Chemical modification of guide rnas with locked nucleic acid for RNA guided nuclease-mediated gene editing

Abstract

Compositions including engineered and / or chemically modified transactivating CRISPR RNA (tracrRNA), guide RNA (gRNA), and / or CRISPR RNA (crRNA) are provided. The chemically modified tracrRNA, gRNA, and / or crRNA incorporate bridged nucleic acid (BNA) modifications and other chemical modifications. BNA modifications include 2',4' locked nucleic acid (LNA) modifications. Additional chemical modifications include 2'-O-methyl (2'-O-Me), 2'-O-methyl 3'phosphorothioate (MS), and phosphorothioate (PS) modifications. The engineered and / or chemically modified tracrRNA, gRNA, and / or crRNA of the disclosure improves gene editing (including base editing and prime editing) efficiency of an RNA-guided nuclease (RGN) system.

Description

[0001] CHEMICAL MODIFICATION OF GUIDE RNAS WITH LOCKED NUCLEIC ACID FOR RNA GUIDED NUCLEASE-MEDIATED GENE EDITING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 373,498, filed August 25, 2022; U.S. Provisional Application No. 63 / 385,887, filed December 2, 2022; and U.S. Provisional Application No. 63 / 517,703, filed August 4, 2023, each of which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY AS AN XML FILE

[0005] The instant application contains a Sequence Listing which has been submitted in xml format via USPTO Patent Center and is hereby incorporated by reference in its entirety. Said xml copy, created on August 24, 2023, is named L103438_1290WO_0235_5_Sequence Listing, and is 2.18 MB in size.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to the field of molecular biology and gene editing.

[0008] BACKGROUND OF THE INVENTION

[0009] Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research, as it allows modification of genomes such as: cutting, deleting, and inserting nucleic acids; substituting nucleotides in nucleic acids; and regulating gene expression at specific locations in a genome, along with many other possible modifications. Genome editing systems that use RNA- guided nucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated (Cas) proteins of the CRISPR-Cas bacterial system, function by complexing a nuclease, an enzyme that cuts a nucleic acid, with a guide RNA. The hybridization of the guide RNA to a particular target sequence allows editing at a specific location in a genome. Thus, genome editing systems that use RNA-guided nucleases (RGN) can be cost-effective and efficient for editing of genome sequences, as a guide RNA is the programmable component of an RGN system, allowing genome editing of specific target sequences by typically straightforward design of the guide RNA. RGN genome editing systems have been adapted from many microbes, and these systems are classified into two classes, 6 types, and multiple subtypes.

[0010] In microbes from which Type II and some Type V RGN systems originate, guide RNAs (gRNAs) are expressed as a two-part RNA system: a CRISPR-RNA (crRNA), containing the spacer sequence which recognizes the target genomic sequence via Watson-Crick base pairing, and scaffold transactivating crRNA (tracrRNA). This two-part guide RNA requires base pairing between regions of a crRNA molecule and a tracrRNA molecule to form a dual guide RNA (dgRNA). For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

[0011] Elements of a guide RNA (e.g., the phosphate backbone, the ribose sugar, the nucleobase) can be chemically modified to, for example, reduce degradation of the guide RNA. Much opportunity exists to delineate the types and / or extent of modifications to guide RNA to improve an RGN system, such as to enhance stability, editing efficiency, and specificity for a target sequence and / or to decrease inflammatory responses associated with toxicity of an RGN system.

[0012] BRIEF SUMMARY OF THE INVENTION

[0013] Provided herein are compositions including chemically modified transactivating CRISPR RNA (tracrRNA), guide RNA (gRNA), and / or CRISPR RNA (crRNA). The chemically modified tracrRNA, gRNA, and / or crRNA incorporate bridged nucleic acid (BNA) modifications and / or other chemical modifications. In some embodiments, BNA modifications include 2', 4' locked nucleic acid modifications of a nucleotide, in which the 2' oxygen is covalently linked to the 4' carbon via a methylene bridge. In some embodiments, additional modifications include 2'-O-methyl (2'-0-Me), 2'- O-methyl 3' phosphorothioate (MS), and phosphorothioate (PS) modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the disclosure improves gene editing efficiency of an RNA-guided nuclease (RGN) system as compared to a reference RGN system comprising tracrRNA, gRNA, and / or crRNA having no BNA modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the disclosure allows a dual guide RNA to be used in applications where otherwise a single guide RNA would be required. In some embodiments, the present disclosure provides for use of BNA modifications and / or other chemical modifications within the first stem of a stem loop 1 of a dual guide RNA to enhance the performance of an RGN system in a cell. In some embodiments, the use of BNA modifications and / or other chemical modifications allows the use of a shortened tracrRNA, gRNA, and / or crRNA. In some embodiments, the cells that are gene edited with an RGN system of the disclosure include primary cells. The chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.

[0014] Also provided are methods for achieving RGN-based gene editing in cells using guide RNAs modified with BNA and for increasing gene editing efficiency.

[0015] In one aspect, the present disclosure provides a nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is within the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification comprises at least two three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive mucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

[0016] In some embodiments of the above tracrRNA aspect, the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is not within a bulge of the tracrRNA. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

[0017] In some embodiments of the above tracrRNA aspect, the at least one BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

[0018] In some embodiments of the above tracrRNA aspect, the tracrRNA further comprises at least one other chemical modification. In some embodiments, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments, the at least one other chemical modification is within the tail of the tracrRNA.

[0019] In some embodiments of the above tracrRNA aspect, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3 'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise MS modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the anti-repeat comprise BNA modifications. In some embodiments, the BNA modifications are LNA modifications.

[0020] In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of about 11 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

[0021] In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above tracrRNA aspect, the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

[0022] In some embodiments of the above tracrRNA aspect, the tracrRNA comprises a total length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

[0023] In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.

[0024] In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

[0025] In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

[0026] In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

[0027] In some embodiments of the above tracrRNA aspect, the tracrRNA is part of a gRNA that is capable of binding to an RGN. In some embodiments, the RGN is a Type II RGN.

[0028] In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

[0029] In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

[0030] In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

[0031] In another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BN A) modification.

[0032] In some embodiments of the above gRNA aspect, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt. In some embodiments of the above gRNA aspect, the gRNA is a dual guide RNA (dgRNA).

[0033] In some embodiments of the above gRNA aspect, the at least one BNA modification is within the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within the second stem of the crRNA repeat.

[0034] In some embodiments of the above gRNA aspect, the at least one BNA modification is within the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

[0035] In some embodiments of the above gRNA aspect, the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within a bulge of the gRNA. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the tail of the tracrRNA. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

[0036] In some embodiments of the above gRNA aspect, at least three terminal nucleotides in the 3' region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti-repeat comprise BNA modification. In some embodiments of the above gRNA aspect, all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the anti -repeat comprise BNA modifications.

[0037] In some embodiments of the above gRNA aspect, the at least one BNA modification is within the spacer. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 5' region of the spacer comprise BNA modifications. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 5' region of the spacer comprise both BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspect, the spacer is 18-30 nucleotides in length.

[0038] In some embodiments of the above gRNA aspect, the at least one BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

[0039] In some embodiments of the above gRNA aspect, the gRNA further comprises at least one other modification. In some embodiments of the above gRNA aspect, the at least one other modification is within the crRNA. In some embodiments of the above gRNA aspect, the at least one other modification is within the 5' region or the 3' region of the crRNA. In some embodiments of the above gRNA aspect, the at least one other modification is within the 5' region and the 3' region of the crRNA.

[0040] In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the crRNA repeat of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the spacer of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tail of the tracrRNA.

[0041] In some embodiments of the above gRNA aspect, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.

[0042] In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti -repeat comprises a total length of about 11 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

[0043] In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat at the 3 ' region or the first stem of the anti -repeat at the 5 ’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5 ’ region comprises a GC-rich nucleotide sequence, wherein the first stem of the crRNA repeat at the 3 ’ region or the first stem of the anti -repeat at the 5 ’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

[0044] In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

[0045] In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as: (a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides; (b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides; (c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides; (d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides; (e) SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides; or (f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments of the above aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.

[0046] In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as (a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides; (b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides; (c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides; (d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides; (e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides; (f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides; (g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides; (h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides; (i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides; j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides; (k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides; (1) SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or (m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

[0047] In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides; (b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides; (c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides; (d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides; (e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides; (f) SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides; and (g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337- 339. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

[0048] In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides; (b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides; (c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides; (d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides; (e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides; (f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides; (g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides; and (h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

[0049] In some embodiments of the above gRNA aspect, the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the antirepeat. In some embodiments, the linker comprises an azide functional group or an alkyne functional group. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG. In some embodiments, the linker has a nucleotide sequence set forth as AAAG.

[0050] In some embodiments of the above gRNA aspect, the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA. In some embodiments of the above gRNA aspect, the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

[0051] In some embodiments of the above gRNA aspect, the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

[0052] In some embodiments of the above gRNA aspect, the gRNA is capable of binding to an RGN. In some embodiments, the RGN is a Type II RGN.

[0053] In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

[0054] In some embodiments of the above gRNA aspect, an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

[0055] In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

[0056] In some embodiments of the above gRNA aspect, the gRNA further comprises an extension comprising an edit template for prime editing.

[0057] In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5 ’ region or 3 ’ region of the crRNA.

[0058] In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5 ’ region or 3 ’ region of the crRNA. In some embodiments, a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.

[0059] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) described hereinabove; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

[0060] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA described hereinabove; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

[0061] In still another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) described hereinabove; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

[0062] In some embodiments of the above RGN systems aspects, the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, or NGG. In some embodiments of the above RGN systems aspects, the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt. In some embodiments of the above RGN systems aspects, the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

[0063] In some embodiments of the above RGN systems aspects, the RGN polypeptide and the gRNA are not found complexed to one another in nature.

[0064] In some embodiments of the above RGN systems aspects, the target sequence is a eukaryotic target sequence. In some embodiments, the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712. In some embodiments of the above RGN systems aspects, the target sequence is within a cell.

[0065] In some embodiments of the above RGN systems aspects, a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence. In some embodiments, the cleavage generates a double-stranded break. In some embodiments, the cleavage generates a single-stranded break.

[0066] In some embodiments of the above RGN systems aspects, the RGN polypeptide is nuclease inactive. In some embodiments of the above RGN systems aspects, the RGN polypeptide is a nickase.

[0067] In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

[0068] In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the above RGN systems aspects, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a detectable label. In some embodiments of the above RGN systems aspects, the RGN system further comprises a donor polynucleotide.

[0069] In some embodiments of the above RGN systems aspects, the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA. In some embodiments, the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter. In some embodiments, the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

[0070] In another aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising an RGN system as described hereinabove.

[0071] In still another aspect, the present disclosure provides a cell comprising a nucleic acid molecule comprising a tracrRNA, a gRNA, a crRNA, an RGN system, or an RNP complex as described hereinabove.

[0072] In some embodiments of the above aspect, the cell comprises a target sequence capable of being bound by a formed gRNA / RGN polypeptide complex of an RGN system, or by an RNP complex, as described hereinabove. In some embodiments, the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

[0073] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a primary cell. In some embodiments, the primary cell is a T cell. In some embodiments, the eukaryotic cell is a plant cell.

[0074] In another aspect, the present disclosure provides a plant comprising a plant cell as described hereinabove.

[0075] In another aspect, the present disclosure provides a seed comprising a plant cell as described hereinabove.

[0076] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tracrRNA, a gRNA, a crRNA, an RGN system, an RNP complex, or a cell as described hereinabove.

[0077] In another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence.

