OMNI 263, 264, 266, 268, 269, 271, 274, 275, 276, 278, 279, 280, 281, 283, 284, 286, 287, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 310,

311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 330,

331, 332, 333, 334, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,

351, 352, 353, 354, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,

371, 372, 373, 375, 376, 377, 378, 380, 381, 382, 383, 384, 385, AND 386 CRISPR NUCLEASES

[0001] This application claims the benefit of U.S. Provisional Application No. 63/281,390, filed November 19, 2021, the contents of which are hereby incorporated by reference.

[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide sequences which are present in the file named “221117_91815-A-PCT_Sequence_Listing_AWG.xml”, which is 4,464 kilobytes in size, and which was created on November 15, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed November 17, 2022 as part of this application.

FIELD OF THE INVENTION

[0004] The present invention is directed to, inter alia, composition and methods for genome editing.

BACKGROUND OF THE INVENTION

[0005] The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR systems have become important tools for research and genome engineering. Nevertheless, many details of CRISPR systems have not been determined and the applicability of CRISPR nucleases may be limited by sequence specificity requirements, expression, or delivery challenges. Different CRISPR nucleases have diverse characteristics such as: size, PAM site, on target activity, specificity, cleavage pattern (e.g. blunt, staggered ends), and prominent pattern of indel formation following cleavage. Different sets of characteristics may be useful for different applications. For example, some CRISPR nucleases may be able to target particular genomic loci that other CRISPR nucleases cannot due to limitations of the PAM site. In addition, some CRISPR nucleases currently in use exhibit pre-immunity, which may limit in vivo applicability. See Charlesworth et al., Nature Medicine (2019) and Wagner et al., Nature Medicine (2019). Accordingly, discovery, engineering, and improvement of novel CRISPR nucleases is of importance.

SUMMARY OF THE INVENTION

[0006] Disclosed herein are compositions and methods that may be utilized for genomic engineering, epigenomic engineering, genome targeting, genome editing of cells, and/or in vitro diagnostics.

[0007] The disclosed compositions may be utilized for modifying genomic DNA sequences. As used herein, genomic DNA refers to linear and/or chromosomal DNA and/or plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, the methods produce double-stranded breaks (DSBs) at predetermined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of a DNA sequence at the target site(s) in a genome.

[0008] Accordingly, in some embodiments, the compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) nucleases. In some embodiments, the CRISPR nuclease is a CRISPR-associated protein.

OMNI CRISPR Nucleases

[0009] Embodiments of the present invention provide for CRISPR nucleases designated as an “OMNI” nuclease as provided in Table 1. [0010] This invention provides a method of modifying a nucleotide sequence at a target site in the genome of a mammalian cell comprising introducing into the cell (i) a composition comprising a CRISPR nuclease having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding a CRISPR nuclease which sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 109-324 and (ii) a DNA-targeting RNA molecule, or a DNA polynucleotide encoding a DNA-targeting RNA molecule, comprising a nucleotide sequence that is complementary to a sequence in the target DNA.

[0011] This invention also provides a non-naturally occurring composition comprising a CRISPR associated system comprising: a) one or more RNA molecules comprising a guide sequence portion linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more RNA molecules; and b) an CRISPR nuclease comprising an amino acid sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and wherein the one or more RNA molecules hybridize to the target sequence, wherein the target sequence is adjacent to the 3’ end of a complimentary sequence of a Protospacer Adjacent Motif (PAM), and the one or more RNA molecules form a complex with the RNA-guided nuclease.

[0012] This invention also provides a non-naturally occurring composition comprising: a) a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and b) one or more RNA molecules, or one or more DNA polynucleotide encoding the one or more RNA molecules, comprising at least one of: i) a nuclease-binding RNA nucleotide sequence capable of interacting with/binding to the CRISPR nuclease; and ii) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence, wherein the CRISPR nuclease is capable of complexing with the one or more RNA molecules to form a complex capable of hybridizing with the target DNA sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figs. 1A-C: The predicted secondary structure of a single guide RNA (sgRNA) (crRNA-tracrRNA) from sgRNA-279, sgRNA-281 and sgRNA-280. Fig. 1A: A representation of a crRNA-tracrRNA duplex for OMNI-279, the crRNA and tracrRNA portions of the sgRNA are noted. Fig. IB: Examples of VI and V2 of sgRNA design for OMNI-281. Fig. 1C: Examples of VI of sgRNA design for OMNI-280 (sgRNA listed in Table 2)

[0014] Figs. 2-109: In vitro TXTL PAM depletion results for OMNI nucleases. The PAM logo is a schematic representation of the ratio of the depleted site (top panel). Depletion ratio (bottom panel, left) of specific PAM sequences (bottom panel, right) from the PAM plasmid library were calculated following NGS of the TXTL reaction. The calculation for each OMNI is based on a 4N window along the 8bp sequence of the PAM library. The required PAM of the tested OMNI and the level of nuclease activity under the reaction conditions is inferred from the depletion ratio. In vitro VAM depletion results for: Fig 2: OMNI-263. Fig 3: OMNI-264. Fig 4: OMNI-266. Fig 5: OMNI-268. Fig 6: OMNI-269. Fig 7: OMNI-271. Fig 8: OMNI-274. Fig 9: OMNL275. Fig 10: OMNI-276. Fig 11 : OMNI-278. Fig 12: OMNI-279. Fig 13: OMNI-280. Fig 14: OMNI-281. Fig 15: OMNI-283. Fig 16: OMNI-284. Fig 17: OMNI-286. Fig 18: OMNI-287. Fig 19: OMNI- 288. Fig 20: OMNI-290. Fig 21 : OMNI-291. Fig 22: OMNI-293. Fig 23: OMNI-294. Fig 24: OMNI-295. Fig 25: OMNI-296. Fig 26: OMNI-297. Fig 27: OMNI-298. Fig 28: OMNI-299. Fig 29: OMNI-300. Fig 30: OMNI-301. Fig 31 : OMNI-302. Fig 32: OMNI-303. Fig 33: OMNI-304. Fig 34: OMNI-305. Fig 35: OMNI-307. Fig 36: OMNI-308. Fig 37: OMNI-309. Fig 38: OMNI- 310. Fig 39: OMNI-311. Fig 40: OMNI-312. Fig 41 : OMNI-313. Fig 42: OMNI-314. Fig 43: OMNI-315. Fig 44: OMNI-316. Fig 45: OMNI-317. Fig 46: OMNI-318. Fig 47: OMNI-319. Fig 48: OMNI-320. Fig 49: OMNI-321. Fig 50: OMNI-322. Fig 51 : OMNI-323. Fig 52: OMNI-324. Fig 53: OMNI-325. Fig 54: OMNI-326. Fig 55: OMNI-327. Fig 56: OMNI-329. Fig 57: OMNI- 330. Fig 58: OMNI-331. Fig 59: OMNI-332. Fig 60: OMNI-333. Fig 61 : OMNI-334. Fig 62: OMNI-336. Fig 63: OMNI-337. Fig 64: OMNI-338. Fig 65: OMNI-339. Fig 66: OMNI-340. Fig 67: OMNI-341. Fig 68: OMNI-342. Fig 69: OMNI-343. Fig 70: OMNI-344. Fig 71 : OMNI-345. Fig 72: OMNI-346. Fig 73: OMNI-347. Fig 74: OMNI-348. Fig 75: OMNI-349. Fig 76: OMNI- 350. Fig 77: OMNI-351. Fig 78: OMNI-352. Fig 79: OMNI-353. Fig 80: OMNI-354. Fig 81 : OMNI-356. Fig 82: OMNI-357. Fig 83: OMNI-358. Fig 84: OMNI-359. Fig 85: OMNI-360. Fig 86: OMNI-361. Fig 87: OMNI-362. Fig 88: OMNI-363. Fig 89: OMNI-364. Fig 90: OMNI-365. Fig 91 : OMNI-366. Fig 92: OMNI-367. Fig 93: OMNI-368. Fig 94: OMNI-369. Fig 95: OMNI- 370. Fig 96: OMNI-371. Fig 97: OMNI-372. Fig 98: OMNI-373. Fig 99: OMNI-375. Fig 100: OMNI-376. Fig 101 : OMNI-377. Fig 102: OMNI-378. Fig 103: OMNI-380. Fig 104: OMNI-381. Fig 105: OMNI-382. Fig 106: OMNI-383. Fig 107: OMNI-384. Fig 108: OMNI-385. Fig 109: OMNI-386.

DETAILED DESCRIPTION

[0015] According to some aspects of the invention, the disclosed compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease and/or a nucleic acid molecule comprising a sequence encoding the same.

[0016] Table 1 lists novel CRISPR nucleases, as well as substitutions at one or more positions within each nuclease which convert the nuclease to a nickase or catalytically dead nuclease.

[0017] Table 2 provides crRNA, tracrRNA, and single-guide RNA (sgRNA) sequences, and portions of crRNA, tracrRNA, and sgRNA sequences, that are compatible with each listed CRISPR nuclease. Accordingly, a crRNA molecule capable of binding and targeting an OMNI nuclease listed in Table 2 as part of a crRNA:tracrRNA complex may comprise any crRNA sequence listed in Table 2. Similarly, a tracrRNA molecule capable of binding and targeting an OMNI nuclease listed in Table 2 as part of a crRNA:tracrRNA complex may comprise any tracrRNA sequence listed in Table 2. Also, a single-guide RNA molecule capable of binding and targeting an OMNI nuclease listed in Table 2 may comprise any sequence listed in Table 2.

[0018] For example, a crRNA molecule of OMNI-263 nuclease (SEQ ID NO: 1) may comprise a sequence of any one of SEQ ID NOs: 326-329; a tracrRNA molecule of OMNI-263 nuclease may comprise a sequence of any one of SEQ ID NOs: 330-338; and a sgRNA molecule of OMNI- 263 nuclease may comprise a sequence of any one of SEQ ID NOs: 325-338. Other crRNA molecules, tracrRNA molecules, or sgRNA molecules for each OMNI nuclease may be derived from the sequences listed in Table 2 in the same manner.

[0019] Any one of these nucleases can target a desired DNA target sequence via a guide RNA molecule. The nuclease-guide complex will also carry any molecule attached to the complex to the target site. Thus, this disclosure also contemplates fusion proteins comprising CRISPR nucleases and a DNA modifying domain (e.g., a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain), as well as the use of such fusion proteins in correcting mutations in a genome (e.g., the genome of a human subject) that are associated with disease, or generating mutations in a genome (e.g., the human genome) to decrease or prevent expression of a gene.

