.: 646, SEQ ID NO.: 686, SEQ ID NO.: 735, SEQ ID NO.: 833, SEQ ID NO.: 847, SEQ ID NO.: 886, SEQ ID NO.: 940, and SEQ ID NO: 1826. 6.3.1.1.1. Fusion proteins

[0207] In some embodiments, the CRISPR-Cas polypeptide is a fusion protein, in which the CRISPR-Cas orthologue nuclease protein sequence is fused to one more amino acid sequences, such as one or more nuclear localization signal peptides, one or more non-native tags, or both. In some embodiments, the polypeptide comprises two or more nuclear localization signals fused to the CRISPR-Cas orthologue nuclease. In some embodiments, the nuclear localization signal comprises an N-terminal nuclear localization signal, a C-terminal nuclear localization signal, or both.

[0208] In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1800-1822. In some embodiments, the nuclear localization signal comprises an amino acid sequence of any one of SEQ ID NOs: 1800-1822.

[0209] Fusion proteins can also comprise an amino acid sequence of, for example, a nucleoside deaminase, a reverse transcriptase, a transcriptional activator, a transcriptional repressor, a histone-modifying protein, an integrase, or a recombinase.

[0210] In some embodiments, the CRISPR-Cas orthologue nuclease protein is fused to one or more protein tags such as SV5 or V5-tags. In some embodiments, the tag peptide comprises an amino acid sequence having a at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1823 or SEQ ID NO: 1824. In some embodiments, the tag peptide comprises an amino acid sequence of SEQ ID NO: 1823 or SEQ ID NO: 1824.

[0211] Additional exemplary fusion partners include, 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 localizationsignals, 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), as described in WO2023118349, the content of which is incorporated herein by reference in its entirety. 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. 6.3.1.2. Guide polynucleotide

[0212] The guide polynucleotide enables target recognition, binding, and optionally cleavage by the Cas endonuclease, and can be a single molecule or a double molecule. In general, a guide polynucleotide is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide polynucleotide is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 140, 150, 180, 200, 220, 250, 300, or more nucleotides in length, including or excluding the spacer sequence. In some embodiments, a guide polynucleotide is less than about 300, 250, 200, 180, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length, including or excluding the spacer sequence. In some embodiments, a guide polynucleotide is between 60-130, 70-280, 80-110, 90-200, or 120-150 in length, including or excluding the spacer sequence. In some embodiments, a guide polynucleotide is between 80-110 nucleotides in length excluding the spacer sequence.

[0213] The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (an RNA-DNA combination sequence). A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a “guide RNA” or “gRNA” (US20150082478, US20150059010, WO2019165168A1, WO2021118626A1, each of which is incorporated herein by reference in its entirety). A guide polynucleotide may be engineered or synthetic.

[0214] In some embodiments, the guide polynucleotide as described herein is a double molecule such as a guide RNA comprising a crRNA and a tracrRNA. The crRNA may comprise a polynucleotide sequence (e.g., a spacer) that recognizes (or hybridizes or complementary to) a target polynucleotide sequence (e.g., protospacer) presence on the target molecule (e.g., genomic locus). In some embodiments, the target polynucleotide sequence (e.g., protospacer) is located adjacent to a PAM such as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0215] In some embodiments, the guide RNA comprises a spacer that is partially or fully complementary to a target mammalian genomic sequence upstream of a Protospacer Adjacent Motif (PAM) sequence in the non-target strand recognized by the polypeptide. Non-limiting examples of polynucleotides are listed in Table 32 and Table 34.

[0216] In some embodiments, the guide RNA capable of complexing with the CRISPR-Cas orthologue nuclease is a sgRNA shown in Table 31 or Table 34. In some embodiments, the sgRNA comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 100 consecutive nucleotides of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377, or any one of SEQ ID NOs: 1450-1483, and SEQ ID NO: 1825 in Table 34.

[0217] In some embodiments, the sgRNA has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377. In some embodiments, the sgRNA comprises the polynucleotide sequence of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377. In some embodiments, the sgRNA comprises the polynucleotide sequence of any one of SEQ ID NOs: 1450-1483, and SEQ ID NO: 1825 in Table 34.

[0218] In some embodiments, the sgRNA comprises at least one modified nucleotide, at least one modified internucleoside linkage, or both. In some embodiments, the sgRNA comprises at least one modified nucleotide selected from 2'-fluoro, 2'-amino or 2'-O-methyl modification on the ribose of pyrimidines, abasic residues, and an inverted base at the 3' end of the RNA. In someembodiments, the sgRNA comprises at least one modified internucleoside linkage selected from phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl inter- sugar linkages, and short chain heteroatomic or heterocyclic inter-sugar linkages.

[0219] In some embodiments, the guide RNA capable of complexing with the CRISPR-Cas orthologue nuclease is a guide RNA having separate crRNA and tracrRNA as shown in Table 29, Table 30, or Table 34.

[0220] In some embodiments, the guide RNA comprises, in 5’ to 3’ order, the crRNA molecule or the tracrRNA molecule comprising at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 100 consecutive nucleotides of any crRNA or tracrRNA as shown in Table 29, Table 30, or Table 34.

[0221] In some embodiments, the crRNA comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 100 consecutive nucleotides of any one of SEQ ID NOs: 1362-1366 in Table 29, or any one of SEQ ID NOs: 1388-1418 in Table 34. In some embodiments, the tracrRNA comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 100 consecutive nucleotides of any one of SEQ ID NOs: 1367-1371 in Table 30, or any one of SEQ ID NOs: 1419-1449 in Table 34.

[0222] In some embodiments, the crRNA molecule or the tracrRNA molecule has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one crRNA or tracrRNA as shown in Table 29, Table 30, or Table 34.

[0223] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1362 or SEQ ID NO: 1367. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1362 and a tracrRNA of SEQ ID NO: 1367.

[0224] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with of SEQ ID NO: 1363 or SEQ ID NO: 1368. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1363 and a tracrRNA of SEQ ID NO: 1368.

[0225] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1364 or SEQ ID NO: 1369. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1364 and a tracrRNA of SEQ ID NO: 1369.

[0226] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1365 or SEQ ID NO: 1370. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1352 and a tracrRNA of SEQ ID NO: 1370.

[0227] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1366 or SEQ ID NO: 1371. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1366 and a tracrRNA of SEQ ID NO: 1371.

[0228] In some embodiments, the guide RNA comprises a crRNA molecule and a tracrRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1366 or SEQ ID NO: 1371. In some embodiments, the guide RNA comprises a crRNA molecule of SEQ ID NO: 1366 and a tracrRNA of SEQ ID NO: 1371.

[0229] In some embodiments, the guide RNA comprises a crRNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1388-1418, and their corresponding tracrRNA molecule has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1419-1449 as shown in Table 34. 6.3.2.2.1. Modified guide RNA

[0230] In some embodiments, the guide RNA is synthetic (i.e., non-naturally occurring). In some embodiments, the guide RNA is modified. Guide RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated, as described in the relevant field. The disclosed gRNA (e.g., sgRNA) molecules can be unmodified or can contain any one or more of an array of chemical modifications.

[0231] While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids theuse 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 CRISPR-Cas orthologue nuclease, 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.

[0232] 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 T m (thus, higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.

[0233] A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligonucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH,~N(CH3)- O-CH2 (known as a methylene(methylimino) or MMI backbone), CH2-O-N (CH3)-CH2, CH2-N (CH3)-N (CH3)-CH2and O-N (CH3)- CH2-CH2backbones, wherein the native phosphodiester backbone is represented as O- P- O- CH,); amide backbones; morpholino backbone structures; 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). 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'.

[0234] 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.

[0235] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; ON; 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 - OCH2 CH2CH3) 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.

[0236] Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group. 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.

[0237] 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).

[0238] 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. A "universal" base, 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 and are aspects of base substitutions.

[0239] Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo- uracil), 4-thiouraci 1 , 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.

[0240] In some embodiments, the modified guide RNA is a cRNA comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1362, SEQ ID NOs: 1363, SEQ ID NOs: 1364, SEQ ID NOs: 1365, and SEQ ID NOs: 1366, or any one of SEQ ID NOs: 1388-1418 as shown in Table 34. In some embodiments, the modified crRNA is capable of complexing with a modified tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1367, SEQ ID NOs: 1368, SEQ ID NOs: 1369, SEQ ID NOs: 1370, and SEQ ID NOs: 1371, or any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

[0241] In some embodiments, the modified guide RNA is a sgRNA comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, or SEQ ID NO: 1376, or any one of SEQ ID NOs: 1450-1483 and SEQ ID NO: 1825 as shown in Table 34. 6.3.1.3. Protospacer Adjacent Motif (PAM)

[0242] A “protospacer adjacent motif” (PAM) refers to a polynucleotide sequence located adjacent (e.g., downstream or upstream) to a target polynucleotide sequence. A PAM sequence is necessary for a CRISPR-Cas protein to bind and cut target genomic DNA. In some aspects, the Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not adjacent to, or near, a PAM sequence.

[0243] A PAM polynucleotide as described herein, or a functional fragment thereof, may comprise a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the PAM polynucleotide, or a functional fragment thereof, have not more than 90%, 95%, 98%, 99%, or 100% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A- 7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0244] A PAM polynucleotide as described herein, or a functional fragment thereof, may comprise a nucleic acid sequence having between 85% and 90%, between 90% and 95%, between 95% and 96%, between 96% and 99%, between 97% and 98%, between 97% and 99%, between 98% and 99%, between 99% and 100%, or 100% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A- 5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16),Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A- 12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, the PAM polynucleotide, or a functional fragment thereof, have between 90% to 95%, between 95% to 99%, or between 95% and 99% nucleic acid sequence identity with at least one PAM nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A- 16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99).

[0245] In some embodiments, a PAM polynucleotide as described herein, or a functional fragment thereof, comprises a consensus nucleic acid sequence as categorized in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). In some embodiments, a PAM polynucleotide as described herein, or a functional fragment thereof, comprises a PAM logo or motif shown in FIGs.3A-3E, FIGs.4A-4C, FIGs. 5A-5B, FIGs.6A-6D, FIGs.7A-7C, FIGs.8A-8B, FIGs.9A-9B, FIG.10, FIGs.11A-11B, FIGs. 12A-12D, FIG.13, FIGs.14A-14B, FIG.15, FIG.16, FIGs.17, FIGs.18A-18B, FIG.19, FIG. 20, FIG.21, FIGs.22A-22B, FIGs.23A-23B, or FIGs.24A-24B. In some embodiments, the PAM is recognized by one or more CRISPR-Cas proteins in a CRISPR-Cas cluster. In some embodiments, a PAM polynucleotide as described herein, or a functional fragment thereof, comprises a consensus nucleic acid sequence as categorized in any one of Table 32 and Table 34. In some embodiments, the PAM is recognized by one or more Cas9 orthologues as described herein. In some embodiments, the PAM is predicted and validated according to methods as described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety.

[0246] In various embodiments, the PAM is found in one or more viral genomes. In some embodiments, the PAM is identified in the viral genomes that have matching or near matching sequences to the spacer sequences of one or more the Cas9 orthologues as described herein. In some embodiments, the PAM has a polynucleotide sequence found, identified, or isolated from aviral genome in the Human Gut virome datasets as described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety. 6.3.1.4. Nucleic acids

[0247] A nucleic acid comprising a polynucleotide encoding the polypeptide capable of complexing with a guide RNA, the sgRNA, the crRNA, or the tracrRNA can be synthesized by chemical means or by in vitro transcription.

[0248] In some embodiments, the human codon optimized polynucleotide encodes a polypeptide, the polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, and SEQ ID NO: 1382. In some embodiments, the polynucleotide is a human codon optimized polynucleotide encoding a CRISPR-Cas orthologue nuclease. In some embodiments, the human codon optimized polynucleotide encodes a polypeptide, the polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0249] In some embodiments, the polynucleotide encodes a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

[0250] In some embodiments, the polynucleotide encodes a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1450-1483, and SEQ ID NO: 1825 as shown in Table 34.

[0251] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1362 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1367.

[0252] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1363 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1368.

[0253] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1364 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1369.

[0254] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1365 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1370.

[0255] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1366 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1371.

[0256] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs: 1388-1418 as shown in Table 34, complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1419-1449 as shown in Table 34. 6.3.1.4.1. Exemplary CRISPR-Cas orthologue nuclease systems

[0257] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 76 or SEQ ID NO: 1387, capable of complexing with a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: SEQ ID NO: 1377, or with a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1366 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1371.

[0258] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease of SEQ ID NO: 76 or SEQ ID NO: 1387, capable of complexing with a sgRNA of SEQ ID NO: 1377, or with a crRNA of SEQ ID NO: 1366 complexed with a tracrRNA of SEQ ID NO: 1371.

[0259] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 362 or SEQ ID NO: 1384, capable of complexing with a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1373, or with a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1363 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1368.

[0260] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease of SEQ ID NO: 362 or SEQ ID NO: 1384, capable of complexing with a sgRNA of SEQ ID NO: 1373, or with a crRNA of SEQ ID NO: 1363 complexed with a tracrRNA of SEQ ID NO: 1368.

[0261] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 444 or SEQ ID NO: 1385, capable of complexing with a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1374, or with a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1364 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1369.

[0262] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease of SEQ ID NO: 444 or SEQ ID NO: 1385, capable of complexing with a sgRNA of SEQ ID NO: 1374, or with a crRNA of SEQ ID NO: 1364 complexed with a tracrRNA of SEQ ID NO: 1369.

[0263] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 721 or SEQ ID NO: 1383, capable of complexing with a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1372, or with a crRNA having at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1362 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1367.

[0264] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease of SEQ ID NO: 721 or SEQ ID NO: 1383, capable of complexing with a sgRNA of SEQ ID NO: 1372, or with a crRNA of SEQ ID NO: 1362 complexed with a tracrRNA of SEQ ID NO: 1367.

[0265] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 768 or SEQ ID NO: 1386, capable of complexing with a sgRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1375 or SEQ ID NO: 1376, or with a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1365 complexed with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1370.

[0266] In some embodiments, the system comprises a CRISPR-Cas9 orthologue nuclease of SEQ ID NO: 768 or SEQ ID NO: 1386, capable of complexing with a sgRNA of SEQ ID NO: 1375 or SEQ ID NO: 1376, or with a crRNA of SEQ ID NO: 1365 complexed with a tracrRNA of SEQ ID NO: 1370. 6.4 Methods for nucleic acid-guided nuclease editing

[0267] In performing nucleic acid-guided nuclease editing, the mined Cas9 orthologue cluster nucleases may be delivered to cells to be edited as a polypeptide; alternatively, a polynucleotide sequence encoding the mined Cas9 orthologue cluster nucleases are transformed or transfected into the cells to be edited. The polynucleotide sequence encoding the mined Cas9 orthologue cluster nuclease may be codon optimized for expression in particular cells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human mammals including non-human primates. The choice of the mined Cas9 orthologue cluster nuclease to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence. The Cas9orthologue cluster series nuclease may be encoded by a DNA sequence on a vector (e.g., the engine vector) and be under the control of a constitutive or inducible promoter. In some embodiments, the sequence encoding the nuclease is under the control of an inducible promoter, and the inducible promoter may be separate from but the same as an inducible promoter controlling transcription of the guide nucleic acid; that is, a separate inducible promoter may drive the transcription of the nuclease and guide nucleic acid sequence / s but the two inducible promoters may be the same type of inducible promoter (e.g., both are pL promoters). Alternatively, the inducible promoter controlling expression of the nuclease may be different from the inducible promoter controlling transcription of the guide nucleic acid; that is, e.g., the nuclease may be under the control of the pBAD inducible promoter, and the guide nucleic acid may be under the control of the pL inducible promoter.

[0268] In general, a guide polypeptide complexes with a compatible nucleic acid-guided nuclease and can then hybridize with a target sequence, thereby directing the nuclease to the target sequence. With the Cas9 orthologue enzymes described herein, the nucleic acid-guided nuclease editing system uses two separate guide nucleic acid components that combine and function as a guide nucleic acid; that is, a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). The gRNA may be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or the coding sequence may reside within an editing cassette and is under the control of a constitutive promoter, or, in some embodiments, an inducible promoter as described herein.

[0269] In the present methods and compositions, the components of the guide polynucleotide is provided as a sequence to be expressed from a plasmid or vector and comprises both the guide sequence and the scaffold sequence as a single transcript under the control of a promoter, and in some embodiments, an inducible promoter. In general, to generate an edit in a target sequence, the gRNA / nuclease complex binds to a target sequence as determined by the guide RNA, and the nuclease recognizes a protospacer adjacent motif (PAM) sequence adjacent to the target sequence. The target sequence can be any polynucleotide endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro. For example, the target sequence can be a polynucleotide residing in the nucleus of a eukaryotic cell. A target sequence can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).

[0270] The guide polynucleotide may be part of an editing cassette that encodes the donor nucleic acid. Alternatively, the guide polynucleotide may not be part of the editing cassette and instead may be encoded on the engine or editing vector backbone. For example, a sequence coding for a guide polynucleotide can be assembled or inserted into a vector backbone first, followed by insertion of the donor nucleic acid in, e.g., the editing cassette. In other cases, thedonor nucleic acid in, e.g., an editing cassette can be inserted or assembled into a vector backbone first, followed by insertion of the sequence coding for the guide nucleic acid. In yet other cases, the sequence encoding the guide polynucleotide and the donor nucleic acid (inserted, for example, in an editing cassette) are simultaneously but separately inserted or assembled into a vector. In yet other embodiments, the sequence encoding the guide polynucleotide and the sequence encoding the donor nucleic acid are both included in the editing cassette.

[0271] The target sequence is associated with a PAM, which is a short nucleotide sequence recognized by the gRNA / nuclease complex. The precise PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-8 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering of the PAM-interacting domain of a nucleic acid- guided nuclease may allow for alteration of PAM specificity, improve fidelity, or decrease fidelity. In certain embodiments, the genome editing of a target sequence both introduces a desired DNA change to a target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a protospacer adjacent motif (PAM) region in the target sequence. Rendering the PAM at the target sequence inactive precludes additional editing of the cell genome at that target sequence, e.g., upon subsequent exposure to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in later rounds of editing. Thus, cells having the desired target sequence edit and an altered PAM can be selected using a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid complementary to the target sequence. Cells that did not undergo the first editing event will be cut rendering a double-stranded DNA break, and thus will not continue to be viable. The cells containing the desired target sequence edit and PAM alteration will not be cut, as these edited cells no longer contain the necessary PAM site and will continue to grow and propagate.

[0272] As mentioned previously, the range of target sequences that nucleic acid-guided nucleases can recognize is constrained by the need for a specific PAM to be located near the desired target sequence. As a result, it often can be difficult to target edits with the precision that is necessary for genome editing. It has been found that nucleases can recognize some PAMs very well (e.g., canonical PAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs). Because the mined Cas9 orthologue cluster nucleases disclosed herein may recognize different PAMs, the mined Cas9 orthologue cluster nucleases increase the number of target sequences that can be targeted for editing; that is, mined Cas9 orthologue cluster nucleases decrease the regions of “PAM deserts” in the genome. Thus, the mined Cas9 orthologue cluster nucleases expand the scope of target sequences that may be edited by increasing the number (variety) of PAM sequences recognized. Moreover, cocktails of mined Cas9 orthologue cluster nucleases may bedelivered to cells such that target sequences adjacent to several different PAMs may be edited in a single editing run.

[0273] Another component of the nucleic acid-guided nuclease system is the donor nucleic acid. In some embodiments, the donor nucleic acid is on the same polynucleotide (e.g., editing vector or editing cassette) as the guide nucleic acid and may be (but not necessarily) under the control of the same promoter as the guide nucleic acid (e.g., a single promoter driving the transcription of both the guide nucleic acid and the donor nucleic acid). For cassettes of this type, see U.S. Pat. Nos.10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715; and 10,465,207. The donor nucleic acid is designed to serve as a template for homologous recombination with a target sequence nicked or cleaved by the nucleic acid-guided nuclease as a part of the gRNA / nuclease complex. A donor nucleic acid polynucleotide may be of any suitable length, such as about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides in length. In certain preferred aspects, the donor nucleic acid can be provided as an oligonucleotide of between 20-300 nucleotides, more preferably between 50-250 nucleotides. The donor nucleic acid comprises a region that is complementary to a portion of the target sequence (e.g., a homology arm). When optimally aligned, the donor nucleic acid overlaps with (is complementary to) the target sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments, the donor nucleic acid comprises two homology arms (regions complementary to the target sequence) flanking the mutation or difference between the donor nucleic acid and the target template. The donor nucleic acid comprises at least one mutation or alteration compared to the target sequence, such as an insertion, deletion, modification, or any combination thereof compared to the target sequence.

[0274] Often the donor nucleic acid is provided as an editing cassette, which is inserted into a vector backbone where the vector backbone may comprise a promoter driving transcription of the gRNA and the coding sequence of the gRNA, or the vector backbone may comprise a promoter driving the transcription of the gRNA but not the gRNA itself. Moreover, there may be more than one, e.g., two, three, four, or more guide nucleic acid / donor nucleic acid cassettes inserted into an engine vector, where each guide nucleic acid is under the control of separate different promoters, separate like promoters, or where all guide nucleic acid / donor nucleic acid pairs are under the control of a single promoter. In some embodiments the promoter driving transcription of the gRNA and the donor nucleic acid (or driving more than one gRNA / donor nucleic acid pair) is an inducible promoter. Inducible editing is advantageous in that isolated cells can be grown for several to many cell doublings to establish colonies before editing is initiated, which increases the likelihood that cells with edits will survive, as the double-strand cuts caused by active editing are largely toxic to the cells. This toxicity results both in cell death in the edited colonies, as well as a lag in growth for the edited cells that do survive but must repair andrecover following editing. However, once the edited cells have a chance to recover, the size of the colonies of the edited cells will eventually catch up to the size of the colonies of unedited cells. See, e.g., U.S. Pat. Nos.10,533,152; 10,550,363; 10,532,324; and U.S. Ser. No. 16 / 597,826, filed 9 Oct.2019; Ser. No.16 / 597,831, filed 9 Oct.2019; Ser. No.16 / 693,630, filed 25 Nov.2019; 16 / 687,640, filed 18 Nov.2019; and 16 / 686,066, filed 15 Nov.2019. Further, a guide nucleic acid may be efficacious directing the edit of more than one donor nucleic acid in an editing cassette; e.g., if the desired edits are close to one another in a target sequence.