[0078] In some embodiments of the above aspect, the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence. In some embodiments of the above aspect, the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence. In some embodiments of the above aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the above aspect, the RGN polypeptide is fused to a base-editing polypeptide. In still another aspect, the present disclosure provides a method for cleaving and / or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

[0079] In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, the assembled RNP complex directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the method aspect, the prime editing polypeptide comprises a DNA polymerase. In some embodiments of the method aspect, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

[0080] In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the method aspect, the prime editing polypeptide comprises a DNA polymerase. In some embodiments of the method aspect, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the polynucleotide encoding the Type II RGN is an mRNA.

[0081] In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) as described hereinabove; and ii) a Type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, the assembled RNP complex directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing.

[0082] In still another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) as described hereinabove; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the polynucleotide encoding the Type II RGN is an mRNA.

[0083] In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a tracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the above method aspects, the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712. In some embodiments of the above method aspects, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

[0084] In another aspect, the present disclosure provides a method of increasing efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or any chemical modification.

[0085] In some embodiments of the above aspect, all nucleotides of the first stem of the anti-repeat of the tracrRNA of an RGN system or of an RNP complex as described hereinabove comprise BNA modifications. In some embodiments of the above aspect, at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications. In some embodiments of the above aspect, the BNA modifications comprise LNA modifications. In some embodiments of the above aspect, the BNA modifications comprise cEt modifications. In some embodiments of the above aspect, the efficiency of cleaving and / or modifying the target sequence is increased by 15-fold to 30-fold. In some embodiments of the above aspect, the efficiency of cleaving and / or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence. In some embodiments of the above aspect, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), mass spectrometry, or a combination thereof.

[0086] In another aspect, the present disclosure provides a method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the crRNA repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the 3’ region of the crRNA repeat and the 5 ’ region of the anti-repeat of the engineered gRNA comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).

[0087] In some embodiments of the above aspect, the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In some embodiments of the above aspect, the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

[0088] In some embodiments of the above aspect, the gRNA is a dgRNA. In some embodiments of the above aspect, the gRNA is a sgRNA.

[0089] In some embodiments of the above aspect, the method further comprises c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.

[0090] In some embodiments of the above aspect, the at least one chemical modification is in the crRNA, the tracrRNA, or both. In some embodiments of the above aspect, the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti-repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti-repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti-repeat, and the tail of the tracrRNA.

[0091] In some embodiments of the above aspect, the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.

[0092] In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3 ’ region of the first stem of the crRNA repeat.

[0093] In some embodiments of the above aspect, the at least one chemical modification comprises a BNA modification. In some embodiments, the BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

[0094] In some embodiments of the above aspect, the editing efficiency of the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the gRNA provided in step a). In some embodiments of the above aspect, the efficiency of cleaving and / or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the RGN system comprising the gRNA provided in step a). In some embodiments of the above aspect, the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence. In some embodiments of the above aspect, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), mass spectrometry, or a combination thereof.

[0095] In another aspect, the present disclosure provides an engineered gRNA produced by the method described hereinabove.

[0096] In still another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0097] In yet another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BN A) modification.

[0098] BRIEF DESCRIPTION OF THE FIGURES

[0099] FIG. 1 provides a schematic of a dual guide RNA (dgRNA) showing the pairing of the crRNA and the tracrRNA.

[0100] FIGs. 2A and 2B show schematics illustrating the parts of a single guide (sgRNA) (FIG. 2A), and of a dgRNA (FIG. 2B). The parts include: a spacer; a stem loop 1 including a first stem, a first bubble, and a second stem; a stem loop 2 comprising a first stem only; an inter stem loop region (ISR); a stem loop 3 including a loop, a first stem, a first bubble, a second stem, a second bubble, and a third stem; and a tail. Stem loop 1 in the sgRNA includes a loop, while stem loop 1 in the dgRNA does not include a loop. The CRISPR RNA (crRNA) repeat anneals to the anti -repeat of the transactivating CRISPR RNA (tracrRNA) to form stem loop 1.

[0101] FIGs. 3A and 3B provide a depiction of the chemical modifications of the dgRNA for APG07433.1 RNA guided nuclease. FIG. 3A shows tracrRNA modification schemes, from left to right: “Stem MS modified”, in which the first stem of stem loop 1 comprises nucleotides with 2'-O- methyl (2'-0-Me) modifications, and with 2'-O-methyl 3'phosphorothioate (MS) modifications at the three terminal nucleotides at the 5' region and MS modifications at the three terminal nucleotides at the 3' region and a 2'-0-Me modification on the fourth nucleotide from the 3’ end of the tracrRNA molecule; “Heavily MS modified” - in addition to the modifications in “Stem MS modified”, adding 2’-0-Me modifications to stem loop 3 through the tail; “Stem LNA Modified” - the first stem of stem loop 1 contains all LNA modifications, and the three terminal nucleotides at the 3' region have MS modifications and the fourth nucleotide from the 3’ end has a 2'-0-Me modification; “Heavily LNA Modified” - in addition to the modifications in “Stem LNA modified”, adding 2'-0-Me modifications throughout most of stem loop 3, with part of the first and second stems of stem loop 3 being LNA modified. FIG. 3B shows a diagram of the modification of crRNAs used in these experiments - both contain 5' and 3' MS modifications, with the version shown on the left also bearing 2'-0-Me modifications on the first stem.

[0102] FIG. 4 shows the efficiency of gene editing, measured by knockout of the CD3 surface marker using flow cytometry, in primary human T cells using different combinations of the modified crRNA and modified tracrRNA. Guide RNAs were designed to target the TRAC gene. These data demonstrate that modifications at stem loop 3 abolishes editing activity, while modifications at the stem loop 1 alone preserve editing ability of the dgRNA. The modifications are shown with the same scheme as in FIGs. 3A and 3B. TracrRNAs are shown diagrammed. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. Mock indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process.

[0103] FIG. 5 demonstrates that LNA modification enhances the editing efficiency of dgRNA in a RNP complex with purified APG07433.1 protein, which reaches similar levels of editing as sgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.

[0104] FIG. 6 shows LNA-modified dgRNA facilitates a high rate of gene disruption, especially relative to dgRNA that is only end modified (the three terminal nucleotides at both the 5' region and 3' region of the dgRNA have MS modification) or dgRNA that has additional 2-0'-Me modifications (MS / PS mod) at stem loop 1, with an mRNA delivery method, using two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.

[0105] FIG. 7 demonstrates the higher potency of LNA-modified dgRNA in gene editing compared to sgRNA, using two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA.

[0106] FIGs. 8A and 8B show that LNA modification enhances the editing efficiency of sgRNAs for two different RNA-guided nucleases (RGNs), with the RGN being delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). (FIG. 8A) APG07433.1 sgRNA. ‘control_RNP’ and ‘control_mRNA’ indicate conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process. (FIG. 8B) APG01604 sgRNA. ‘control TRAC’ and ‘control_B2M’ indicate conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The two different spacer names in the controls indicate that antibodies against TRAC (2275) or B2M (1989) were used in the flow cytometry to establish a no-editing readout. The sgRNAs for each RGN used two exemplary spacers. (-): control sgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. (+): sgRNA with additional LNA modifications as depicted in each schematic. Each guide RNA schematic shows the LNA modifications in a region of the antirepeat forming the first stem and the MS modifications of the 3 terminal nucleotides at the 5' and 3' regions of the sgRNAs. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker for editing of the TRAC target sequence or immunostaining B2M for editing of the B2M target sequence using flow cytometry.

[0107] FIGs. 9A and 9B show that MS modification of stem loop 1 does not enhance editing efficiency. FIG. 9A depicts eight scenarios of 2'-0-Me and / or MS modifications in stem loop 1 and / or stem loop 3 for APG07433.1 sgRNA. FIG. 9B shows that none of the tested sgRNAs with 2'-0-Me and / or MS modifications enhance sgRNA editing as compared to a control sgRNA. Chemical modification at stem loop 3 abolishes sgRNA activity. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control’ indicates conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.

[0108] FIGs. 10A-10C show that the amount of LNA modifications correlates with guide RNA editing efficiency in primary human T cells as measured by knockout of the CD3 surface marker (for TRAC target sequence) or immunostaining B2M (for B2M target sequence) using flow cytometry. (FIG. 10A) Gene editing efficiency of APG07433.1 dgRNA with 1, 3, 6, or 11 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG07433.1 dgRNA show: the ‘0 LNA @ stem loop 1’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified. The highest editing was achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified. (FIG. 10B) Gene editing efficiency of APG01604 dgRNA with 3 or 7 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG01604 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA- modified. (FIG. 10C) Gene editing efficiency of APG05586 dgRNA with 4 or 9 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG05586 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified. Gene editing was improved for the LNA-modified APG01604 dgRNA and APG05586 dgRNA as compared to the ‘0 LNA’ dgRNA. The dgRNAs for each RGN used two exemplary spacers. Each RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). ‘control_TRAC’ and ‘control_B2M’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.

[0109] FIGs. 11A-11C show that LNA modification maintains or increases gene editing efficiency for shortened APG07433. 1 sgRNAs. (FIG. 11A) Top: the full-length APG07433. 1 sgRNA was shortened by a combination of truncations in various regions of the sgRNA: 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1 and 6 nt deleted from the tail (-10 first stem SL1, -6 tail); 5 nt pairs (10 nt) deleted from the first stem of stem loop 1, 4 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -4 tail, -2 first stem SL3); and 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1, 6 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -6 tail, -2 first stem SL3). These shortened APG07433.1 sgRNAs have MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA and serve as controls to assess additional chemical modifications introduced into the first stem of stem loop 1. Bottom: the shortened APG07433.1 sgRNAs as depicted at top but including LNA and MS modifications in the first stem of stem loop 1. (FIG. 1 IB) Gene editing efficiency for shortened APG07433.1 sgRNAs chemically modified as illustrated in FIG. 11A as compared to control full-length APG07433. 1 sgRNA or control shortened APG07433.1 sgRNA. The control full-length and shortened sgRNAs each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not contain any chemical modifications at stem loop 1. ‘Mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method. The sgRNAs were used at a dilution factor of 1. (FIG. 11C) LNA modification increases editing potency for shortened APG07433.1 sgRNAs. A serial dilution of the sgRNAs were conducted. Data were collected at day 4. The ‘3MS’ full-length and ‘3MS’ shortened sgRNAs are as depicted in FIG. 11A, top. The modified shortened sgRNAs are as depicted in FIG. 11A, bottom. DF = dilution factor. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

[0110] FIGs. 12A and 12B show that LNA modification maintains or increases gene editing efficiency for shortened APG07433.1 dgRNAs with RNP delivery. The ‘M’ shortening and chemical modification scheme performs the best for sgRNA and dgRNA. (FIG. 12A) Chemical modification and shortening schemes for crRNA and tracrRNA. The crRNA is shortened by 5 terminal nt at the 3' region: left, MS modifications at the three terminal nucleotides at the 5' and 3' regions (O and Q represent two exemplary spacers used); right, MS modifications at the three terminal nucleotides at the 5' and 3' regions plus 2’-O-Me modifications within the crRNA repeat forming the first stem of stem loop 1 (P and R represent two exemplary spacers used). tracrRNA: ‘tracr(L)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 6 nt, and the three terminal nucleotides at the 3' region comprise MS modifications; ‘tracr(M)’, the anti -repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 4 nt, 1 nt pair (2 nt) is deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3’ region comprise MS modifications; ‘tracr(N)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the antirepeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 6 nt, 1 nt pair (2 nt) is deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3' region comprise MS modifications. (FIG. 12B) Gene editing efficiency for shortened APG07433.1 dgRNAs chemically modified as illustrated in FIG. 12A and for shortened APG07433. 1 sgRNAs chemically modified as illustrated in FIG. 11A. gRNAs with non-shortened backbones: 1, full-length sgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region but not having any chemical modifications elsewhere in the sgRNA; 2, dgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 3, dgRNA with 1881 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 4, dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see FIGs. 3A and 3B), with 1880 spacer; and 5, dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see FIGs. 3A and 3B), with 1881 spacer. sgRNAs with shortened backbones (see FIG. 11A): 6, shortened (L) sgRNA without chemical modifications in the first stem of stem loop 1 ; 7, shortened (M) sgRNA without chemical modifications in the first stem of stem loop 1; 8, shortened (N) sgRNA without chemical modifications in the first stem of stem loop 1; 9, shortened (L) sgRNA with additional chemical modifications in the first stem of stem loop 1; 10, shortened (M) sgRNA with additional chemical modifications in the first stem of stem loop 1; and 11, shortened (N) sgRNA with additional chemical modifications in the first stem of stem loop 1. dgRNAs with shortened backbones: shortened and chemically modified crRNAs (O, Q, P, and R) and tracrRNAs (L, M, N) are as described in FIG. 12A. ‘control’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process, for each delivery method, ‘mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method. The sgRNAs and dgRNAs used two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

[0111] FIG. 13 shows the designs for testing gene editing efficiency of a shortened (‘M’ backbone, see FIG. 11A), chemically modified APG07433.1 gRNA in a sgRNA format. Top: the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA / PS; 3 conditions) and has no additional chemical modifications at the first stem of stem loop 1. Bottom: the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA / PS; 3 conditions) and includes MS and / or LNA modifications at the first stem of stem loop 1.