[0020] In some embodiments, any of the CRISPR nucleases provided herein may be fused to a protein that has an enzymatic activity. In some embodiments, the enzymatic activity modifies a target DNA. In some embodiments, the enzymatic activity is nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity. In some cases, the enzymatic activity is nuclease activity. In some cases, the nuclease activity introduces a double strand break in the target DNA. In some cases, the enzymatic activity modifies a target polypeptide associated with the target DNA. In some cases, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some cases, the target polypeptide is a histone and the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity or deubiquitinating activity.

[0021] Thus, any one of the CRISPR nucleases, nickases, or dead-nucleases may be fused (e.g. directly fused or fused via a linker) to another DNA modulating or DNA modifying enzyme, including, but not limited to, base editors such as a deaminase, a reverse transcriptase (e.g. for use in prime editing, see Anzaolone et al. (2019)), an enzyme that modifies the methylation state of DNA (e.g. a methyltransferase), or a modifier of histones (e.g. a histone acetyl transferase). Indeed, the OMNI-50 nucleases, nickases, inactive nucleases described herein may be fused to a DNA modifying enzyme or an effector domain thereof. Examples of DNA modifiers include but are not limited to: a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a reverse transcriptase, an helicase, an integrase, a ligase, a transposase, a demethylase, a phosphatase, a transcriptional activator, or a transcriptional repressor. In some embodiments, any of the CRISPR nucleases provided herein are fused to a protein that has an enzymatic activity. In some embodiments, the enzymatic activity modifies a target DNA molecule. In some embodiments, the CRISPR nucleases described herein or fusion proteins thereof, may be used to correct or generate one or more mutations in a gene associated with disease, or to increase, correct, decrease or prevent expression of a gene.

[0022] The invention provides a non-naturally occurring composition comprising a CRISPR nuclease comprising a sequence having at least 90% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 1-12, and 14-108, or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease.

[0023] In some embodiments, the composition further comprises one or more RNA molecules, or a DNA polynucleotide encoding any one of the one or more RNA molecules, wherein the one or more RNA molecules and the CRISPR nuclease do not naturally occur together and the one or more RNA molecules are configured to form a complex with the CRISPR nuclease and/or target the complex to a target site.

[0024] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 325-338.

[0025] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 326-329. [0026] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 330-338.

[0027] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 325-338.

[0028] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 339-352.

[0029] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 340-343.

[0030] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 344-352.

[0031] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 339-352.

[0032] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 353-368.

[0033] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 354-357. [0034] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 358-368.

[0035] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 353-368.

[0036] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 4 and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 369-380.

[0037] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 4 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 370-373 and 114.

[0038] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 374-380.

[0039] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 4 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 369-380.

[0040] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 5, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 381-395.

[0041] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 5 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 382-385. [0042] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 386-395.

[0043] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 5 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 381-395.

[0044] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 6 and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 396-409.

[0045] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 6 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 397-400.

[0046] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 401-409.

[0047] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 6 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 396-409.

[0048] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 7 and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 410-433.

[0049] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 7 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 411-414. [0050] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 415-433.

[0051] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 7 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 410-433.

[0052] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8 and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 434-447.

[0053] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 435-437.

[0054] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 438-447.

[0055] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 434-447.

[0056] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 9 and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 448-461.

[0057] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 9 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 449-452. [0058] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 453-461.

[0059] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 9 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 448-461.

[0060] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 462-475.

[0061] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 463-466.

[0062] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 467-475.

[0063] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 462-475.

[0064] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 11, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 476-491.

[0065] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 11 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 477-480. [0066] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 481-491.

[0067] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 11 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 476-491.

[0068] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 12, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 492-512.

[0069] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 12 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 493-496 and 509-512.

[0070] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 497-507.

[0071] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 12 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 492-512.

[0072] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 513-535.

[0073] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 514-517 and 528-531. [0074] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 518-526 and 532-535.

[0075] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 513-535.

[0076] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 14, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 536-549.

[0077] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 14 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 537-540.

[0078] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 541-549.

[0079] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 14 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 536-549.

[0080] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 15, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 550-575.

[0081] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 15 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 551-553. [0082] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 554-563, 565-569, and 571-575.

[0083] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 15 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 550-575.

[0084] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 576-598.

[0085] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 577-580.

[0086] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 581-589 and 591-598.

[0087] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 576-598.

[0088] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 599-612.

[0089] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 600-603 and 201. [0090] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 604-612.

[0091] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 599-612.

[0092] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 613-633.

[0093] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 614-617 and 630.

[0094] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 618-628 and 631-633.

[0095] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 613-633.

[0096] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 634-651.

[0097] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 635-638. [0098] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 639-647 and 649-651.

[0099] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 634-651.

[00100] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 20, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 652-665.

[00101] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 20 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 653-656.

[00102] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 657-665.

[00103] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 20 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 652-665.

[00104] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 21, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 666-683 and UUGCUACAA.

[00105] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 21 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 667-670 and 680. [00106] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 671-678, 681-683, and UUGCUACAA.

[00107] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 21 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 666-683 and UUGCUACAA.

[00108] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 22, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 684-707.

[00109] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 22 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 685-688.

[00110] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 689-697, 699-703, and 705-707.

[00111] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 22 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 684-707.

[00112] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 23, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 708-728.

[00113] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 23 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 709-712 and 721-724. [00114] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 713-719 and 725-728.

[00115] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 23 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 708-728.

[00116] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 729-742.

[00117] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 730-733.

[00118] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 734-742.

[00119] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 729-742.

[00120] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 25, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 743-761.

[00121] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 25 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 744-747. [00122] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 748-756 and 758-761.

[00123] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 25 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 743-761.

[00124] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 26, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 762-775.

[00125] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 26 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 763-766.

[00126] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 767-775.

[00127] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 26 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 762-775.

[00128] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 27, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 776-798.

[00129] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 27 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 777-780. [00130] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 781-789, 791-794, and 796-798.

[00131] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 27 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 776-798.

[00132] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 799-816.

[00133] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 800-803.

[00134] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 804-812 and 814-816.

[00135] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 799-816.

[00136] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 29, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 817-830.

[00137] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 29 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 818-821. [00138] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 822-830.

[00139] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 29 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 817-830.

[00140] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 30, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 831-844.

[00141] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 30 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 832-835.

[00142] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 836-844.

[00143] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 30 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 831-844.

[00144] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 31, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 845-862.

[00145] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 31 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 846-849. [00146] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 850-858 and 860-862.

[00147] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 31 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 845-862.

[00148] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 863-883.

[00149] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 864-867 and 880-883.

[00150] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 868-878.

[00151] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 863-883.

[00152] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 33, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 884-901 and UUACUACAA.

[00153] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 33 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 885-888 and 898. [00154] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 889-896, 899-901, and UUACUACAA.

[00155] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 33 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 884-901 and UUACUACAA.

[00156] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 34, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 902-915.

[00157] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 34 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 903-906.

[00158] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 907-915.

[00159] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 34 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 902-915.

[00160] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 35, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 916-931.

[00161] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 35 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 917-920 and 931. [00162] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 921-929.

[00163] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 35 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 916-931.

[00164] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 36, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 932-947.

[00165] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 36 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 933-936.

[00166] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 937-947.

[00167] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 36 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 932-947.

[00168] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 37, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 948-962.

[00169] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 37 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 949-952. [00170] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 953-962.

[00171] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 37 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 948-962.

[00172] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 38, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 963-985.

[00173] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 38 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 964-967 and 978-981.

[00174] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 968-976 and 982-985.

[00175] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 38 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 963-985.

[00176] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 39, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 986-1010.

[00177] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 39 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 987-990 and 1001-1004. [00178] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 991-999 and 1005-1010.

[00179] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 39 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 986-1010.

[00180] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1011-1029.

[00181] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1012-1015 and 1026-1029.

[00182] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1016-1024.

[00183] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1011-1029.

[00184] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 41, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1030-1050, UUACUAAAA, and UUACUAGAA.

[00185] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 41 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1031-1034 and 1044-1047. [00186] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1035-1042, 1048-1050, UUACUAAAA, and UUACUAGAA.

[00187] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 41 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1030-1050, UUACUAAAA, and UUACUAGAA.

[00188] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 42, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1051-1080.

[00189] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 42 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1052-1055 and 1068-1071.

[00190] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1056-1066, 1072-1074, and 1076-1079.

[00191] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 42 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1051-1080.

[00192] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 43, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1081-1101, UUACUAAAA, and UUACUAGAA.

[00193] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 43 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1082-1085 and 1095-1098.

[00194] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1086-1093, 1099-1101, UUACUAAAA, and UUACUAGAA.

[00195] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 43 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1081-1101, UUACUAAAA, and UUACUAGAA.

[00196] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 44, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1102-1115.

[00197] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 44 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1103-1106.

[00198] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1107-1115.

[00199] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 44 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1102-1115.

[00200] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1116-1135.

[00201] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1117-1120.

[00202] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1121-1130 and 1132-1135.

[00203] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1116-1135.

[00204] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 46, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1136-1167.

[00205] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 46 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1137-1140 and 1151-1154.

[00206] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1141-1149, 1155-1158, 1160-1163, and 1167-1167.

[00207] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 46 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1136-1167.

[00208] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 47, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1168-1186.

[00209] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 47 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1169-1172.

[00210] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1173-1181 and 1183-1186.

[00211] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 47 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1168-1186.

[00212] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1187-1211.

[00213] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1188-1191 and 1204-1207.

[00214] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1192-1202 and 1208-1211.

[00215] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1187-1211.

[00216] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 49, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1212-1243.

[00217] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 49 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1213-1216 and 1227-1230.

[00218] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1217-1225, 1231-1234, 1236-1239, and 1241-1243.

[00219] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 49 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1212-1243.

[00220] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 50, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1244-1255.

[00221] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 50 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1245-1247.

[00222] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1248-1255.

[00223] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 50 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1244-1255.

[00224] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 51, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1256-1284.

[00225] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 51 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1257-1260 and 1271-1274.

[00226] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1261-1269, 1276-1279, and 1281-1284.

[00227] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 51 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1256-1284.

[00228] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 52, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1285-1298.

[00229] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 52 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1286-1289.

[00230] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1290-1298.

[00231] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 52 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1285-1298.

[00232] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 53, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1299-1312.

[00233] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 53 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1300-1303.

[00234] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1304-1312.

[00235] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 53 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1299-1312.

[00236] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 54, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1313-1342.

[00237] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 54 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 177 and 178.

[00238] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1314-1315 and 1334-1335.

[00239] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 54 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1316-1326, 1328-1332, and 1336-1342.

[00240] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 55, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1343-1362.

[00241] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 55 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1344-1347.

[00242] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1348-1356 and 1358-1362.