[0275] In addition to the donor nucleic acid, an editing cassette may comprise one or more primer sites. The primer sites can be used to amplify the editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the editing cassette.

[0276] In addition, the editing cassette may comprise a barcode. A barcode is a unique DNA sequence that corresponds to the donor DNA sequence such that the barcode can identify the edit made to the corresponding target sequence. The barcode typically comprises four or more nucleotides. In some embodiments, the editing cassettes comprise a collection of donor nucleic acids representing, e.g., gene-wide or genome-wide libraries of donor nucleic acids. The library of editing cassettes is cloned into vector backbones where, e.g., each different donor nucleic acid is associated with a different barcode.

[0277] Additionally, in some embodiments, an expression vector or cassette encoding components of the nucleic acid-guided nuclease system further encodes one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the nuclease comprises NLSs at or near the amino-terminus of the mined Cas9 orthologue cluster RGN, NLSs at or near the carboxy-terminus of the mined Cas9 orthologue cluster RGN, or a combination.

[0278] The engine and editing vectors comprise control sequences operably linked to the component sequences to be transcribed. As stated above, the promoters driving transcription of one or more components of the mined Cas9 orthologue cluster nuclease editing system may be inducible, and an inducible system is likely employed if selection is to be performed. A number of gene regulation control systems have been developed for the controlled expression of genes in plant, microbe, and animal cells, including mammalian cells, including the pL promoter (induced by heat inactivation of the CI857 repressor), the pBAD promoter (induced by the addition of arabinose to the cell growth medium), and the rhamnose inducible promoter (induced by the addition of rhamnose to the cell growth medium). Other systems include the tetracycline- controlled transcriptional activation system (Tet-On / Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70- 72 (1992); U.S. Pat. No.4,833,080), the ecdysone-inducible gene expression system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al., BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible gene expression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) as well as others.

[0279] Typically, performing genome editing in live cells entails transforming cells with the components necessary to perform nucleic acid-guided nuclease editing. For example, the cells may be transformed simultaneously with separate engine and editing vectors; the cells may already be expressing the mined Cas9 orthologue cluster nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the mined Cas9 orthologue cluster nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells; or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing.

[0280] A variety of delivery systems can be used to introduce (e.g., transform or transfect) nucleic acid-guided nuclease editing system components into a host cell. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Alternatively, molecular trojan horse liposomes may be used to deliver nucleic acid- guided nuclease components across the blood brain barrier. Of particular interest is the use of electroporation, particularly flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system) as described in, e.g., U.S. Pat. Nos. 10,435,713; 10,443,074; 10,323,1223; and 10,415,058.

[0281] After the cells are transformed with the components necessary to perform nucleic acid- guided nuclease editing, the cells are cultured under conditions that promote editing. For example, if constitutive promoters are used to drive transcription of the mined Cas9 orthologue cluster nucleases and / or gRNA, the transformed cells need only be cultured in a typical culture medium under typical conditions (e.g., temperature, CO2 atmosphere, etc.) Alternatively, if editing is inducible—by, e.g., activating inducible promoters that control transcription of one or more of the components needed for nucleic acid-guided nuclease editing, such as, e.g., transcription of the gRNA, donor DNA, nuclease, or, in the case of bacteria, a recombineering system—the cells are subjected to inducing conditions.6.4.1 Recombinant Constructs for Transformation of Cells

[0282] Several aspects of the disclosure relate to vector systems comprising one or more vectors, or vectors as such. Vectors can be designed for expression of guide polynucleotides, Cas polypeptide (e.g., Cas endonucleases), or guide polynucleotide / Cas polypeptide systems disclosed herein, or any one combination thereof, in prokaryotic or eukaryotic cells. For example, guide polynucleotides, Cas polypeptide (e.g., Cas endonucleases), or guide polynucleotide / Cas polypeptide systems disclosed herein, or any one combination thereof can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, mammalian cells, or plant cells. Exemplary mammalian cells include, but not limit to human cells, mouse cells, rat cells, rabbit cells, dog cells, or non-human primate cells. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0283] Standard recombinant DNA and molecular cloning techniques used herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, recombinant DNA, and transformation methods (see e.g., US8697359B1, WO2019165168A1, each of which is incorporated herein by reference in its entirety).

[0284] Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, or regulatory sequences. In some examples a recognition site and / or target site can be comprised within an intron, coding sequence, 5' UTRs, 3' UTRs, and / or regulatory regions.

[0285] Vectors may be introduced and propagated in a prokaryote. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separationof the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A. respectively, to the target recombinant protein.

[0286] In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.

[0287] In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding one of the Cas9 orthologues proteins described in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), or functional fragments, or modified versions thereof. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

[0288] In some embodiments, the expression vector comprises a human codon optimized polynucleotide encoding a polypeptide, the polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, and SEQ ID NO: 1382. In some embodiments, the polynucleotide is a human codon optimized polynucleotide encoding a CRISPR-Cas orthologue nuclease. In some embodiments, the expression vector comprises a human codon optimized polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

[0289] In some embodiments, the expression vector comprises a polynucleotide encoding a human codon optimized polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of the nucleases having a SEQ ID NO as shown in Table 34.

[0290] In some embodiments, the expression vector comprises a sgRNA comprising a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377, or any one of SEQ ID NOs: 1450-1483, and SEQ ID NO: 1825 as shown in Table 34.

[0291] In some embodiments, the expression vector comprises a crRNA comprising a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1362-1366, and the crRNA is capable of complexing with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with any one of SEQ ID NOs: 1367-1371.

[0292] In some embodiments, the polynucleotide encodes a crRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs: 1388-1418 as shown in Table 34, and the crRNA is capable of complexing with a tracrRNA having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

[0293] In some embodiments, an enzyme coding sequence of a Cas9 orthologue is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res.28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, commercially available codon optimization tools are used (e.g., Gensmart, IDT). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid. 6.5 Methods of altering a cell

[0294] The disclosure further provides methods of using the CRISPR-Cas orthologue nuclease, gRNAs, nucleic acids (including pluralities of nucleic acids), systems, and particles (including pluralities of particles) of the disclosure for altering cells.

[0295] In one aspect, a method of altering a cell comprises contacting a eukaryotic cell (e.g., a human cell) with a polypeptide, nucleic acid, particle, system or pharmaceutical composition described herein.

[0296] 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, systemor pharmaceutical composition. In some embodiments, the methods can further comprise returning or implanting the contacted cell or a progeny thereof to the subject.

[0297] CRISPR-Cas orthologue nuclease, as well as nucleic acids encoding CRISPR-Cas orthologue nuclease and gRNAs can be delivered to a cell by, for example, by viral or non-viral delivery vehicles, electroporation or lipid nanoparticles.

[0298] A polynucleotide encoding CRISPR-Cas orthologue nuclease 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.

[0299] 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).

[0300] 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.

[0301] CRISPR-Cas orthologue nuclease 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 AAVrhl 0 serotype), or by another viral vector.

[0302] 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 CRISPR-Cas orthologue nuclease 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 AAVrhl 0 serotype) are used to deliver both a sgRNA and a CRISPR-Cas orthologue nuclease. In some embodiments, a CRISPR-Cas orthologue nuclease and a sgRNA are delivered using separate vectors. In other embodiments, aCRISPR-Cas orthologue nuclease and a sgRNA are delivered using a single vector. CRISPR-Cas orthologue nuclease with their relatively small size, can be delivered with a gRNA (e.g., sgRNA) using a single AAV vector.

[0303] Compositions and methods for delivering CRISPR-Cas orthologue nuclease 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.

[0304] 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 CRISPR-Cas orthologue nuclease.

[0305] 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.

[0306] 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.

[0307] 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 favoredDNA end joining repair outcome. In some instances, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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.6.5.1. Particles and cells

[0313] The disclosure further provides particles comprising a CRISPR-Cas polypeptide protein of the disclosure (e.g., a Cas9 orthologue nuclease), 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.

[0314] 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 CRISPR-Cas orthologue nuclease proteins to cells (e.g., complexed together as a RNP).

[0315] The disclosure provides particles (e.g., virus particles) comprising a nucleic acid encoding a CRISPR-Cas orthologue nuclease protein of the disclosure. The particles can further comprise a nucleic acid encoding a gRNA. Alternatively, a nucleic acid encoding a CRISPR-Cas orthologue nuclease protein can further encode a gRNA.

[0316] The disclosure further provides pluralities of particles (e.g., pluralities of virus particles). Such pluralities can include a particle encoding a CRISPR-Cas orthologue nuclease protein and a different particle encoding a gRNA. For example, a plurality of particles can comprise a virus particle (e.g., a AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhI O virus particle) encoding a CRISPR-Cas orthologue nuclease protein and a second virus particle (e.g., a AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO virus particle) encoding a gRNA. Alternatively, a plurality of particles can comprise a plurality of virus particles where each particle encodes a CRISPR-Cas orthologue nuclease protein and a gRNA.

[0317] The disclosure further provides cells and populations of cells (e.g., ex vivo cells and populations of cells) that can comprise a CRISPR-Cas orthologue nuclease protein (e.g., introduced to the cell as a RNP) or a nucleic acid encoding the CRISPR-Cas orthologue nuclease 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 CRISPR-Cas orthologue nuclease protein) or a nucleic acid encoding the gRNA (e.g., DNA or mRNA) (optionally also encoding a CRISPR-Cas orthologue nuclease 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. Methods for introducing proteins and nucleic acids to cells are known in the art. For example, a RNP can be produced by mixing a CRISPR-Cas orthologuenuclease 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.

[0318] 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. 6.6 Methods for CRISPR-Cas Protein PAM Identification

[0319] In certain aspects, methods for predicting a PAM sequence recognized by one or more (e.g., some or all) CRISPR-Cas proteins in a CRISPR-Cas protein cluster are described. Predicting a PAM sequence can comprise (a) mapping spacer sequences of CRISPR arrays corresponding to a CRISPR-Cas protein cluster to a set of viral genome sequences to identify putative protospacer sequences in the viral genomes and their flanking upstream and downstream sequences in the viral genomes; (b) for each spacer sequence mapped to one or more viral genome sequences, aligning the putative protospacer sequences and their flanking upstream and downstream viral genome sequences to generate a set of aligned putative protospacer and flanking sequences for each mapped spacer sequence; and (c) predicting a PAM sequence recognized by one or more CRISPR- Cas proteins in the cluster from the sets of aligned putative protospacer and flanking sequences. The method is described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety.

[0320] The method can be computer implemented. Computer implemented methods can comprise executing, in a computer system having one or more processors coupled to a memory storing one or more computer readable instructions for execution by the one or more processors, the one or more computer readable instructions comprising instructions for steps (a)-(c) as described in the preceding paragraph. 6.6.1 PAM predictions for Cas9 clusters

[0321] Interrogation of massive metagenomic datasets combined with a newly developed computational method allows for the identification of a vast number of unreported CRISPR- Cas loci and their respective PAM requirements. FIG.1 depicts an exemplary schematic description of the pipeline used to generate PAM predictions using proprietary methods and systems as described in Ciciani et al.2022, which is incorporated herein by reference in its entirety.

[0322] Referring to FIG.1, from a compendium of 825,698 bacterial and archaeal genomes reconstructed via metagenomic assembly of human, host-associated, and non-host associatedenvironmental microbiomes (see Methods section: Catalog of reference and metagenomic- assembled genomes) and 1222,670 genomes from microbial isolates are retrieved from the NCBI database, the sequences of 92,140 CRISPR-Cas9 loci can be identified. As shown in FIG.1, the pipeline can perform Cas9 orthologues proteins clustering at multiple sequence identity levels (from 95% to 100%) and apply a PAM-prediction procedure on each cluster. For predicting the PAMs, identified sequences adjacent to protospacers are identified, for instance, by aligning 613,478 unique spacers to phage genomes of the human microbiome: 142,809 viral genomes from the Gut Phage Database (Camarillo-Guerrero et al. Cell.2021;184:1098–1109.e9.189,680), from the Metagenomic Gut Viral catalog (Nayfach S, et al. Nat. Microbiol.2021;6:960–970), and from de-novo assembled gut phages from highly enriched viromes as profiled via ViromeQC ( Zolfo M, et al. Detecting contamination in viromes using ViromeQC. Nat. Biotechnol.2019;37:1408–1412). This procedure mostly focused on human-associated bacterial and viral genomes as they are largely overrepresented in the dataset, and in particular the human gut has been sufficiently densely sampled to permit multiple reliable host-virus associations via spacer matching (Supplementary FIG.1 in Ciciani et al.2022). Only full-length, near-perfect matches were retained (at most 4 nucleotide variations), resulting in a total of 39,109,402 putative protospacers. Cas9 clusters with less than 10 mapped spacers are discarded to retain only highly reliable PAM predictions. Upstream and downstream sequences flanking the matches, up to 30 nt, are retrieved. For each Cas9 cluster, sequences flanking the same spacer are realigned and the multiple sequence alignment is collapsed into a single consensus flanking sequence. 6.6.2 Mapping spacer sequences to viral genomes

[0323] The set of viral genome sequences can comprise, for example, a set of phage genomes, for example phage genomes of the human microbiome. Various sources of viral genomes can be used, for example the Gut Phage Database and the Metagenomic Gut Virus catalog (Nayfach et al., 2021 Nat. Microbiol.6:960–970). Viral genomes can also be assembled de novo, for example using BLASTN.

[0324] In some embodiments, the set of viral genomes contains at least 100,000 viral genomes, at least 200,000 viral genomes at least 300,000 viral genomes and / or up to 1 million viral genomes, up to 750,000 viral genomes, up to 500,000 viral genomes, or up to 400,000 viral genomes.

[0325] Spacer sequences of CRISPR arrays corresponding to one or more CRISPR-Cas proteins of a CRISPR-Cas protein cluster can be mapped to a set of viral genome sequences by performing sequence alignments of a given spacer sequence to the viral genome sequences, for example by using a sequence alignment algorithm such as BLASTN. A given spacer sequence can be considered mapped to a viral genome sequence when, for example, the spacer sequencealigns over its full length to a viral genome sequence with a near-perfect or perfect nucleotide match. In some embodiments, a spacer is considered mapped to a viral genome sequence when there are no more than four nucleotide mismatches or gaps (e.g., 4, 3, 2, 1, or 0 nucleotide mismatches or gaps) in an alignment of the full-length spacer sequence and a viral genome sequence.

[0326] The viral genome sequences to which a given spacer sequence is mapped can be identified as putative protospacer sequences. Following identification of putative protospacer sequences corresponding to a given spacer sequence, the viral genomic sequences flanking the putative protospacer sequences both upstream and downstream can be retrieved. The upstream and downstream sequences are retrieved because true CRISPR-Cas protospacer sequences are adjacent to a PAM sequence. Because the PAM is adjacent to the protospacer, it is only necessary to retrieve a relatively small number of flanking nucleotides. In some embodiments, up to 40 nucleotides upstream and up to 40 nucleotides downstream of the putative protospacer sequences are retrieved. In some embodiments, up to 30 nucleotides upstream and up to 30 nucleotides downstream of the putative protospacer sequences are retrieved. In some embodiments, up to 20 nucleotides upstream and up to 20 nucleotides downstream of the putative protospacer sequences are retrieved. In some embodiments, up to 10 nucleotides upstream and up to 10 nucleotides downstream of the putative protospacer sequences are retrieved.

[0327] In FIG.2, the CRISPR-Cas protein cluster is shown as having sequences for three CRISPR-Cas proteins, “Cas protein 1”, “Cas protein 2”, and “Cas protein 3”. It should be understood that the features illustrated in FIG.2 are only exemplary. For example, a CRISPR- Cas cluster can have a few to many CRISPR-Cas protein sequences, for example at least one, at least 10, or at least 100 sequences, and / or up to 100, 1000, 2000, 4000, 5000, 10,000 or more than 10,000 sequences. FIG.2 shows three spacers of CRISPR arrays corresponding to the CRISPR-Cas protein cluster (specifically, “Spacer 1”, “Spacer 2”, and “Spacer 3” from CRISPR arrays corresponding to Cas protein 1, Cas protein 2, and Cas protein 3, respectively). The spacer sequences can be mapped to a set of viral genome sequences. FIG.2 shows that the spacer sequences map to viral genomes 1-9 (specifically, Spacer 1 maps to viral genomes 1-3; Spacer 2 maps to viral genomes 4-6; and Spacer 3 maps to viral genomes 7-9). By aligning the spacer sequences to the viral genomes, putative protospacer sequences in the viral genomes (illustrated in FIG.2 as PP1 to PP9) and their flanking upstream and downstream sequences (illustrated in FIG.2 as black bars flanking PP1 to PP9) can be identified. Then, the putative protospacer sequences and their flanking sequences can be aligned, and consensus sequences generated for each set of alignments. In FIG.2, PP1, PP2, and PP3 and their flanking sequences are aligned and consensus sequence C1 is generated from the alignment because Spacer 1 maps to the viral genome sequences corresponding to PP1, PP2, and PP3. Similarly, PP4, PP5, and PP6 and theirflanking sequences are aligned and consensus sequence C2 is generated from the alignment because Spacer 2 maps to the viral genome sequences corresponding to PP4, PP5, and PP6; and PP7, PP8, and PP9 and their flanking sequences are aligned and a consensus sequence C3 is generated because Spacer 3 maps to the viral genome sequences corresponding to PP7, PP8, and PP9. Consensus sequences can be generated, for example, by taking the most frequent base at each position in an alignment. Columns in an alignment that contain many gaps (e.g., >30% gaps, >40% gaps, >50% gaps) can, in some embodiments, be discarded. Then, the PAM sequence for the CRIPSR-Cas protein cluster can be predicted by identifying conserved nucleotides in the consensus sequences, as one or more conserved nucleotides in either the upstream or downstream flanking sequences are indicative of a PAM sequence. In FIG.2, nucleotides conserved among consensus sequences C1, C2, and C3 are identified by asterisks. Conserved nucleotides can be identified, for example, by calculating nucleotide frequencies at each position for the consensus sequences and identifying positions where a particular nucleotide is present at a relatively high frequency. For example, a nucleotide can be considered conserved at a position if its frequency at the position is larger (e.g., significantly larger) than the frequencies of nucleotides at other positions in the upstream and downstream flanking sequences. In some embodiments, a sequence logo can be generated (e.g., for example by Logomaker (Tareen & Kinney, 2020, Bioinformatics 36:2272–2274)) to represent the nucleotide frequencies at each position. 6.6.3 Predicting PAM sequences from putative protospacer sequences and flanking sequences

[0328] Putative protospacer sequences and their flanking upstream and downstream sequences can be aligned to generate a set of aligned putative protospacer and flanking sequences. For example, a multiple sequence alignment tool such as MUSCLE (Edgar, 2004, Nucleic Acids Research 32(5):1792-97; Edgar, 2004, BMC Bioinformatics 5:113) or MAFFT (Katoh et al., 2002, Nucleic Acids Research 30(14):3059–3066) can be used. In some embodiments, MUSCLE is used. In other embodiments, MAFFT is used.

[0329] As protospacer sequences are adjacent to PAM sequences in viral genomes, the sets of aligned putative protospacer and flanking sequences generated for the different spacer sequences can be used to predict the PAM sequence for the CRISPR-Cas proteins of a CRISPR-Cas protein cluster. For example, a consensus sequence can be generated from each set of aligned putative protospacer and flanking sequences (each set corresponding to a single spacer), for example by taking the most frequent base at each position in the alignment, discarding columns composed of many or composed mostly of (e.g., >50%) gaps. The spacer sequence can be aligned to the consensus sequence to define the upstream and downstream regions of the consensus sequence.

[0330] A consensus sequence can be generated from each set of aligned putative protospacer and flanking sequences, for example as illustrated in FIG.2. The nucleotide frequencies at each position in the upstream and downstream flanking sequences in the consensus sequences can be computed to identify one or more conserved nucleotides, which are indicative of a PAM sequence for a CRISPR-Cas protein cluster. For example, CRISPR-Cas protein clusters generated at a sequence homology of at least 98%, 99%, or 100% having the same predicted PAM are used for generating the PAM consensus sequence. In other words, a PAM sequence for a cluster (and, by extension, each CRISPR- Cas protein whose sequence is in the cluster) can be predicted by analyzing the nucleotide frequencies at nucleotide positions in the upstream and downstream sequences to identify nucleotides present at a relatively high frequency (e.g., outliers), because nucleotides present at a relatively high frequency (e.g., outliers) are likely to correspond to nucleotides of the PAM. In some embodiments, a sequence logo can be generated from the nucleotide frequencies at positions in the upstream and downstream flanking sequences, which can provide a graphical representation of the sequence conservation. In some embodiments, a nucleotide in a sequence logo can be considered a conserved nucleotide when it has more than one bit of information. In some embodiments, a nucleotide is considered conserved when it has more than one bit of information and its bit level is larger than the median bit level in both flanking regions plus 1.5 times the interquartile range of bit levels in both flanking regions. Tools for generating sequence logos include Logomaker (Tareen & Kinney, 2020, Bioinformatics 36:2272–2274). In some embodiments, the methods comprise generating a report with the sequence logo in a computerized system. For example, nucleotide frequencies in the consensus flanking sequences are computed and represented as sequence logos, as illustrated in FIGs.3A-3E, FIGs.4A-4C, FIGs.5A-5B, FIGs.6A-6D, FIGs.7A-7C, FIGs.8A-8B, FIGs.9A-9B, FIG.10, FIGs.11A-11B, FIGs.12A- 12D, FIG.13, FIGs.14A-14B, FIG.15, FIG.16, FIG.17, FIGs.18A-18B, FIG.19, FIG.20, FIG. 21, FIGs.22A-22B, FIGs.23A-23B, or FIGs.24A-24B.