[0112] FIG. 14 shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). The ‘MS / LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and / or LNA chemical modifications in the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA / PS modifications, respectively.

[0113] FIG. 15. shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications. A serial dilution of the sgRNAs were conducted. Data were collected at day 4. The ‘3MS’, ‘3LNA’, ‘3LNA PS’ shortened ‘M’ APG07433.1 sgRNAs are as depicted in FIG. 13. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). The ‘MS / LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and / or LNA chemical modifications at the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA / PS modifications, respectively.

[0114] FIGs. 16A and 16B. Gene editing efficiencies of a dgRNA with various chemical modifications at the three terminal nucleotides at the 5' region and 3' region. (FIG. 16A, left) A design for testing gene editing efficiency of a wild-type (WT, full-length), chemically modified APG07433. 1 gRNA in a dgRNA format. The WT APG07433.1 gRNA is shown with possible chemical modifications: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the crRNA; LNA modifications at all nucleotides of the first stem of the anti-repeat; and MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 3' region of the tracrRNA. (FIG. 16A, right) a table showing the 18 total conditions tested given various combinations of 3MS, 3LNA, or 3LNA+PS in the crRNA, 3MS, 3LNA, or 3LNA+PS in the tracrRNA, and two delivery modes of the RGN (RNP and mRNA). (FIG. 16B) Gene editing efficiency for APG07433. 1 dgRNAs with various combinations of chemical modifications as shown in FIG. 16A. All tested dgRNAs have LNA modifications at all nucleotides of the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used. ‘control_TRAC’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process, for each delivery method. The control dgRNA (‘dgl880’ and ‘dgl881’) has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

[0115] FIGs. 17A and 17B show strategies for rescuing gene editing of RGN systems having dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1. FIG. 17A shows a strategy that includes lengthening the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of APG05586 pre-crRNA and LNA modification of all nucleotides in the lengthened first stem of the anti -repeat. FIG. 17B show strategies to lengthen the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA or APG08167 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of the respective pre-crRNAs. FIG. 17B highlights the G:C rich characteristic of the APG07433.1 nucleotide pairs most distal to the first bubble in the first stem of stem loop 1 (i.e. the nucleotides most proximal to the 3’ region of the crRNA and the 5’ region of the tracrRNA). WT APG07433.1 dgRNA having LNA modifications at all 11 nucleotides of the first stem of the anti-repeat achieves the highest gene editing (see FIG. 10A). Therefore, nucleotide sequence from APG07433. 1 most distal to the first bubble of stem loop 1 will be used to lenghthen APG05586 and APG08167 as an alternative approach.

[0116] FIG. 18 shows that gene editing is rescued for RGN systems having WT (original) dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1 by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiments, and there were two repeats per target gene. The ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘Unmod’ indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. ‘LNA’ indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the antirepeat. ‘Native seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA. ‘APG07433.1 seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA). A schematic of a APG01604 gRNA shortened in the first stem of stem loop 1 (APG01604.1, 81 nt backbone length) below the graph illustrates the ‘unmod’ APG01604.1 gRNA. The nucleotide sequences above the data points indicate the sequence of the 4 nucleotides at the 5’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0117] FIG. 19 shows that lengthening the first stem at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) using either nucleotide sequences from a native pre-crRNA or from APG07433. 1 gRNA and modifying all nucleotides of the first stem of the anti-repeat with LNA rescues gene editing for RGN systems having WT (original) dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1. The dgRNAs were lengthened to 11 nucleotide pairs or 13 nucleotide pairs in the first stem of stem loop 1. ‘native’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA. ‘APG07433.1’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA). The nucleotide sequences above the data points indicate the sequence of the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of the lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0118] FIG. 20 shows a strategy to improve gene editing efficiency by a shortened dgRNA. Left: schematic of a WT APG07433.1 crRNA. Center: schematic of a shortened ‘M’ APG07433.1 crRNA and tracrRNA. Right: the 3’ 3 terminal nucleotides of the crRNA and the 5’ 2 terminal nucleotides of the tracrRNA are substituted with C and G nucleotides, respectively (the starred nucleotides). For both the shortened (M) and the engineered shortened (M), the first stem of the anti-repeat is LNA modified.

[0119] FIG. 21 shows that gene editing efficiency is improved for a shortened ‘M’ APG07433. 1 dgRNA by substituting nucleotides as shown in FIG. 20. Two spacers were tested. ‘Original’ indicates the shortened ‘M’ APG07433.1 dgRNA without nucleotide substitutions. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0120] FIGs. 22A and 22B show strategies for LNA modification of the anti-repeat forming stem loop 1 of the tracrRNA in a gRNA. FIG. 22A shows a modified APG07433.1 dgRNA that performs well in gene editing, having all 11 nucleotides LNA modified in the first stem (FS) of the anti-repeat (Tracr(J); see FIG. 10A). FIG. 22B shows APG07433.1 tracrRNA modified at all nucleotides in the FS and in the second stem (SS) of the anti-repeat (Tracr(Jb); FS+SS); modified at all nucleotides in the SS of the anti-repeat (Tracr(Jc); SS); and modified at all nucleotides in the anti-repeat, including nucleotides of the first stem, the bubble, and the second stem (Tracr(Jd); full stem loop 1).

[0121] FIG. 23 shows that having LNA modification of all nucleotides of the first stem of the antirepeat in the tracrRNA of a gRNA is most effective for gene editing as compared to other LNA modification strategies for the anti-repeat. Gene editing efficiencies are shown for APG07433.1 dgRNAs having LNA-modified tracrRNA (right-hand side of the graph; Tracr(J), Tracr(Jb), Tracr(Jc), and Tracr(Jd)), as shown in FIGs. 22A and 22B. The crRNA had MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS), as shown in FIG. 22A. Two exemplary spacers (1880 and 1881) were used. ‘control_TRAC’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The control dgRNAs on the left-hand side of the graph has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0122] FIG. 24 shows that LNA modification of all nucleotides of the first stem of the crRNA repeat in the crRNA of a gRNA is effective for gene editing as long as all nucleotides of the first stem of the anti -repeat in the tracrRNA of the gRNA are also LNA-modified (see top graph, right side). Having LNA modification of all nucleotides of the second stem of the crRNA repeat in the crRNA of a gRNA worsens gene editing for a gRNA having LNA modification of all nucleotides of the first stem of the anti-repeat (see bottom graph, right side). Gene editing efficiencies are shown for APG07433.1 dgRNAs having LNA modifications at all nucleotides of the first stem or the second stem of the crRNA repeat: upper left and left side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS); upper right and right side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat; lower left and left side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with 3MS; and lower right and right side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used, ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0123] FIG. 25 shows that LNA modification increases editing potency for APG05586 sgRNAs. A serial dilution of the sgRNAs were conducted. The ‘unmod’ indicates sgRNAs having MS modifications at the three terminal nucleotides at both the 5' region and 3' region and no other chemical modifications (3MS). The ‘LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, along with 3MS. The ‘MS / LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, MS modifications at the three terminal nucleotides of the crRNA repeat most proximal to the loop of stem loop 1, and 3MS. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control (TRAC)’ and ‘control (B2M)’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.

[0124] FIG. 26 shows that the amount of LNA modification at the first stem of the anti -repeat correlates with guide RNA editing efficiency and melting temperature (Tm) of a DNA / tracrRNA antirepeat heteroduplex. Schematic of APG07433.1 dgRNA showing 1, 3, 6, or 11 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. The dgRNA includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA. The highest editing and highest Tm were achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified. The amount of LNA modification is shown on the x-axis, and the Tm and gene editing efficiency are shown on the vertical axes. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Two spacers (1062 and 1881) were tested in the gene editing experiments.

[0125] FIG. 27 shows that gene editing is rescued for an APG07991 RGN system having WT (original) dgRNA with < 11 (6) nucleotide pairs in the first stem of stem loop 1 (see left schematic) by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiments, and there were two repeats per target gene. The ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘Unmod’ indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. ‘LNA’ indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the anti-repeat. ‘Native’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the APG07991pre-crRNA. ‘APG07433.1’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti -repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti-repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA). The nucleotide sequences above the data points indicate the sequence of the 6 nucleotides at the 5 ’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0126] FIG. 28 shows that gene editing efficiency for APG07991 dgRNA can be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘sgRNA’ indicates an APG07991 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA. The numbers 6, 8, 10, 11, 12, and 13 indicate the length of the first stem of stem loop 1. ‘Native Seq’ indicates that native sequence from the APG07991 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length. ‘-’ and ‘+’ indicate whether all nucleotides of the first stem of the anti-repeat are modified with LNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0127] FIG. 29 shows that gene editing efficiency for Streptococcus pyogenes Cas9 (SpyCas9) dgRNA, which works with APG07991 RGN for gene editing, can also be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. The first stem of stem loop 1 of WT (original) SpyCas9 dgRNA has 4 nucleotide pairs (see left schematic) and has very low gene editing with APG07991 RGN. ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘sgRNA’ indicates a SpyCas9 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA. The numbers 4, 8, 11, and 13 indicate the length of the first stem of stem loop 1. ‘Native Seq’ indicates that native sequence from the SpyCas9 pre- crRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (targeting the TRAC and B2M genes) were used.

[0128] FIG. 30 shows that LNA modifications of all nucleotides of the anti-repeat forming the first stem of stem loop 1 confer more stability to an sgRNA. Left schematic: (A) End modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the sgRNA; (B) LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions and LNA modifications at all nucleotides of the first stem of the anti-repeat; (C) MS / LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions, MS modifications of the three terminal nucleotides at the 3 ’ region of the crRNA repeat, and LNA modifications at all nucleotides of the first stem of the anti-repeat; and (D) MS / LNA modified dgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications of the three terminal nucleotides at the 3’ region of the tracrRNA. MS / LNA modified dgRNA enables effective gene editing with co-delivery of the mRNA and gRNA components, but is not as stable as the chemically modified sgRNAs, as demonstrated in the case of staggered delivery.

[0129] FIG. 31 shows that base editing efficiency is increased using gRNAs having LNA modifications. The tested gRNAs are as described in FIG. 30. The A, B, C, and D of the graphs correspond to the A, B, C, and D depicted in the left schematic.

[0130] FIG. 32 shows that base editing efficiency is increased using gRNAs having LNA modifications and also shortened gRNAs having LNA modifications. The No LNA, LNA, and MS / LNA of the graphs correspond to what is depicted in the left schematic, ‘shrt’ indicates a shortened sgRNA. gRNAs with two exemplary spacers, SGN001880 and SGN001881, were used.