[00243] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 55 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1343-1362.

[00244] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1363-1378.

[00245] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1364-1367.

[00246] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1368-1378.

[00247] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1363-1378.

[00248] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 57, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1379-1397.

[00249] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 57 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1380-1383.

[00250] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1384-1394 and 1396-1397.

[00251] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 57 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1379-1397.

[00252] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 58, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1398-1413.

[00253] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 58 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1399-1402.

[00254] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1403-1413.

[00255] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 58 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1398-1413.

[00256] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 59, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1414-1437.

[00257] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 59 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1415-1418 and 1431-1434.

[00258] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1419-1429 and 1436-1437.

[00259] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 59 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1414-1437.

[00260] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 60, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1438-1462.

[00261] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 60 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1439-1442 and 1455-1458.

[00262] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1443-1453 and 1459-1462.

[00263] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 60 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1438-1462.

[00264] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 61, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1463-1495.

[00265] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 61 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1464-1467 and 1478-1481.

[00266] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1468-1476, 1482-1490, and 1492-1495.

[00267] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 61 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1463-1495.

[00268] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 62, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1496-1533.

[00269] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 62 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1497-1500 and 1515-1518.

[00270] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1501-1513, 1519-1527, and 1529-1532.

[00271] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 62 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1496-1533.

[00272] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 63, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1534-1556.

[00273] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 63 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1535-1538.

[00274] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1539-1549, 1551-1553, and 1555-1556.

[00275] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 63 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1534-1556.

[00276] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1557-1572.

[00277] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1558-1561.

[00278] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1562-1572.

[00279] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1557-1572.

[00280] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 65, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1573-1617.

[00281] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 65 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1574-1577 and 1592-1595.

[00282] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1578-1590, 1596-1605, 1607-1612, and 1614-1617.

[00283] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 65 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1573-1617.

[00284] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 66, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1618-1634.

[00285] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 66 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1619-1622.

[00286] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1623-1631 and 1633-1634.

[00287] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 66 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1618-1634.

[00288] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 67, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1635-1655.

[00289] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 67 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1636-1639.

[00290] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1640-1652 and 1654-1655.

[00291] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 67 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1635-1655.

[00292] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 68, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1656-1674.

[00293] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 68 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1657-1660.

[00294] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1661-1671 and 1673-1674.

[00295] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 68 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1656-1674.

[00296] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 69, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1675-1701.

[00297] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 69 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1676-1679.

[00298] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1680-1692, 1694-1697, and 1699-1701.

[00299] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 69 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1675-1701.

[00300] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1702-1729.

[00301] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1703-1706.

[00302] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1707-1719, 1721-1722, 1724-1727, and 1729.

[00303] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1702-1729.

[00304] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 71, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1730-1747.

[00305] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 71 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1731-1734.

[00306] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1735-1747.

[00307] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 71 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1730-1747.

[00308] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1748-1774.

[00309] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1749-1752.

[00310] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1753-1763, 1765-1770, and 1772-1774.

[00311] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1748-1774.

[00312] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 73, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1775-1801.

[00313] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 73 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1776-1779 and 1792-1795.

[00314] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1780-1790 and 1796-1801.

[00315] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 73 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1775-1801.

[00316] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 74, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1802-1828.

[00317] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 74 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1803-1806.

[00318] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1807-1817, 1819-1820, 1822-1825, and 1827-1828.

[00319] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 74 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1802-1828.

[00320] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 75, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1829-1844.

[00321] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 75 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1830-1833.

[00322] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1834-1844.

[00323] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 75 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1829-1844.

[00324] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 76, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1845-1870.

[00325] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 76 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1846-1849.

[00326] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1850-1860, 1862-1863, 1685-1868, and 1870.

[00327] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 76 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1845-1870.

[00328] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 77, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1871-1895.

[00329] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 77 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1872-1875 and 1888-1891.

[00330] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1876-1886 and 1892-1895.

[00331] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 77 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1871-1895.

[00332] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 78, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1896-1911.

[00333] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 78 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1897-1900.

[00334] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1901-1911.

[00335] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 78 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1896-1911.

[00336] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 79, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1912-1930.

[00337] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 79 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1913-1916.

[00338] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1917-1927 and 1929-1930.

[00339] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 79 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1912-1930.

[00340] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1931-1942.

[00341] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1932-1934.

[00342] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1935-1942.

[00343] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1931-1942.

[00344] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 81, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1943-1971.

[00345] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 81 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1944-1947 and 1960-1963.

[00346] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1948-1958 and 1964-1971.

[00347] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 81 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1943-1971.

[00348] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 82, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1972-1999.

[00349] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 82 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1973-1976.

[00350] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 1977-1989, 1991-1995, and 1997-1998.

[00351] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 82 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 1972-1999.

[00352] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 83, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2000-2024.

[00353] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 83 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2001-2004.

[00354] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2005-2017, 2019-2020, and 2022-2023.

[00355] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 83 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2000-2024.

[00356] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 84, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2025-2045.

[00357] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 84 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2026-2029.

[00358] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2030-2042 and 2044-2045.

[00359] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 84 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2025-2045.

[00360] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 85, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2046-2063.

[00361] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 85 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2047-2050.

[00362] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2051-2063.

[00363] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 85 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2046-2063.

[00364] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 86, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2064-2088.

[00365] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 86 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2065-2068.

[00366] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2069-2081, 2083-2084, and 2086-2087.

[00367] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 86 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2064-2088.

[00368] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 87, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2089-2109.

[00369] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 87 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2090-2093.

[00370] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2094-2106 and 2108-2109.

[00371] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 87 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2089-2109.

[00372] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2110-2131.

[00373] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2111-2114.

[00374] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2115-2127, 2129, and 2131.

[00375] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2110-2131.

[00376] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 89, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2132-2156.

[00377] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 89 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2133-2136.

[00378] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2137-2149, 2151-2152, and 2154-2155.

[00379] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 89 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2132-2156.

[00380] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 90, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2157-2178.

[00381] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 90 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2158-2161.

[00382] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2162-2174 and 2176-2178.

[00383] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 90 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2157-2178.

[00384] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 91, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2179-2203.

[00385] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 91 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2180-2183.

[00386] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2184-2192, 2194-2196, and 2198-2203.

[00387] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 91 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2179-2203.

[00388] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 92, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2204-2230.

[00389] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 92 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2205-2208.

[00390] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2209-2221, 2223-2226, and 2228-2230.

[00391] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 92 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2204-2230.

[00392] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 93, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2231-2256.

[00393] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 93 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2232-2235.

[00394] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2236-2248, 2250-2252, and 2254-2255.

[00395] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 93 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2231-2256.

[00396] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 94, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2257-2277.

[00397] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 94 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2258-2261.

[00398] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2262-2274 and 2276-2277.

[00399] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 94 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2257-2277.

[00400] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 95, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2278-2304.

[00401] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 95 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2279-2282.

[00402] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2283-2295, 2297-2299, and 2301-2304.

[00403] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 95 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2278-2304.

[00404] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2305-2333.

[00405] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2306-2309.

[00406] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2310-2320, 2322-2325, 2327-2329, and 2331-2333.

[00407] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2305-2333.

[00408] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 97, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2334-2349.

[00409] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 97 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2335-2338.

[00410] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2339-2349.

[00411] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 97 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2334-2349.

[00412] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 98, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2350-2368.

[00413] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 98 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2351-2354.

[00414] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2355-2365 and 2367-2368.

[00415] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 98 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2350-2368.

[00416] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 99, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2369-2390.

[00417] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 99 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2370-2373.

[00418] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2374-2384 and 2386-2390.

[00419] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 99 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2369-2390.

[00420] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 100, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2391-2411.

[00421] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 100 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2392-2395.

[00422] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2396-2408 and 2410-2411.

[00423] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 100 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2391-2411.

[00424] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 101, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2412-2436.

[00425] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 101 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2413-2416 and 2429-2432.

[00426] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2417-2427 and 2433-2436.

[00427] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 101 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2412-2436.

[00428] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 102, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2437-2452.

[00429] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 102 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2438-2441.

[00430] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2442-2452.

[00431] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 102 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2437-2452.

[00432] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 103, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 24523-2468.

[00433] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 103 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2454-2457.

[00434] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2458-2468.

[00435] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 103 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 24523-2468.

[00436] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2469-2484.

[00437] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2470-2473.

[00438] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2474-2484.

[00439] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2469-2484.

[00440] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 105, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2485-2500.

[00441] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 105 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2486-2489.

[00442] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2490-2500.

[00443] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 105 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2485-2500.

[00444] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 106, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2501-2536.

[00445] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 106 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2502-2505 and 2520-2523.

[00446] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2506-2518, 2524-2529, 2531-2534, and 2536.

[00447] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 106 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2501-2536.

[00448] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 107, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2537-2552.

[00449] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 107 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2538-2541.

[00450] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2542-2552.

[00451] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 107 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2537-2552.

[00452] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 108, and at least one RNA molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 2553-2574.

[00453] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 108 and at least one RNA molecule is a CRISPR RNA (crRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2554-2557.

[00454] In some embodiments, the composition further comprises a transactivating CRISPR RNA (tracrRNA) molecule comprising a sequence set forth in the group consisting of SEQ ID NOs: 2558-2566, 2568-2570, and 2572-2574.

[00455] In some embodiments, the CRISPR nuclease comprises a sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 108 and at least one RNA molecule is a single-guide RNA (sgRNA) molecule comprising a guide sequence portion and a sequence selected from the group consisting of SEQ ID NOs: 2553-2574.

[00456] In some embodiments, the CRISPR nuclease is a nickase having an inactivated RuvC domain created by an amino acid substitution at a position provided for the CRISPR nuclease in column 5 of Table 1.

[00457] In some embodiments, the CRISPR nuclease is a nickase having an inactivated HNH domain created by an amino acid substitution at a position provided for the CRISPR nuclease in column 6 of Table 1. [00458] In some embodiments, the CRISPR nuclease is a catalytically dead nuclease having an inactivated RuvC domain and an inactivated HNH domain created by substitutions at the positions provided for the CRISPR nuclease in column 7 of Table 1.

[00459] For example, a nickase may be generated for the OMNI-263 nuclease by inactivating its RuvC domain by substituting an aspartic acid residue (D) in position 9 of the amino acid sequence of OMNI-263 (SEQ ID NO: 1) for another amino acid e.g. alanine (A). Substitution to any other amino acid is permissible for each of the amino acid positions indicated in columns 5-7, unless otherwise indicated in Table 1. Other nickases or catalytically dead nucleases can be generated using the same notation in Table 1.