[0331] A PAM is predicted for a Cas9 cluster if there is at least one conserved position in either the upstream or the downstream flanking sequence. Repeating the PAM-prediction procedure on the different Cas9 clustering identity thresholds, the highest reliability is obtained at 98% identity clustering. At this clustering stringency, PAM predictions for 2546 out of 2779 Cas9 clusters (representing 61,095 Cas9 sequences) with more than 10 mapped spacersare obtained. Detailed methods of PAM predictions for Cas9 clusters are described in Methods section in Ciciani et al.2022, which incorporated herein by reference in its entirety. 6.6.4 Cas9s sequence predictions corresponding to the predicted PAMs

[0332] The PAM prediction approach can be validated. Gene sequences coding for proteins with high sequence identity (> 98%) to previously characterized Cas9s are searched for in thedataset: SpCas9 (Jinek et al., 2021, Science 337:816–821), SaCas9 (Ran et al., 2015, Nature 520:186–191), St1Cas9(Garneau et al., 2010, Nature 468:67–71), St3Cas9 (Horvath et al., 2008, J. Bacteriol.190:1401–1412) and SmCas9 (Shields et al., 2020, PLOS Pathog.16:e1008344). For these Cas9s sequence predictions corresponding to the described PAMs is obtained. The method can be further validated by cross checking the PAM predictions obtained with the pipeline with the sequences experimentally identified and recently reported and characterized in Gasiunas et al., 2020, Nat. Commun.11:5512. Of the 79 Cas9s reported, 21 can be used for the evaluation here as they have a close orthologue in the dataset (> 98% identity), and for them the accuracy of the prediction strategy was confirmed by obtaining PAM logos with high identity (assessed by Jensen- Shannon distance on nucleotide frequencies). The accuracy of PAM predictions generated by the method is as high as 85%. 6.6.5 Identification of Cas9 subtypes capable of recognizing the predicted PAM

[0333] To further evaluate the reliability and potential of the PAM prediction pipeline in expanding the Cas9 toolbox from the databank, Cas9 candidates can be searched for using parameters favoring the identification of functionally active enzymes (with preserved domain structures and located in complete CRISPR-Cas loci) and with reduced molecular size (< 1,100 amino acids), thus potentially more convenient for genome editing applications. Their PAM logos can be predicted and subsequently validated through an in vitro assay (Karvelis et al., 2019, Methods in Enzymology 616:219–240). Overall, the method allows PAM prediction for the vast majority of Cas9 orthologues proteins identified in the databank with 10 or more mapped spacers, across all Cas9 subtypes (93.6% for A, 93.0% for B and 87.9% for C). 6.6.6 Generation of Cas9 orthologues protein clusters capable of recognizing the predicted PAMs

[0334] The PAM predictor method is then applied to the metagenomically extended set of 14909 Cas9 orthologues protein families (98% identity clustering) to identify PAM requirements and explore whether specific PAM clusters may exist. For each Cas9 orthologue cluster, a PAM preference may be generated (FIGs.3A-3E, FIGs.4A-4C, FIGs.5A-5B, FIGs.6A-6D, FIGs.7A- 7C, FIGs.8A-8B, FIGs.9A-9B, FIG.10, FIGs.11A-11B, FIGs.12A-12D, FIG.13, FIGs.14A- 14B, FIG.15, FIG.16, FIGs.17, FIGs.18A-18B, FIG.19, FIG.20, FIG.21, FIGs.22A-22B, FIGs.23A-23B, or FIGs.24A-24B). Interestingly, the most prevalent PAM sequences represented only a small fraction of all possible PAMs. Therefore, even though the PAM variability is high for Type II Cas9 (Vink et al., 2021, Genome Biol.22:281), only definite combinations of nucleotides were identified.6.6.7 CRISPR-Cas protein clusters

[0335] CRISPR-Cas protein clusters can be generated from an initial set of CRISPR-Cas proteins (e.g., an initial set of CRISPR-Cas proteins having at least 10,000, at least 50,000, at least 75,000, at least 90,000 and / or up to 1 million, up to 750,000, up to 500,000, up to 200,000, or up to 100,000 CRISPR-Cas protein sequences) by use of a clustering algorithm that clusters amino acid sequences by percent identity. For example, in some embodiments UCLUST (Edgar, 2010, Bioinformatics 26:2460–2461) or CD-HIT (Weizhong & Godzik, 2006, Bioinformatics 22(13):1658-1659) are used. In some embodiments, UCLUST is used to cluster CRISPR-Cas proteins at the 95%, 96%, 97%, 98%, 99%, or 100% identity level. In some embodiments, UCLUST is used to cluster CRISPR-Cas proteins at the 98% identity level. PAM prediction methods of the disclosure comprise a step of generating one or more CRISPR-Cas protein clusters. Alternatively, PAM prediction can be performed using one or more previously generated CRISPR-Cas protein clusters. Methods for generating CRISPR-Cas protein clusters are described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety.

[0336] An initial set of CRISPR-Cas proteins used to make a CRISPR-Cas protein cluster can be obtained from bacterial and / or archaeal genomes. For example, genomes (e.g., at least 100,000, 200,000, 500,000, 800,000, 1 million, or more, and / or up to 2 million genomes) can be retrieved from a database, for example NCBI, and an algorithm can be used to identity CRISPR-Cas loci, for example CRISPR-Cas9 loci. Algorithms for identifying CRISPR-Cas loci are known in the art and include, for example, CRISPRCasTyper (Russel et al., 2020, CRISPR J.3:462–469). PAM prediction methods of the disclosure comprise a step of generating an initial set of CRISPR-Cas protein sequences, which is then used to generate one or more CRISPR-Cas protein clusters. Alternatively, a pre-existing set of CRISPR-Cas protein sequences can be used to generate one or more CRISPR-Cas protein clusters.

[0337] The methods of the disclosure can be performed using various types of CRISPR-Cas protein sequences. For example,` the CRISPR-Cas proteins in a CRISPR-Cas protein cluster can comprise or consist of Class II Cas proteins, e.g., Type II Cas proteins (e.g., Cas9), Type V Cas proteins (e.g., Cas12a), or Type VI Cas proteins (e.g., Cas13) (for reviews of Class II Cas proteins, see Makarova et al., 2020, Nat Rev Microbiol 18(2):67-83; Tong et al., 2020, Front Cell Dev Biol.8:622103; and Chylinksi et al., 2014 Nucleic Acids Res.42(10):6091-6105, the contents of which are incorporated herein by reference in their entireties). Type II Cas proteins include, for example, Type II-A, Type II-B, and Type II-C. Type V Cas proteins include, for example, Type V-A, Type V-B, Type V-C, Type V-D, Type V-E, Type V-F, Type V-G, Type V- H, Type V-I, Type V-J, and Type V-K. In some embodiments, the CRISPR-Cas proteins are Type II Cas proteins, for example Cas9 orthologues proteins. In other embodiments, the CRISPR-Cas proteins are Type V Cas proteins (e.g., Cas12a).

[0338] An initial set of CRISPR-Cas proteins (e.g., Type II Cas proteins such as Cas9 proteins) contains protein sequences having a minimum and / or maximum sequence length. For example, a minimum length can be 150, 200, 400, 500, 600, 700, 800, 900, 950, 1000, 1100, 1200 or 1300 amino acids. A maximum length can be, for example, 2200, 2100, 2000, 1900, 1800, 1232, 1600, 1500, 1400, 1300, 1200 or 1100 amino acids. Selecting a maximum length, for example of 1100 amino acids, can be used, for example, to limit a set of CRISPR-Cas proteins to those which can be packaged together with a gRNA in a single AAV vector genome. In other embodiments, an initial set of CRISPR-Cas proteins is not limited to those having a particular amino acid sequence length.

[0339] The number of CRISPR-Cas protein sequences in a CRISPR-Cas protein cluster can vary, for example depending on the number of CRISPR-Cas protein sequences in an initial set of CRISPR-Cas proteins subjected to clustering and / or depending on how divergent the sequences in the initial set of CRISPR-Cas protein are from each other. A CRISPR-Cas protein cluster can contain, for example, at least 1, at least 10, or at least 100 CRISPR-Cas protein sequences.

[0340] Members in a CRISPR-Cas protein cluster may have at least one PAM preferences. Members in a CRISPR-Cas protein cluster may have different PAM preferences, e.g., 1, 5, 10, 20, 50, or more PAM preferences.

[0341] To increase reliability of PAM predictions, clusters having a low number of mapped spacers can in some embodiments be discarded. For example, in some embodiments, cluster having fewer than 50, fewer than 25, or fewer than 10 mapped spacers are discarded. In some embodiments, clusters having fewer than 10 mapped spacers are discarded. In some embodiments, clusters having fewer than 5 mapped spacers are discarded.

[0342] In some embodiments, a PAM prediction method of the disclosure is repeated using a different CRISPR-Cas protein cluster(s) and / or a different set of viral genomes from those used initially. Repeating the method using a different CRISPR-Cas protein cluster(s) and / or a different set of viral genomes can be useful, for example, to update a CRISPR-Cas protein library with additional CRISPR-Cas protein sequences and / or to update a CRISPR-Cas protein library after a new set of viral genomes becomes available.

[0343] The methods can further comprise a step of generating a report with the predicted PAM for a CRISPR-Cas protein cluster. The report can include, for example, identifying information for one or more CRISPR-Cas proteins (e.g., CRISPR-Cas protein name and / or ID number and / or CRISPR-Cas protein amino acid sequences and / or species information) and their corresponding predicted PAM sequences. The information in a report can be included in a CRISPR-Cas protein library of the disclosure.

[0344] Following prediction of a PAM for a CRISPR-Cas protein cluster, the predicted PAM can optionally be validated, for example by an in vitro assay using one or more CRISPR-Cas proteins of the cluster, for example as described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety.

[0345] To validate a PAM in vitro, tracrRNA sequences for the one or more CRISPR-Cas proteins to be used for PAM validation can be determined so that an appropriate gRNA for the in vitro assay can be designed. tracrRNA sequences can be identified computationally, for example, as described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety. tracrRNA sequences, which contain an anti-repeat and a Rho-independent terminator (RIT), can be identified by aligning CRISPR repeats to sequences flanking a CRISPR-Cas locus (e.g., up to 1000 nucleotides) (for example, using BLASTN) to identify putative anti-repeats. RITs can be predicted using RNIE software (Gardner et al., 2011, Nucleic Acids Res.39:5845–5852). Other methods for predicting tracrRNAs can also be used, for example as described in Chyour and Brown 2019, RNA Biol.16(4):423-434 or Dooley et al., 2021 CRISPR J.4(3):438-447, each of which is incorporated herein by reference in its entirety.

[0346] When PAMs are predicted for a plurality of CRISPR-Cas protein clusters, the CRISPR- Cas protein sequences of the CRISPR-Cas protein clusters can be clustered according to their predicted PAM sequences to generate PAM clusters. PAM clustering can be performed, for example, by computing an all-to-all PAM prediction distance matrix using a clustering tool such as usearch (Edgar, 2010, Bioinformatics 26:2460–2461). Hierarchical clustering can be performed on a PAM cluster, for example as described in Example 1. PAM clustering can be useful for measuring the diversity of a library, and for studying the hierarchical relationship between CRISPR-Cas proteins in a library.

[0347] As described herein, by interrogating an extended microbiome databank with an accurate computational pipeline, a large variety of new Cas9 nucleases can be identified accompanied by their PAM requirements. This analysis reveals that PAM sequences follow defined nucleotide patterns which are associated with specific Cas9 subtypes and overlap with 98.6% of the pathogenic mutations reported in ClinVar (Landrum et al., 2018, Nucleic Acids Res.46:D1062–D1067; Ciciani et al., 2022). This approach opens to the expansion of the genome editing toolbox with mutation-tailored nucleases and supports the strategy of an application-specific search for suitable natural prokaryotic genome editing tools requiring minimal or no engineering. 6.7 Libraries of CRISPR-Cas Proteins with Predicted PAMs

[0348] In another aspect, the disclosure provides methods of selecting a CRISPR-Cas protein of interest, for example a previously uncharacterized CRISPR-Cas protein. The method is based onthe prediction and validation that a CRISPR-Cas protein (e.g., Cas9 orthologue) is capable of recognizing a putative PAM sequence (e.g., a consensus PAM sequence) as described in Ciciani et al., 2022, which is incorporated herein by reference in its entirety. Such methods can comprise identifying a CRISPR-Cas protein in a library of the disclosure having relatively high (or highest) amino acid sequence identity with the CRISPR-Cas protein of interest (e.g., greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100% identity), and predicting that the PAM sequence of the CRISPR-Cas protein of interest is the same as the PAM sequence of the CRISPR-Cas protein in the library. In some embodiments, such methods are computer implemented. After a PAM is predicted for the CRISPR-Cas protein of interest, the prediction can optionally be validated in vitro.

[0349] In another aspect, the disclosure provides methods for selecting a CRISPR-Cas protein for editing a genomic sequence comprising a PAM of interest, comprising (a) identifying, in a CRISPR-Cas library of the disclosure, one or more CRISPR-Cas proteins whose predicted PAM sequences correspond to the PAM sequence of interest; (b) evaluating the ability of the one or more CRISPR-Cas proteins to edit the genomic sequence (e.g., in an in vitro gene editing assay); and (c) selecting a CRISPR-Cas protein from the one or more CRISPR-Cas proteins following the evaluation. The selected CRISPR-Cas protein(s) can be, for example, the CRISPR-Cas protein(s) having the highest editing activity and / or highest fidelity in an in vitro gene editing assay. In some embodiments, step (a) is computer implemented.

[0350] In another aspect, the disclosure provides methods for selecting a genomic sequence having a pathogenic mutation for editing with a CRISPR-Cas protein, comprising (a) identifying, in a CRISPR-Cas library of the disclosure, a PAM sequence associated with a pathogenic mutation (e.g., a PAM sequence created by the pathogenic mutation); and (b) selecting a genomic sequence that includes that PAM sequence for editing with a CRISPR- Cas protein. For example, predicted PAM sequences in a library of the disclosure can be aligned with a set of genomic sequences having pathogenic or likely pathogenic mutations, for example mutations in the ClinVar database (Landrum et al., 2018, Nucleic Acids Res.46: D1062–D1067) to identify genomic sequences that include a PAM sequence present in a library of the disclosure. Genomic sequences that include a sequence that matches exactly to a PAM sequence in the library can be selected for editing. CRISPR-Cas proteins in the library having that PAM sequence can be selected and evaluated for their ability to edit the selected genome sequence. For autosomal dominant diseases, if the PAM sequence is present in the mutant allele but not the wild-type allele, allele specific editing can be achieved.

[0351] In another aspect, the disclosure provides methods of designing a guide RNA (gRNA) molecule for a CRISPR-Cas protein in a CRISPR-Cas protein library of the disclosure. ThegRNAs can be designed, for example, for a CRISPR-Cas protein selected by a method of the disclosure to edit a genomic sequence of interest, for example, a genomic sequence having a PAM of interest, as described herein. Design of a gRNA can be computer implemented. For example, design of a gRNA can comprise identifying a tracrRNA sequence for a selected CRISPR-Cas protein, and selecting targeting sequence that is complementary to a target genomic sequence and adjacent to the PAM for the selected CRISPR-Cas protein. The designed gRNA can comprise separate crRNA and tracrRNA or can comprise an sgRNA.

[0352] In another aspect, the disclosure provides methods for editing a genomic sequence. Such methods can comprise contacting a cell (e.g., a mammalian cell such as a human cell) with a system comprising a CRISPR-Cas protein selected according to a method described herein and a gRNA for editing the genomic sequence. In some embodiments, the contacting is performed ex vivo. In other embodiments, the contacting is performed in vivo. In some embodiments, the method comprises contacting a cell with a nucleic acid (e.g., an AAV genome) encoding both the CRISPR-Cas protein and the gRNA. In other embodiments, the method comprises contacting a cell with a nucleic acid (e.g., an AAV genome) encoding the CRISPR-Cas protein and a different nucleic acid (e.g., an AAV genome) encoding the gRNA. When the nucleic acid is an AAV genome, the method can comprise contacting the cell with an AAV particle comprising the AAV genome. In other embodiments, the method comprises contacting a cell with a ribonucleoprotein complex comprising the CRISPR-Cas protein and gRNA. 6.8 Methods of treatment

[0353] In another aspect the invention provides a method of treating a subject in need thereof, the method comprising administering a polypeptide (e.g., a CRISPR-Cas orthologue nuclease and / or a gRNA polypeptide), system, nucleic acid, expression vector, or pharmaceutical composition as described herein to the subject. In another aspect, the invention provides a polypeptide (e.g., a CRISPR-Cas orthologue nuclease and / or a gRNA polypeptide), system, nucleic acid, expression vector, or pharmaceutical composition as described herein for use in therapy. In yet another aspect, invention provides a polypeptide (e.g., a CRISPR-Cas orthologue nuclease and / or a gRNA polypeptide), system, nucleic acid, expression vector, or pharmaceutical composition as described herein for use in a method of treating or preventing a disease or condition caused by a genetic defect (e.g., a disease or condition caused by a protein deficiency or over-abundance), the method comprising administering said polypeptide (e.g., a CRISPR-Cas orthologue nuclease and / or a gRNA polypeptide), system, nucleic acid, expression vector, or pharmaceutical composition to the subject. In some embodiments, the invention does not comprise a process for modifying the germ line genetic identity of human beings, and / or the use of human embryo for industrial or commercial purposes.7. FURTHER EMBODIMENTS