[0131] FIG. 33 shows that gene editing efficiency for a dgRNA chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of a LNA-modified dgRNA. The gene editing efficiency of APG07433. 1 dgRNA with 11 LNA-modified nucleotides within the anti-repeat forming the first stem of stem loop 1 (depicted in upper left schematic, see also FIG. 10A) was compared to that of APG07433. 1 dgRNA with 11 S-constrained ethyl (cEt) -modified nucleotides within the anti-repeat forming the first stem of stem loop 1 (depicted in lower left schematic). ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07433.1 RGN was delivered as mRNA encoding the APG07433.1 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0132] FIG. 34 shows that gene editing efficiency for a dgRNA lengthened at the first stem distal to the first bubble of stem loop 1 and chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of the same lengthened dgRNA that has been LNA-modified. APG05586 dgRNA was lengthened at the first stem distal to the first bubble of stem loop 1 to 11 nucleotide pairs (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA; see FIGs. 18 and 19) and all nucleotides of the first stem of the anti-repeat were modified with either LNA (depicted in upper left schematic) or with S- constrained ethyl (cEt) (depicted in lower left schematic). ‘Native seq for APG05586’ indicates that native sequence from the APG05586 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides. ‘Original’ indicates WT APG05586 dgRNA having 9 nucleotide pairs in the first stem of stem loop 1 that has not been lengthened. ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG05586 RGN was delivered as mRNA encoding the APG05586 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0133] FIG. 35 shows that LNA modification improved gene editing efficiency of a dgRNA having an extension. The top of FIG. 35 shows schematics illustrating three chemically modified dgRNAs where the extensions are at the tail of the tracrRNA: (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat , and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail of the tracrRNA (i.e. stem loop 3 for the depicted system), and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA corresponding to a dgRNA that does not have the extension, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without the extension. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

[0134] FIGs. 36A-36C show schematics of guide RNAs having extensions and details of the engineering of a shortened guide RNA. FIG. 36A, a non-engineered guide RNA + extension has: a crRNA length of 46 nt (a spacer + 21 nt crRNA repeat); and a tracrRNA length of 79 nt (85 nt WT length minus 6 nt from the tail). FIG. 36B, a shortened guide RNA + extension has a shortened crRNA / tracrRNA backbone and has: a crRNA length of 41 nt (a spacer + 16 nt shortened crRNA repeat); and a tracrRNA length of 72 nt. FIG. 36C, schematic of the engineered shortened crRNA and tracrRNA (except without the extension), showing the 3' 3 terminal nucleotides of the crRNA and the 5' 2 terminal nucleotides of the tracrRNA are substituted with C and G nucleotides (the starred nucleotides). For both dgRNAs + extensions shown in FIGs. 36A and 36B, the first stem of the antirepeat is LNA modified.

[0135] FIG. 37 shows that LNA modification improved gene editing efficiency of shortened dgRNAs + extensions that are engineered with nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA. a, dgRNA + extension with engineered shortened backbone and having MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); and b, dgRNA + extension with engineered shortened backbone and having 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

[0136] FIG. 38 shows that LNA modification at the first stem of the anti-repeat is the key for editing efficiency of dgRNA + extension, (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti -repeat, and MS modifications at the three terminal nucleotides at the 3 ' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail of the tracrRNA (stem loop 3 in this system), and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (d) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (e) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the antirepeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (f) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of stem loop 3, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (g) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (h) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension, (g) and (h) dgRNAs + extensions have the shortened backbone engineered as described in FIG. 36C. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

[0137] DETAILED DESCRIPTION

[0138] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0139] I. Overview

[0140] The present disclosure provides, inter alia, compositions and methods related to modified guide RNA (gRNA) for use in RNA-guided nuclease (RGN) systems, and systems and methods related thereto. In various embodiments, inclusion of one or more bridged nucleic acid, such as, e.g., locked nucleic acid, within certain regions of the gRNA (but in some embodiments, not within other regions) improves editing efficiency of RGN systems. Additionally, such modifications unlock editing potential in mRNA-based dual guide systems not previously possible with unmodified dual guide RNAs available in the prior art. This is a critical advancement in the art, as new editing modalities, such as prime editing (also known as reverse transcriptase editing or, RT-editing), require very long gRNA templates that are difficult if not impossible to synthesize at scale in single guide format using current manufacturing processes. Thus, lack of manufacturing feasibility in the art significantly limits the commercialization potential of these mRNA-based therapeutics. These and other advancements are presented in the present disclosure.

[0141] An RGN system allows for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RGN systems have been used for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene, for example. An RGN system involves a complex of an RGN with a gRNA. The hybridization of the gRNA to a particular target sequence allows targeting of the guide RNA / RGN complex to a specific location in a genome for editing.

[0142] In RGN systems, gRNAs can exist as a two-part gRNA or as a single gRNA. The two-part gRNA system includes a CRISPR RNA (crRNA) containing a spacer sequence which recognizes the target genomic sequence via Watson-Crick base pairing, and a scaffold transactivating crRNA (tracrRNA). The crRNA hybridizes to the tracrRNA to form a dual guide RNA (dgRNA) held together as a duplex at a crRNA IracrRNA annealing region. For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

[0143] There are advantages and disadvantages to both the dgRNA and the sgRNA. sgRNAs generally provide relatively good editing efficiency. However, a sgRNA, while somewhat convenient as a single chemical species, is relatively long - generally at or above 100 nt. A common method to produce gRNAs is through solid phase oligonucleotide synthesis, which is a serial synthetic route. With increasing length, yield and purity of the full-length product are low despite high coupling efficiency of each individual step (Reese, Org Biomol Chem, 2005; Beaucage & Reese, Curr Protoc Nucleic Acid Chem, 2009; Beaucage, Curr Opin Drug Di De, 2008; Shiba, Nucleic Acids, 2WT, LeProust Nuc. Acids Res. 2010). Therefore, the ability to use shorter gRNAs and / or dgRNAs has some utility in reducing the maximum length of oligonucleotides to be synthesized. Another advantage of using a dgRNA is that the tracrRNA is paired with different crRNAs that contain varied spacer sequences, so tracrRNA scaffolds can be made in large batches and paired with individual crRNAs for specific genomic targets.

[0144] However, a dgRNA has relatively low gene editing efficiency, especially in a delivery method where mRNA encoding an RGN is introduced to a cell for expression of the RGN. The additional exposed 5' and 3' ends of the crRNA and tracrRNA and likely weaker duplex strength relative to sgRNA (in which intramolecular components are hybridizing) may result in lower RNA stability and hence poor performance. In some embodiments, strengthening the duplex can enhance and rescue the effectiveness of dgRNA.

[0145] A number of chemical modifications including 2'-fluoro-ribose (2'-F), 2'-O-methyl (2'-O- Me), 3' phosphorothioate (PS), 2'-O-Me 3' phosphorothioate (MS), along with other modifications, have been used to improve gRNA stability and thus increase editing efficiency in cells ex vivo and in vivo. However, these methods fail to enable effective editing when transfecting RGN-encoding mRNA with dgRNA. Instead, they require long sgRNAs to be produced. With new technologies such as prime editing using additional guide extensions, sgRNAs can be infeasible to produce at scale and with high levels of purity. Additionally, while the aforementioned modifications protect the RNA from nuclease degradation, it is possible that other mechanisms can lead to guide degradation or inactivation, notably disruption of the secondary structure. A bridged nucleic acid (BNA) includes a nucleotide analog having a restricted conformation due to an intramolecular bond or crosslink. A type of BNA, a locked nucleic acid (LNA), has been used, for example, in the design of PCR probes to shorten the length of the probes and to increase hybridization strength. An LNA includes a covalent linkage between the 2' oxygen to the 4' carbon on the ribose sugar of a nucleotide and has been shown to contribute to highly efficient complementary pairing to improve mismatch discrimination, as well as to improve nuclease resistance (Y on et al. Nucleic Acids Res., 2006; Vester & Wengel, J Biochemistry, 2004). Provided herein are methods for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications. Data in this application demonstrate that such guide RNAs modified with BNA modifications are improved over prior art methods, significantly enhancing the potency of the guide RNA and enabling the use of dual guide RNA in applications where it otherwise would not be suitable, such as when gRNA is co-transfected with RGN-encoding mRNA. Without being limited by conjecture, we believe that these benefits are at least because the BNA modifications stabilize the stem loop formed by hybridization between a crRNA repeat and a tracr RNA anti-repeat. In some instances, the BNA is LNA. In some instances, the BNA is S- constrained ethyl (cEt). In some instances, methods are provided herein for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications and / or other chemical modifications. In various embodiments, the present disclosure incorporates BNA (e.g., LNA and / or cEt) or other chemical modifications in tracrRNA, gRNA, and / or crRNA to generate chemically modified tracrRNA, gRNA, and / or crRNA for use in RGN-based gene editing systems. In some embodiments, the crRNA and tracrRNA both comprise BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, either the crRNA or the tracrRNA comprise BNA (e.g., LNA and / or cEt) modifications. In some embodiments, the tracrRNA comprises BNA modifications whereas the crRNA does not comprise BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, the tracrRNA comprises BNA modifications and the crRNA comprises MS modifications. In embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the disclosure improves gene editing efficiency of an RGN system as compared to a reference RGN system with tracrRNA, gRNA, and / or crRNA comprising only MS modifications at the three terminal nucleotides at the 5 ’ and 3’ regions. In embodiments, the modified tracrRNA, gRNA, and / or crRNA of the disclosure allows a dgRNA to be used in applications where otherwise a sgRNA would be more desirable. In some embodiments, the present disclosure provides for use of BNA (e.g., LNA and / or cEt) modifications within the crRNA: tracrRNA annealing region of a dgRNA to enhance the performance (e.g., editing efficiency) of an RGN system in a cell. In some embodiments, BNA (e.g., LNA and / or cEt) modifications allows a shortened crRNA IracrRNA annealing region. In some embodiments, BNA (e.g., LNA) modifications in combination with engineering of the crRNA:tracrRNA annealing region of a dgRNA enhances performance of an RGN system in a cell. In some embodiments, the cells that are gene edited include primary cells. The modified tracrRNA, gRNA, and / or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.

[0146] Without being bound by any one theory, chemical modifications to nucleotides in gRNAs may enhance stability by interfering with the degradation of the gRNAs by endogenous nucleases and / or stabilize RNA-RNA interactions in the chemically modified region. II. Guide RNA

[0147] The present disclosure provides guide RNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and / or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and / or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and / or cEt) modification in the first stem of the anti-repeat of the tracrRNA. The term “guide RNA” is known in the art and generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a RNA-guided nuclease (RGN) and aid in targeting the RGN to a specific location within a target polynucleotide (e.g., a DNA or an mRNA molecule) such as, e.g., a genomic locus. In some embodiments, the guide RNA comprises a nucleotide sequence (i.e., a spacer) having sufficient complementarity with a target strand nucleotide sequence to hybridize with the target strand and direct sequence -specific binding of an RGN to the target nucleotide sequence. In some embodiments, when the target nucleotide sequence is double-stranded as is the case with DNA, the target nucleotide sequence comprises a non-target strand (which comprises the PAM sequence) and the target strand, which hybridizes with the spacer of the guide RNA. In these embodiments, the guide RNA has sufficient complementarity with the target strand of a double-stranded target sequence (e.g., target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequencespecific binding of an associated RGN to the target sequence (e.g., target DNA sequence). Therefore, in some embodiments, a guide RNA includes a spacer that is identical to the sequence of the nontarget strand except that uracil (U) replaces thymidine (T) in the guide RNA.

[0148] An RGN’s respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target strand and / or the non-target strand. In general, a guide RNA comprises a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA).

[0149] The term “guide RNA” also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules. Native guide RNAs that comprise both a crRNA and a tracrRNA generally comprise two separate RNA molecules that hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. In certain embodiments, the crRNA and tracrRNA are linked together by a linker. A "linker" can be any kind of chemical linkage that covalently connects two molecules together, for example, a linkage formed by click chemistry or any other chemical reactions, a polynucleotide, a polymer, or any entity that can link two molecules. In some embodiments, a linker connecting a crRNA and a tracrRNA comprises a multi-nucleotide linker (e.g., a four-nucleotide linker) to form a single guide RNA molecule, wherein the crRNA and the tracrRNA hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. Thus, a guide RNA encompasses a single-guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule or strand.