[00460] In some embodiments, the CRISPR nuclease utilizes a protospacer adj acent motif (PAM) sequence provided for the CRISPR nuclease in any one of columns 2-7 of Table 3. A CRISPR nuclease utilizes a PAM sequence as a targeting component in addition to a spacer sequence of a guide molecule.

[00461] The invention also provides a method for modifying a nucleotide sequence at a DNA target site in a cell-free system or the genome of a cell comprising introducing into the cell any one of the compositions described above. In some embodiments, the composition comprises a CRISPR nuclease and a crRNA:tracrRNA complex or a sgRNA molecule.

[00462] In some embodiments, the CRISPR nuclease effects a DNA break in a DNA strand adjacent to a protospacer adjacent motif (PAM) sequence provided for the CRISPR nuclease in any one of columns 2-7 of Table 3, and effects a DNA break in a DNA strand adjacent to a sequence that is complementary to the PAM sequence. For example, the OMNI-263 nuclease with, the appropriate targeting sgRNA or crRNA:tracrRNA complex, is capable of forming a DNA break in strand adjacent to a NNNRCM or NNNRCC sequence and in a DNA strand adjacent to a sequence that is complementary to a NNNRCM or NNNRCC sequence. In another example, the OMNI-281 nuclease, with the appropriate targeting sgRNA or crRNA:tracrRNA complex, is capable of forming a DNA break in strand adjacent to a NNGRNNNN, NNGRVNNN, or NNGAVNNN sequence and in a DNA strand adjacent to a sequence that is complementary to a NNGRNNNN, NNGRVNNN, or NNGAVNNN sequence.

[00463] In some embodiments, the DNA strand is within a nucleus of a cell. [00464] In some embodiments, the CRISPR nuclease is a nickase having an inactivated RuvC domain created by an amino acid substitution at a position provided for the CRISPR nuclease in column 5 of Table 1, and effects a DNA break in a DNA strand adjacent to a sequence that is complementary to the PAM sequence.

[00465] In some embodiments, the CRISPR nuclease is a nickase having an inactivated HNH domain created by an amino acid substitution at a position provided for the CRISPR nuclease in column 6 of Table 1, and effects a DNA break in a DNA strand adjacent to the PAM sequence.

[00466] In some embodiments, the CRISPR nuclease is a catalytically dead nuclease having an inactivated RuvC domain and an inactivated HNH domain created by substitutions at the positions provided for the CRISPR nuclease in column 7 of Table 1, and effects a DNA break in a DNA strand adjacent to the PAM sequence.

[00467] In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell.

[00468] In some embodiments, the cell is a mammalian cell.

[00469] In some embodiments, the cell is a human cell.

[00470] In some embodiments, the CRISPR nuclease comprises an amino acid sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, or 82% amino acid sequence identity to a CRISPR nuclease as set forth in any of SEQ ID NOs: 1-108. In an embodiment the sequence encoding the CRISPR nuclease has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 109- 324.

[00471] According to some aspects of the invention, the disclosed compositions comprise DNA constructs or a vector system comprising nucleotide sequences that encode the CRISPR nuclease or variant CRISPR nuclease. In some embodiments, the nucleotide sequence that encode the CRISPR nuclease or variant CRISPR nuclease is operably linked to a promoter that is operable in the cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments the cell of interest is a mammalian cell. In some embodiments, the nucleic acid sequence encoding the engineered CRISPR nuclease is codon optimized for use in cells from a particular organism. In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for A. coli. In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for eukaryotic cells. In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for mammalian cells.

[00472] In some embodiments, the composition comprises a recombinant nucleic acid, comprising a heterologous promoter operably linked to a polynucleotide encoding a CRISPR enzyme having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% identity to any of SEQ ID NOs: 1-108. Each possibility represents a separate embodiment.

[00473] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 1 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 109 and 217.

[00474] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 2 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110 and 218.

[00475] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 3 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 111 and 219.

[00476] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 4 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 112 and 220.

[00477] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 5 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 113 and 221. [00478] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 6 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 114 and 222.

[00479] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 7 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 115 and 223.

[00480] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 8 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 116 and 224.

[00481] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 9 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 117 and 225.

[00482] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 10 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 118 and 226.

[00483] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 11 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 119 and 227. [00484] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 12 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 120 and 228.

[00485] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 13 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 121 and 229.

[00486] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 14 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122 and 230.

[00487] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 15 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 123 and 231.

[00488] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 16 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 124 and 232.

[00489] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 17 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 125 and 233. [00490] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 18 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 126 and 234.

[00491] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 19 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127 and 235.

[00492] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 20 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 128 and 236.

[00493] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 21 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 129 and 237.

[00494] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 22 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 130 and 238.

[00495] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 23 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 131 and 239. [00496] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 24 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 132 and 240.

[00497] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 25 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 133 and 241.

[00498] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 26 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 134 and 242.

[00499] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 27 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135 and 243.

[00500] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 28 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 136 and 244.

[00501] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 29 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137 and 245. [00502] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 30 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 138 and 246.

[00503] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 31 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 139 and 247.

[00504] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 32 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 140 and 248.

[00505] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 33 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 141 and 249.

[00506] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 34 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 142 and 250.

[00507] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 35 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 143 and 251. [00508] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 36 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144 and 252.

[00509] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 37 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 145 and 253.

[00510] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 38 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 146 and 254.

[00511] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 39 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 147 and 255.

[00512] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 40 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 148 and 256.

[00513] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 41 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 149 and 257. [00514] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 42 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 150 and 258.

[00515] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 43 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 151 and 259.

[00516] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 44 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 152 and 260.

[00517] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 45 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 153 and 261.

[00518] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 46 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 154 and 262.

[00519] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 47 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 155 and 263. [00520] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 48 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 156 and 264.

[00521] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 49 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 157 and 265.

[00522] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 50 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 158 and 266.

[00523] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 51 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 159 and 267.

[00524] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 52 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 160 and 268.

[00525] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 53 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 161 and 269. [00526] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 54 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 162 and 270.

[00527] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 55 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 163 and 271.

[00528] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 56 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 164 and 272.

[00529] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 57 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 165 and 273.

[00530] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 58 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 166 and 274.

[00531] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 59 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 167 and 275. [00532] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 60 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 168 and 276.

[00533] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 61 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 169 and 277.

[00534] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 62 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 170 and 278.

[00535] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 63 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 171 and 279.

[00536] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 64 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 172 and 280.

[00537] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 65 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 173 and 281. [00538] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 66 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 174 and 282.

[00539] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 67 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 175 and 283.

[00540] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 68 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 176 and 284.

[00541] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 69 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 177 and 285.

[00542] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 70 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 178 and 286.

[00543] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 71 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 179 and 287. [00544] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 72 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 180 and 288.

[00545] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 73 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 181 and 289.

[00546] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 74 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182 and 290.

[00547] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 75 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183 and 291.

[00548] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 76 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 184 and 292.

[00549] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 77 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 185 and 293. [00550] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 78 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 186 and 294.

[00551] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 79 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 187 and 295.

[00552] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 80 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 188 and 296.

[00553] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 81 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 189 and 297.

[00554] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 82 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 190 and 298.

[00555] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 83 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 191 and 299. [00556] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 84 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 192 and 300.

[00557] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 85 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 193 and 301.

[00558] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 86 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194 and 302.

[00559] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 87 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195 and 303.

[00560] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 88 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 196 and 304.

[00561] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 89 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 197 and 305. [00562] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 90 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 198 and 306.

[00563] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 91 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199 and 307.

[00564] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 92 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 200 and 308.

[00565] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 93 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 201 and 309.

[00566] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 94 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 202 and 310.

[00567] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 95 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 203 and 311. [00568] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 96 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 204 and 312.

[00569] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 97 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 205 and 313.

[00570] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 98 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 206 and 314.

[00571] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 99 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 207 and 315.

[00572] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 100 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 208 and 316.

[00573] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 101 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 209 and 317. [00574] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 102 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 210 and 318.

[00575] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 103 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 211 and 319.

[00576] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 104 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 212 and 320.

[00577] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 105 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 213 and 321.

[00578] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 106 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 214 and 322.

[00579] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 107 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 215 and 323. [00580] In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 108 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 216 and 324.

[00581] According to some embodiments, there is provided an engineered or non-naturally occurring composition comprising a CRISPR nuclease comprising a sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease. Each possibility represents a separate embodiment.

In an embodiment, the CRISPR nuclease is engineered or non-naturally occurring. The CRISPR nuclease may also be recombinant. Such CRISPR nucleases are produced using laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.

[00582] In an embodiment, the CRISPR nuclease of the invention exhibits increased specificity to a target site compared to a SpCas9 nuclease when complexed with the one or more RNA molecules.

[00583] In an embodiment, the complex of the CRISPR nuclease of the invention and one or more RNA molecules exhibits at least maintained on-target editing activity of the target site and reduced off-target activity compared to SpCas9 nuclease.

[00584] In an embodiment, the CRISPR nuclease further comprises an RNA-binding portion capable of interacting with a DNA-targeting RNA molecule (gRNA) and an activity portion that exhibits site-directed enzymatic activity.

[00585] In an embodiment, the composition further comprises a DNA-targeting RNA molecule or a DNA polynucleotide encoding a DNA-targeting RNA molecule, wherein the DNA-targeting RNA molecule comprises a guide sequence portion, i.e. a nucleotide sequence that is complementary to a sequence in a target region, wherein the DNA-targeting RNA molecule and the CRISPR nuclease do not naturally occur together. [00586] In an embodiment, the DNA-targeting RNA molecule further comprises a nucleotide sequence that can form a complex with a CRISPR nuclease.

[00587] This invention also provides a non-naturally occurring composition comprising a CRISPR associated system comprising: a) one or more RNA molecules comprising a guide sequence portion linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more RNA molecules; and b) a CRISPR nuclease comprising an amino acid sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; wherein the one or more RNA molecules hybridize to the target sequence, wherein the target sequence is 3' of a Protospacer Adjacent Motif (PAM), and the one or more RNA molecules form a complex with the RNA-guided nuclease.

[00588] In an embodiment, the composition further comprises an RNA molecule comprising a nucleotide sequence that can form a complex with a CRISPR nuclease (e.g. a tracrRNA molecule) or a DNA polynucleotide comprising a sequence encoding an RNA molecule that can form a complex with the CRISPR nuclease.

[00589] In an embodiment, the composition further comprises a donor template for homology directed repair (HDR).

[00590] In an embodiment, the composition is capable of editing the target region in the genome of a cell.