[0354] Further embodiments are provided in the following numbered embodiments. 1. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas polypeptide comprising the following components: an amino acid sequence having at least about 90% identity to any one of the amino acid sequences set forth in Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A- 10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. 2. The CRISPR-Cas polypeptide according to embodiment 1, wherein the CRISPR-Cas polypeptide amino acid sequence and the corresponding PAM polynucleotide nucleic acid sequence are as shown in any one of Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A- 6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). 3. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas polypeptide system comprising the following components: an amino acid sequence having at least about 90% identity to any one of the amino acid sequences set forth in Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A- 10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof; and a single guide RNA polynucleotide.4. The CRISPR-Cas polypeptide system according to embodiment 3, wherein the CRISPR-Cas polypeptide amino acid sequence and the corresponding PAM polynucleotide nucleic acid sequence are as shown in any one of Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). 5. The CRISPR-Cas polypeptide system according to embodiment 3 or 4, wherein the single guide RNA polynucleotide comprises: a spacer sequence comprising a nucleotide sequence that is complementary to / hybridizes with a portion of a target polynucleotide located upstream of the PAM polynucleotide. 6. The CRISPR-Cas polypeptide system according to any one of embodiments 3-5, wherein the guide polynucleotide comprises: (a) a CRISPR RNA (crRNA) comprising a 3' region and a 5' region, wherein the 3' region comprises at least about 10 consecutive nucleotides of a CRISPR repeat comprising the nucleotide sequence of the crRNA from the CRISPR-Cas polypeptide, and / or a functional fragment thereof, and the 5' region comprises at least about 10 consecutive nucleotides of a spacer sequence located upstream of the repeat; and (b) a trans-activating crRNA (tracrRNA) comprising a 5' and 3' region wherein at least a portion of the 5' region of the tracrRNA is complementary to the 3' region of the crRNA, wherein the tracrRNA comprises the nucleotide sequence of the tracrRNA from the CRISPR-Cas polypeptide. 7. A protein-polynucleotide complex comprising: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas polypeptide comprising an amino acid sequence having at least about 90% identity to any one of the amino acid sequences set forth in Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A- 9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof; anda single guide RNA polynucleotide, 8. The protein-polynucleotide complex according to embodiment 7, wherein the guide polynucleotide comprises: (a) a spacer sequence comprising a nucleotide sequence that is complementary to a portion of a target polynucleotide located upstream of the PAM polynucleotide. 9. The protein-polynucleotide complex according to embodiment 8, wherein the spacer sequence recognizes / hybridizes with the portion of the target polynucleotide located upstream of the PAM polynucleotide. 10. The protein-polynucleotide complex according to any one of embodiments 7-9, wherein the guide polynucleotide comprises: (a) a CRISPR RNA (crRNA) comprising a 3' region and a 5' region, wherein the 3' region comprises at least about 10 consecutive nucleotides of a CRISPR repeat comprising the nucleotide sequence of the crRNA from the CRISPR-Cas polypeptide, and / or a functional fragment thereof, and the 5' region comprises at least about 10 consecutive nucleotides of a spacer sequence located upstream of the repeat; and (b) a trans-activating crRNA (tracrRNA) comprising a 5' and 3' region wherein at least a portion of the 5' region of the tracrRNA is complementary to the 3' region of the crRNA, wherein the tracrRNA comprises the nucleotide sequence of the tracrRNA from the CRISPR-Cas polypeptide. 11. The protein-polynucleotide complex according to any one of embodiments 7-10, wherein the CRISPR-Cas polypeptide amino acid sequence and the corresponding PAM polynucleotide nucleic acid sequence are as shown in any one of Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). 12. A method of cleaving a polynucleotide, the method comprising: contacting a target polynucleotide having a target nucleotide sequence with the protein- polynucleotide complex according to any one of embodiments 7-11, wherein the PAM polynucleotide is recognized by the protein-polynucleotide complex; wherein the guide polynucleotide comprises a spacer sequence that is complementary to a portion of a target polynucleotide located upstream of the PAM polynucleotide and the spacernucleotide sequence hybridizes with the portion of a target polynucleotide sequence to form a double stranded polynucleotide; wherein hybridization of the spacer sequence with the portion of the target polynucleotide sequence allows for site specific cleaving of the target polynucleotide; and thereby cleaving the target polynucleotide with the CRISPR-Cas polypeptide. 13. A method of cleaving a polynucleotide, the method comprising: contacting a target polynucleotide with the protein-polynucleotide complex according to any one of embodiments 7-11, wherein the target polynucleotide comprises (a) a protospacer sequence comprising a nucleotide sequence that is at least 80% complementary to the spacer sequence in the crRNA of the protein-polynucleotide complex; and (b) a PAM polynucleotide comprising a nucleotide sequence as categorized in any one of Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A- 21B (Cluster 41), and Table 22 (Cluster 99) adjacent to the protospacer sequence, thereby cleaving the polynucleotide in the region defined by complementary binding of the protospacer sequence to the spacer sequence in the crRNA of the protein-polynucleotide complex. 14. A nuclease system for performing nucleic acid-guided nuclease site specific genome targeting of a target polynucleotide, wherein the nuclease system comprises: (a) a nuclease having amino acid sequence identity of at least 90% to any one of the amino acid sequences set forth in Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof; and (b) at least one guide RNA molecule. 15. The nuclease system according to embodiment 14, wherein the nuclease has an amino acid sequence of 92% identity to any one of the amino acid sequences set forth in Tables 1A- 1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. 16. The nuclease system according to embodiment 14 or 15, wherein the nuclease has an amino acid sequence of 95% identity to any one of the amino acid sequences set forth in Tables 1A- 1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. 17. The nuclease system according to any one of embodiments 14-16, wherein the nuclease has an amino acid sequence of 99% identity to any one of the amino acid sequences set forth in Tables 1A-1E (Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15 (Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99), and / or a functional fragment thereof. 18. The nuclease system according to any one of embodiments 14-17, wherein the nuclease is encoded by a human codon optimized sequence. 19. The nuclease system according to any one of embodiments 14-18, wherein the guide RNA molecule recognizes (hybridizes with) a portion of the target polynucleotide upstream of the PAM polynucleotide. 20. The nuclease system according to embodiment 19, wherein hybridization of the guide RNA molecule with the target polynucleotide allows for site specific genome editing or cleaving of the target polynucleotide. 21. The nuclease system according to embodiment 20, wherein the nuclease is a Cas9 nickase variant or a dCas9.22. The nuclease system according to any one of embodiments 14-21, wherein the guide RNA molecule is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a trans- activating small RNA (tracrRNA) linked by a loop. 23. The nuclease system according to embodiment 22, wherein the crRNA and the tracrRNA are linked by a synthetic loop. 24. The nuclease system according to embodiment 22 or embodiment 23, wherein the crRNA and tracrRNA respectively comprises: (a) the crRNA comprises a 3' region and a 5' region, wherein the 3' region comprises at least about 10 consecutive nucleotides of a CRISPR repeat comprising the nucleotide sequence of the crRNA from the nuclease, and / or a functional fragment thereof, and the 5' region comprises at least about 10 consecutive nucleotides of a spacer sequence located upstream of the repeat; and (b) the tracrRNA comprises a 5' and 3' region wherein at least a portion of the 5' region of the tracrRNA is complementary to the 3' region (CRISPR repeat) of the crRNA, wherein the tracrRNA comprises the nucleotide sequence of the tracrRNA from the nuclease. 25. The nuclease system according to any one of embodiments 22-24, wherein the sgRNA has a sequence with a length of from about 10 to about 250 nucleotides (nt), or from about 80 to about 110 nt, or from about 15 to about 30 nt, or from about 20 to about 53 nt, or from about 25 to about 53 nt, or from about 29 to about 53 nt, or from about 40 to about 50 nt. 26. The nuclease system according to any one of embodiments 14-25, wherein the system is configured to perform specific genome targeting of a target polynucleotide in a prokaryotic cell or a eukaryotic cell. 27. The nuclease system according to embodiment 26, wherein the prokaryotic cell is a bacterial cell. 28. The nuclease system according to embodiment 27, wherein the eukaryotic cell is a mammalian cell. 29. The nuclease system according to embodiment 28, wherein the mammalian cell is a human cell. 30. An expression vector comprising a nucleic acid molecule encoding the polypeptide according to any one of embodiments 1-2, a nucleic acid molecule encoding the polypeptide according to any one of embodiments 3-6, a nucleic acid molecule encoding the protein of the protein- polynucleotide complex according to any one of embodiments 7-11, or a nucleic acid molecule encoding the nuclease according to any one of embodiments 14-29.31. An expression vector comprising a nucleic acid molecule encoding the guide polynucleotide according to any one of embodiments 1-2, a nucleic acid molecule encoding the guide polynucleotide according to any one of embodiments 3-6, a nucleic acid molecule encoding the guide polynucleotide according to any one of embodiments 7-11, or a nucleic acid molecule encoding the guide RNA molecule according to any one of embodiments 14-29. 32. A cell comprising the expression vector according to embodiment 30 or embodiment 31. 33. The cell according to embodiment 32, wherein the cell is a prokaryotic cell or a eukaryotic cell. 34. The cell according to embodiment 33, wherein the prokaryotic cell is a bacterial cell. 35. The cell according to embodiment 33, wherein the eukaryotic cell is a mammalian cell. 36. The cell according to embodiment 35, wherein the mammalian cell is a human cell. 37. A method of cleaving a DNA target in a cell comprising providing to the cell the nuclease system according to any one of embodiments 14-29. 38. The method according to embodiment 37, comprising contacting a target molecule with the nuclease system, wherein the target molecule comprises a protospacer sequence; the nuclease recognizes a protospacer adjacent motif (PAM) polynucleotide sequence (downstream) of the protospacer sequence; the guide RNA molecule is a sgRNA sequence comprising a spacer sequence that is complementary to the protospacer sequence, wherein the spacer sequence hybridizes with the protospacer sequence to form a double stranded nucleotide sequence; and the nuclease cleaves the target molecule. 39. A method of modulating translation in a cell comprising administering to the cell an effective amount of the nuclease system according to any one of embodiment 14-29. 40. An in vivo method for site-specifically editing a cellular genome at a desired target sequence, the method comprising: administering to a subject the system of any one of embodiment 14-29. 41. A method for site-specifically editing a cellular genome at a desired target sequence, the method comprising: introducing into the cell the system of any one of embodiment 14-29. 8. EXAMPLES8.1 Example 1: Identification of Cas9 orthologues proteins and corresponding PAMs

[0355] This Example illustrates identification of Cas9 orthologues proteins and corresponding PAMs, using the novel nucleases (e.g., Cas9 orthologues proteins) mined / identified using a version of the strategy outlined in FIGs.1 and 2 above. The method mined / identified 14909 Cas9 families in the database. For example, members in a Cas9 family have sequence identity of at least 98%. The mined / identified Cas9 orthologues proteins were blasted against known Cas9 orthologues, and any Cas9 orthologues proteins having greater than 90% identity to a previously disclosed orthologue in the literature were discarded, which reduced the number of hits to 9554 families, each sequence having less than 90% identity to known Cas9 orthologues proteins. The remaining Cas9 orthologues protein families were clustered by amino acid sequence identity (e.g., at least 90%) into 5897 clusters. The Cas9 clusters having a single member were discarded, resulting in 1406 clusters. The Cas9 orthologue / PAM pairs can be validated using either an in vitro assay. Both methods required the nuclease to cut a specific target sequence that is then sequenced by next-generation sequencing (NGS). 8.1.2. Results