[0150] The crRNA and tracrRNA of a guide RNA can be linked by an organic molecule, group, polymer, or chemical moiety. In some embodiments, the crRNA and tracrRNA of a guide RNA is linked by click chemistry. Click chemistry involves the rapid generation of compounds by joining small units together via heteroatom links (C-X-C). The main objective of click chemistry is to develop a set of powerful, selective, and modular “blocks” that are useful for small- and large-scale applications. Click chemistry reactions are fast, modular, efficient, often do not produce toxic waste products, can be done with water as a solvent, and can be set up to be stereospecific.

[0151] Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecules can be involved, and labeling with small molecules, such as fluorescent dyes, biotin, and other groups can be readily achieved. Click chemistry reaction takes place between two components: an azide functional group and an alkyne functional group. Azide is a linear, polyatomic anion with the formula Ns’ and structure “N=N+=N“. It is the conjugate base of hydrazoic acid HN3. Organic azides are organic compounds with the formula RN3, containing the azide functional group. An alkyne is an unsaturated hydrocarbon containing at least one carbon — carbon triple bond (-C=C-; e.g., terminal acetylene). The simplest acyclic alkynes with only one triple bond and no other functional groups form a homologous series with the general chemical formula CnHsn-2. Terminal alkynes have the formula RC2H. An example is methylacetylene (propyne using IUPAC nomenclature). Both azido and alkyne groups are nearly never encountered in natural biomolecules. Hence, the reaction can occur within biological systems without interfering with other cellular processes (i.e. highly bioorthogonal) and specific.

[0152] A well-known click reaction is the Huisgen 1,3-dipolar cycloaddition of azides and alkynes. This reaction, yielding triazoles, has become the gold standard of click chemistry for its reliability, specificity, and biocompatibility. Such cycloadditions need high temperatures or pressures when the reaction involves simpler alkene or azides, since the activation energies are high (AGt»+26 kcal / mol). Cu(I) catalysts expedite the reaction of terminal alkynes and azides, thereby affording 1,4- disubstituted-I,2,3-triazoles. This reaction is an ideal click reaction and is widely employed in material science, medicinal chemistry, and chemical biology.

[0153] However, the cytotoxic nature of transition metals, employed as catalysts for the click reactions, preclude their use for in vivo applications. Alternative approaches with lower activation barriers and copper-free reactions have been established. Such reactions are referred to as “copper-free click chemistry”. Instead of using copper to activate the alkyne, the alkyne is instead introduced in a strained difluorooctyne (DIFO), in which the electron-withdrawing, propargylic, gemfluorines act together with the ring strain to greatly destabilize the alkyne (Agard et al. (2006) ACS Chem. Biol. 1(10): 644-648). This destabilization increases the reaction driving force, and the desire of the cycloalkyne to relieve its ring strain. Copper-free click chemistry proceeds as a concerted [3+2] cycloaddition in the same mechanism as the Huisgen 1,3-dipolar cycloaddition. Substituents other than fluorines, such as benzene rings, are also allowed on the cyclooctyne.

[0154] The reactive groups in click chemistry (e.g., azides, terminal alkynes, strained alkynes (e.g., dibenzocyclooctyne (DBCO)) can be introduced into any form of nucleic acid molecule and can be introduced enzymatically or chemically. Alkyne-modified and azide-modified oligonucleotides can be ordered from an oligo-synthesizing facility or company. An azide-modified version of a nucleotide can be introduced during RNA synthesis into a first RNA molecule, and an alkyne-modified version of a nucleotide can be introduced during RNA synthesis into a second RNA molecule. The resulting Click-functionalized nucleic acid molecules can be isolated and purified to remove any unreacted reagents or byproducts that might interfere with the subsequent click reaction. The purified Click- functionalized nucleic acid molecules can be mixed together in a reaction buffer that supports the click reaction. This can include a copper catalyst to facilitate the reaction between the azide and alkyne or can be copper-free. The azide and alkyne functional groups react to form a covalent bond, linking the two nucleic acid molecules together. Linked nucleic acid molecules can be further purified, and analytical techniques such as gel electrophoresis or mass spectrometry can be used to verify the successful linking of the nucleic acid molecules and assess the purity of the product.

[0155] Click chemistry is further described, for example, in: Kumar et al. (2007) J. Am. Chem. Soc, 129:6859-6864; El-Sagheer and Brown (2010) Chem. Soc. Rev. 39: 1388-1405; Haque and Peng (2014) Sci. China Chem. 57:215-231; Wittig and Krebs 1961 Chem. Ber. 1961, 94, 3260-3275; US 7,375,234; US 7,070,941; US2013 / 0046084, the contents of each of which are hereby incorporated by reference in its entirety.

[0156] Linking of a crRNA and a tracrRNA can proceed in the following manner. A 3' amino modifer is included on the crRNA and a 5' amino modifier is included on the tracrRNA by solid phase synthesis. After synthesis, deprotection, and purification of the RNA oligonucleotides, an NHS ester of azidobutyrate (4-azido-butan-l-oic acid A-hydroxysuccinimide ester; available from, e.g., Glen Research cat no. 50-1904-24) is used to install an azide group at the 5' region of the tracrRNA and an NHS ester of DBCO (dibenzocyclooctyne-PEG4-JV-hydroxysuccinimidyl ester; available, for example, from Sigma Aldrich cat. no. 764019) is used to install the cyclooctyne group at the 3' region of the crRNA. Assembly in aqueous media (with optionally optimization of ionic strength, pH and reagent concentration) allows the strain promoted azide-alkyne Huisgen cycloaddition ("copper free click chemistry") to proceed.

[0157] In some embodiments, a crRNA and tracrRNA linked by click chemistry are linked by chemical moieties. In some embodiments, a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the anti-repeat of the tracrRNA and an alkyne group at one or more nucleotides at the crRNA repeat of the crRNA. In some embodiments, a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the crRNA repeat of the crRNA and an alkyne group at one or more nucleotides at the anti-repeat of the tracrRNA. One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the crRNA repeat of the guide RNA. One or more azide- modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the anti-repeat of the guide RNA. In some embodiments, a crRNA and tracrRNA linked by chemical moieties is a single guide RNA and comprises an azide-modified nucleotide or an alkyne- modified nucleotide at one or more nucleotides in the nucleotide loop connecting the crRNA repeat and the anti-repeat.

[0158] A variety of further chemical reactions and the corresponding modifications are available to the skilled person to link nucleic acid molecules (e.g., crRNA and tracrRNA) to each other in a covalent way. These modifications include a variety of crosslinkers, such as thiol modifications, like a thioctic acid N-hydroxy succinimide (NHS) ester, chemical groups that react with primary amines ( — NH2). These primary amines are positively charged at physiologic pH and nucleophilic; this makes them easy to target for conjugation with several reactive groups. There are numerous synthetic chemical groups that will form chemical bonds with primary amines. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfo-NHS esters containing a sulfonate ( — SO3) group, for example, bis(sulfosuccinimidyl)suberate (BS3), sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, such as, for example l-ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), anhydrides, and fluorophenyl esters.

[0159] As described herein, a guide RNA can comprise a crRNA and a tracrRNA, wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one BNA modification. In some embodiments, the anti -repeat is capable of hybridizing to the crRNA repeat to form a stem loop comprising a first stem and a second stem.

[0160] As described herein, the present disclosure also provides a nucleic acid molecule comprising a tracrRNA, wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one BNA modification. In some embodiments, the antirepeat of the tracrRNA is capable of hybridizing to a crRNA repeat of a crRNA to form a stem loop comprising a first stem and a second stem. In some embodiments, a gRNA comprising the tracrRNA is capable of binding to an RGN.

[0161] As described herein, the present disclosure also provides a nucleic acid molecule comprising a crRNA comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA. In some embodiments, the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a stem loop comprising a first stem and a second stem. In some embodiments, a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.

[0162] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0163] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0164] The present invention provides, inter alia, CRISPR RNAs (crRNAs) or polynucleotides encoding CRISPR RNAs that comprise at least one BNA (e.g., LNA and / or cEt) modification. As used herein, the term “crRNA” refers to an RNA molecule or portion thereof that includes a spacer, which is the nucleotide sequence that directly hybridizes with the target strand of a target sequence, and a CRISPR repeat that comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule. As used herein, the term “tracrRNA” or “transactivating crRNA” refers to an RNA molecule that comprises an anti-repeat sequence that has sufficient complementarity to hybridize to at least a portion of the CRISPR repeat of a crRNA to form a structure that is recognized by an RGN molecule. In some embodiments, additional secondary structure(s) (e.g., stem-loops) within the tracrRNA molecule is required for binding to an RGN.

[0165] In some embodiments, the crRNAs comprise at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, the at least one modification is a BNA (e.g., LNA and / or cEt) modification. In some embodiments, the BNA modification comprises a 2', 4' BNA modification. In certain embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is an LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification. In some embodiments, the at least one chemical modification is a 2'-0-Me modification. In certain embodiments, the at least one chemical modification is an MS modification.

[0166] A crRNA comprises a spacer and a CRISPR repeat. The “spacer” is a nucleotide sequence that directly hybridizes with the target strand of a target sequence (e.g., target DNA sequence) of interest. The spacer is engineered to have full or partial complementarity with the target strand of a target sequence of interest. In some embodiments, the spacer can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In some embodiments, the spacer is about 30 nucleotides in length. In some embodiments, the spacer is 30 nucleotides in length. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the spacer can be identical in sequence to the non-target strand of a target sequence. In some of those embodiments wherein the target sequence is a target DNA sequence, the spacer can be identical in sequence to the non-target strand of the target DNA sequence, with the exception of the thymidines (Ts) in the nontarget strand are replaced by uracils (Us) in the spacer. In embodiments, the spacer is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(l):23-24).

[0167] In some embodiments, a spacer of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or to the phosphate backbone of the spacer. In certain embodiments, a spacer of the disclosure includes at least one chemical modification. In some embodiments, the at least one modification is selected from the group consisting of: 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, a spacer of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, a spacer of the disclosure includes at least one LNA modification. In some embodiments, a spacer of the disclosure includes at least one 2'-0-Me modification. In some embodiments, a spacer of the disclosure includes at least one MS modification.

[0168] In some embodiments, a spacer of the disclosure includes at least one 2'-0-Me modification and at least one MS modification. In certain embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one other chemical modification (e.g., 2'-0-Me or MS). In some embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one PS modification. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14 or that differs from SEQ ID NO: 14 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14.

[0169] For clarification, as used herein, when a nucleotide sequence “differs from a SEQ ID NO by a certain number of nucleotides” or “has a certain percentage identity to a SEQ ID NO”, the difference only occurs in the nucleotide sequence, and chemical modifications or lack thereof remain the same.

[0170] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15 or differs from SEQ ID NO: 15 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 15 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 15 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15.

[0171] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17.

[0172] In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89 or that differs from SEQ ID NO: 89 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89.

[0173] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91 or that differs from SEQ ID NO: 91 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91.

[0174] In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90 or that differs from SEQ ID NO: 90 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90.

[0175] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92 or that differs from SEQ ID NO: 92 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92.

[0176] In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111 or that differs from SEQ ID NO: 111 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111.

[0177] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113 or that differs from SEQ ID NO: 113 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113.

[0178] In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112 or that differs from SEQ ID NO: 112 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112.

[0179] In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114.

[0180] Along with a spacer, a crRNA further comprises a CRISPR RNA (crRNA) repeat. The crRNA repeat comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule. In embodiments, the crRNA repeat can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In embodiments, the crRNA repeat is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0181] In some embodiments, a crRNA repeat of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or to the phosphate backbone of the crRNA repeat. In certain embodiments, a crRNA repeat of the disclosure includes at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, a crRNA repeat of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4’-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one LNA modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one cEt modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification. In some embodiments, a crRNA repeat of the disclosure includes at least one MS modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification and at least one MS modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one other chemical modification (e.g., 2’- O-Me or MS). In some embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA In certain embodiments, a crRNA repeat of the disclosure includes at least one LNA modification.) modification and at least one PS modification.