[00591] According to some embodiments, there is provided a non-naturally occurring composition comprising:

(a) a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, comprising: an RNA-binding portion; and an activity portion that exhibits site-directed enzymatic activity, wherein the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to any of SEQ ID NOs: 1-108; and (b) one or more RNA molecules or a DNA polynucleotide encoding the one or more RNA molecules comprising: i) a DNA-targeting RNA sequence, comprising a nucleotide sequence that is complementary to a sequence in a target DNA sequence; and ii) a protein-binding RNA sequence, capable of interacting with the RNA-binding portion of the CRISPR nuclease, wherein the DNA targeting RNA sequence and the CRISPR nuclease do not naturally occur together. Each possibility represents a separate embodiment.

[00592] In some embodiments, there is provided a single RNA molecule comprising the DNA- targeting RNA sequence and the protein-binding RNA sequence, wherein the RNA molecule can form a complex with the CRISPR nuclease and serve as the DNA targeting module. In some embodiments, the RNA molecule has a length of up to 1000 bases, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases. Each possibility represents a separate embodiment. In some embodiments, a first RNA molecule comprising the DNA-targeting RNA sequence and a second RNA molecule comprising the protein-binding RNA sequence interact by base pairing or alternatively fused together to form one or more RNA molecules that complex with the CRISPR nuclease and serve as the DNA targeting module.

[00593] This invention also provides a non-naturally occurring composition comprising: a) a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and b) one or more RNA molecules, or one or more DNA polynucleotide encoding the one or more RNA molecules, comprising at least one of: i) a nuclease-binding RNA nucleotide sequence capable of interacting with/binding to the

CRISPR nuclease; and ii) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence, wherein the CRISPR nuclease is capable of complexing with the one or more RNA molecules to form a complex capable of hybridizing with the target DNA sequence. [00594] In an embodiment, the CRISPR nuclease and the one or more RNA molecules form a CRISPR complex that is capable of binding to the target DNA sequence to effect cleavage of the target DNA sequence.

[00595] In an embodiment, the CRISPR nuclease and at least one of the one or more RNA molecules do not naturally occur together.

[00596] In an embodiment: a) the CRISPR nuclease comprises an RNA-binding portion and an activity portion that exhibits site-directed enzymatic activity; b) the DNA-targeting RNA nucleotide sequence comprises a nucleotide sequence that is complementary to a sequence in a target DNA sequence; and c) the nuclease-binding RNA nucleotide sequence comprises a sequence that interacts with the RNA-binding portion of the CRISPR nuclease.

[00597] In an embodiment, the nuclease-binding RNA nucleotide sequence and the DNA- targeting RNA nucleotide sequence are on a single guide RNA molecule (sgRNA), wherein the sgRNA molecule can form a complex with the CRISPR nuclease and serve as the DNA targeting module.

[00598] In an embodiment, the nuclease-binding RNA nucleotide sequence is on a first RNA molecule and the DNA-targeting RNA nucleotide sequence is on a second RNA molecule, and wherein the first and second RNA molecules interact by base-pairing or are fused together to form a RNA complex or sgRNA that forms a complex with the CRISPR nuclease and serves as a DNA targeting module.

[00599] In an embodiment, the sgRNA has a length of up to 1000 bases, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases.

[00600] In an embodiment, the composition further comprises a donor template for homology directed repair (HDR).

[00601]

[00602] In an embodiment, the CRISPR nuclease is non-naturally occurring. [00603] In an embodiment, the CRISPR nuclease is engineered and comprises unnatural or synthetic amino acids.

[00604] In an embodiment, the CRISPR nuclease is engineered and comprises one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags.

[00605] In an embodiment, the CRISPR nuclease comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of a CRISPR complex comprising the CRISPR nuclease in a detectable amount in the nucleus of a eukaryotic cell.

[00606] This invention also provides a method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell any of the compositions of the invention.

[00607] In an embodiment, the cell is a eukaryotic cell.

[00608] In another embodiment, the cell is a prokaryotic cell.

[00609] In some embodiments, the one or more RNA molecules further comprises an RNA sequence comprising a nucleotide molecule that can form a complex with the RNA nuclease (tracrRNA) or a DNA polynucleotide encoding an RNA molecule comprising a nucleotide sequence that can form a complex with the CRISPR nuclease.

[00610] In an embodiment, the CRISPR nuclease comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near carboxyterminus, or a combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the aminoterminus and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near carboxy-terminus. In an embodiment 1-4 NLSs are fused with the CRISPR nuclease. In an embodiment, an NLS is located within the open-reading frame (ORF) of the CRISPR nuclease.

[00611] Methods of fusing an NLS at or near the amino-terminus, at or near carboxy -terminus, or within the ORF of an expressed protein are well known in the art. As an example, to fuse an NLS to the amino-terminus of a CRISPR nuclease, the nucleic acid sequence of the NLS is placed immediately after the start codon of the CRISPR nuclease on the nucleic acid encoding the NLS- fused CRISPR nuclease. Conversely, to fuse an NLS to the carboxy-terminus of a CRISPR nuclease the nucleic acid sequence of the NLS is placed after the codon encoding the last amino acid of the CRISPR nuclease and before the stop codon. [00612] Any combination of NLSs, cell penetrating peptide sequences, and/or affinity tags at any position along the ORF of the CRISPR nuclease is contemplated in this invention.

[00613] The amino acid sequences and nucleic acid sequences of the CRISPR nucleases provided herein may include NLS and/or TAGs inserted so as to interrupt the contiguous amino acid or nucleic acid sequences of the CRISPR nucleases.

[00614] In an embodiment, the one or more NLSs are in tandem repeats.

[00615] In an embodiment, the one or more NLSs are considered in proximity to the N- or C- terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.

[00616] As discussed, the CRISPR nuclease may be engineered to comprise one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags.

[00617] In an embodiment, the CRISPR nuclease exhibits increased specificity to a target site compared to the wild-type of the CRISPR nuclease when complexed with the one or more RNA molecules.

[00618] In an embodiment, the complex of the CRISPR nuclease and one or more RNA molecules exhibits at least maintained on-target editing activity of the target site and reduced off-target activity compared to the wild-type of the CRISPR nuclease.

[00619] In an embodiment, the composition further comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to the nucleotide acid molecule comprising the sequence encoding the CRISPR nuclease.

[00620] In an embodiment, the CRISPR nuclease or nucleic acid molecule comprising a sequence encoding the CRISPR nuclease is non-naturally occurring or engineered.

[00621] This invention also provides a non-naturally occurring or engineered composition comprising a vector system comprising the nucleic acid molecule comprising a sequence encoding any of the CRISPR nucleases of the invention.

[00622] This invention also provides use of any of the compositions of the invention for the treatment of a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subject. [00623] This invention provides a method of modifying a nucleotide sequence at a target site in the genome of a mammalian cell comprising introducing into the cell (i) a composition comprising a CRISPR nuclease having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108 or a nucleic acid molecule comprising a sequence encoding a CRISPR nuclease which sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 109-324 and (ii) a DNA-targeting RNA molecule, or a DNA polynucleotide encoding a DNA-targeting RNA molecule, comprising a nucleotide sequence that is complementary to a sequence in the target DNA.

[00624] In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo. In some embodiments, some steps of the method are performed ex vivo and some steps are performed in vivo. In some embodiments the mammalian cell is a human cell.

[00625] In an embodiment, the method further comprises introducing into the cell: (iii) an RNA molecule comprising a tracrRNA sequence or a DNA polynucleotide encoding an RNA molecule comprising a tracrRNA sequence.

[00626] In an embodiment, the DNA-targeting RNA molecule comprises a crRNA repeat sequence.

[00627] In an embodiment, the RNA molecule comprising a tracrRNA sequence is able to bind the DNA-targeting RNA molecule.

[00628] In an embodiment, the DNA-targeting RNA molecule and the RNA molecule comprising a tracrRNA sequence interact to form an RNA complex, and the RNA complex is capable of forming an active complex with the CRISPR nuclease.

[00629] In an embodiment, the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-binding RNA sequence are fused in the form of a single guide RNA molecule that is suitable to form an active complex with the CRISPR nuclease.

[00630] In an embodiment, the guide sequence portion comprises a sequence complementary to a protospacer sequence. [00631] In an embodiment, the CRISPR nuclease forms a complex with the DNA-targeting RNA molecule and effects a double strand break in a region that is 3’ or 5’ of a Protospacer Adjacent Motif (PAM).

[00632] In an embodiment of any of the methods described herein, the method is for treating a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subject.

[00633] In an embodiment, the method comprises first selecting a subject afflicted with a disease associated with a genomic mutation and obtaining the cell from the subject.

[00634] This invention also provides a modified cell or cells obtained by any of the methods described herein. In an embodiment these modified cell or cells are capable of giving rise to progeny cells. In an embodiment these modified cell or cells are capable of giving rise to progeny cells after engraftment.

[00635] This invention also provides a composition comprising these modified cells and a pharmaceutically acceptable carrier. Also provided is an in vitro or ex vivo method of preparing this, comprising mixing the cells with the pharmaceutically acceptable carrier.

DNA-targeting RNA molecules

[00636] The “guide sequence portion” of an RNA molecule refers to a nucleotide sequence that is capable of hybridizing to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence which is partially or fully complementary to the DNA sequence being targeted along the length of the guide sequence portion. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length, or approximately 17-50, 17-49,

17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35,

17-34, 17-33, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-22, 17-21, 18-25, 18-24,

18-23, 18-22, 18-21, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-22, 18-20, 20-21, 21-22, or 17-

20 nucleotides in length. The entire length of the guide sequence portion is fully complementary to the DNA sequence being targeted along the length of the guide sequence portion. The guide sequence portion may be part of an RNA molecule that can form a complex with a CRISPR nuclease with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex. When the DNA molecule having the guide sequence portion is present contemporaneously with the CRISPR molecule the RNA molecule is capable of targeting the CRISPR nuclease to the specific target DNA sequence. Each possibility represents a separate embodiment. An RNA molecule can be custom designed to target any desired sequence. Accordingly, a molecule comprising a “guide sequence portion” is a type of targeting molecule. Throughout this application, the terms “guide molecule,” “RNA guide molecule,” “guide RNA molecule,” and “gRNA molecule" are synonymous with a molecule comprising a guide sequence portion, and the term “spacer” is synonymous with a “guide sequence portion.”

[00637] In embodiments of the present invention, the CRISPR nuclease has its greatest cleavage activity when used with an RNA molecule comprising a guide sequence portion having 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[00638] A single-guide RNA (sgRNA) molecule may be used to direct a CRISPR nuclease to a desired target site. The single-guide RNA comprises a guide sequence portion as well as a scaffold portion. The scaffold portion interacts with a CRISPR nuclease and, together with a guide sequence portion, activates and targets the CRISPR nuclease to a desired target site. A scaffold portion may be further engineered, for example, to have a reduced size.