[0356] In total, 46 clusters covering 932 unique Cas9 orthologue proteins were identified. Each cluster having Cas9 orthologues proteins having at least 90% amino acid sequence similarity. Among the identified clusters, 27 clusters have at most 1250 amino acids and 19 clusters have at least 1250 amino acids. Exemplary amino acid sequences of the mined / identified Cas9 orthologues proteins were determined and set forth in Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A-5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A-12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). Each of the Cas9 orthologues proteins was found to have at least one PAM polynucleotide (e.g., a PAM ID) and having a nucleic acid sequence (consensus sequence, e.g., motif) as shown in Tables 1A-1E(Cluster 9), Tables 2A-2C (Cluster 10), Tables 3A-3B (Cluster 11), Tables 4A-4D (Cluster 12), Tables 5A- 5C (Cluster 13), Tables 6A-6B (Cluster 14), Tables 7A-7D (Cluster 15), Table 8 (Cluster 16), Tables 9A-9B (Cluster 17), Tables 10A-10D (Cluster 18), Table 11 (Cluster 20), Tables 12A- 12B (Cluster 21), Table 13 (Cluster 22), Table 14 (Cluster 23), Table 15(Cluster 25), Tables 16A-16B (Cluster 28), Table 17 (Cluster 29), Table 18 (Cluster 32), Table 19 (Cluster 33), Tables 20A-20B (Cluster 38), Tables 21A-21B (Cluster 41), and Table 22 (Cluster 99). The predicted PAM logos or motifs were presented in FIGs.3A-3E (correspondence to Nuclease IDshown in Tables 1A-1E, respectively), FIGs.4A-4C (correspondence to Nuclease ID shown in Tables 2A-2C respectively), FIGs.5A-5B (correspondence to Nuclease ID shown in Tables 3A- 3B, respectively), FIGs.6A-6D (correspondence to Nuclease ID shown in Tables 4A-4D, respectively), FIGs.7A-7C (correspondence to Nuclease ID shown in Tables 5A-5C, respectively), FIGs.8A-8B (correspondence to Nuclease ID shown in Tables 6A-6B, respectively), FIGs.9A-9D (correspondence to Nuclease ID shown in Tables 7A-7D, respectively), FIG.10 (correspondence to Nuclease ID shown in Table 8), FIGs.11A-11B (correspondence to Nuclease ID shown in Tables 9A-9B, respectively), FIGs.12A-12D (correspondence to Nuclease ID shown in Tables 10A-10d, respectively), FIG.13 (correspondence to Nuclease ID shown in Table 11), FIGs.14A-14B (correspondence to Nuclease ID shown in Tables 12A-12B, respectively), FIG.15 (correspondence to Nuclease ID shown in Table 13), FIG.16 (correspondence to Nuclease ID shown in Table 14), FIG.17 (correspondence to Nuclease ID shown in Table 15), FIGs.18A-18B (correspondence to Nuclease ID shown in Tables 16A-16B, respectively), FIG.19 (correspondence to Nuclease ID shown in Table 17), FIG.20 (correspondence to Nuclease ID shown in Table 18), FIG.21 (correspondence to Nuclease ID shown in Table 19), FIGs.22A-22B (correspondence to Nuclease ID shown in Tables 20A-20B, respectively), FIGs.23A-23B (correspondence to Nuclease ID shown in Tables 21A-21B, respectively), and FIGs.24A-24B (correspondence to Nuclease ID shown in Table 22). Table 1A (cluster 9_1) Nuclease ID PAM Motif (5’-3’) Cas9 orthologue nuclease amino acid sequence SEQ ID NO.: AJCV NNNNAA SEQ ID NO.: 1AJGW ANATACNNT SEQ ID NO.: 2 DOEQ NNANAC SEQ ID NO.: 3 DKQW - SEQ ID NO.: 4 GBWG - SEQ ID NO.: 5 GCCE NNGNAA SEQ ID NO.: 6 AJDO - SEQ ID NO.: 7 DKQC - SEQ ID NO.: 8 DKRM - SEQ ID NO.: 9 Table 1B (Cluster 9_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BCIM NTGANNA SEQ ID NO.: 10 CFXD NTGANNA SEQ ID NO.: 11 AWDL NTGANNA SEQ ID NO.: 12 DFBQ NTGANNA SEQ ID NO.: 13 GEDZ NNGAA SEQ ID NO.: 14 CKMT NNGAAGA SEQ ID NO.: 15 AIMC - SEQ ID NO.: 16 EDIJ NTGANGA SEQ ID NO: 18Table 1B (Cluster 9_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BBGG - SEQ ID NO.: 17 Table 1C (Cluster 9_10)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:BBSJ NNGNAASEQ ID NO.: 27BBSD NNGNAASEQ ID NO.: 28BBRE NNGNAACSEQ ID NO.: 29DQSW NNATAASEQ ID NO.: 30BBZD NNANAAMSEQ ID NO.: 31BKGA NNANAASEQ ID NO.: 32BBQY -SEQ ID NO.: 33DOGZ -SEQ ID NO.: 34DQWJ -SEQ ID NO.: 35Table 1D (Cluster 9_14)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:AFXD NNNNCCSEQ ID NO.: 36AFZL NNNNCCSEQ ID NO.: 37ADKA NNNNCCSEQ ID NO.: 38DJTA NNNNCCSEQ ID NO.: 39ACJO NNNNCCSEQ ID NO.: 40FQCM NNNGCCSEQ ID NO.: 41DJKN -SEQ ID NO.: 42AGDD -SEQ ID NO.: 43FWNI -SEQ ID NO.: 44Table 1E (Cluster 9_17)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:FHJJ NGCCATSEQ ID NO.: 45FJNI NNNNACSEQ ID NO.: 46FOHR NNCNNTSEQ ID NO.: 47FMZW -SEQ ID NO.: 48FHUL NGNNNTSEQ ID NO.: 49FPJO -SEQ ID NO.: 50FNEJ -SEQ ID NO.: 51FISJ -SEQ ID NO.: 52FHVU -SEQ ID NO.: 53Table 2A (Cluster 10_5)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:AEGP NNGAAA SEQ ID NO.: 54 EMAN NNGAAA SEQ ID NO.: 55 FKUJ NNAAAA SEQ ID NO.: 56 AEAZ NNAAAA SEQ ID NO.: 57Table 2A (Cluster 10_5)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:AAPN NNGNAAA SEQ ID NO.: 58 ANWG NNGAAA SEQ ID NO.: 59 CZSW NNATAA SEQ ID NO.: 60 FLFB - SEQ ID NO.: 61 AYTO NNACAAA SEQ ID NO.: 62 ATCF - SEQ ID NO.: 63 Table 2B (Cluster 10_7)Nuclease ID PAM Motif (5’-3’)SEQ ID NO.:ATJR NNGCAC SEQ ID NO.: 64 DJTP - SEQ ID NO.: 65 DWAS NNNCAC SEQ ID NO.: 66 AVTS - SEQ ID NO.: 67 DJGG - SEQ ID NO.: 68 FQBP NNNNAC SEQ ID NO.: 69 GMLJ NNNCAC SEQ ID NO.: 70 DLTB NNNCAC SEQ ID NO.: 71 AVUM - SEQ ID NO.: 72 DIXY - SEQ ID NO.: 73 Table 2C (Cluster 10_8) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: GDZM NNNCCC SEQ ID NO.: 74 DJXL NNNCCC SEQ ID NO.: 75 DUUZ NNNCCC SEQ ID NO.: 76 AOHY NNNCCC SEQ ID NO.: 77 DQMD - SEQ ID NO.: 78 AOMM NNNCCC SEQ ID NO.: 79 BNJN NNNCCC SEQ ID NO.: 80 DZSG - SEQ ID NO.: 81 AOLW - SEQ ID NO.: 82 CCPM - SEQ ID NO.: 83 Table 3A (Cluster 11_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BAGF NNNNNNAA SEQ ID NO.: 106 BAGO NNNNCC SEQ ID NO.: 107 DYYL NNNNNNAA SEQ ID NO.: 108 CHBN - SEQ ID NO.: 109 BAIW NNNNCC SEQ ID NO.: 110 BAHB NNNACC SEQ ID NO.: 111 FRRP - SEQ ID NO.: 112 DMQH - SEQ ID NO.: 113 FRCB - SEQ ID NO.: 114Table 3A (Cluster 11_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FUYG - SEQ ID NO.: 115 BSDN - SEQ ID NO.: 116 Table 3B (Cluster 11_10) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ABWM - SEQ ID NO.: 128 ACWK NNNNNTTA SEQ ID NO.: 129 DMOP NNNNNCNAA SEQ ID NO.: 130 BMTS - SEQ ID NO.: 131 DNPM - SEQ ID NO.: 132 DKRR - SEQ ID NO.: 133 ABYM - SEQ ID NO.: 134 DLIR - SEQ ID NO.: 135 DJXY - SEQ ID NO.: 136 DKPY - SEQ ID NO.: 137 DJCE - SEQ ID NO.: 138 Table 4A (Cluster 12_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: DHKL - SEQ ID NO.: 139 AYLK NNGNNNTA SEQ ID NO.: 140 AYXV NNNNNNAA SEQ ID NO.: 141 DGMW NNNNNCAA SEQ ID NO.: 142 BBMH - SEQ ID NO.: 143 EBRA - SEQ ID NO.: 144 BEDO - SEQ ID NO.: 145 BBLQ - SEQ ID NO.: 146 BAVZ - SEQ ID NO.: 147 BFGY - SEQ ID NO.: 148 BEFG - SEQ ID NO.: 149 DOBC - SEQ ID NO.: 150 Table 4B (Cluster 12_4) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ECNC - SEQ ID NO.: 151 CCWE NNG SEQ ID NO.: 152 DSSF NNGT SEQ ID NO.: 153 BKMI NNGT SEQ ID NO.: 154 DPIE - SEQ ID NO.: 155 BIMB NNGCAA SEQ ID NO.: 156 AWYD NNGTNT SEQ ID NO.: 157 AWYB - SEQ ID NO.: 158 BUDD NNGTNT SEQ ID NO.: 159 AWZA NNGTNT SEQ ID NO.: 160Table 4B (Cluster 12_4) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: COVC NNGTNT SEQ ID NO.: 161 AWYM NNG SEQ ID NO.: 162 Table 4C (Cluster 12_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ADAE NNNANT SEQ ID NO.: 163 ACZA NNNAAA SEQ ID NO.: 164 ADCM NNNAAA SEQ ID NO.: 165 COEB NNNANT SEQ ID NO.: 166 ADLN NNTAAA SEQ ID NO.: 167 ADDC - SEQ ID NO.: 168 ACLI NNNAAAA SEQ ID NO.: 169 BJQI - SEQ ID NO.: 170 FXQV - SEQ ID NO.: 171 DFKB - SEQ ID NO.: 172 ACJQ - SEQ ID NO.: 173 ADPP NANAAC SEQ ID NO.: 174 Table 4D (Cluster 12_11) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BRSR NNGAAGT SEQ ID NO.: 175 BOLV NNGNAC SEQ ID NO.: 176 BBHD NNGAAGT SEQ ID NO.: 177 AWDO NNGAACT SEQ ID NO.: 178 AWDQ NNGNAC SEQ ID NO.: 179 AKIM - SEQ ID NO.: 180 FUAP NNAAANC SEQ ID NO.: 181 FTFE NNGAAGT SEQ ID NO.: 182 GBZD NNGAAGT SEQ ID NO.: 183 CULT NNGAAG SEQ ID NO.: 184 DNAK - SEQ ID NO.: 185 BBFC NNGRACT SEQ ID NO.: 186 Table 5A (Cluster 13_2)CLOO NANNCC SEQ ID NO.: 200 AFWN NNNNTA SEQ ID NO.: 201 AGED - SEQ ID NO.: 202 AFXJ NAANCC SEQ ID NO.: 203 AMWQ NAAGC SEQ ID NO.: 204 FTWV NANNT SEQ ID NO.: 205 CQXO - SEQ ID NO.: 206 AFZP NGGNT SEQ ID NO.: 207Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: CVBG - SEQ ID NO.: 208 FWIN - SEQ ID NO.: 209 AGBY - SEQ ID NO.: 210 AGEO - SEQ ID NO.: 211 CDAT - SEQ ID NO.: 212 Table 5B (Cluster 13_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AKPD NNAAAT SEQ ID NO.: 213 DRIM NNGATT SEQ ID NO.: 214 AKPZ NNANTT SEQ ID NO.: 215 DZEC NNGATT SEQ ID NO.: 216 AKOU NNAANT SEQ ID NO.: 217 AKOK NNAANT SEQ ID NO.: 218 AZDK NNGATT SEQ ID NO.: 219 BOLT - SEQ ID NO.: 220 BUFU NNAAAT SEQ ID NO.: 221 AKPU NNNATT SEQ ID NO.: 222 AWEG NNAAAT SEQ ID NO.: 223 DKWZ NNGATT SEQ ID NO.: 224 BPUG - SEQ ID NO.: 225 Table 5C (Cluster 13_5) PAM ID PAM Motif (5’-3’) SEQ ID NO.: AHNN NAANA SEQ ID NO.: 239 AHOJ - SEQ ID NO.: 240 ABWH - SEQ ID NO.: 241 APSS NAANC SEQ ID NO.: 242 ABXR - SEQ ID NO.: 243 FVDQ - SEQ ID NO.: 244 AHRO - SEQ ID NO.: 245 FXMV NANNC SEQ ID NO.: 246 CERK - SEQ ID NO.: 247 DPWE - SEQ ID NO.: 248 FUHC - SEQ ID NO.: 249 CQRM - SEQ ID NO.: 250 CAUH NAANCC SEQ ID NO.: 251 Table 6A (Cluster 14_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ABJA - SEQ ID NO.: 252 FOEF - SEQ ID NO.: 253 BCRS NAANC SEQ ID NO.: 254 FIWJ - SEQ ID NO.: 255 FOCM - SEQ ID NO.: 256 FIHX - SEQ ID NO.: 257Table 6A (Cluster 14_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BCRT - SEQ ID NO.: 258 FJDK - SEQ ID NO.: 259 ABKS NAA SEQ ID NO.: 260 FOLJ - SEQ ID NO.: 261 FPED - SEQ ID NO.: 262 FMPK - SEQ ID NO.: 263 FJEW - SEQ ID NO.: 264 FOAG - SEQ ID NO.: 265 Table 6B (Cluster 14_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BEWC NNNNGAAA SEQ ID NO.: 266 AROQ NNNNCNAC SEQ ID NO.: 267 ARQR NNNNCTAT SEQ ID NO.: 268 ARSY NNNNCGAC SEQ ID NO.: 269 ARQA NNNNCNAT SEQ ID NO.: 270 DTUL NNNNCTAT SEQ ID NO.: 271 ARUO NNNNGNAT SEQ ID NO.: 272 ADSF NNANNTAA SEQ ID NO.: 273 BGHQ NNNKRTAA SEQ ID NO.: 274 AROX NNNNCGAC SEQ ID NO.: 275 ARUF NNNNCAAA SEQ ID NO.: 276 CPLT - SEQ ID NO.: 277 DUAP - SEQ ID NO.: 278 FUQB NNNNCAAA SEQ ID NO.: 279 Table 7A (Cluster 15_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ECFV NNGCNA SEQ ID NO.: 280 EDAQ NNGCNA SEQ ID NO.: 281 AWYH NNGC SEQ ID NO.: 282 AWYJ NNGCNA SEQ ID NO.: 283 CKAC NNGCNA SEQ ID NO.: 284 AINM NNGCNA SEQ ID NO.: 285 AWYT NNGCNA SEQ ID NO.: 286 AWYQ NNGCNA SEQ ID NO.: 287 DXAN NNGCNA SEQ ID NO.: 288 DVQQ NNGTNT SEQ ID NO.: 289 DYOL - SEQ ID NO.: 290 BXLY - SEQ ID NO.: 291 DLEQ - SEQ ID NO.: 292 AWYL NNGCNANA SEQ ID NO.: 293 GBHF NNGCNA SEQ ID NO.: 294Table 7B (Cluster 15_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AXHH NNNCCC SEQ ID NO.: 295 EGNW NNNNCC SEQ ID NO.: 296 AAGZ NNNCCC SEQ ID NO.: 297 AODJ NNNCCC SEQ ID NO.: 298 AKWQ NNNCCC SEQ ID NO.: 299 ACFY - SEQ ID NO.: 300 FFNF - SEQ ID NO.: 301 ACDJ NNNCCC SEQ ID NO.: 302 AAEZ NNNCCC SEQ ID NO.: 303 BQTP - SEQ ID NO.: 304 DJUS - SEQ ID NO.: 305 EWOE - SEQ ID NO.: 306 DUOT NNNTCCNNT SEQ ID NO.: 307 BOEE NNNCCC SEQ ID NO.: 308 ACEG - SEQ ID NO.: 309 Table 7C (Cluster 15_4) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FIYZ NNNNCC SEQ ID NO.: 310 FJDM NNNNAT SEQ ID NO.: 311 ENJG NNGNCAC SEQ ID NO.: 312 AWKM - SEQ ID NO.: 313 FJNU - SEQ ID NO.: 314 FMZC NCGNC SEQ ID NO.: 315 FLEU NNGCCA SEQ ID NO.: 316 DNBM - SEQ ID NO.: 317 FKPU - SEQ ID NO.: 318 FMNN - SEQ ID NO.: 319 FLME - SEQ ID NO.: 320 FQFB NNGNNAC SEQ ID NO.: 321 FHHK - SEQ ID NO.: 322 FHJK - SEQ ID NO.: 323 FNJI - SEQ ID NO.: 324 Table 7D (Cluster 15_6) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FHLM NNNT SEQ ID NO.: 325 ARUE NNGCCC SEQ ID NO.: 326 FKCF NNGNCNC SEQ ID NO.: 327 ARNR NNGTC SEQ ID NO.: 328 FHLH - SEQ ID NO.: 329 FPSO - SEQ ID NO.: 330 FJOH - SEQ ID NO.: 331 CZAS - SEQ ID NO.: 332Table 7D (Cluster 15_6) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FPCW - SEQ ID NO.: 333 FNBB - SEQ ID NO.: 334 FNXQ - SEQ ID NO.: 335 CXNZ - SEQ ID NO.: 336 FLPS - SEQ ID NO.: 337 FPGU - SEQ ID NO.: 338 FMEO - SEQ ID NO.: 339 Table 8 (Cluster 16_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ARWH NNNNNNNA SEQ ID NO.: 340 CKGB NNNNGNA SEQ ID NO.: 341 ARWC - SEQ ID NO.: 342 DJVL NNNNGNTAA SEQ ID NO.: 343 ARVL - SEQ ID NO.: 344 ARVU - SEQ ID NO.: 345 BMHV - SEQ ID NO.: 346 BQVK - SEQ ID NO.: 347 DJVT - SEQ ID NO.: 348 EFPB NNNGNCAA SEQ ID NO.: 349 CRAY - SEQ ID NO.: 350 ADKF - SEQ ID NO.: 351 DJUL - SEQ ID NO.: 352 GCPW - SEQ ID NO.: 353 DICM - SEQ ID NO.: 354 BMNQ - SEQ ID NO.: 355 Table 9A (Cluster 17_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BBHX NNAAAA SEQ ID NO.: 356 DZUQ NNGGG SEQ ID NO.: 357 BPXU NNGGG SEQ ID NO.: 358 DNBN NNGGNG SEQ ID NO.: 359 DUGK NNAAAA SEQ ID NO.: 360 BLAM NNGAAA SEQ ID NO.: 361 DNDB NNGGNG SEQ ID NO.: 362 BBEH NNGGNG SEQ ID NO.: 363 BBHG NNGGG SEQ ID NO.: 364 DIXT NNGGNG SEQ ID NO.: 365 AIMT NNGGNG SEQ ID NO.: 366 CEDA NNGGG SEQ ID NO.: 367 EDAG NNANAA SEQ ID NO.: 368 BDKD NNGGNG SEQ ID NO.: 369 GDZB NNGAAA SEQ ID NO.: 370Table 9A (Cluster 17_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: GAXE - SEQ ID NO.: 371 DYVX - SEQ ID NO.: 372 Table 9B (Cluster 17_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AVWK NNNA SEQ ID NO.: 373 AVZC - SEQ ID NO.: 374 AAWX - SEQ ID NO.: 375 CQJG - SEQ ID NO.: 376 DJTW NNGAAA SEQ ID NO.: 377 AVXN - SEQ ID NO.: 378 FMJL - SEQ ID NO.: 379 AVVC - SEQ ID NO.: 380 BPPN - SEQ ID NO.: 381 AVVP - SEQ ID NO.: 382 CTIZ - SEQ ID NO.: 383 DKCT - SEQ ID NO.: 384 FZEK NRNA SEQ ID NO.: 385 GFLD - SEQ ID NO.: 386 BJPW - SEQ ID NO.: 387 CVYR - SEQ ID NO.: 388 AWAU - SEQ ID NO.: 389 Table 10A (Cluster 18_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AHNE NGNNGC SEQ ID NO.: 390 AHPT NGNNA SEQ ID NO.: 391 AHNI NNANCC SEQ ID NO.: 392 AHPG - SEQ ID NO.: 393 AHQJ NNANCC SEQ ID NO.: 394 AHOO NAANGA SEQ ID NO.: 395 CPNL - SEQ ID NO.: 396 FQYH - SEQ ID NO.: 397 DNZR - SEQ ID NO.: 398 FVFE - SEQ ID NO.: 399 BYMS - SEQ ID NO.: 400 DKSQ - SEQ ID NO.: 401 FUFN - SEQ ID NO.: 402 AICY - SEQ ID NO.: 403 FRPB - SEQ ID NO.: 404 FTXY - SEQ ID NO.: 405 FXPN NARNAC SEQ ID NO.: 406 FUHD - SEQ ID NO.: 407Table 10B (Cluster 18_5) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BQCN NNNNNTAAC SEQ ID NO.: 408 AAFR NNNNNTAAC SEQ ID NO.: 409 AHIG NNNNNNAAA SEQ ID NO.: 410 GCCZ NNNNNTAAC SEQ ID NO.: 411 AOEF NNNNNNAAC SEQ ID NO.: 412 ACFU NNNNNNAAC SEQ ID NO.: 413 BZXR - SEQ ID NO.: 414 AVUU - SEQ ID NO.: 415 DRBY - SEQ ID NO.: 416 GLAO NNNNNTAAC SEQ ID NO.: 417 ACWW NNNNNTNAC SEQ ID NO.: 418 AAFI NNNNNNAAA SEQ ID NO.: 419 GBZM - SEQ ID NO.: 420 EBPB - SEQ ID NO.: 421 ACWH - SEQ ID NO.: 422 BNHM NNNNNNAAC SEQ ID NO.: 423 AODS - SEQ ID NO.: 424 DOFK NNNNNNNA SEQ ID NO.: 425 Table 10C (Cluster 18_6) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FKTI NGG SEQ ID NO.: 426 AIYH NGG SEQ ID NO.: 427 FJGB NGG SEQ ID NO.: 428 FJMJ NGG SEQ ID NO.: 429 FOCY NGG SEQ ID NO.: 430 FIFQ NGGC SEQ ID NO.: 431 FPLC NGG SEQ ID NO.: 432 FHYV NGG SEQ ID NO.: 433 FPJL NGGNCG SEQ ID NO.: 434 FKSG NGGC SEQ ID NO.: 435 FQLK NGG SEQ ID NO.: 436 FMRE NGG SEQ ID NO.: 437 FNMJ - SEQ ID NO.: 438 FPHI - SEQ ID NO.: 439 FGKU NGGC SEQ ID NO.: 440 PBS NGG SEQ ID NO.: 441 FNYL NGG SEQ ID NO.: 442 AGXC NGGNC SEQ ID NO.: 443 Table 10D (Cluster 18_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: GMIE NNNNNCC SEQ ID NO.: 444 AJFU - SEQ ID NO.: 445Table 10D (Cluster 18_7) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AEHL - SEQ ID NO.: 446 FKAG - SEQ ID NO.: 447 FHZJ - SEQ ID NO.: 448 ADSC - SEQ ID NO.: 449 CXKV - SEQ ID NO.: 450 FIUY - SEQ ID NO.: 451 FKDD - SEQ ID NO.: 452 FIAP - SEQ ID NO.: 453 FMZA - SEQ ID NO.: 454 FKUY - SEQ ID NO.: 455 FPFX - SEQ ID NO.: 456 AJFT - SEQ ID NO.: 1025 AKBM - SEQ ID NO.: 457 AJFQ - SEQ ID NO.: 458 FLET - SEQ ID NO.: 459 ADTK - SEQ ID NO.: 460 Table 11 (Cluster 20_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ASFR NNAACA SEQ ID NO.: 461 CJRI NNGG SEQ ID NO.: 462 ASFQ ANGG SEQ ID NO.: 463 AJAA NNAANA SEQ ID NO.: 464 CGJO NNAAAG SEQ ID NO.: 465 ASCT - SEQ ID NO.: 466 BPSL NNAAAA SEQ ID NO.: 467 ASEW - SEQ ID NO.: 468 ASDT NNGATA SEQ ID NO.: 469 ASGS NNAAAG SEQ ID NO.: 470 ASGI - SEQ ID NO.: 471 EDKN - SEQ ID NO.: 472 BWNO - SEQ ID NO.: 473 ASER - SEQ ID NO.: 474 ASHI - SEQ ID NO.: 475 ASCI NAAAAA SEQ ID NO.: 476 DKFU - SEQ ID NO.: 477 ASCM - SEQ ID NO.: 478 ASFY - SEQ ID NO.: 479 BNMB NNAACA SEQ ID NO.: 480 Table 12A (Cluster 21_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AWNH NNNGCC SEQ ID NO.: 481 AWOC NNGANT SEQ ID NO.: 482Table 12A (Cluster 21_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ADFC - SEQ ID NO.: 483 DIBA - SEQ ID NO.: 484 ACYV - SEQ ID NO.: 485 AWKF - SEQ ID NO.: 486 BOQC - SEQ ID NO.: 487 FPZY - SEQ ID NO.: 488 DEOC - SEQ ID NO.: 489 FRUE - SEQ ID NO.: 490 BVHZ NNNAAA SEQ ID NO.: 491 ACKO NNNAAAA SEQ ID NO.: 492 COZD - SEQ ID NO.: 493 CUOG - SEQ ID NO.: 494 DSWH NNNNCC SEQ ID NO.: 495 BYHD NNNA SEQ ID NO.: 496 GFAR - SEQ ID NO.: 497 BJTY - SEQ ID NO.: 498 BLWD NNAAAC SEQ ID NO.: 499 BWBY - SEQ ID NO.: 500 AWHZ - SEQ ID NO.: 501 Table 12B (Cluster 21_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FKAB NNNTTT SEQ ID NO.: 502 FPAU NNNTTT SEQ ID NO.: 503 CYKN - SEQ ID NO.: 504 FPPO - SEQ ID NO.: 505 ARIZ - SEQ ID NO.: 506 CXCL - SEQ ID NO.: 507 FPWK - SEQ ID NO.: 508 FHXE - SEQ ID NO.: 509 FIQV - SEQ ID NO.: 510 FPWD - SEQ ID NO.: 511 FLXN - SEQ ID NO.: 512 FKUS - SEQ ID NO.: 513 CZAB - SEQ ID NO.: 514 FOMM - SEQ ID NO.: 515 FLUZ - SEQ ID NO.: 516 FOJY - SEQ ID NO.: 517 FPID - SEQ ID NO.: 518 DCUM - SEQ ID NO.: 519 FJYT - SEQ ID NO.: 520 ARIG NNGTTT SEQ ID NO.: 521 ARMU - SEQ ID NO.: 522Table 13 (Cluster 22_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AKLB NGG SEQ ID NO.: 523 AWLC NNGGNA SEQ ID NO.: 524 AWIR NGGNAA SEQ ID NO.: 525 AWJK NGGNNA SEQ ID NO.: 526 AWIE - SEQ ID NO.: 527 CJXD NGG SEQ ID NO.: 528 AWHK NGGNNA SEQ ID NO.: 529 BHUP NGGNNA SEQ ID NO.: 530 AWKR - SEQ ID NO.: 531 AWIG NGGNAA SEQ ID NO.: 532 AKMR NGGNNA SEQ ID NO.: 533 FOPN NGGNA SEQ ID NO.: 534 AKNI NGGNNA SEQ ID NO.: 535 DLBV NGGNAA SEQ ID NO.: 536 AWKK - SEQ ID NO.: 537 AWKU - SEQ ID NO.: 538 BQUS - SEQ ID NO.: 539 BWHD - SEQ ID NO.: 540 DLME - SEQ ID NO.: 541 FSJL - SEQ ID NO.: 1026 CGBU - SEQ ID NO.: 542 FTET NGG SEQ ID NO.: 543 Table 14 (Cluster 23_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BCZK NNNAAGT SEQ ID NO.: 544 DIUM NNNATT SEQ ID NO.: 545 DQUF NNAAAG SEQ ID NO.: 546 GCKA NNNAAG SEQ ID NO.: 547 APHH NNNATA SEQ ID NO.: 548 DLJC - SEQ ID NO.: 549 BCYV - SEQ ID NO.: 550 BWST - SEQ ID NO.: 551 DSGB NNNAC SEQ ID NO.: 552 DXDQ NNNACC SEQ ID NO.: 553 BDAA - SEQ ID NO.: 554 BCXN - SEQ ID NO.: 555 BACQ - SEQ ID NO.: 556 BADR - SEQ ID NO.: 557 DNWP NNNACNT SEQ ID NO.: 558 DKAS - SEQ ID NO.: 559 BDLA - SEQ ID NO.: 560 BCXK - SEQ ID NO.: 561 GEDU - SEQ ID NO.: 562Table 14 (Cluster 23_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FKGU - SEQ ID NO.: 563 DQRR - SEQ ID NO.: 564 DNWZ - SEQ ID NO.: 565 FLBV - SEQ ID NO.: 566 Table 15 (Cluster 25_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ARYS NNNNNNAT SEQ ID NO.: 593 ARYR NNNNNNAA SEQ ID NO.: 594 ARWS NNNNNNA SEQ ID NO.: 595 BLJG NNNNGNA SEQ ID NO.: 596 AXWP NNNNGNAT SEQ ID NO.: 597 ARYD NNNNGAAC SEQ ID NO.: 598 ARZL NNNNGNAT SEQ ID NO.: 599 GCKE NNNNGAAC SEQ ID NO.: 600 ASAG NNNNNNA SEQ ID NO.: 601 DIUZ NNNNGCAT SEQ ID NO.: 602 BDKY NNNNGAA SEQ ID NO.: 603 ACKX NNNNGNAA SEQ ID NO.: 604 CVMH NNNNNNAT SEQ ID NO.: 605 AYCJ - SEQ ID NO.: 606 AXZQ - SEQ ID NO.: 607 ARXH NNNNGAAT SEQ ID NO.: 608 AYFH NNNNCNAA SEQ ID NO.: 609 GDNS - SEQ ID NO.: 610 DQJJ NNNNGCAT SEQ ID NO.: 611 DOLK NNNNGCAT SEQ ID NO.: 612 ADRM - SEQ ID NO.: 613 DKER - SEQ ID NO.: 614 BDMB - SEQ ID NO.: 615 BOJR - SEQ ID NO.: 616 ADSR - SEQ ID NO.: 617 Table 16A (Cluster 28_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FMVE NNNNTAAAA SEQ ID NO.: 618 ALHZ - SEQ ID NO.: 619 FJUL - SEQ ID NO.: 620 FPVO - SEQ ID NO.: 621 FLXA - SEQ ID NO.: 622 FNXW - SEQ ID NO.: 623 FJYV - SEQ ID NO.: 624 ALIB - SEQ ID NO.: 625 FIYY - SEQ ID NO.: 626Table 16A (Cluster 28_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ALID - SEQ ID NO.: 627 FLLN - SEQ ID NO.: 628 FKXC - SEQ ID NO.: 629 FODX - SEQ ID NO.: 630 FJCA - SEQ ID NO.: 631 FMOM - SEQ ID NO.: 632 FQJS - SEQ ID NO.: 633 FLML - SEQ ID NO.: 634 FKCW - SEQ ID NO.: 635 FOJK - SEQ ID NO.: 636 FMUY - SEQ ID NO.: 637 FKBX - SEQ ID NO.: 638 ALIR - SEQ ID NO.: 639 FKPS - SEQ ID NO.: 640 FNEF - SEQ ID NO.: 641 FNFK - SEQ ID NO.: 642 ALHX - SEQ ID NO.: 643 FIPC - SEQ ID NO.: 644 FPBX - SEQ ID NO.: 645 Table 16B (Cluster 28_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AKKA NNNNAA SEQ ID NO.: 646 BADU NNGAAA SEQ ID NO.: 647 DPZO NNAAAA SEQ ID NO.: 648 CBYK NNANAG SEQ ID NO.: 649 BWMP NNAAAA SEQ ID NO.: 650 EDEI NNAAAA SEQ ID NO.: 651 FQVK NNAAAA SEQ ID NO.: 652 AQSN NNAAAAT SEQ ID NO.: 653 CIFH NNGAAA SEQ ID NO.: 654 AWDN NNGAAA SEQ ID NO.: 655 BBHM NNNAAAT SEQ ID NO.: 656 BCFX NNANAAC SEQ ID NO.: 657 BBGB NNAAAA SEQ ID NO.: 658 DKFA - SEQ ID NO.: 659 BCIX NNGAAA SEQ ID NO.: 660 FWBW NNGAAA SEQ ID NO.: 661 BNIB - SEQ ID NO.: 662 DOEI NNGAAAC SEQ ID NO.: 663 AKIS NNANNG SEQ ID NO.: 664 GMWT NNGNAAT SEQ ID NO.: 665 FZJV - SEQ ID NO.: 666 GCTE NNAAAAT SEQ ID NO.: 667Table 16B (Cluster 28_3) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: FUCK - SEQ ID NO.: 668 DNLB NNGAAAT SEQ ID NO.: 669 GHME NNGAAA SEQ ID NO.: 670 AKKI NNANAA SEQ ID NO.: 671 CVCR NNGAAAC SEQ ID NO.: 672 BURC - SEQ ID NO.: 673 Table 17 (Cluster 29_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: CEBH NNTA SEQ ID NO.: 674 DMMA NNNANC SEQ ID NO.: 675 ECNG NNAANC SEQ ID NO.: 676 AJXR NNTA SEQ ID NO.: 677 BKHU NNNANC SEQ ID NO.: 678 DWMG NNTA SEQ ID NO.: 679 DRCI - SEQ ID NO.: 680 CAUM NNNANC SEQ ID NO.: 681 CQSL NATA SEQ ID NO.: 682 AJXG NANANC SEQ ID NO.: 683 DSKU NGTA SEQ ID NO.: 684 CPAN NNNA SEQ ID NO.: 685 BTTL NNTA SEQ ID NO.: 686 AIUL NATA SEQ ID NO.: 687 CICL NNTANC SEQ ID NO.: 688 BOUE NANANC SEQ ID NO.: 689 DTYC - SEQ ID NO.: 690 GEYR NATA SEQ ID NO.: 691 BUWF - SEQ ID NO.: 692 GAMT - SEQ ID NO.: 693 DVSF - SEQ ID NO.: 694 AJYB NNTA SEQ ID NO.: 695 DYCG - SEQ ID NO.: 696 EDOJ - SEQ ID NO.: 697 DUCE - SEQ ID NO.: 698 BWNE NNTA SEQ ID NO.: 700 AIUT NNNANC SEQ ID NO.: 701 AJXE NNNNNC SEQ ID NO.: 702 Table 18 (Cluster 32_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ASQK NNNNGNAT SEQ ID NO.: 703 ASPK NNNNGCAT SEQ ID NO.: 704 ASOV NNNNGNAT SEQ ID NO.: 705 ASRS NNNNGCAA SEQ ID NO.: 706Table 18 (Cluster 32_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ASSY NNNNCCAT SEQ ID NO.: 707 ASUC NNNNCCA SEQ ID NO.: 708 FYVA NNNNCCA SEQ ID NO.: 709 CDUE NNNNGCA SEQ ID NO.: 710 FXCB NNNNCAAT SEQ ID NO.: 711 ASSQ NNNNGTAT SEQ ID NO.: 712 BWOF NNNNCNAC SEQ ID NO.: 713 BONU NNNNGAAT SEQ ID NO.: 714 CODW NNNNCCA SEQ ID NO.: 715 ASSW - SEQ ID NO.: 716 BLSZ NNNNCTAT SEQ ID NO.: 717 ASSI NNNTNAAA SEQ ID NO.: 718 CFWS - SEQ ID NO.: 719 ASUE NNNNCCA SEQ ID NO.: 720 FRDB NNNNGCAC SEQ ID NO.: 721 FTGX NNNNGCA SEQ ID NO.: 722 FVUO - SEQ ID NO.: 723 BHMG - SEQ ID NO.: 724 ASTT NNNNCAAT SEQ ID NO.: 725 DLLV NNNNGCAT SEQ ID NO.: 726 GCEU NNNNGCAA SEQ ID NO.: 727 FYOO NNNNCAAA SEQ ID NO.: 728 BOVG NNNNCCAC SEQ ID NO.: 729 FRTD NNNNCTA SEQ ID NO.: 730 BCGW NNNTCAAT SEQ ID NO.: 731 FUJU - SEQ ID NO.: 732 ASPF - SEQ ID NO.: 733 FUWN - SEQ ID NO.: 734 Table 19 (Cluster 33_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BLAF NNAG SEQ ID NO.: 735 AMZI - SEQ ID NO.: 736 BMDT - SEQ ID NO.: 737 CBJL - SEQ ID NO.: 738 AOHG - SEQ ID NO.: 739 CGTU - SEQ ID NO.: 740 EPDC - SEQ ID NO.: 741 FYFR - SEQ ID NO.: 742 DLEZ - SEQ ID NO.: 743 DUUD - SEQ ID NO.: 744 AMQC - SEQ ID NO.: 745 DKQM - SEQ ID NO.: 746 CEYY - SEQ ID NO.: 747Table 19 (Cluster 33_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AMQE - SEQ ID NO.: 748 DKNR - SEQ ID NO.: 749 FSRX - SEQ ID NO.: 750 DNJU - SEQ ID NO.: 751 AIJW - SEQ ID NO.: 752 DBKC - SEQ ID NO.: 753 DLWG - SEQ ID NO.: 754 BCYA - SEQ ID NO.: 755 FSVL - SEQ ID NO.: 756 FWCE - SEQ ID NO.: 757 DKGU - SEQ ID NO.: 758 FXZZ - SEQ ID NO.: 759 BTHY - SEQ ID NO.: 760 DBNW - SEQ ID NO.: 761 FWOH - SEQ ID NO.: 762 DRCR - SEQ ID NO.: 763 DMXV - SEQ ID NO.: 764 CCQT - SEQ ID NO.: 765 DKKR - SEQ ID NO.: 766 BPNX - SEQ ID NO.: 767 Table 20A (Cluster 38_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ARHP NNAANC SEQ ID NO.: 768 ARIC NTGAAA SEQ ID NO.: 769 ARKB NTAAA SEQ ID NO.: 770 ARIE NNAAANA SEQ ID NO.: 771 ARMA NTGGANT SEQ ID NO.: 772 CSSN NNAA SEQ ID NO.: 773 AGAE NTGAAA SEQ ID NO.: 774 BPLN NNAG SEQ ID NO.: 775 AGAQ NNAAANA SEQ ID NO.: 776 DOXP NNAANNC SEQ ID NO.: 777 BAFL NNAAAA SEQ ID NO.: 778 AFXE NNAAA SEQ ID NO.: 779 FZDJ NNAAG SEQ ID NO.: 780 EGQL NNAAA SEQ ID NO.: 781 GGVS NTAA SEQ ID NO.: 782 ACDY NTGGA SEQ ID NO.: 783 ARMQ NTAACANNNT SEQ ID NO.: 784 DNGP NTAAANA SEQ ID NO.: 785 CEQO NTGAC SEQ ID NO.: 786 AGCU - SEQ ID NO.: 787 BADF NTAAAG SEQ ID NO.: 788Table 20A (Cluster 38_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: DJVS NNANAA SEQ ID NO.: 789 CQDS NTAAAA SEQ ID NO.: 790 BDMX NNANAC SEQ ID NO.: 791 BDBV ANGCNC SEQ ID NO.: 792 BVTB NYAAC SEQ ID NO.: 793 CUDL NTGAGA SEQ ID NO.: 794 DXAT - SEQ ID NO.: 795 DZED NNAANNC SEQ ID NO.: 796 AGFT - SEQ ID NO.: 797 ARHQ - SEQ ID NO.: 798 AGDO - SEQ ID NO.: 799 DPEL - SEQ ID NO.: 800 DJWX NTGAAA SEQ ID NO.: 801 GCIG NTAAA SEQ ID NO.: 802 BYBF - SEQ ID NO.: 803 DSGV NCAAA SEQ ID NO.: 804 ARLP NCAACAA SEQ ID NO.: 805 Table 20B (Cluster 38_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AQKN NNNNAT SEQ ID NO.: 806 AVCE NNNAAA SEQ ID NO.: 807 AAXC NNAAAT SEQ ID NO.: 808 AQGR NNNNNT SEQ ID NO.: 809 BUML NNTATT SEQ ID NO.: 810 BXEQ NNNANT SEQ ID NO.: 811 AQHQ NNNNCT SEQ ID NO.: 812 AQKJ NNCNAT SEQ ID NO.: 813 AQLF NNNAA SEQ ID NO.: 814 AQKO NNNNNT SEQ ID NO.: 815 AVDS NACNCC SEQ ID NO.: 816 AVBN NNCAAC SEQ ID NO.: 817 AQJC NNTCAT SEQ ID NO.: 818 EAJU NNCAAA SEQ ID NO.: 819 DMLJ NNTANC SEQ ID NO.: 820 ACGJ NNNNC SEQ ID NO.: 821 ACKZ - SEQ ID NO.: 822 DPTF NANNAT SEQ ID NO.: 823 DOPI NNNCAT SEQ ID NO.: 824 BKHX - SEQ ID NO.: 825 DNPI NNNCAT SEQ ID NO.: 826 AQLC NNTATC SEQ ID NO.: 827 FSTR NNNRCCNNNC SEQ ID NO.: 828 GAUZ NANAC SEQ ID NO.: 829Table 20B (Cluster 38_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AVEE NNNAAT SEQ ID NO.: 830 GEFL NNNNCC SEQ ID NO.: 831 AQKT NNGNNT SEQ ID NO.: 832 CFWW NNNNAT SEQ ID NO.: 833 AQHP NNNCAT SEQ ID NO.: 834 BADD - SEQ ID NO.: 835 BTCP - SEQ ID NO.: 836 DWBW - SEQ ID NO.: 837 BVGL - SEQ ID NO.: 838 AQIQ NGGNNT SEQ ID NO.: 839 FWWL NACGCCGNT SEQ ID NO.: 840 CYMJ - SEQ ID NO.: 841 DITT NNNAA SEQ ID NO.: 842 AQFV NNNCAT SEQ ID NO.: 843 Table 21A (Cluster 41_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: ADQI - SEQ ID NO.: 844 BINZ - SEQ ID NO.: 845 FGYA - SEQ ID NO.: 846 ADLG NNNNNNNA SEQ ID NO.: 847 ADLU - SEQ ID NO.: 848 CGQH - SEQ ID NO.: 849 DZBV NNNNNNAA SEQ ID NO.: 850 DVLZ - SEQ ID NO.: 851 ADPD NNNNCC SEQ ID NO.: 852 ADPY NNNNNNAAA SEQ ID NO.: 853 ADQK - SEQ ID NO.: 854 ECZI NNNNGNAA SEQ ID NO.: 855 DYZX NNNNCC SEQ ID NO.: 856 BFPC - SEQ ID NO.: 857 ADLS - SEQ ID NO.: 858 CAUK NNNNNNNAA SEQ ID NO.: 859 DYSA - SEQ ID NO.: 860 DSXE - SEQ ID NO.: 861 FQQT NNNNNNAAC SEQ ID NO.: 862 ADMZ - SEQ ID NO.: 863 FZKB - SEQ ID NO.: 864 ADQN NNNNGNANA SEQ ID NO.: 865 DQFM - SEQ ID NO.: 866 ADQF - SEQ ID NO.: 867 BHBP NNNNNNAAC SEQ ID NO.: 868 EBWW - SEQ ID NO.: 869 ADQP - SEQ ID NO.: 870Table 21A (Cluster 41_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: DOGV - SEQ ID NO.: 871 DULX - SEQ ID NO.: 872 ADOY NNNNGNAA SEQ ID NO.: 873 ADQJ - SEQ ID NO.: 874 BRTC NNNNCC SEQ ID NO.: 875 BGXI - SEQ ID NO.: 876 DZLD - SEQ ID NO.: 877 ECME - SEQ ID NO.: 878 EBRK - SEQ ID NO.: 879 EDEP - SEQ ID NO.: 880 EAMW - SEQ ID NO.: 881 DTDH - SEQ ID NO.: 882 DYSX NNNNGNAA SEQ ID NO.: 883 ADQH NNNNNNTAA SEQ ID NO.: 884 Table 21B (Cluster 41_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: BAFP NNAAC SEQ ID NO.: 885 AKKH NNAAC SEQ ID NO.: 886 BBHO NNAAANC SEQ ID NO.: 887 CQRI NNAANNC SEQ ID NO.: 888 DPUW NNAAA SEQ ID NO.: 889 BBHC NTAAC SEQ ID NO.: 890 BUTP NNAANA SEQ ID NO.: 891 AWDG ANAAANC SEQ ID NO.: 892 BHUB NCAAANC SEQ ID NO.: 893 AWDD NNAACA SEQ ID NO.: 894 BBIK NNAAC SEQ ID NO.: 895 BBEN NTAAC SEQ ID NO.: 896 AKJT NTAANNC SEQ ID NO.: 897 GBIR NNAAAC SEQ ID NO.: 898 BQKK NNGAAGAA SEQ ID NO.: 899 FSDV NNAAA SEQ ID NO.: 900 BJWF - SEQ ID NO.: 901 CKRU NTAA SEQ ID NO.: 902 DUXV NNAAANCA SEQ ID NO.: 903 BXOD NNAAAAA SEQ ID NO.: 904 GEEM NTAANNC SEQ ID NO.: 905 BHLD NTAAAAA SEQ ID NO.: 906 FOPJ - SEQ ID NO.: 907 BNEU - SEQ ID NO.: 908 DRJH NNAAANC SEQ ID NO.: 909 CPEM NCAAANA SEQ ID NO.: 910 DNBA - SEQ ID NO.: 911Table 21B (Cluster 41_2) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: DLXC - SEQ ID NO.: 912 DQAM NYAAATC SEQ ID NO.: 913 BBHZ NNAAC SEQ ID NO.: 914 DKWB NCAANNC SEQ ID NO.: 915GMTH NNAAAAA SEQ ID NO.: 916BBGU - SEQ ID NO.: 917 AKKQ - SEQ ID NO.: 918 CENT NNAAAAA SEQ ID NO.: 919 CIFB - SEQ ID NO.: 920 BCZP - SEQ ID NO.: 921 DTEA NNAAANC SEQ ID NO.: 922 BBGA NNAAC SEQ ID NO.: 923 AKKJ - SEQ ID NO.: 924 BBHP - SEQ ID NO.: 925 Table 22 (Cluster 99_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: AOTX NNTAT SEQ ID NO.: 926 AIDC NNNANC SEQ ID NO.: 927 AOBL NNTAT SEQ ID NO.: 928 AOBX NNNAA SEQ ID NO.: 929 AOPX NNNAA SEQ ID NO.: 930 AORM NNTANT SEQ ID NO.: 931 AORE NNTAT SEQ ID NO.: 932 AOQJ NNNAAA SEQ ID NO.: 933 FWVY NNTANT SEQ ID NO.: 934 AOSL NNTATT SEQ ID NO.: 935 CVYC NNNANT SEQ ID NO.: 936 BDWW NNNAA SEQ ID NO.: 937 BDUT NNNANC SEQ ID NO.: 938 AOCA NNCAAG SEQ ID NO.: 939 BKEY NNTANC SEQ ID NO.: 940 AOQM NNCACC SEQ ID NO.: 941 BDVC NNNAAC SEQ ID NO.: 942 DWYB NNTANT SEQ ID NO.: 943 EGPY NNCAAG SEQ ID NO.: 944 BXNS NNNAAA SEQ ID NO.: 945 CNZL NNTAT SEQ ID NO.: 946 CBRG NNTAT SEQ ID NO.: 947 BKPY NNCATT SEQ ID NO.: 948 BDTY NNNAAT SEQ ID NO.: 949 BDUJ NNNANT SEQ ID NO.: 950 BDXB NNTAT SEQ ID NO.: 951 BDXG NNTATT SEQ ID NO.: 952Table 22 (Cluster 99_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: CJST - SEQ ID NO.: 953 BVEH NNTATT SEQ ID NO.: 954 BQEO NNTAT SEQ ID NO.: 955 BCXJ NACAAG SEQ ID NO.: 956 CVHZ NACACC SEQ ID NO.: 957 CWOB NNTAT SEQ ID NO.: 958 DNVC NNNAA SEQ ID NO.: 959 GCLA NNTATY SEQ ID NO.: 960 EGZF NNTA SEQ ID NO.: 961 AONG NNNAAA SEQ ID NO.: 962 GFPG NNNANT SEQ ID NO.: 963 BDUF NACAAANNC SEQ ID NO.: 964 BRPK NNTANT SEQ ID NO.: 965 BDWY - SEQ ID NO.: 966 BKPO NNTA SEQ ID NO.: 967 AOTM NNNAATNA SEQ ID NO.: 968 CCUX NNTATT SEQ ID NO.: 969 DPDH NNTAT SEQ ID NO.: 970 BJGN NNTAT SEQ ID NO.: 971 BKFK NNNANC SEQ ID NO.: 972 DRJT NNTATT SEQ ID NO.: 973 DFOW NNCAAC SEQ ID NO.: 974 DKWW - SEQ ID NO.: 975 EILI NNTA SEQ ID NO.: 976 AOQP NNCAAG SEQ ID NO.: 977 AOUJ NNNAA SEQ ID NO.: 978 CSGT NNTAT SEQ ID NO.: 979 BXBX - SEQ ID NO.: 980 BTRE NNTAT SEQ ID NO.: 981 CNRP NNTA SEQ ID NO.: 982 DTCZ NATA SEQ ID NO.: 983 DRYB - SEQ ID NO.: 984 DFKZ NACACC SEQ ID NO.: 985 DFOE NNTA SEQ ID NO.: 986 AORT - SEQ ID NO.: 987 BMMM - SEQ ID NO.: 988 CKWG - SEQ ID NO.: 989 DNDC NNNANT SEQ ID NO.: 990 DPAY NNNAAA SEQ ID NO.: 991 DTOD NNNAA SEQ ID NO.: 992 DPHK NNTAT SEQ ID NO.: 993 AOOO NNCAAC SEQ ID NO.: 994 AOPA NNNAAT SEQ ID NO.: 995 DUMV - SEQ ID NO.: 996Table 22 (Cluster 99_1) Nuclease ID PAM Motif (5’-3’) SEQ ID NO.: DKLZ - SEQ ID NO.: 997 CIDI NNCAAA SEQ ID NO.: 998 DSIC - SEQ ID NO.: 999 FUOP - SEQ ID NO.: 1000 DNQQ NNTA SEQ ID NO.: 1001 DPLN NNNAAT SEQ ID NO.: 1002 BHCX NNNAANC SEQ ID NO.: 1003 CQBV NGYAAT SEQ ID NO.: 1004 DDYG NNNAAC SEQ ID NO.: 1005 AJBH - SEQ ID NO.: 1006 AOPO NACAATT SEQ ID NO.: 1007 FZZM NNTA SEQ ID NO.: 1008 GCJE NNTAT SEQ ID NO.: 1009 EDEB NNNAAC SEQ ID NO.: 1010 DYJX NNTAT SEQ ID NO.: 1011 BDXS NNTAAT SEQ ID NO.: 1012 DLGZ NNNANC SEQ ID NO.: 1013 DJYK - SEQ ID NO.: 1014 BSSZ - SEQ ID NO.: 1015 AORS NNCAA SEQ ID NO.: 1016 AOUS - SEQ ID NO.: 1017 BXBF NATA SEQ ID NO.: 1018 DYCE NNCAA SEQ ID NO.: 1019 DUXH - SEQ ID NO.: 1020 DNPN NNCACC SEQ ID NO.: 1021 DMUR NNTARTCA SEQ ID NO.: 1022 ADNG NNTATT SEQ ID NO.: 1023 BDZZ NANAAC SEQ ID NO.: 1024 8.2 Example 2: Identification of Cas9 orthologues proteins and corresponding PAMs