[0182] In some embodiments, the crRNA repeat comprises the nucleotide sequence of any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or an active variant or fragment thereof, that when comprised within a guide RNA, is capable of directing the sequence-specific binding of an associated RNA-guided nuclease provided herein to a presently disclosed target sequence. In some embodiments, an active crRNA repeat variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. In some embodiments, an active crRNA repeat fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 2 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 2 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 2. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70 or differs from SEQ ID NO: 70 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 70 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 70 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94 or differs from SEQ ID NO: 94 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 94 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 94 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241 or differs from SEQ ID NO: 241 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 241 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 241 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253 or differs from SEQ ID NO: 253 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 253 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 253 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538 or differs from SEQ ID NO: 538 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538.

[0183] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39.

[0184] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384.

[0185] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385.

[0186] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as (c) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 386.

[0187] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387.

[0188] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300.

[0189] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304.

[0190] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308.

[0191] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312.

[0192] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320.

[0193] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344.

[0194] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348.

[0195] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352.

[0196] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356.

[0197] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360.

[0198] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388.

[0199] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389.

[0200] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390.

[0201] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324.

[0202] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328.

[0203] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332.

[0204] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336.

[0205] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391.

[0206] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392.

[0207] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393.

[0208] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397.

[0209] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465.

[0210] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469.

[0211] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473.

[0212] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477.

[0213] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481.

[0214] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508.

[0215] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512.

[0216] In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516.

[0217] In some embodiments, the crRNA is not naturally-occurring. In some embodiments, the specific crRNA repeat sequence is not linked to the engineered spacer sequence in nature and the crRNA repeat sequence is considered heterologous to the spacer sequence. In some embodiments, the spacer sequence is an engineered sequence that is not naturally occurring.

[0218] In some embodiments, a crRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or to the phosphate backbone of the crRNA. In certain embodiments, a crRNA of the disclosure includes at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'- fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'- O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, a crRNA of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'- O,4’-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, a crRNA of the disclosure includes at least one LNA modification. In some embodiments, a crRNA of the disclosure includes at least one 2'-O-Me modification. In some embodiments, a crRNA of the disclosure includes at least one MS modification. In some embodiments, a crRNA of the disclosure includes at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one other chemical modification (e.g., 2'-O-Me or MS). In some embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one PS modification. In some embodiments, a chemically modified crRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337- 339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 71. In some embodiments, a chemically modified crRNA has the nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 96.

[0219] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 4.

[0220] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 5.

[0221] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 6.

[0222] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 7.

[0223] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 9.

[0224] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 708.

[0225] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 292, and wherein with reference to SEQ ID NO: 292.

[0226] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 293.

[0227] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 73.

[0228] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 75.

[0229] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 301.

[0230] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 302.

[0231] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 303.

[0232] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 305.

[0233] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 307.

[0234] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 309.

[0235] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 310.

[0236] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 311.

[0237] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 313.

[0238] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 315.

[0239] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 321.

[0240] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 322.

[0241] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 323.

[0242] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 345.

[0243] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 347.

[0244] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 349.

[0245] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 350.

[0246] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 351.

[0247] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 353.

[0248] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 355.

[0249] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 357.

[0250] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 358.

[0251] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 359.

[0252] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 361.

[0253] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 363.

[0254] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 97.

[0255] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 98.

[0256] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 99.

[0257] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 325.

[0258] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 327.

[0259] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 329.

[0260] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 330.

[0261] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 331.

[0262] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 333.

[0263] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 335.

[0264] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 337.

[0265] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 338.

[0266] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 339.

[0267] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 42.

[0268] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 44.

[0269] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 380.

[0270] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 381.

[0271] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 382.

[0272] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 399.

[0273] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 401.

[0274] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 466.

[0275] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 467.

[0276] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 468.

[0277] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 470.

[0278] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 472.

[0279] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 474.

[0280] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 475.

[0281] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 476.

[0282] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 478.

[0283] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 480.

[0284] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 482.

[0285] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 483.

[0286] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 484.

[0287] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 509.

[0288] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 511.

[0289] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 513.

[0290] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 514.

[0291] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 515.

[0292] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 517.

[0293] In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 519.

[0294] Presently disclosed guide RNAs comprise a crRNA and a trans-activating CRISPR RNA (tracrRNA). A tracrRNA molecule comprises a nucleotide sequence comprising a region, referred to herein as the anti-repeat, that has sufficient complementarity to hybridize to a crRNA repeat. In embodiments, the tracrRNA molecule further comprises a region with secondary structure (e.g., stemloop). In some embodiments, secondary structure includes nucleotides that are in one of two states, paired or unpaired, where nucleotide or base pairing includes base-base hydrogen bonding interactions (e.g., adenine (A) pairs with uracil (U), cytosine (C) pairs with guanine (G)) between two complementary nucleic acid strands to form a helix. In some embodiments, the combination of one or more helical elements interspersed with unpaired, single-stranded nucleotides constitutes an RNA structure.

[0295] A “stem loop” as used herein refers to a form of secondary structure comprising at least one “stem” and at least one “loop”, “bulge”, or “bubble” found in polynucleotides. A stem loop can form intramolecularly (within one molecule, e.g., within a tracrRNA or a sgRNA) or intermolecularly (between two distinct nucleic acids, e.g., in a dgRNA by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA). Stem loops are created when there is at least some complementarity between two nucleic acid sequences to form a paired double helix. The paired double helix region with full complementarity or sometimes including a G:U wobble base pair (or I:U, I:A, or I:C, where I refers to inosine) is referred to as a “stem”. The term “loop”, “bulge”, or “bubble” refers to a single stranded region within the “stem loop” structure where there is no complementarity between nucleotides, excluding G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine). Thus, “loops”, “bulges” and “bubbles” include nucleotides that are not paired. In some embodiments, a “loop” is distinguished from a “bulge” or “bubble” by being located at one end of the “stem loop” structure, while a “bulge” or a “bubble” is located between two “stems” in the “stem loop” structure.

[0296] In certain embodiments, a stem loop structure comprises a stem and a loop at one end of the stem. In some embodiments, a stem loop structure comprises a first stem and a second stem with a bubble in between the stems. In some embodiments, a stem loop structure comprises a loop, multiple stems and multiple bubbles in between the stems. In this circumstance, the bubbles in the order of closeness to the loop are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc., and the stems in the order of closeness to the loop are referred to as a “first stem”, a “second stem”, a “third stem”, etc. In embodiments of dgRNA, the stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA does not include a loop, and thus the bubbles in the order of closeness to the 5’ end of the tracrRNA (or 3’ end of the crRNA) are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc., and the stems in the order of closeness to the 5’ end of the tracrRNA (or 3 ’ end of the crRNA) are referred to as a “first stem”, a “second stem”, a “third stem”, etc.

[0297] The term “first stem of a crRNA repeat of a crRNA”, “first stem of a crRNA repeat”, or “first stem of a crRNA” means the region in the crRNA repeat of the crRNA that forms the first stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA. The term “second stem of a crRNA repeat of a crRNA”, “second stem of a crRNA repeat”, or “second stem of a crRNA” means the region in the crRNA repeat of the crRNA that forms the second stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA. Similarly, the term “first stem of an anti-repeat of a tracrRNA”, “first stem of an anti -repeat”, or “first stem of a tracrRNA” means the region in the anti-repeat of the tracrRNA that forms the first stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA. The term “second stem of an anti-repeat of a tracrRNA”, “second stem of an anti-repeat”, or “second stem of a tracrRNA” means the region in the anti-repeat of the tracrRNA that forms the second stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA.

[0298] In some embodiments, a stem loop formed intramolecularly is a hairpin stem loop. Base pairings occur in the stem part of a stem loop and typically involve guanine-cytosine base pairing and adenine-uracil(thymidine) base pairing, although guanine -uracil base pairing is possible. Base stacking interactions promote helix formation. The loop part of a stem loop includes bases that are not paired. In some embodiments, a loop is the point at which a nucleic acid strand turns back on itself for nucleotide pairing to create a stem. In some embodiments, loops that are less than three bases long are sterically impossible and do not form. In some embodiments, optimal loop length is about 4-8 bases long. Common loops with four nucleotide sequences such as GAAA, AAAG, ACUU, or UUCG are known as the "tetraloop" and are particularly stable due to the base-stacking interactions of its component nucleotides.

[0299] In some embodiments, the region of the tracrRNA that is fully or partially complementary to a crRNA repeat is at the 5' end of the molecule and the 3' end of the tracrRNA comprises secondary structure. This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat. The nexus forms the core of the interactions between the guide RNA and the RGN, and is at the intersection between the guide RNA, the RGN, and the target sequence. The nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs. In embodiments, guide RNAs or RGN systems of the disclosure use tracrRNAs that comprise non- canonical sequences in the base of the hairpin stem of their nexus hairpins, including UNANNG and CNANNC. In some embodiments, a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of UNANNG. In some embodiments, a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of CNANNC. There are often terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc, doi: 10. 1101 / pdb.top090902, and U.S. Publication No. 2017 / 0275648, each of which is herein incorporated by reference in its entirety.

[0300] In some embodiments, a tracrRNA of the disclosure includes additional hairpin or stem loop structures in addition to the nexus hairpin. In some embodiments, a tracrRNA includes at least one stem loop. In some embodiments, a tracrRNA includes at least one stem loop proximal to the antirepeat and at least one stem loop proximal to the 3’ end of the tracrRNA. “Proximal” refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides of a region or an end of a nucleic acid molecule. In certain embodiments, “proximal” refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides of a region or an end of a nucleic acid molecule. “Most proximal” refers to being the nearest to a region or to an end of a nucleic acid molecule. For example, a stem loop most proximal to the tail of a tracrRNA is the first stem loop nearest the tail of the tracrRNA. “Distal” refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a region or an end of a nucleic acid molecule. In some embodiments, “distal” refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a structure of a nucleic acid molecule (e.g., bubble, loop). For example, the first stem of the anti -repeat of a dual guide RNA lengthened at the end distal to the first bubble of the stem loop is lengthened from the 3 ’ terminal nucleotide of the crRNA and from the 5’ terminal nucleotide of the tracrRNA. A tracrRNA also forms secondary structure upon hybridizing with its corresponding crRNA. The anti-repeat region of a tracrRNA is fully or partially complementary to the crRNA repeat of a crRNA. In some embodiments, a portion of the anti-repeat of a tracrRNA and a portion of a crRNA repeat hybridize and form a stem. In some embodiments, the crRNA:tracrRNA stem includes at least one nucleotide pair (i.e. base pair) because these portions of the anti-repeat and crRNA repeat are complementary. As described elsewhere herein, a portion of the anti-repeat of a tracrRNA forming a first stem is the first stem of the anti-repeat, a portion of the anti-repeat of a tracrRNA forming a second stem is the second stem of the anti-repeat, a portion of the anti-repeat of a tracrRNA forming a third stem is the third stem of the anti-repeat, etc. As described elsewhere herein, a portion of the crRNA repeat of a crRNA forming a first stem is the first stem of the crRNA repeat, a portion of the crRNA repeat of a crRNA forming a second stem is the second stem of the crRNA repeat, a portion of the crRNA repeat of a crRNA forming a third stem is the third stem of the crRNA repeat, etc. In some embodiments, a portion of the anti-repeat of a tracrRNA and a portion of the crRNA repeat are not complementary with each other and thus do not hybridize to form base pairs. In some embodiments, the region of non-complementarity between the anti -repeat and the crRNA repeat forms a bulge or a bubble. In some embodiments, hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem. In some embodiments, hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem and at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes two stems and one bubble in between.