[00639] According to some aspects of the invention, the disclosed methods comprise a method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell the composition of any one of the embodiments described herein.

[00640] In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell.

[00641] According to some aspects of the invention, the disclosed methods comprise a use of any one of the compositions described herein for the treatment of a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subj ect.

[00642] According to some aspects of the invention, the disclosed methods comprise a method of treating subject having a mutation disorder comprising targeting any one of the compositions described herein to an allele associated with the mutation disorder. [00643] In some embodiments, the mutation disorder is related to a disease or disorder selected from any of a neoplasia, age-related macular degeneration, schizophrenia, neurological, neurodegenerative, or movement disorder, Fragile X Syndrome, secretase-related disorders, prion- related disorders, ALS, addiction, autism, Alzheimer’s Disease, neutropenia, inflammation-related disorders, Parkinson’s Disease, blood and coagulation diseases and disorders, beta thalassemia, sickle cell anemia, cell dysregulation and oncology diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, dermatological diseases and disorders, neurological and neuronal diseases and disorders, and ocular diseases and disorders.

Diseases and therapies

[00644] Certain embodiments of the invention target a nuclease to a specific genetic locus associated with a disease or disorder as a form of gene editing, method of treatment, or therapy. For example, to induce editing or knockout of a gene, a novel nuclease disclosed herein may be specifically targeted to a pathogenic mutant allele of the gene using a custom designed guide RNA molecule. The guide RNA molecule is preferably designed by first considering the PAM requirement of the nuclease, which as shown herein is also dependent on the system in which the gene editing is being performed. For example, a guide RNA molecule designed to target an OMNI- 263 nuclease to a target site is designed to contain a spacer region complementary to a DNA strand of a DNA double-stranded region that neighbors a OMNI-263 PAM sequence, e.g. “NNNRCM” or “NNNRCC.” The guide RNA molecule is further preferably designed to contain a spacer region (i.e. the region of the guide RNA molecule having complementarity to the target allele) of sufficient and preferably optimal length in order to increase specific activity of the nuclease and reduce off-target effects.

[00645] As a non-limiting example, the guide RNA molecule may be designed to target the nuclease to a specific region of a mutant allele, e.g. near the start codon, such that upon DNA damage caused by the nuclease a non-homologous end joining (NHEJ) pathway is induced and leads to silencing of the mutant allele by introduction of frameshift mutations. This approach to guide RNA molecule design is particularly useful for altering the effects of dominant negative mutations and thereby treating a subject. As a separate non-limiting example, the guide RNA molecule may be designed to target a specific pathogenic mutation of a mutated allele, such that upon DNA damage caused by the nuclease a homology directed repair (HDR) pathway is induced and leads to template mediated correction of the mutant allele. This approach to guide RNA molecule design is particularly useful for altering haploinsufficiency effects of a mutated allele and thereby treating a subject.

[00646] Non-limiting examples of specific genes which may be targeted for alteration to treat a disease or disorder are presented herein below. Specific disease-associated genes and mutations that induce a mutation disorder are described in the literature. Such mutations can be used to design a DNA-targeting RNA molecule to target a CRISPR composition to an allele of the disease associated gene, where the CRISPR composition causes DNA damage and induces a DNA repair pathway to alter the allele and thereby treat the mutation disorder.

[00647] Mutations in the ELANE gene are associated with neutropenia. Accordingly, without limitation, embodiments of the invention that target ELANE may be used in methods of treating subjects afflicted with neutropenia.

[00648] CXCR4 is a co-receptor for the human immunodeficiency virus type 1 (HIV-1) infection. Accordingly, without limitation, embodiments of the invention that target CXCR4 may be used in methods of treating subjects afflicted with HIV-1 or conferring resistance to HIV-1 infection in a subject.

[00649] Programmed cell death protein 1 (PD-1) disruption enhances CAR-T cell mediated killing of tumor cells and PD-1 may be a target in other cancer therapies. Accordingly, without limitation, embodiments of the invention that target PD-1 may be used in methods of treating subjects afflicted with cancer. In an embodiment, the treatment is CAR-T cell therapy with T cells that have been modified according to the invention to be PD-1 deficient.

[00650] In addition, BCL11A is a gene that plays a role in the suppression of hemoglobin production. Globin production may be increased to treat diseases such as thalassemia or sickle cell anemia by inhibiting BCL11A. See for example, PCT International Publication No. WO 2017/077394 A2; U.S. Publication No. US2011/0182867A1; Humbert et al. Sci. Transl. Med. (2019); and Canver et al. Nature (2015). Accordingly, without limitation, embodiments of the invention that target an enhancer of BCL11 A may be used in methods of treating subjects afflicted with beta thalassemia or sickle cell anemia. [00651] Embodiments of the invention may also be used for targeting any disease-associated gene, for studying, altering, or treating any of the diseases or disorders listed in Table A or Table B below. Indeed, any disease-associated gene may be studied, altered, or targeted by any one of the nucleases disclosed herein to treat the disease caused by the disease-associated gene. Nonlimiting examples are those listed in U.S. Publication No. 2018/0282762A1 and European Patent No. EP3079726B1.

Table A - Diseases, Disorders and their associated genes

Table B - Diseases, Disorders and their associated genes

[00652] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[00653] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[00654] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application. [00655] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[00656] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

[00657] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[00658] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonueleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, in Irons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. [00659] The term "nucleotide analog" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions), in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)), in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate. Each of the RNA sequences described herein may comprise one or more nucleotide analogs.

[00660] As used herein, the following nucleotide identifiers are used to represent a referenced nucleotide base(s):

[00661 ] As used herein, the term “targeting sequence” or “targeting molecule” refers a nucleotide sequence or molecule comprising a nucleotide sequence that is capable of hybridizing to a specific target sequence, e.g., the targeting sequence has a nucleotide sequence which is at least partially complementary to the sequence being targeted along the length of the targeting sequence. The targeting sequence or targeting molecule may be part of a targeting RNA molecule that can form a complex with a CRISPR nuclease with the targeting sequence serving as the targeting portion of the CRISPR complex. When the molecule having the targeting sequence is present contemporaneously with the CRISPR molecule, the RNA molecule is capable of targeting the CRISPR nuclease to the specific target sequence. Each possibility represents a separate embodiment. A targeting RNA molecule can be custom designed to target any desired sequence.

[00662] The term “targets” as used herein, refers to preferential hybridization of a targeting sequence or a targeting molecule to a nucleic acid having a targeted nucleotide sequence. It is understood that the term “targets” encompasses variable hybridization efficiencies, such that there is preferential targeting of the nucleic acid having the targeted nucleotide sequence, but unintentional off-target hybridization in addition to on-target hybridization might also occur. It is understood that where an RNA molecule targets a sequence, a complex of the RNA molecule and a CRISPR nuclease molecule targets the sequence for nuclease activity.

[00663] In the context of targeting a DNA sequence that is present in a plurality of cells, it is understood that the targeting encompasses hybridization of the guide sequence portion of the RNA molecule with the sequence in one or more of the cells, and also encompasses hybridization of the RNA molecule with the target sequence in fewer than all of the cells in the plurality of cells. Accordingly, it is understood that where an RNA molecule targets a sequence in a plurality of cells, a complex of the RNA molecule and a CRISPR nuclease is understood to hybridize with the target sequence in one or more of the cells, and also may hybridize with the target sequence in fewer than all of the cells. Accordingly, it is understood that the complex of the RNA molecule and the CRISPR nuclease introduces a double strand break in relation to hybridization with the target sequence in one or more cells and may also introduce a double strand break in relation to hybridization with the target sequence in fewer than all of the cells. As used herein, the term “modified cells” refers to cells in which a double strand break is affected by a complex of an RNA molecule and the CRISPR nuclease as a result of hybridization with the target sequence, i.e. on- target hybridization.

[00664] As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. Accordingly, as used herein, where a sequence of amino acids or nucleotides refers to a wild type sequence, a variant refers to variant of that sequence, e.g., comprising substitutions, deletions, insertions. In embodiments of the present invention, an engineered CRISPR nuclease is a variant CRISPR nuclease comprising at least one amino acid modification (e.g., substitution, deletion, and/or insertion) compared to the CRISPR nuclease of any of the CRISPR nucleases indicated in Table 1.

[00665] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate human manipulation. The terms, when referring to nucleic acid molecules or polypeptides may mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[00666] As used herein the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or I, optical isomers, and amino acid analogs and peptidomimetics.

[00667] As used herein, “genomic DNA” refers to linear and/or chromosomal DNA and/or to plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, the methods produce double-stranded breaks (DSBs) at pre-determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the target site(s) in a genome.

[00668] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

[00669] The term "nuclease" as used herein refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acid. A nuclease may be isolated or derived from a natural source. The natural source may be any living organism. Alternatively, a nuclease may be a modified or a synthetic protein which retains the phosphodiester bond cleaving activity.

[00670] The term “PAM” as used herein refers to a nucleotide sequence of a target DNA located in proximity to the targeted DNA sequence and recognized by the CRISPR nuclease. The PAM sequence may differ depending on the nuclease identity.

[00671] The term “mutation disorder” or “mutation disease” as used herein refers to any disorder or disease that is related to dysfunction of a gene caused by a mutation. A dysfunctional gene manifesting as a mutation disorder contains a mutation in at least one of its alleles and is referred to as a “disease-associated gene.” The mutation may be in any portion of the disease-associated gene, for example, in a regulatory, coding, or non-coding portion. The mutation may be any class of mutation, such as a substitution, insertion, or deletion. The mutation of the disease-associated gene may manifest as a disorder or disease according to the mechanism of any type of mutation, such as a recessive, dominant negative, gain-of-function, loss-of-function, or a mutation leading to haploinsufficiency of a gene product.

[00672] A skilled artisan will appreciate that embodiments of the present invention disclose RNA molecules capable of complexing with a nuclease, e.g. a CRISPR nuclease, such as to associate with a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM). The nuclease then mediates cleavage of target DNA to create a double-stranded break within the protospacer.

[00673] In embodiments of the present invention, a CRISPR nuclease and a targeting molecule form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence. A CRISPR nuclease may form a CRISPR complex comprising the CRISPR nuclease and RNA molecule without a further, separate tracrRNA molecule. Alternatively, CRISPR nucleases may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule.

[00674] The term “protein binding sequence” or “nuclease binding sequence” refers to a sequence capable of binding with a CRISPR nuclease to form a CRISPR complex. A skilled artisan will understand that a tracrRNA capable of binding with a CRISPR nuclease to form a CRISPR complex comprises a protein or nuclease binding sequence.