[0357] CRISPR tools have significantly accelerated the development of curative therapies for genetic diseases. There is a need of nucleases with different PAM preferences for targeting awide range of genetic sequences in the human genome. These advancements benefit both industrial applications and basic research. 8.2.1. Methods 8.2.1.1. Identification of Cas9 variants from metagenomic data

[0358] New Cas9 variants were identified by screening 154723 bacterial and archaeal metagenome-assembled genomes (MAGs) reconstructed from the human microbiome. Briefly, the microbiome genomes were annotated using a protein annotation tool (Prokka version 1.12). CRISPR-associated genes cas1, cas2 and cas9 genes were identified and its corresponding CRISPR arrays were identified using MinCED version 0.4.2 (with default parameters). Only loci having a CRISPR array, cas1, cas2, and / or cas9 genes within a maximum distance of 10k bp from each other were considered. Loci containing coding sequence of Cas9 orthologous proteins having shorter than 950 aa were discarded. Cas9 orthologous proteins (variants) from the same species, having similar length but slightly different sequence, were compared by multiple sequence alignment to identify coding sequence of the functional domains. Cas9 orthologous proteins presenting deletions in nuclease domains were discarded. The remaining proteins were compared for sequencing coverage and the ortholog with the highest coverage was selected for each species. The sequences of exemplary new Cas9 orthologue nuclease amino acid sequences and their corresponding PAM motifs are shown in Tables 32 and 34 below. 8.2.1.2. Prediction of tracrRNA for CRISPR-Cas9 orthologous polypeptides

[0359] Prediction of tracrRNAs for CRISPR-Cas9 orthologous polypeptides was performed with a method based on the work by Chyou and Brown. Starting from unique direct repeats in the CRISPR array, BLAST version 2.2.31 (with parameters -task blastn-short -gapopen 2 -gapextend 1 -penalty -1 -reward 1 -evalue 1 -word_size 8) was used to identify anti-repeats within a 3000 bp window flanking the CRISPR-Cas9 locus. A custom version of RNIE 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). sgRNAs lacking the functional modules, namely the repeat:anti-repeat duplex, nexus and 3’ hairpin-like folds, were discarded. 8.2.1.3. Mammalian expression plasmids and constructs

[0360] CRISPR-Cas polypeptides (i.e. Cas9 orthologs) were expressed in mammalian cells from a plasmid vector characterized by an EF1alpha-driven cassette. Each CRISPR-Cas polypeptide 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 relative sgRNAs were expressed from a U6-driven cassette located on an independent plasmid construct. The human codon-optimized coding sequence of the CRISPR- Cas polypeptide, as well as the sgRNA scaffolds, were obtained as synthetic fragments from Twist Bioscience. Spacer sequences were cloned into the sgRNA plasmid as annealed DNA oligonucleotides (Eurofins Genomics) using double BsaI sites present in the plasmid. The list of spacer sequences and relative cloning oligonucleotides used in the present example is listed in Table 23. 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. Table 23. Spacer sequences and oligo used in the EGFP reporter assay Name Spacer SEQ ID NO: Oligo 1 SEQ ID NO: Oligo 2 SEQ ID NO: (5'→3') (5'→3')* (5'→3')* sgRNA- CGTCGCCG SEQ ID NO: caccgCGT SEQ ID NO: cagcCCTG SEQ ID NO: FRDB- TCCAGCTC 1300 CGCCGT 1316 GTCGAGC 1331 EGFP-g1 GACCAGG CCAGCT TGGACGG CGACCA CGACGc GG sgRNA- GAACTTCA SEQ ID NO: caccGAA SEQ ID NO: cagcTACG SEQ ID NO: FRDB- GGGTCAGC 1301 CTTCAG 1317 GCAAGCT 1332 EGFP-g2 TTGCCGTA GGTCAG GACCCTG CTTGCC AAGTTC GTA sgRNA- AGGTGGCA SEQ ID NO: caccgAGG SEQ ID NO: cagcCGAG SEQ ID NO: FRDB- TCGCCCTC 1302 TGGCAT 1318 GGCGAGG 1333 EGFP-g3 GCCCTCG CGCCCT GCGATGC CGCCCT CACCTc CG sgRNA- GCAAGGGC SEQ ID NO: caccGCA SEQ ID NO: gaacGGTG SEQ ID NO: DNDB- GAGGAGCT 1304 AGGGCG 1319 AACAGCT 1334 EGFP-g1 GTTCACC AGGAGC CCTCGCC TGTTCA CTTGC CC sgRNA- GGTGCCCA SEQ ID NO: caccGGTG SEQ ID NO: gaacCCAG SEQ ID NO: DNDB- TCCTGGTC 1305 CCCATC 1320 CTCGACC 1335 EGFP-g2 GAGCTGG CTGGTC AGGATGG GAGCTG GCACC G sgRNA- CCGCCATG SEQ ID NO: caccgCCG SEQ ID NO: gaacGACG SEQ ID NO: DNDB- CCCGAAGG 1306 CCATGC 1321 TAGCCTT 1336 EGFP-g3 CTACGTC CCGAAG CGGGCAT GCTACG GGCGGc TC sgRNA- GTCCAGCT SEQ ID NO: caccGTCC SEQ ID NO: caacTGCCC SEQ ID NO: GMIE- CGACCAGG 1307 AGCTCG 1322 ATCCTGG 1337 EGFP-g1 ATGGGCA ACCAGG TCGAGCT GGACTable 23. Spacer sequences and oligo used in the EGFP reporter assay Name Spacer SEQ ID NO: Oligo 1 SEQ ID NO: Oligo 2 SEQ ID NO: (5'→3') (5'→3')* (5'→3')* ATGGGC A sgRNA- GAAGTTCA SEQ ID NO: caccGAA SEQ ID NO: caacTTGCC SEQ ID NO: GMIE- TCTGCACC 1308 GTTCAT 1323 GGTGGTG 1338 EGFP-g2 ACCGGCAA CTGCAC CAGATGA CACCGG ACTTC CAA sgRNA- GACGTAAA SEQ ID NO: caccGAC SEQ ID NO: caacCTGA SEQ ID NO: GMIE- CGGCCACA 1309 GTAAAC 1324 ACTTGTG 1339 EGFP-g3 AGTTCAG GGCCAC GCCGTTT AAGTTC ACGTC AG sgRNA- GCTGGACG SEQ ID NO: caccGCTG SEQ ID NO: aaacGGCC SEQ ID NO: ARHP- GCGACGTA 1310 GACGGC 1325 GTTTACG 1340 EGFP-g1 AACGGCC GACGTA TCGCCGT AACGGC CCAGC C sgRNA- GGGCGAGG SEQ ID NO: caccGGG SEQ ID NO: aaacCGTA SEQ ID NO: ARHP- GCGATGCC 1311 CGAGGG 1326 GGTGGCA 1341 EGFP-g2 ACCTACG CGATGC TCGCCCT CACCTA CGCCC CG sgRNA- CCTGAAGT SEQ ID NO: caccgCCT SEQ ID NO: aaacCGGT SEQ ID NO: ARHP- TCATCTGC 1312 GAAGTT 1327 GGTGCAG 1342 EGFP-g3 ACCACCG CATCTG ATGAACT CACCAC TCAGGc CG sgRNA- GCGAGGAG SEQ ID NO: caccGCG SEQ ID NO: caccCACC SEQ ID NO: DUUZ- CTGTTCAC 1313 AGGAGC 1328 CCGGTGA 1343 EGFP-g1 CGGGGTG TGTTCA ACAGCTC CCGGGG CTCGC TG sgRNA- GTCCAGCT SEQ ID NO: caccGTCC SEQ ID NO: caccGTGC SEQ ID NO: DUUZ- CGACCAGG 1314 AGCTCG 1329 CCATCCT 1344 EGFP-g2 ATGGGCAC ACCAGG GGTCGAG ATGGGC CTGGAC AC sgRNA- AGTTCATC SEQ ID NO: caccgAGT SEQ ID NO: caccCTTGC SEQ ID NO: DUUZ- TGCACCAC 1315 TCATCT 1330 CGGTGGT 1345 EGFP-g3 CGGCAAG GCACCA GCAGATG CCGGCA AACTc AG (*) The cloning overhang is reported in lowercase. Nucleotides highlighted in bold represent 5’-G appended to favor transcription from canonical U6 Pol III promoters.