[0301] In certain embodiments, a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include BNA (e.g., LNA and / or cEt) modifications. In some embodiments, a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include any chemical modifications.

[0302] In certain embodiments, the nucleotides in a loop, bulge, or bubble do not include BNA (e.g., LNA) modifications. In some embodiments, the nucleotides in a loop, bulge, or bubble do not include any chemical modifications.

[0303] In some embodiments, the anti-repeat of the tracrRNA that is fully or partially complementary to the crRNA repeat comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the stem formed by the tracrRNA anti-repeat and the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the stem formed by the tracrRNA anti-repeat and the crRNA repeat is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0304] In some embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. In some embodiments, the tracrRNA comprises a total length of 60 to 80 nucleotides, 80 to 100 nucleotides, 100 to 120 nucleotides, 120 to 140 nucleotides, 140 to 160 nucleotides, 160 to 180 nucleotides, or more than 180 nucleotides. For example, the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, or more nucleotides in length. In embodiments, the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides in length. In some embodiments, the tracrRNA is about 70 to about 105 nucleotides in length, including about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, and about 105 nucleotides in length. In some embodiments, the tracrRNA is 70 to 105 nucleotides in length, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105 nucleotides in length. In some embodiments, the tracrRNA is about 90 to about 125 nucleotides in length, including about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, and about 125 nucleotides in length. In some embodiments, the tracrRNA is 90 to 125 nucleotides in length, including 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and 125 nucleotides in length.

[0305] In some embodiments, a tracrRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the tracrRNA. In certain embodiments, a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In certain embodiments, a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of: 2'- O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In certain embodiments, the BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the BNA modification is an LNA modification. Thus, in some embodiments, the tracrRNA comprises at least one LNA modification. In some embodiments, the BNA modification is a cEt modification. Thus, in some embodiments, the tracrRNA comprises at least one cEt modification. In certain embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'- O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one LNA modification and at least one PS modification. In certain embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one PS modification.

[0306] In some embodiments, the tracrRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or an active variant or fragment thereof, that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA- guided nuclease provided herein to a presently disclosed target sequence. In some embodiments, an active tracrRNA sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51 - 53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713. In embodiments, an active tracrRNA sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713.

[0307] In some embodiments, a tracrRNA comprises at least one chemical modification at its 5' region or at its 3' region. In some embodiments, a tracrRNA comprises at least one chemical modification at both its 5' region and 3' region. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 539.

[0308] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity toSEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 10.

[0309] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 12.

[0310] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 709.

[0311] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 713.

[0312] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 294. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 294.

[0313] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 295. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 295.

[0314] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 80.

[0315] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 81.

[0316] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 364.

[0317] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 365.

[0318] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 366.

[0319] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 367.

[0320] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 369.

[0321] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 375.

[0322] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 376.

[0323] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 377.

[0324] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 378.

[0325] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 379.

[0326] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 102.

[0327] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 103.

[0328] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 370.

[0329] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 371.

[0330] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 372.

[0331] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 373.

[0332] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 710.

[0333] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 711.

[0334] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 51.

[0335] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 52.

[0336] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 53.

[0337] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 383.

[0338] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 499.

[0339] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 500.

[0340] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 501.

[0341] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 504.

[0342] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 505.

[0343] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 534.

[0344] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 535.

[0345] In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 537.

[0346] The term “derived from” as used herein in the context of a polynucleotide molecule refers to a molecule generated or synthesized using a parent molecule or information from that parent molecule. For example, a tracrRNA, gRNA, or crRNA of the disclosure comprising at least one BNA (e.g., LNA) modification is derived from its respective unmodified tracrRNA, gRNA, or crRNA by having at least one of its nucleotides modified with a BNA (e.g., LNA) modification. In some embodiments, the tracrRNA, gRNA, or crRNA comprising at least one BNA (e.g., LNA) modification derived from its respective unmodified parent tracrRNA, gRNA, or crRNA has the same polynucleotide sequence as the parent molecule. The term “unmodified” in the context of a crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA refers to a conventional crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA that does not include any modified nucleotides, BNA modifications, modified sugars, modified nucleobases, and / or modified phosphate backbones, or any chemical modifications.

[0347] Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Likewise, an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to a target sequence under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g. , greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

[0348] The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500 / L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and / or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and / or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and / or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley- Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0349] The term “sequence specific” can also refer to the binding of a RGN polypeptide to a target sequence at a greater affinity than binding to a randomized background sequence.

[0350] The guide RNA can be a single guide RNA (sgRNA) or a dual -guide RNA (dgRNA). A sgRNA comprises the crRNA and tracrRNA on a single molecule of RNA, whereas a dgRNA comprises a crRNA and a tracrRNA present on two distinct RNA molecules, hybridized to one another through at least a portion of the crRNA repeat of the crRNA and at least a portion of the antirepeat of the tracrRNA, which may be fully or partially complementary to each other. Hybridization of the anti-repeat of a tracrRNA to the crRNA repeat of a crRNA forms a stem loop comprising the anti-repeat and the crRNA repeat. In some embodiments, the stem loop includes one or more stems formed by the anti-repeat and the crRNA repeat. In some embodiments wherein the guide RNA is a sgRNA, the crRNA and tracrRNA are separated by a linker nucleotide sequence. In general, the linker nucleotide sequence is one that does not include bases complementary within itself or to other parts of the sgRNA in order to avoid the formation of secondary structure within or comprising nucleotides of the linker nucleotide sequence. In certain embodiments, the linker forms a loop at one end of the first stem in the stem loop structure comprising the crRNA repeat and the anti-repeat. In some embodiments, the linker nucleotide sequence between the crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length. In certain embodiments, the linker nucleotide sequence of a sgRNA is at least 4 nucleotides in length. In certain embodiments, the linker nucleotide sequence includes a nucleotide sequence set forth as any of AAAG, GAAA, ACUU, and CAAAGG.

[0351] The total length of a guide RNA can comprise about 100 nt to 120 nt, about 120 nt to 140 nt, about 140 nt to about 160 nt, about 160 nt to about 180 nt, about 180 nt to about 200 nt, or more. In some embodiments, the total length of a guide RNA is 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt. 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 171 nt, 172 nt, 173 nt, 174 nt, 175 nt, 176 nt, 177 nt, 178 nt, 179 nt, 180 nt, 181 nt, 182 nt, 183 nt, 184 nt, 185 nt, 186 nt, 187 nt, 188 nt, 189 nt, 190 nt, 191 nt, 192 nt, 193 nt, 194 nt, 195 nt, 196 nt, 197 nt, 198 nt, 199 nt, 200 nt, or more.

[0352] In some embodiments, a chemically modified sgRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

[0353] The sgRNA or dgRNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence -specific binding between an RGN and a guide RNA are known in the art and include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads). A control guide RNA with an unrelated sequence or structure to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA.

[0354] In some embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be chemically synthesized.

[0355] In embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RGN polypeptide.

[0356] The guide RNA directs an associated RGN to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target sequence of interest. The target sequence can be bound (and in some embodiments, cleaved) by an RGN in vitro or in a cell. A target sequence can comprise DNA, RNA, or a combination of both and can be single -stranded or doublestranded. In some embodiments, a target sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, episomal DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). In those embodiments wherein the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plastid or mitochondrial chromosomal sequence. In the presently disclosed compositions and methods, the target sequence is within a target nucleic acid molecule that is double-stranded (e.g., a target DNA sequence). In some embodiments, the target sequence is unique in the target genome. In some embodiments, the target sequence comprises a target strand and a non-target strand, and the target sequence (i.e., the sequence on the non-target strand) has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

[0357] The target sequence is adjacent to a protospacer adjacent motif (PAM) and the non-target strand of the target sequence is the strand that comprises the PAM. The PAM is immediately adjacent to the target sequence and often comprise Ns, which represent any nucleotide. In some embodiments, the PAM comprises about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns. In some embodiments, a PAM comprises 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. The PAM can be 5' or 3' of the target sequence on its non-target strand. In some embodiments, the PAM is 3' of the target sequence on its non-target strand for the presently disclosed guide RNAs and RGN systems. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in some embodiments, it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.

[0358] In some embodiments, a PAM sequence adjacent to a presently disclosed target sequence on its non-target strand comprises the consensus sequence set forth as any one of the PAM sequences in Table 1. In some embodiments, a PAM sequence adjacent to the presently disclosed target sequence on its non-target strand includes the consensus sequence set forth as any one of NNNNCC, NNGRR, NNRYA, and NGG. In some embodiments, the PAM sequence is 3' of the target sequence on its non- target strand.

[0359] It is well-known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015) Genome Biol 16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo.

[0360] Upon recognizing its corresponding PAM sequence, the RGN can cleave one or both strands of a target sequence at a specific cleavage site. As used herein, a cleavage site is made up of the two particular nucleotides within a target sequence between which the target strand, non-target strand, or both strands of a target sequence are cleaved by an RGN. The cleavage site can comprise the 1stand 2nd, 2ndand 3rd, 3rdand 4th, 4thand 5th, 5thand 6th, 7thand 8th, or 8thand 9thnucleotides from the PAM in either the 5' or 3' direction. In embodiments, the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction. As RGNs can cleave a target sequence resulting in staggered ends, in embodiments, the cleavage site is defined based on the distance of the two nucleotides from the PAM on the non-target strand of the target sequence, and for the target strand, the distance of the two nucleotides from the complement of the PAM.

[0361] III. Chemical modifications and length modifications to guide RNA

[0362] Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA of the disclosure can in some embodiments comprise at least one BNA (e.g., LNA) modification. In some embodiments, the at least one BNA (e.g., LNA) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA) modification in the first stem of the anti -repeat of the tracrRNA. Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA of the disclosure can in some embodiments include a modification in the ribose (e.g., sugar) group, phosphate group, nucleobase, or any combination thereof. The term "chemical modification" in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5' end modifications or 3' end modifications, (b) nucleobase (or "base") modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2', 3', and / or 4' positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages. The term "modified nucleotide" generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates. The terms “modification” and “chemical modification” are used interchangeably herein.

[0363] In some embodiments, a modified nucleotide includes a sugar modification. Non-limiting examples of sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'- deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5 '-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'- deoxyuridine-5'- triphosphate), 2'-O-alkyl oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2 '-O- methylcytidine-5'-triphosphate, 2'-methyluridine-5 '-triphosphate), 2'-C-alkyl oligoribonucleotide, and isomers thereof (2'-aracytidine-5'-triphosphate, 2'- arauridine-5'-triphosphate), azidotriphosphate (2'- azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate), and combinations thereof.

[0364] In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more 2'-fluoro, 2'-amino and / or 2'-thio modifications. In some embodiments, the modification is a 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'- amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, 5 -amino-allyl- uridine, 5- bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl- pyrene -uridine, 5-fluoro-cytidine, and / or 5 -fluoro-uridine. There are more than 96 naturally occurring nucleoside modifications found on mammalian RNA. See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994). A nucleoside includes a purine or pyrimidine base linked to a sugar (i.e., nucleotides without a phosphate group). The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art and described in, e.g., U.S. Patent Nos. 4,373,071; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319; 5,262,530; and 5,700,642. Numerous modified nucleosides and modified nucleotides that are suitable for use in the present disclosure are commercially available. The nucleoside can be an analogue of a naturally occurring nucleoside. In some embodiments, a nucleoside analogue includes dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxy cytodine, and deoxyuridine.