[00675] An “RNA binding portion” of a CRISPR nuclease refers to a portion of the CRISPR nuclease which may bind to an RNA molecule to form a CRISPR complex, e.g. the nuclease binding sequence of a tracrRNA molecule. An “activity portion” or “active portion” of a CRISPR nuclease refers to a portion of the CRISPR nuclease which effects a double strand break in a DNA molecule, for example when in complex with a DNA-targeting RNA molecule.

[00676] An RNA molecule may comprise a sequence sufficiently complementary to a tracrRNA molecule so as to hybridize to the tracrRNA via basepairing and promote the formation of a CRISPR complex. (See U.S. Patent No. 8,906,616). In embodiments of the present invention, the RNA molecule may further comprise a portion having a tracr mate sequence.

[00677] In embodiments of the present invention, the targeting molecule may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of the guide portion of the RNA molecule (gRNA or crRNA) and the trans-activating crRNA (tracrRNA), together forming a single guide RNA (sgRNA). (See Jinek et al., Science (2012)). Embodiments of the present invention may also form CRISPR complexes utilizing a separate tracrRNA molecule and a separate RNA molecule comprising a guide sequence portion. In such embodiments the tracrRNA molecule may hybridize with the RNA molecule via base pairing and may be advantageous in certain applications of the invention described herein.

[00678] In embodiments of the present invention an RNA molecule may comprise a “nexus” region and/or “hairpin” regions which may further define the structure of the RNA molecule. (See Briner et al., Molecular Cell (2014)).

[00679] As used herein, the term “direct repeat sequence” refers to two or more repeats of a specific amino acid sequence of nucleotide sequence.

[00680] As used herein, an RNA sequence or molecule capable of “interacting with” or “binding” with a CRISPR nuclease refers to the RNA sequence or molecules ability to form a CRISPR complex with the CRISPR nuclease.

[00681] As used herein, the term “operably linked” refers to a relationship (i.e. fusion, hybridization) between two sequences or molecules permitting them to function in their intended manner. In embodiments of the present invention, when an RNA molecule is operably linked to a promoter, both the RNA molecule and the promotor are permitted to function in their intended manner.

[00682] As used herein, the term “heterologous promoter” refers to a promoter that does not naturally occur together with the molecule or pathway being promoted.

[00683] As used herein, a sequence or molecule has an X% “sequence identity” to another sequence or molecule if X% of bases or amino acids between the sequences of molecules are the same and in the same relative position. For example, a first nucleotide sequence having at least a 95% sequence identity with a second nucleotide sequence will have at least 95% of bases, in the same relative position, identical with the other sequence.

Nuclear Localization Sequences

[00684] The terms "nuclear localization sequence" and "NLS" are used interchangeably to indicate an amino acid sequence/peptide that directs the transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier. The term "NLS" is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier. NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide. In addition, a polypeptide having an NLS coupled by its N- or C-terminus to amino acid side chains located randomly along the amino acid sequence of the polypeptide will be translocated. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. Non- limiting examples of NLSs include an NLS sequence derived from: the SV40 virus large T-antigen, nucleoplasmin, c-myc, the hRNPAl M9 NLS, the IBB domain from importin-alpha, myoma T protein, human p53, mouse c- abl IV, influenza vims NS1, Hepatitis virus delta antigen, mouse Mxl protein, human poly(ADP- ribose) polymerase, and the steroid hormone receptors (human) glucocorticoid.

Delivery

[00685] The CRISPR nuclease or CRISPR compositions described herein may be delivered as a protein, DNA molecules, RNA molecules, Ribonucleoproteins (RNP), nucleic acid vectors, or any combination thereof. In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2'-0-methyl (M), 2'-0-m ethyl, 3'phosphorothioate (MS) or 2'-0-m ethyl, 3 'thioPACE (MSP), pseudouridine, and 1- methyl pseudo-uridine. Each possibility represents a separate embodiment of the present invention.

[00686] The CRISPR nucleases and/or polynucleotides encoding same described herein, and optionally additional proteins (e.g., ZFPs, TALENs, transcription factors, restriction enzymes) and/or nucleotide molecules such as guide RNA may be delivered to a target cell by any suitable means. The target cell may be any type of cell e.g., eukaryotic or prokaryotic, in any environment e.g., isolated or not, maintained in culture, in vitro, ex vivo, in vivo or in planta. [00687] In some embodiments, the composition to be delivered includes mRNA of the nuclease and RNA of the guide. In some embodiments, the composition to be delivered includes mRNA of the nuclease, RNA of the guide and a donor template. In some embodiments, the composition to be delivered includes the CRISPR nuclease and guide RNA. In some embodiments, the composition to be delivered includes the CRISPR nuclease, guide RNA and a donor template for gene editing via, for example, homology directed repair. In some embodiments, the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA. In some embodiments, the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA and a donor template. In some embodiments, the composition to be delivered includes the CRISPR nuclease DNA-targeting RNA and the tracrRNA. In some embodiments, the composition to be delivered includes the CRISPR nuclease, DNA-targeting RNA and the tracrRNA and a donor template for gene editing via, for example, homology directed repair.

[00688] Any suitable viral vector system may be used to deliver RNA compositions. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids and/or CRISPR nuclease in cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer nucleic acids encoding and/or CRISPR nuclease protein to cells in vitro. In certain embodiments, nucleic acids and/or CRISPR nuclease are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or pol oxamer. For a review of gene therapy procedures, see Anderson, Science (1992); Nabel and Feigner, TIBTECH (1993); Mitani and Caskey, TIBTECH (1993); Dillon, TIBTECH (1993); Miller, Nature (1992); Van Brunt, Biotechnology (1988); Vigne et al., Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin (1995); Haddada et al., Current Topics in Microbiology and Immunology (1995); and Yu et al., Gene Therapy 1 : 13- 26 (1994).

[00689] Methods of non-viral delivery of nucleic acids and/or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, artificial virions, and agent- enhanced uptake of nucleic acids or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus. See, e.g., Chung et al. Trends Plant Sci. (2006). Sonoporation using, e.g., the Sonitron 2000 system (Rich- Mar) can also be used for delivery of nucleic acids. Cationic-lipid mediated delivery of proteins and/or nucleic acids is also contemplated as an in vivo or in vitro delivery method. See Zuris et al., Nat. Biotechnol. (2015), Coelho et al., N. Engl. J. Med. (2013); Judge et al., Mol. Ther. (2006); and Basha et al., Mol. Ther. (2011). Non-viral vectors, such as transposon-based systems e.g., recombinant Sleeping Beauty transposon systems or recombinant PiggyBac transposon systems, may also be delivered to a target cell and utilized for transposition of a polynucleotide sequence of a molecule of the composition or a polynucleotide sequence encoding a molecule of the composition in the target cell.

[00690] Additional exemplary nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc., (see for example U.S. Patent No. 6,008,336). Lipofection is described in e.g., U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787; and U.S. Patent No. 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM., Lipofectin.TM. and Lipofectamine.TM. RNAiMAX). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in PCT International Publication Nos. WO/1991/017424 and WO/1991/016024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

[00691] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science (1995); Blaese et al., Cancer Gene Ther. (1995); Behr et al., Bioconjugate Chem. (1994); Remy et al., Bioconjugate Chem. (1994); Gao and Huang, Gene Therapy (1995); Ahmad and Allen, Cancer Res., (1992); U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[00692] Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGenelC delivery vehicles (ED Vs). These ED Vs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiamid et al., Nature Biotechnology (2009)).

[00693] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, recombinant retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. However, an RNA virus is preferred for delivery of the RNA compositions described herein. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. Nucleic acid of the invention may be delivered by non-integrating lentivirus. Optionally, RNA delivery with Lentivirus is utilized. Optionally the lentivirus includes mRNA of the nuclease, RNA of the guide. Optionally the lentivirus includes mRNA of the nuclease, RNA of the guide and a donor template. Optionally, the lentivirus includes the nuclease protein, guide RNA. Optionally, the lentivirus includes the nuclease protein, guide RNA and/or a donor template for gene editing via, for example, homology directed repair. Optionally the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA. Optionally the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA, and a donor template. Optionally, the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA. Optionally, the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA, and a donor template for gene editing via, for example, homology directed repair.

[00694] As mentioned above, the compositions described herein may be delivered to a target cell using a non-integrating lentiviral particle method, e.g. a LentiFlash® system. Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell. See also PCT International Publication Nos. WO2013/014537, WO2014/016690, WO2016185125, WO2017194902, and WO2017194903.

[00695] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher Panganiban, J. Virol. (1992); Johann et al., J. Virol. (1992); Sommerfelt et al., Virol. (1990); Wilson et al., J. Virol. (1989); Miller et al., J. Virol. (1991); PCT International Publication No. WO/1994/026877A1).

[00696] At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.

[00697] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood (1995); Kohn et al., Nat. Med. (1995); Malech et al., PNAS (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. (1997); Dranoff et al., Hum. Gene Ther. (1997).

[00698] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, AAV, and psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced at clinical scale using baculovirus systems (see U.S. Patent No. 7,479,554).

[00699] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virustarget cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.

[00700] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. In some embodiments, delivery of mRNA in vivo and ex vivo, and RNPs delivery may be utilized.

[00701] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via reinfusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with an RNA composition, and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, “Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010)) and the references cited therein for a discussion of how to isolate and culture cells from patients).

[00702] Suitable cells include but not limited to eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e g., CHO— S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Agl4, HeLa, HEK293 (e g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, any plant cell (differentiated or undifferentiated) as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. In certain embodiments, the cell line is a CHO- Kl, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with the nucleases (e.g. ZFNs or TALENs) or nuclease systems (e.g. CRISPR). Suitable primary cells include peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.

[00703] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in-vitro or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-gamma. and TNF-alpha are known (as a non-limiting example see, Inaba et al., J. Exp. Med. (1992)).

[00704] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (as a non-limiting example see Inaba et al., J. Exp. Med. (1992)). Stem cells that have been modified may also be used in some embodiments.

[00705] Notably, any one of the CRISPR nucleases described herein may be suitable for genome editing in post-mitotic cells or any cell which is not actively dividing, e.g., arrested cells. Examples of post-mitotic cells which may be edited using a CRISPR nuclease of the present invention include, but are not limited to, myocyte, a cardiomyocyte, a hepatocyte, an osteocyte and a neuron.

[00706] Vectors (e.g., retroviruses, liposomes, etc.) containing therapeutic RNA compositions can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked RNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[00707] Vectors suitable for introduction of transgenes into immune cells (e.g., T-cells) include non-integrating lentivirus vectors. See, for example, U.S. Patent Publication No. 2009/0117617.