[0361] Exemplary amino acid sequences of nuclear localization signal or SV5 tag are listed in Table 24 and Table 25 below. Table 24. Exemplary amino acid sequences of nuclear localization signals Sequence SEQ ID NO: RTADGSEFESPKKKRKV SEQ ID NO: 1800 KRTADGSEFESPKKKRKV SEQ ID NO: 1801Table 24. Exemplary amino acid sequences of nuclear localization signals Sequence SEQ ID NO: PKKKRKV SEQ ID NO: 1802 PKKKRRV SEQ ID NO: 1803 KRPAATKKAGQAKKKK SEQ ID NO: 1804 YGRKKRRQRRR SEQ ID NO: 1805 RKKRRQRRR SEQ ID NO: 1806 PAAKRVKLD SEQ ID NO: 1807 RQRRNELKRSP SEQ ID NO: 1808 VSRKRPRP SEQ ID NO: 1809 PPKKARED SEQ ID NO: 1810 PQPKKKPL SEQ ID NO: 1811 SALIKKKKKMAP SEQ ID NO: 1812 PKQKKRK SEQ ID NO: 1813 RKLKKKIKKL SEQ ID NO: 1814 REKKKFLKRR SEQ ID NO: 1815 KRKGDEVDGVDEVAKKKSKK SEQ ID NO: 1816 RKCLQAGMNLEARKTKK SEQ ID NO: 1817 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY SEQ ID NO: 1818 RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV SEQ ID NO: 1819 PAAKKKKLD SEQ ID NO: 1820 PAAKRVKLD SEQ ID NO: 1821 KRPAATKKAGQAKKKKLD SEQ ID NO: 1822 Table 25. Exemplary amino acid sequence of SV5 / V5 tag Sequence SEQ ID NO: GKPIPNPLLGLDST SEQ ID NO: 1823 IPNPLLGLD SEQ ID NO: 1824 8.2.1.4. In vitro assays for Cas9 PAM identification

[0362] The in vitro PAM evaluation of the Cas9 orthologous polypeptides identified in Section 6.2.1.3. above was performed according to the protocol from Karvelis et al., 2019 (Karvelis et al. 2019. A pipeline for characterization of novel Cas9 orthologs, Methods in Enzymology, 616, pp. 219–240). In brief: the identified Cas9 orthologs coding sequences were modified by the addition of an SV5 tag at the N-terminus and two nuclear localization signals (1 at the N-term and 1 at the C-term) and were human codon-optimized. The constructs for the coding sequences were obtained as synthetic fragments from Twist Bioscience and cloned into an expression vector for in vitro transcription and translation (IVT) (pT7-N-His-GST, Thermo Fisher Scientific).

[0363] The sgRNAs used to identify Cas9 PAMs were in vitro transcribed (HighYield T7 RNA Synthesis Kit, Jena Bioscience) starting from the amplification (Phusion HF DNA polymerase, Thermo Fisher Scientific) of the plasmid bearing sgRNA. The primers used to generate the IVT templates are listed in Table 24. In vitro transcribed sgRNAs were subsequently purified using the MEGAClear Transcription Clean-up kit (Thermo Fisher Scientific). The in vitro transcription and translation reaction for Cas9 expression was performed according to the manufacturer’s protocol (1-Step Human High-Yield Mini IVT Kit, Thermo Fisher Scientific). The hairpin structure of the sgRNA was generated for visualization after in silico folding using RNA folding form v2.3 (unafold.org) of the sgRNA scaffolds (not including the spacer sequence).

[0364] The Cas9 orthologous nuclease-guide RNA ribonucleoprotein (RNP) complex was assembled by combining 20 μL of the supernatant containing soluble Cas9 orthologous polypeptides proteins with 1μL of RiboLock RNase Inhibitor (Thermo Fisher Scientific) and 2μg of guide RNA (sgRNA, previously transcribed in vitro). The RNP complex was used to digest 1ug 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. Sequences of the primers used for PCR amplification of gRNAs used as templates for in vitro transcription are listed in Table 26. Table 26. Sequences of the primers used for PCR amplification of gRNAs used as templates for in vitro transcription Primer name Sequence (5’ → 3’)SEQ ID NODNDB-sgRNA_IVT-F cctcTAATACGACTCACTATAGGTCGTCGCCCTCG SEQ ID NO: 1346 AACTTCACCTGTTCTTGAGGAGAAATCTGGAAG DNDB-sgRNA_IVT-R AAAAAATGAGATAGCCACAATTAGC SEQ ID NO: 1347 DUUZ-sgRNA_IVT-F cctcTAATACGACTCACTATAGGTCGTCGCCCTCG SEQ ID NO: 1348 AACTTCACCTGTTGTGGTTTGAGAAATCTTATCA C DUUZ-sgRNA_IVT-R AAAAAACTCCCACCGAAGC SEQ ID NO: 1349 FRDB-sgRNA_IVT-F cctcTAATACGACTCACTATAGGTCGTCGCCCTCG SEQ ID NO: 1350 AACTTCACCTGCTGTAGTTCCCGAAAGGTTAT FRDB-sgRNA_IV-R AAAAAAGATAATCCCATTCCGAAGAAT SEQ ID NO: 1351 GMIE-sgRNA_IVT-F cctcTAATACGACTCACTATAGGTCGTCGCCCTCG SEQ ID NO: 1352 AACTTCACCTGTTGTGGTTTGGAAACATATCACA GMIE-sgRNA_IVT-R AAAAAAGCCTCTCCGAAGAGAG SEQ ID NO: 1353 ARHP-sgRNA_IVT-F cctcTAATACGACTCACTATAGGTCGTCGCCCTCG SEQ ID NO: 1354 AACTTCACCTGTTTTGCTACCAGAAATGACCT ARHP-sgRNA_IVT-R AAAAAAGAAAAGGCTCCTTATAGGA SEQ ID NO: 1355

[0365] A double stranded DNA adapter (Table 27) was ligated to the DNA ends generated by the targeted Cas9 cleavage and the final ligation product was purified using a GeneJet PCR Purification Kit (Thermo Fisher Scientific).Table 27. Sequences of the two oligonucleotides used to prepare the dsDNA adapter for the in vitro PAM assay Primer name Sequence (5’ → 3’) SEQ ID NO Oligo UP CGGCATTCCTGCTGAACCGCTCTTCCGATCT SEQ ID NO: 1356 Oligo BOTTOM GATCGGAAGAGCGGTTCAGCAGGAATGCCG SEQ ID NO: 1357

[0366] 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 28). 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). The library was analysed with a 71-bp single read sequencing, using a flow cell v2 micro, on an Illumina MiSeq sequencer. Table 28. Sequences of the primers used for NGS library preparation in the in vitro PAM assay Primer name Sequence (5’ → 3’) SEQ ID NOF4aTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGCTSEQ ID NO: 1358 GAACCGCTCTTCCGATCF4bTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAAGASEQ ID NO: 1359 CTGCTGAACCGCTCTTCCGATCF4cTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTAGSEQ ID NO: 1360 ACCTAATGTGATCTGCTGAACCGCTCTTCCGATCR3GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTGSEQ ID NO: 1361 CGTTCTGATTTAATCTGTATCAGGC

[0367] 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. 8.2.1.5. Cell lines and cell culture conditions

[0368] 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). 8.2.1.6. Cell lines transfections

[0369] To perform editing experiments, 200,000 U2OS-EGFP cells were nucleofected with 500 ng of nuclease-expressing plasmid and 250 ng of sgRNA-expressing plasmid containing a guide designed to target EGFP using the 4D-Nucleofector™ SE Kit (Lonza), DN-100 program, according to the manufacturer’s protocol. After electroporation, cells were plated in a 24-well plate. EGFP knock-out was analyzed 4 days after nucleofection using a BD FACSymphony A1 (BD) flow cytometer.

[0370] For each Cas9 ortholog, 3 different sgRNAs targeting the EGFP coding sequence were tested. Loss of EGFP fluorescence, expressed as % of EGFP-negative cells, was measured by cytofluorimetry. Streptococcus pyogenes Cas9 (SpCas9) was used as reference for indel formation. 8.2.2. Results 8.2.2.1. Identification of Cas9 orthologous polypeptides from metagenomic data

[0371] A custom bioinformatics pipeline was used to explore a curated collection of assembled bacterial and archaeal metagenomes. This pipeline aimed to identify Cas9 orthologs with low sequence similarity to previously published and characterized Cas9 proteins. The discovered Cas9 orthologs were filtered based on: i) the length of their coding sequence, discarding those amino acid sequences having less than 950; ii) their origin from putative unknown species, and iii) the presence of intact nuclease domains. Cas9 orthologous proteins with high sequence similarity were clustered together and the orthologs with the greater sequence representation in the original metagenomic library were selected for each cluster. Among the identified Cas9, a set of particular interest was identified. The capitalized four letter identifier assigned to each CRISPR-Cas polypeptide is arbitrary and unique: • FRDB CRISPR-Cas polypeptide, originating from an unclassified bacterium from the Lachnospiraceae family, 1159 aa long. • DNDB CRISPR-Cas polypeptide, originating from Eubacterium sp. AF22-8LB, 1355 aa long. • GMIE CRISPR-Cas polypeptide, originating from Alloprevotella tannerae, 1223 aa long. • ARHP CRISPR-Cas polypeptide, originating from Catenibacterium sp. AM22-15, 1113 aa long.• DUUZ CRISPR-Cas polypeptide, originating from an unclassified bacterium from the Prevotella genus, 1202 aa long. 8.2.2.2. Identification of crRNAs and tracrRNAs of Cas9 orthologous polypeptides

[0372] We identified crRNAs and tracrRNAs corresponding to the Cas9 orthologous polypeptides identified in Example 1 within the same metagenomic data. This identification was achieved using a custom-built bioinformatic pipeline (see Methods for details). sgRNAs were designed for each CRISPR-Cas polypeptide by combining the respectively identified tracrRNAs with the corresponding crRNAs extracted from the CRISPR arrays (see Tables 29-31). We used the predicted hairpin structure of the full length sgRNA molecules to determine how to trim the sgRNA scaffolds at the level of the repeat:anti-repeat loop regions, resulting in more compact sgRNA molecules represented in FIGs.25A-25C and 26A-26B. The nucleic acid sequences of the sgRNA molecules are listed in Table 31. Table 29. Sequences of crRNAs of novel CRISPR-Cas polypeptides Cas9 orthologous Sequence SED ID NO: nuclease ID FRDB (SEQ ID NO: GCUGUAGUUCCCUAUUAUUUCUUGGUAUGGUAUAAU SEQ ID NO: 1362 721 or SEQ ID NO: 1383) DNDB GUUCUUGAGGAUUGAAUUUAUUUGUAUUAUUUGAA SEQ ID NO: 1363 (SEQ ID NO: 362 or C SEQ ID NO: 1384) GMIE GUUGUGGUUUGAUGUAGAAGUAAGAUAUAA SEQ ID NO: 1364 (SEQ ID NO: 444 or SEQ ID NO: 1385) ARHP GUUUUGCUACCAUCUAUUAUUUUGCUAGAAGAUGAC SEQ ID NO: 1365 (SEQ ID NO: 768 or SEQ ID NO: 1386) DUUZ GUUGUGGUUUGAUGUAGGAAUCAAAAGAUAUACAAC SEQ ID NO: 1366 (SEQ ID NO: 76 or SEQ ID NO: 1387) Table 30. Nucleic acid sequences of tracrRNAs of novel CRISPR-Cas polypeptides Cas9 orthologous Sequence SED ID NO: nuclease ID FRDB AUUAUACUAUACCAAGAAAUAAUCGGUUAUUGCAGUAU SEQ ID NO: 1367 (SEQ ID NO: 721 or GGUAAGCAAAACCGAAAAGCUCUAAUCCCAUUCUUCGGA SEQ ID NO: 1383) AUGGGAUUAUCUUUU DNDB UCAAAUAAUACAAAUGAAUUUAGUCUGGAAGAAUAAGA SEQ ID NO: 1368 (SEQ ID NO: 362 or UUUAAAUUCGAAAACUAUUAGAGAUAGCUAAUUGUGGC SEQ ID NO: 1384) UAUCUCAUUUUTable 30. Nucleic acid sequences of tracrRNAs of novel CRISPR-Cas polypeptides Cas9 orthologous Sequence SED ID NO: nuclease ID GMIE AAAAGGAUAUUCUACAUCAUAUCACAAUAAGGCUAUAU SEQ ID NO: 1369 (SEQ ID NO: 444 or GCCGAAGGAUUUUAUUCCCAUGGCCUCUCUUCGGAGAGG SEQ ID NO: 1385) CUUU ARHP GUUAUAAUCUAGCAAAAUAAUAUAUGACCUAGCAAAAC SEQ ID NO: 1370 (SEQ ID NO: 768 or AAGGGUUUAUCCCGGAUUCGGCUCCUAUAAGGAGCCUUU SEQ ID NO: 1386) UCUUUUU DUUZ UUCCUACAUCUUAUCACAAUAAGGCAGUAAUUGCCGAAG SEQ ID NO: 1371 (SEQ ID NO: 76 or GUGUUAAACCUAUACUCCCGCUUCGGUGGGAGUUU SEQ ID NO: 1387) Table 31. Nucleic acid sequences of sgRNAs of novel CRISPR-Cas polypeptides Cas9 orthologous Sequence SED ID NO: nuclease ID FRDB GCUGUAGUUCCCGAAAGGUUAUUGCAGUAUGGUAAGCA SEQ ID NO: 1372 (SEQ ID NO: 721 or AAACCGAAAAGCUCUAAUCCCAUUCUUCGGAAUGGGAU SEQ ID NO: 1383) UAUCUUUUUU DNDB GUUCUUGAGGAGAAAUCUGGAAGAAUAAGAUUUAAAU SEQ ID NO: 1373 (SEQ ID NO: 362 or UCGAAAACUAUUAGAGAUAGCUAAUUGUGGCUAUCUCA SEQ ID NO: 1384) UUUUUU GMIE GUUGUGGUUUGGAAACAUAUCACAAUAAGGCUAUAUGC SEQ ID NO: 1374 (SEQ ID NO: 444 or CGAAGGAUUUUAUUCCCAUGGCCUCUCUUCGGAGAGGC SEQ ID NO: 1385) UUUUUU ARHP GUUUUGCUACCAGAAAUGACCUAGCAAAACAAGGGUUU SEQ ID NO: 1375 (SEQ ID NO: 768 or AUCCCGGAUUCGGCUCCUAUAAGGAGCCUUUUCUUUUU SEQ ID NO: 1386) U ARHP (alternative GUUUUGCUACCAGAAAUGACCUAGCAAAACAAGGGUUU SEQ ID NO: 1376 trimmed) AUCCCGGAUUCGGCUCCUAUAAGGAGCCUUUUUU (SEQ ID NO: 768 or SEQ ID NO: 1386) DUUZ GUUGUGGUUUGAGAAAUCUUAUCACAAUAAGGCAGUAA SEQ ID NO: 1377 (SEQ ID NO: 76 or UUGCCGAAGGUGUUAAACCUAUACUCCCGCUUCGGUGG SEQ ID NO: 1387) GAGUUUUUU 8.2.2.3. Determination of PAM specificity of the identified Cas9 orthologs

[0373] Based on the sgRNA requirements for the selected CRISPR-Cas polypeptide proteins, we discovered the PAM sites recognized by each nuclease. The in vitro PAM assay is described above (see Methods). Briefly, the assay uses in vitro translated CRISPR-Cas polypeptide 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 were identified and sequenced by next generation sequencing. Table 32 below shows the PAM preferences as determined basedon the assay outcome. The PAM logos and the PAM heatmaps depicting the nucleotide preferences for specific positions along the PAMs are shown in FIGs.27A-27F and 28A-28D. Table 32. Cas9 orthologue nuclease ID and corresponding PAM sequences Cas9 orthologue nuclease ID SEQ ID NO In vitro-determined PAM sequence FRDB SEQ ID NO: 721 NNNNRYA NNNNGYA NNNNACY DNDB SEQ ID NO: 362 NNGG NNGGNG GMIE SEQ ID NO: 444 NNNNNCC ARHP SEQ ID NO: 768 NYAAR NYAGG DUUZ SEQ ID NO: 76 NNNCCC NNNTCC

[0374] The human codon optimized nucleic acid sequences encoding the Cas9 orthologue nuclease ID and their amino acid sequences are listed in Table 33.Table 33. Exemplary human codon optimized nucleic acid sequences and amino acid sequences of Cas9 orthologue nucleases Cas9 Human codon optimized sequence (including SEQ ID Amino acid sequence SEQ ID orthologue 2xNLS, 1 C-term + 1 N-term and detection NO: NO: nuclease ID tag) FRDB ATGGGCAAACCTATCCCCAATCCCCTGCT SEQ ID MGKPIPNPLLGLDS SEQ ID GGGCCTGGACAGCACCAAGCGGACTGCC NO: TKRTADGSEFESPK NO: GACGGCAGTGAGTTCGAGTCTCCCAAGA 1378 KKRKVGSGEMSKT 1383 AAAAGAGGAAAGTGggatccGGCGAGATGT NMDRHRSYRIGLDI CCAAAACCAACATGGACAGACACAGAA GIASVGWAVLENN GCTACAGAATCGGCCTGGATATCGGCAT SQDEPIRILDLGVRI TGCCAGCGTGGGATGGGCCGTGTTAGAG FEAAEDSQTGAAL AACAACAGCCAGGACGAACCTATCAGGA AAKRRDARTARRR TCTTGGACCTGGGCGTGCGGATCTTCGA IRRRRHRLERIKWL GGCCGCAGAGGACAGCCAGACCGGCGCC FEKEGLIQIDSFMER GCCCTGGCCGCTAAGCGGCGGGACGCCA YHSPNLPEVYGLRY GAACAGCCAGAAGAAGAATCAGACGGC EGLERKLSNEEFAQ GGAGACACCGGCTGGAAAGAATTAAGTG VLLHIAKHRGFRST GCTGTTCGAGAAGGAGGGCCTGATCCAG RKAELKEGDNGKV ATTGACTCATTCATGGAGAGATACCACA LKATEENRKRMEE GCCCTAACCTGCCCGAGGTGTATGGCCT HGYRTVGEMIYKD GCGGTACGAGGGACTGGAAAGAAAGCT AYFHTECAWADNG GAGTAACGAGGAGTTCGCCCAGGTCCTG YLLTPRNKADDYS CTGCACATCGCTAAGCACCGGGGCTTCA HTMLRALLEEEVHI GATCCACCCGGAAAGCCGAACTGAAGGA LFDSQRKFGNEKAT GGGCGATAATGGCAAGGTGCTTAAAGCC EKLEQEYLEIMLSQ ACCGAGGAGAACCGGAAGCGGATGGAG RSFDMGPGQQADG GAACATGGCTACCGGACCGTGGGCGAGA TPSPYAMEGFGNK TGATCTACAAAGACGCCTACTTCCACAC VGYCTLERESGERR AGAGTGCGCCTGGGCCGATAATGGCTAT AAKATYTAELFVA CTCCTCACCCCTAGAAACAAAGCCGATG LQKITHMKLVHMD ACTACAGCCACACTATGCTGAGGGCCCT GTSRFFTEEERNILL GCTGGAAGAGGAAGTGCACATCCTGTTT EMLSSKKEIKYAAV GATAGCCAAAGAAAGTTTGGCAATGAAA RKKLNLPLEEKFNT AGGCCACAGAGAAGCTGGAACAGGAGT LNYNMKRKGKDN ACCTGGAAATTATGCTGAGCCAGAGATC EEEEVDEAAIIAAT TTTTGATATGGGCCCCGGCCAGCAAGCC EKATFVSMSNCYE GACGGCACCCCTAGCCCTTACGCTATGG YRKRIGDYLDTLPE AGGGCTTTGGCAACAAGGTGGGATATTG EEQQDLLDEIGTILT TACACTGGAGCGGGAGTCTGGCGAACGG CYKNDDSRMNRLT AGAGCCGCCAAGGCCACATACACCGCCG EAGLSKELADSLLE AGCTGTTCGTGGCCCTGCAGAAGATCAC LTPAKFQHLSIKAM CCACATGAAGCTGGTGCACATGGACGGC KKIMPYMKEGMVY ACATCCAGATTCTTCACAGAGGAAGAGC DKACEAAGYDFKN GCAACATCCTGTTGGAGATGCTGAGCTC DNHGKKMKLLKGE TAAGAAAGAGATAAAGTACGCCGCTGTG NITEIINEITNPVVK AGAAAGAAACTGAATCTGCCCCTGGAGG RSVSQTVKVINAIIL AAAAATTCAACACCCTGAACTACAACAT KYGSPQAVSIELAR GAAGCGGAAGGGCAAGGATAACGAAGA EMSKNFEERKKAE AGAGGAGGTGGACGAGGCTGCTATCATC KQIEENRKKNEMA GCCGCCACCGAAAAAGCGACATTCGTGA KKEIQELGKLSPNG GCATGTCTAACTGCTACGAGTACAGAAA QDILKYRLWHDQQ ACGGATCGGAGACTACCTGGACACACTG GICLYTGKKIPLEEL CCAGAAGAAGAGCAGCAGGACCTGCTGG FQPGYDIDHILPYSI ACGAGATTGGCACAATCCTGACCTGTTA TFDDSYRNKVLVTS CAAGAACGATGACAGCCGGATGAACCGC QANREKGNRTPYE CTGACAGAGGCTGGCCTGAGCAAGGAGC YFGQDEVHWSAYE TGGCTGACTCTCTGCTGGAGCTGACACCT ARVNHFIKDYKKR GCCAAGTTCCAGCACCTGAGCATCAAGG RNLLKQGFTKEDRTable 33. Exemplary human codon optimized nucleic acid sequences and amino acid sequences of Cas9 orthologue nucleases Cas9 Human codon optimized sequence (including SEQ ID Amino acid sequence SEQ ID orthologue 2xNLS, 1 C-term + 1 N-term and detection NO: NO: nuclease ID tag) CCATGAAAAAGATCATGCCCTACATGAA REFKERNLNDTKYI AGAAGGCATGGTGTACGACAAGGCCTGC TRVIYNMIRQNLEM GAGGCCGCTGGCTACGATTTCAAGAACG EPLNREDRKKQVF ACAACCACGGCAAGAAAATGAAGCTGCT AVNGSITSYLRKR GAAAGGGGAGAACATCACCGAGATCATC WGLPTKDRSTDTH AACGAGATCACAAACCCTGTGGTTAAGA HAMDAVVVACCT GAAGTGTGTCCCAGACCGTGAAGGTCAT DGMIQKISRSTQYR CAATGCCATCATCCTGAAGTACGGCTCTC EVLYKGQSHPDYEI CTCAGGCTGTGTCTATCGAGCTGGCCAG VDVETGEIISLEELS GGAAATGTCCAAGAACTTCGAGGAGAGA REEWDKCYGAQVP AAGAAAGCCGAGAAGCAGATCGAGGAA PPWNHFREELKVR AATAGAAAGAAGAACGAGATGGCCAAG MGSDPKNDLDAHP AAGGAAATCCAAGAACTGGGCAAGCTGA DMYQLFRYPEDVY GCCCCAA...

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas orthologue nuclease having an amino acid sequence with at least 80% identity to any one of SEQ ID NO: 721, SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

2. The polypeptide of claim 1, wherein the polypeptide is a fusion protein.

3. The polypeptide of claim 2, wherein the polypeptide comprises a nuclear localization signal peptide, a tag peptide, or both fused to the CRISPR-Cas orthologue nuclease.

4. The polypeptide of claim 3, wherein the polypeptide comprises two or more nuclear localization signals fused to the CRISPR-Cas orthologue nuclease.

5. The polypeptide of claim 4, wherein the nuclear localization signal comprises an N-terminal nuclear localization signal, a C-terminal nuclear localization signal, or both.

6. The polypeptide of claim 5, wherein the nuclear localization signal comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 1800-1822.

7. The polypeptide of any one of claims 2-6, wherein the tag peptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1823 or SEQ ID NO: 1824.

8. The polypeptide of any one of claims 1-7, wherein the CRISPR-Cas orthologue nuclease has at least 90% identity with any one of SEQ ID NO: 721, SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

9. The polypeptide of any one of claims 1-8, wherein the CRISPR-Cas orthologue nuclease has at least 95% identity with any one of SEQ ID NO: 721, SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

10. The polypeptide of any one of claims 1-9, wherein the CRISPR-Cas orthologue nuclease is any one of SEQ ID NO: 721, SEQ ID NO: 76, SEQ ID NO: 362, SEQ ID NO: 444, SEQ ID NO: 768, SEQ ID NO: 1383, SEQ ID NO: 1384, SEQ ID NO: 1385, SEQ ID NO: 1386, and SEQ ID NO: 1387.

11. The polypeptide of any one of claims 1-10, wherein the CRISPR-Cas orthologue nuclease is SEQ ID NO: 76 or SEQ ID NO: 1387.

12. The polypeptide of any one of claims 1-10, wherein the CRISPR-Cas orthologue nuclease is SEQ ID NO: 362 or SEQ ID NO: 1384.

13. The polypeptide of any one of claims 1-10, wherein the CRISPR-Cas orthologue nuclease is SEQ ID NO: 444 or SEQ ID NO: 1385.

14. The polypeptide of any one of claims 1-10, wherein the CRISPR-Cas orthologue nuclease is SEQ ID NO: 721 or SEQ ID NO: 1383.

15. The polypeptide of any one of claims 1-10, wherein the CRISPR-Cas orthologue nuclease is SEQ ID NO: 768 or SEQ ID NO: 1386.

16. The polypeptide of any one of claims 1-15, wherein the CRISPR-Cas orthologue nuclease is capable of complexing with a guide RNA.

17. The polypeptide of claim 16, wherein the guide RNA comprises: (i) a crRNA comprising a spacer and a crRNA scaffold wherein the spacer is positioned 5’ to the crRNA scaffold; and (ii) a tracrRNA.

18. The polypeptide of claim 16, wherein the guide RNA is a single guide RNA (sgRNA) comprising a spacer and a sgRNA scaffold, wherein the spacer is positioned 5’ to the sgRNA scaffold.

19. A guide RNA capable of complexing with the polypeptide according to any one of claims 1- 15.

20. The guide RNA of claim 19, further comprises a spacer.

21. The guide RNA of claim 20, wherein the spacer comprises a nucleic acid sequence that is partially or fully complementary to a target mammalian genomic sequence.

22. The guide RNA of claim 21, wherein the target mammalian genomic sequence is upstream of a Protospacer Adjacent Motif (PAM) sequence in the non-target strand recognized by the polypeptide.

23. The guide RNA of claim 22, wherein the PAM sequence comprises a PAM nucleic acid sequence as set forth in Table 32.

24. The guide RNA of any one of claims 19-23, wherein the guide RNA comprises separate crRNA and tracrRNA molecules.

25. The guide RNA of claim 24, wherein the crRNA comprises, in 5’ to 3’ order, the spacer and a crRNA scaffold.

26. The guide RNA of claim 25, wherein the guide RNA comprises, in 5’ to 3’ order, (i) the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1362, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1367; (ii) the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1363, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1368; (iii) the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1364, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1369; (iv) the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1365, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1370; or (v) the crRNA molecule comprising at least 20 consecutive nucleotides of SEQ ID NO: 1366, and the tracrRNA molecule comprising at least 50 consecutive nucleotides of SEQ ID NO: 1371.

27. The guide RNA of claim 26, wherein the guide RNA comprises, in 5’ to 3’ order, (i) the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1362, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1367; (ii) the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1363, and the tracrRNA molecule comprising comprises at least 80 consecutive nucleotides of SEQ ID NO: 1368;(iii) the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1364, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1369; (iv) the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1365, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1370; or (v) the crRNA molecule comprising at least 30 consecutive nucleotides of SEQ ID NO: 1366, and the tracrRNA molecule comprising at least 80 consecutive nucleotides of SEQ ID NO: 1371.

28. The guide RNA of any one of claims 24-27, wherein (i) the crRNA molecule has at least 90% identity with SEQ ID NO: 1362, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1367; (ii) the crRNA molecule has at least 90% identity with SEQ ID NO: 1363, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1368; (iii) the crRNA molecule has at least 90% identity with SEQ ID NO: 1364, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1369; (iv) the crRNA molecule has at least 90% identity with SEQ ID NO: 1365, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1370; or (v) the crRNA molecule has at least 90% identity with SEQ ID NO: 1366, and the tracrRNA molecule has at least 90% identity with SEQ ID NO: 1371.

29. The guide RNA of claim 28, wherein (i) the crRNA molecule has at least 95% identity with SEQ ID NO: 1362, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1367; (ii) the crRNA molecule has at least 95% identity with SEQ ID NO: 1363, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1368; (iii) the crRNA molecule has at least 95% identity with SEQ ID NO: 1364, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1369;(iv) the crRNA molecule has at least 95% identity with SEQ ID NO: 1365, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1370; or (v) the crRNA molecule has at least 95% identity with SEQ ID NO: 1366, and the tracrRNA molecule has at least 95% identity with SEQ ID NO: 1371.

30. The guide RNA of any one of claims 19-23, wherein the guide RNA is a sgRNA.

31. The sgRNA of claim 30, wherein the sgRNA comprises, in 5’ to 3’ order, a spacer and a sgRNA scaffold.

32. The sgRNA of claim 30 or 31, wherein the sgRNA comprises at least one modified nucleotide or modified internucleoside linkage.

33. The sgRNA of claim 32, wherein the sgRNA comprises at least one modified nucleotide selected from 2'-fluoro, 2'-amino or 2'-O-methyl modification on the ribose of pyrimidines, abasic residues, and an inverted base at the 3' end of the RNA.

34. The sgRNA of claim 32, wherein sgRNA comprises at least one modified internucleoside linkage selected from phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl inter-sugar linkages, and short chain heteroatomic or heterocyclic inter-sugar linkages.

35. The sgRNA of any one of claims 30-34, wherein the sgRNA comprises at least 50 consecutive nucleotides of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

36. The sgRNA of claim 35, wherein the sgRNA comprises at least 80 consecutive nucleotides of any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

37. The sgRNA of any one of claims 30-36, wherein the sgRNA has at least 90% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

38. The sgRNA of claim 37, wherein the sgRNA has at least 95% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

39. A system, comprising:a polypeptide comprising the CRISPR-Cas9 orthologue nuclease according to any one of claims 1-15, and a guide RNA according to any one of claims 19-38, wherein the CRISPR-Cas9 orthologue nuclease is capable of complexing with the guide RNA.

40. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 76 or SEQ ID NO: 1387, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1366, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1371.

41. The system of claim 40, wherein the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1366, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1371.

42. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 76 or SEQ ID NO: 1387, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1377.

43. The system of claim 42, wherein the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1377.

44. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 362 or SEQ ID NO: 1384, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1363, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1368.

45. The system of claim 44, wherein the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1363; and the tracrRNA polynucleotide has at least 90% identity to SEQ ID NO: 1368.

46. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 362 or SEQ ID NO: 1384, andwherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1373.

47. The system of claim 46, wherein the sgRNA comprises a polynucleotide having at least 90% identity to SEQ ID NO: 1373.

48. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 444 or SEQ ID NO: 1385, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1364, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1369.

49. The system of claim 48, wherein the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1364; and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1369.

50. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 444 or SEQ ID NO: 1385, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1374.

51. The system of claim 50, wherein the sgRNA comprises a polynucleotide having at least 90% identity to SEQ ID NO: 1374.

52. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 721 or SEQ ID NO: 1383, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1362, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1367.

53. The system of claim 52, wherein the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1362, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1367.

54. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 721 or SEQ ID NO: 1383, andwherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1372.

55. The system of claim 54, wherein the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1372.

56. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 768 or SEQ ID NO: 1386, and wherein the guide RNA comprises a crRNA polynucleotide comprising, in 5’ to 3’ order, at least 20 consecutive nucleotides of SEQ ID NO: 1365, and a tracrRNA polynucleotide comprising, in 5’ to 3’ order, at least 50 consecutive nucleotides of SEQ ID NO: 1370.

57. The system of claim 56, wherein the crRNA polynucleotide has at least 90% identity with SEQ ID NO: 1365, and the tracrRNA polynucleotide has at least 90% identity with SEQ ID NO: 1370.

58. The system of claim 39, wherein the CRISPR-Cas9 orthologue nuclease has at least 90% identity with SEQ ID NO: 765 or SEQ ID NO: 1386, and wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 50 consecutive nucleotides of SEQ ID NO: 1375 or SEQ ID NO: 1376.

59. The system of claim 58, wherein the sgRNA comprises a polynucleotide having at least 90% identity with SEQ ID NO: 1375 or SEQ ID NO: 1376.

60. A nucleic acid comprising a polynucleotide encoding the polypeptide according to any one of claims 1-18, or the polypeptide in the system according to any one of claims 39-59.

61. The nucleic acid of claim 60, wherein the polynucleotide has at least 80% identity with any one of SEQ ID NO: 1378, SEQ ID NO: 1379, SEQ ID NO: 1380, SEQ ID NO: 1381, and SEQ ID NO: 1382.

62. A nucleic acid comprising a polynucleotide encoding the guide RNA according to any one of claims 19-38, wherein the guide RNA comprises:(i) a polynucleotide encoding the crRNA has at least 90% identity with SEQ ID NO: 1362, and a polynucleotide encoding the tracrRNA has at least 90% identity with SEQ ID NO: 1367; (ii) a polynucleotide encoding the crRNA has at least 90% identity with SEQ ID NO: 1363, and a polynucleotide encoding the tracrRNA has at least 90% identity with SEQ ID NO: 1368; (iii) a polynucleotide encoding the crRNA has at least 90% identity with SEQ ID NO: 1364, and a polynucleotide encoding the tracrRNA has at least 90% identity with SEQ ID NO: 1369; (iv) a polynucleotide encoding the crRNA has at least 90% identity with SEQ ID NO: 1365, and a polynucleotide encoding the tracrRNA has at least 90% identity with SEQ ID NO: 1370; or (v) a polynucleotide encoding the crRNA has at least 90% identity with SEQ ID NO: 1366, and a polynucleotide encoding the tracrRNA has at least 90% identity with SEQ ID NO: 1371.

63. A nucleic acid comprising a polynucleotide encoding the guide RNA according to any one of claims 19-38, wherein the guide RNA is a sgRNA comprising a polynucleotide having at least 90% identity with any one of SEQ ID NO: 1372, SEQ ID NO: 1373, SEQ ID NO: 1374, SEQ ID NO: 1375, SEQ ID NO: 1376, and SEQ ID NO: 1377.

64. The nucleic acid comprising the polynucleotide encoding the polypeptide according to claim 60 or 61, or the nucleic acids encoding the crRNA according to claim 62; or the nucleic acids encoding the tracrRNA according to claim 62; or the nucleic acids encoding the sgRNA according to claim 63, wherein the nucleic acid is obtained by in vitro transcription.

65. The nucleic acid comprising the polynucleotide encoding the polypeptide according to claim 60 or 61, or the nucleic acids encoding the crRNA according to claim 62; or the nucleic acids encoding the tracrRNA according to claim 62; or the nucleic acids encoding the sgRNA according to claim 63, wherein the nucleic acid is operably linked to a promoter.

66. The nucleic acid comprising the polynucleotide encoding the polypeptide according to claim 60 or 61, or the nucleic acids encoding the crRNA according to claim 62; or the nucleic acidsencoding the tracrRNA according to claim 62; or the nucleic acids encoding the sgRNA according to claim 63, wherein the nucleic acid is synthetic.

67. The nucleic acid comprising the polynucleotide encoding the polypeptide according to claim 60 or 61, or the nucleic acids encoding the crRNA according to claim 62; or the nucleic acids encoding the tracrRNA according to claim 62; or the nucleic acids encoding the sgRNA according to claim 63, wherein the nucleic acid is human codon optimized.

68. An expression vector comprising the nucleic acids encoding the polypeptide according to claim 60, 61, or 64-67.

69. An expression vector comprising the nucleic acids encoding the crRNA of any one of claims 62 or 64-67.

70. An expression vector comprising the nucleic acids encoding the tracrRNA of any one of claims 62 or 64-67.

71. An expression vector comprising the nucleic acids encoding the sgRNA according to claim 63 or 64-67.

72. A cell comprising the system according to any one of claims 39-59.

73. The cell of claim 72, wherein the cell is a prokaryotic cell or eukaryotic cell.

74. The cell of claim 73, wherein the prokaryotic cell is a bacterial cell.

75. The cell of claim 73, wherein the eukaryotic cell is a mammalian cell.

76. The cell of claim 75, wherein the cell is a human cell.

77. A pharmaceutical composition comprising: (i) the polypeptide according to any one of claims 1-18, or the polypeptide in the system according to any one of claims 39-59; and / or the guide RNA according to any one of claims 19-38, wherein the guide RNA comprises separate crRNA and tracrRNA, or wherein the guide RNA comprises a sgRNA; or (ii) the system according to any one of claims 39-59; or (iii) the nucleic acid according to any one of claims 60-67; or(iv) the expression vector according to any one of claims 68-71; and a pharmaceutically acceptable excipient.

78. A method of site-specifically cleaving a DNA target in a cell, the method comprising contacting the cell with: (i) the polypeptide according to any one of claims 1-18 or the polypeptide in the system according to any one of claims 39-59, and the guide RNA in the system according to any one of claims 39-59; or (ii) the system according to any one of claims 39-59; or (iii) the nucleic acid according to any one of claims 60-67; (iv) the expression vector according to any one of claims 68-71; and / or (v) the pharmaceutical composition of claim 77.

79. A polypeptide comprising: a CRISPR-Cas orthologue nuclease having an amino acid sequence with at least 80% identity to any nuclease capable of complexing with a guide RNA as shown in Table 34.

80. The polypeptide of claim 79, wherein the nuclease comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO.: 1, SEQ ID NO.: 28, SEQ ID NO.: 55, SEQ ID NO.: 106, SEQ ID NO.: 129, SEQ ID NO.: 163, SEQ ID NO.: 217, SEQ ID NO.: 242, SEQ ID NO.: 254, SEQ ID NO.: 270, SEQ ID NO.: 297, SEQ ID NO.: 325, SEQ ID NO.: 341, SEQ ID NO.: 373, SEQ ID NO.: 391, SEQ ID NO.: 409, SEQ ID NO.: 426, SEQ ID NO.: 462, SEQ ID NO.: 481, SEQ ID NO.: 503, SEQ ID NO.: 523, SEQ ID NO.: 552, SEQ ID NO.: 593, SEQ ID NO.: 646, SEQ ID NO.: 686, SEQ ID NO.: 735, SEQ ID NO.: 833, SEQ ID NO.: 847, SEQ ID NO.: 886, SEQ ID NO.: 940, and SEQ ID NO: 1826.

81. The polypeptide of claim 79 or 80, wherein the polypeptide is a fusion protein.

82. A guide RNA capable of complexing with the polypeptide of claim 79 or 80, wherein the guide RNA is a sgRNA.

83. The guide RNA of claim 82, wherein the sgRNA comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOs: 1450-1483 and SEQ ID NO: 1825 as shown in Table 34.

84. The guide RNA of claim 82, wherein the guide RNA comprises separate crRNA and tracrRNA.

85. The guide RNA of claim 84, wherein the crRNA comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NOs: 1388-1418 as shown in Table 34, and the crRNA is capable of complexing with a tracrRNA comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1419-1449 as shown in Table 34.

86. A system comprising the polypeptide according to claim 79 or 80, and their corresponding guide RNA as shown in Table 34; or the guide RNA according to any one of claims 82-85, and their corresponding complexing nuclease as shown in Table 34.

87. A nucleic acid comprising a polynucleotide encoding the polypeptide according to claim 79 or 80, or the polypeptide in the system according to claim 86.

88. A nucleic acid comprising a polynucleotide encoding the guide RNA according to any one of claims 82-85, or the guide RNA in the system according to claim 86.

89. An expression vector comprising the nucleic acid of claim 87 or 88.

90. A cell comprising the system of claim 86.

91. A pharmaceutical composition comprising: the polypeptide according to claim 79 or 80 or the polypeptide in the system according to claim 86; and / or the guide RNA according to any one of claims 82-85, or the guide in the system according to claim 86, and a pharmaceutically acceptable excipient.

92. A method of site-specifically cleaving a DNA target in a cell, the method comprising contacting the cell with: (i) the polypeptide according to claim 79 or 80, and the polypeptide in the system according to claim 86; or(ii) the system according to claim 86; or (iii) the nucleic acid according to claim 87 or 88; (iv) the expression vector of claim 89; and / or the pharmaceutical composition of claim 91.

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