[00708] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

DNA Repair by Homologous Recombination

[00709] The term "homology-directed repair" or "HDR" refers to a mechanism for repairing DNA damage in cells, for example, during repair of double-stranded and single- stranded breaks in DNA. HDR requires nucleotide sequence homology and uses a "nucleic acid template" (nucleic acid template or donor template used interchangeably herein) to repair the sequence where the doublestranded or single break occurred (e.g., DNA target sequence). This results in the transfer of genetic information from, for example, the nucleic acid template to the DNA target sequence. HDR may result in alteration of the DNA target sequence (e.g., insertion, deletion, mutation) if the nucleic acid template sequence differs from the DNA target sequence and part or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence. In some embodiments, an entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is integrated at the site of the DNA target sequence.

[00710] The terms "nucleic acid template" and “donor”, refer to a nucleotide sequence that is inserted or copied into a genome. The nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid or may be used to modify the target sequence. A nucleic acid template sequence may be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), preferably between about 100 and 1,000 nucleotides in length (or any integer there between), more preferably between about 200 and 500 nucleotides in length. A nucleic acid template may be a single stranded nucleic acid, a double stranded nucleic acid. In some embodiment, the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position. In some embodiment, the nucleic acid template comprises a ribonucleotide sequence, e.g., of one or more ribonucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position. In some embodiment, the nucleic acid template comprises modified ribonucleotides.

[00711] Insertion of an exogenous sequence (also called a "donor sequence," donor template” or "donor"), for example, for correction of a mutant gene or for increased expression of a wild-type gene can also be carried out. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest. [00712] The donor polynucleotide can be DNA or RNA, single-stranded and/or doublestranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 2010/0047805; 2011/0281361; 2011/0207221; and 2019/0330620. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends. See, for example, Chang and Wilson, Proc. Natl. Acad. Sci. USA (1987); Nehls et al., Science (1996). Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[00713] Accordingly, embodiments of the present invention using a donor template for repair may use a DNA or RNA, single-stranded and/or double-stranded donor template that can be introduced into a cell in linear or circular form. In embodiments of the present invention a geneediting composition comprises: (1) an RNA molecule comprising a guide sequence to affect a double strand break in a gene prior to repair and (2) a donor RNA template for repair, the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule. In some embodiments, the guide RNA molecule and template RNA molecule are connected as part of a single molecule.

[00714] A donor sequence may also be an oligonucleotide and be used for gene correction or targeted alteration of an endogenous sequence. The oligonucleotide may be introduced to the cell on a vector, may be electroporated into the cell, or may be introduced via other methods known in the art. The oligonucleotide can be used to correct' a mutated sequence in an endogenous gene (e.g., the sickle mutation in beta globin), or may be used to insert sequences with a desired purpose into an endogenous locus.

[00715] A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by recombinant viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

[00716] The donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted. However, it will be apparent that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.

[00717] The donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to the transgene) or none of the endogenous sequences are expressed, for example as a fusion with the transgene. In other embodiments, the transgene (e.g., with or without additional coding sequences such as forthe endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus, for example a CCR5 gene, a CXCR4 gene, a PPPlR12c (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Publication Nos. 2008/0159996; 20100/0218264; 2010/0291048; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983 and 2013/0177960 and U.S. Provisional Application No. 61/823,689).

[00718] When endogenous sequences (endogenous or part of the transgene) are expressed with the transgene, the endogenous sequences may be full-length sequences (wild-type or mutant) or partial sequences. Preferably the endogenous sequences are functional. Non-limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier.

[00719] Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

[00720] In certain embodiments, the donor molecule comprises a sequence selected from the group consisting of a gene encoding a protein (e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein), a regulatory sequence and/or a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.

[00721] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment. For example, it is understood that any of the RNA molecules or compositions of the present invention may be utilized in any of the methods of the present invention.

[00722] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

[00723] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[00724] 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 subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[00725] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, Sambrook et al., "Molecular Cloning: A laboratory Manual" (1989); Ausubel, R. M. (Ed.), "Current Protocols in Molecular Biology" Volumes I-III (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.), "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); Methodologies as set forth in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; Cellis, J. E. (Ed.), "Cell Biology: A Laboratory Handbook", Volumes I-III (1994); Freshney, "Culture of Animal Cells - A Manual of Basic Technique" Third Edition, Wiley -Liss, N. Y. (1994); Coligan J. E. (Ed.), "Current Protocols in Immunology" Volumes I-III (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (Eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); Clokie and Kropinski (Eds.), "Bacteriophage Methods and Protocols", Volume 1 : Isolation, Characterization, and Interactions (2009), all of which are incorporated by reference. Other general references are provided throughout this document.

[00726] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

EXPERIMENTAL DETAILS

[00728] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

[00729] CRISPR repeat (crRNA), trans-activating RNA (tracrRNA), nuclease polypeptide (OMNI), and protospacer adjacent motif (PAM) sequences were predicted from different metagenomic databases of sequences of environmental samples.

Construction of OMNI nuclease polypeptides

[00730] For construction of novel nuclease polypeptides (OMNIs), the open reading frame of several identified OMNIs were codon optimized for human cell line expression. The ORF was cloned into the bacterial expression plasmid pET9a and into the mammalian expression plasmid pmOMNI (Table 4).

Prediction and construction of sgRNA

[00731] For each OMNI the single guide RNA (sgRNA) was predicted by detection of the CRISPR repeat array sequence and a tracrRNA in the respective bacterial genome. The native premature crRNA and tracrRNA sequences were connected in silico with a tetra-loop ‘gaaa’ sequence and the secondary structure elements of the duplex were predicted using an RNA secondary structure prediction tool.

[00732] The predicted secondary structures of the full duplex RNA elements (crRNA-tracrRNA chimera) were used for identification of possible tracrRNA sequences for the design of a sgRNA. Several possible sgRNA scaffolds versions were constructed by shortening the duplex at the upper stem at different locations (sgRNA designs of all OMNIs are listed in Table 2). Additionally, to overcome potential transcriptional and structural constraints and to assess the plasticity of the sgRNA scaffold in the human cellular environmental context, small changes in the nucleotide sequence of the possible sgRNA were made in some cases (Fig. 1, Table 2). Finally, up to four versions of possible designed scaffolds were synthesized for each OMNI and connected downstream to a 22-nucleotide universal unique spacer sequence (T2, SEQ ID NO: 268) and cloned into a bacterial expressing plasmid under an inducible T7 promoter combined with a U6 promoter for mammalian expression (pShuttl eGuide, Table 4).

T2 - GGAAGAGCAGAGCCTTGGTCTC (SEQ ID NO: 2782)

In-vitro Depletion assay by TXTL

[00733] Depletion of PAM sequences in vitro was followed as described by Maxwell et al, Methods. 2018. Briefly, linear DNA expressing the OMNI nucleases and an sgRNA under T7 promoter were added to a cell-free transcription-translation in vitro system (TXTL mix, Arbor Bioscience) together with a linear construct expressing T7 polymerase. RNA expression and protein translation by the TXTL mix result in the formation of a ribonucleoprotein (RNP) complex. Since linear DNA was used, Chi6 DNA sequences were added to the TXTL reaction mix to inhibit the exonuclease activity of RecBCD, thereby protecting the linear DNA from degradation. The sgRNA spacer is designed to target a library of plasmids containing the target protospacer (pbPOS T2 library, Table 4) flanked by an 8N randomized set of potential PAM sequences. Depletion of PAM sequences from the library was measured by high-throughput sequencing using PCR to add the necessary adapters and indices to both the cleaved library and to a control library expressing a non-targeting gRNA. Following deep sequencing, the in vitro activity was confirmed by the fraction of the depleted sequences having the same PAM sequence relative to their occurrence in the control, indicating functional DNA cleavage by the OMNI nuclease (Figs. 2-109, Table 3).

Activity in human cells on endogenous genomic targets

[00734] OMNIs were also assayed for their ability to promote editing on specific genomic locations in human cells. To this end, per each OMNI, a corresponding OMNI-P2A-mCherry expression vector (pmOMNI, Table 4) was transfected into HeLa cells together with an sgRNA designed to target a specific location in the human genome (pShuttle guide - Table 4, spacer sequence - Table 5). At 72h, cells were harvested, and their genomic DNA content was used in a PCR reaction, amplifying the corresponding putative genomic targets. Amplicons were subjected to next-generation sequence (NGS) and the resulting sequences were then used to calculate the percentage of editing events in each target site. Short insertions or deletions (indels) around the cut site are the typical outcome of DNA repair following nuclease induced DNA cleavage. The calculation of editing percent was therefore deduced from the fraction of indel-containing sequences within each amplicon. [00735] Genomic activity of each ONMI was assessed using a panel of six (6) unique sgRNAs each designed to target a different genomic location. The results of these experiments are summarized in Table 5. As can be seen in the table (column 6, “% indels”), many OMNIs exhibited high and significant editing levels compared to the negative control (not shown) in most or some of the target sites tested.

Table 1 - OMNI CRISPR nuclease sequences

Table 1. OMNI nuclease sequences: Table 1 lists the OMNI name, its corresponding nuclease protein sequence, its native DNA sequence, its human optimized DNA sequence, alternative positions to be substituted to generate a nickase having an inactivated RuvC domain, alternative positions to be substituted to generate a nickase having an inactivated HNH domain, and alternative positions to be substituted to generate a catalytically dead nuclease having inactivated RuvC and HNH domains. Substitution to any other amino acid is permissible for each of the amino acid positions indicated in columns 5-7, except a substitution of aspartic acid (D) to glutamic acid (E) or glutamic acid (E) to aspartic acid (D) in the HNH domain, in order to achieve inactivation.

Table 2 - OMNI Guide RNA and Scaffold RNA Sequences

Table 3 - OMNI PAM Sequences showing activity for each tested sgRNA

Table 4 - Plasmids used in assays

Table 4 Appendix - Details of construct elements

Table 5 - Nuclease activity in endogenous context in mammalian cells

Table 5. Nuclease activity in endogenous context in mammalian cells: OMNI nucleases were expressed in mammalian cell system (HeLa) by DNA transfection together with an sgRNA expressing plasmid. Cell lysates were used for site specific genomic DNA amplification and NGS. The percentage of indels was measured and analyzed to determine the editing level. Each sgRNA is composed of the tracrRNA (see Table 2) and the spacer detailed here. The spacer 3’ genomic sequence contains the expected PAM relevant for each OMNI nuclease. All tests were performed in triplicates. OMNI nuclease only (no guide) transfected cells served as a negative control. REFERENCES

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