[0365] In some cases, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, atracrRNA, an anti-repeat, or a guide RNA) includes a nucleobase-modified ribonucleotide, i.e., a ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Non-limiting examples of modified nucleobases which can be incorporated into modified nucleosides and modified nucleotides include m5C (5 -methylcytidine), m5U (5 - methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1- methyl adenosine), m2A (2- methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2- methylthio-N6-methyladenosine), i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio- N6isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2- methylthio- N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycinylcarbamoyladenosine), t6A (N6-threonyl carbamoyladenosine), ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine), m6t6A (N6-methyl- N6-threonylcarbamoyladenosine), hn6A (N6-hydroxynorvalylcarbamoyl adenosine), ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine), Ar(p) (2'-O-ribosyladenosine(phosphate)), I (inosine), mil (1 -methylinosine), m'lm (l,2'-O-dimethylinosine), m3C (3 -methylcytidine), Cm (2T-O- methylcytidine), s2C (2 -thiocytidine), ac4C (N4-acetylcytidine), f5C (5-fonnylcytidine), m5Cm (5,2- O-dimethylcytidine), ac4Cm (N4acetyl2TOmethylcytidine), k2C (lysidine), mlG (1- methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-0- methylguanosine), m22G (N2,N2-dimethylguanosine), m2Gm (N2,2'-O-dimethylguanosine), m22Gm (N2,N2,2'-O-trimethylguanosine), Gr(p) (2'-O-ribosylguanosine(phosphate)), yW (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW* (undermodified hydroxywybutosine), imG (wyosine), mimG (methylguanosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galtactosyl-queuosine), manQ (mannosyl- queuosine), preQo (7-cyano-7- deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G (archaeosine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2 -thiouridine), s2Um (2-thio- 2'-0-methyluridine), acp3U (3-(3-amino-3- carboxypropyl)uridine), ho5U (5 -hydroxyuridine), mo5U (5 -methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5- (carboxyhydroxymethyl)uridine)), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5 -methoxycarbonyl methyluridine), mcm5Um (S-methoxycarbonyhnethyl-2- O-methyluridine), mcm5s2U (5 -methoxy carbonylmethyl -2 -thiouridine), nm5s2U (5- aminomethyl-2- thiouridine), mnm5U (5 -methylaminomethyluridine), mnm5s2U (5- methylaminomethyl-2- thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5 -carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-0-methyluridine), cmnm5U (5- carboxymethylaminomethyluridine), cnmm5Um (5 -carboxymethylaminomethyl- 2-L- Omethyluridine), cmnm5s2U (5 -carboxymethylaminomethyl -2 -thiouridine), m62A (N6,N6- dimethyladenosine), Tm (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2-O- dimethylcytidine), hm5C (5 -hydroxymethylcytidine), m3U (3 -methyluridine), cm5U (5- carboxymethyluridine), m6Am (N6,T-O-dimethyladenosine), m62Am (N6,N6,0-2- trimethyladenosine), m2'7G (N2,7-dimethylguanosine), m2'2'7G (N2,N2,7- trimethylguanosine), m3Um (3,2T-O-dimethyluridine), m5D (5 -methyldihydrouridine), f5Cm (5 -formyl -2'-0- methylcytidine), mlGm (l,2'-O-dimethylguanosine), m'Am (1,2-0- dimethyl adenosine)irinomethyluridine), tm5s2U (S-taurinomethyl-2-thiouridine)), imG-14 (4-demethyl guanosine), imG2 (isoguanosine), or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo- adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2- thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-Cg)-alkyluracil, 5 -methyluracil, 5-(C2-Cg)- alkenyluracil, 5-(C2-Cg)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5- fluorouracil, 5 -bromouracil, 5 -hydroxy cytosine, 5-(Ci- Cg)-alkylcytosine, 5 -methylcytosine, 5-(C2-Cg)-alkenylcytosine, 5-(C2-Cg)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5- bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8- azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-Cg)alkynylguanine, 7-deaza-8-substituted guanine, 8- hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4- diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7- substituted purine, 7-deaza-8-substituted purine, and combinations thereof.

[0366] In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more modifications in the phosphate backbone. The modification can include one or more of phosphorothioate, phosphorodithioate, phosphoramidate (e.g., N3'- P5'-phosphoramidate (NP)), and / or methylphosphonate linkages. In some embodiments, a backbone modification includes a neutral backbone modification including: phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA) modifications. In some embodiments, all stereoisomers of these backbone modifications are useful in the present disclosure.

[0367] In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2’ position of the ribose sugar. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl (2'-0-Me) modification (“B” indicates “base” in chemical structures herein):

[0368] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O- methoxy-ethyl (2'-M0E) modification:

[0369] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-fluoro (2'- F) modification:

[0370] In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2' position and 4' position of the ribose sugar. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'F-4'Ca-OMe modification:

[0371] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2',4'-di-Ca- OMe modification:

[0372] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a phosphorothioate (PS) modification (e.g., in the backbone): In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2' position of the ribose sugar and the phosphate backbone. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'- phosphorothioate (MS) modification:

[0373] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'thiophosphonoacetate (MSP; 2'-O-methyl 3'thioPACE) modification:

[0374] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'phosphonoacetate (MP) modification:

[0375] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2’-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3 ’thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA) includes one or more MS modifications and one or more 2’,4’-BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more 2’,4’-BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more LNA modifications. In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more cEt modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more LNA modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more cEt modifications.

[0376] In some embodiments, a modification includes a bridged nucleic acid (BNA) modification. The term "bridged nucleic acid" refers to a nucleic acid having a structure wherein the degree of freedom of the nucleic acid is restricted through an intramolecular bond or crosslink. In certain embodiments, a BNA modification includes a 2', 4' BNA modification. In some embodiments, the 2' oxygen and 4' carbon of the ribose are linked through a “bridge”.

[0377] First generation BNA modifications include locked nucleic acid (LNA) modifications. LNA nucleotides comprise conformationally-restricted RNA nucleotides in which the 2' oxygen in the ribose forms a covalent bond to the 4' carbon, inducing N-type (C3'-endo) sugar puckering and preference for an A-form helix (Y ou et al. (2006) Nucleic Acids Res 34(8):e60), depicted as follows:

[0378] LNAs display improved base stacking and thermal stability compared to RNA, resulting in highly efficient binding to complementary nucleic acids and improved mismatch discrimination, as well as nuclease resistance (You et al. (2006) Nucleic Acids Res 34(8):e60; Vester & Wengel (2004) Biochemistry 43(42): 13233-13241). They have been successfully used in numerous applications ranging from SNP detection assays to siRNA (Vester & Wengel (2004) Biochemistry 43(42): 13233- 13241; Elmen et al. (2005) Nucleic Acids Res 33(l):439-447). N-methyl substituted bridged nucleic acids (BNANC[N-Me]) have been designed to improve upon the original first generation LNA scaffold by introducing more conformational flexibility for DNA binding, even greater nuclease resistance due to steric bulk, and reduced cellular toxicity (Rahman et al. (2008) J Am Chem Soc 130(14):4886-4896).

[0379] A number of bridged nucleic acids are known to those of skill in the art and are available from commercial sources (e.g., Biosynthesis, Inc.). In some embodiments, BNAs include: 2'-O,4'-C- ethylene BNA (2',3'-ENA); 2'-O,4'-C-methylenecytidine; 2'-O,4'-C-methyleneuridine; 2',4'-BNA-l- isoquinolone; 2',4'-BNA-2-pyridone; 2',4'-BNA-TeNA; 2',4'-BNA-TrNA; 2',4'-BNAC0C; 2', 4'- BNANC[NBn]; 2',4'-BNANC[NH]; 2',4'-BNANC[NMe]; 3'-amino-2',4'-BNA; AmNA; DpNA (3,4- dihydro-2H-pyran bridge moiety; EoNA; GuNA (guanidine BNA); HxNA; PrNA; scpBNA (2'-O,4'- C-spirocycloprepylene BNA); six-membered AmNA; SuNA; urea-BNA; a-L-LNA; 5-methyl-2'-O,4'- C-methyleneuridine; 5-bromo-2'-O,4'-C-methyleneuridine; 3'-O-benzyl-5'-O-mesyl-5-methyl-2'-O,4'- C-methyleneuridine; and benzylidene acetal -type BNA (BA-BNA).

[0380] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a BNANC[N-Me] modification:

[0381] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification:

[0382] In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a S- constrained ethyl (cEt) modification:

[0383] Other BNAs are disclosed in: U.S. Patent Nos. US 6,770,748; US 6,770,748; 8,153,365; 8,080,644; 7,060,809; 7,084,125; 7,060,809; 7,053,207; 6,670,461; 6,436,640; 6,316,198; and 7,427,672, each of which is herein incorporated by reference in its entirety.

[0384] In certain embodiments, ...

Claims

THAT WHICH IS CLAIMED:

1. A nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises:(a) an anti-repeat;(b) a tail; and(c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

2. The nucleic acid molecule of claim 1, wherein the at least one BNA modification is within the anti-repeat.

3. The nucleic acid molecule of claim 1 or 2, wherein the at least one BNA modification is within the first stem of the anti-repeat.

4. The nucleic acid molecule of claim 3, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive mucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti -repeat.

5. The nucleic acid molecule of claim 3 or 4, wherein all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

6. The nucleic acid molecule of claim 1 or 2, wherein the at least one BNA modification is not within the second stem of the anti -repeat.

7. The nucleic acid molecule of any one of claims 1-6, wherein the at least one BNA modification is not within a bulge of the tracrRNA.

8. The nucleic acid molecule of any one of claims 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications.

9. The nucleic acid molecule of any one of claims 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

10. The nucleic acid molecule of any one of claims 1-9, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

11. The nucleic acid molecule of claim 10, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4’-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

12. The nucleic acid molecule of claim 10 or 11, wherein the 2', 4' BNA is a LNA modification.

13. The nucleic acid molecule of claim 10 or 11, wherein the 2', 4' BNA is a cEt modification.

14. The nucleic acid molecule of any one of claims 1-13, wherein the tracrRNA further comprises at least one other chemical modification.

15. The nucleic acid molecule of claim 14, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

16. The nucleic acid molecule of claim 14 or 15, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

17. The nucleic acid molecule of claim 14, wherein the at least one other chemical modification is within the tail of the tracrRNA.

18. The nucleic acid molecule of any one of claims 14-17, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di- Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

19. The nucleic acid molecule of claim 18, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications.

20. The nucleic acid molecule of claim 18, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the anti -repeat comprise BNA modifications.

21. The nucleic acid molecule of claim 20, wherein the BNA modifications comprise LNA modifications.

22. The nucleic acid molecule of claim 20, wherein the BNA modifications comprise cEt modifications.

23. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

24. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

25. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of about 11 nucleotides.

26. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

27. The nucleic acid molecule of any one of claims 1-26, wherein the first stem of the antirepeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

28. The nucleic acid molecule of claim 27, wherein the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

29. The nucleic acid molecule of any one of claims 1-28, wherein the tracrRNA comprises a total length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

30. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383, 709, and 713.

31. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

32. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 102, 103, and 370-373, 710, and 711.

33. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

34. The nucleic acid molecule of any one of claims 1-33, wherein the tracrRNA is part of a gRNA that is capable of binding to an RGN.

35. The nucleic acid molecule of claim 34, wherein the RGN is a Type II RGN.

36. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

37. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

38. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93.

39. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

40. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

41. The gRNA of claim 40, wherein the gRNA is a single guide RNA (sgRNA).

42. The gRNA of claim 41, wherein the sgRNA comprises a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

43. The gRNA of claim 40, wherein the gRNA is a dual guide RNA (dgRNA).

44. The gRNA of any one of claims 40-43, wherein the at least one BNA modification is within the crRNA repeat.

45. The gRNA of any one of claims 40-43, wherein the at least one BNA modification is within the first stem of the crRNA repeat.

46. The gRNA of any one of claims 40-43, wherein the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat.

47. The gRNA of claim 45 or 46, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications.

48. The gRNA of claim 45 or 46, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications.

49. The gRNA of any one of claims 40-48, wherein the at least one BNA modification is not within the second stem of the crRNA repeat.

50. The gRNA of any one of claims 40-44, wherein the at least one BNA modification is within the anti-repeat.

51. The gRNA of claim 50, wherein the at least one BNA modification is within the first stem of the anti-repeat.

52. The gRNA of claim 51, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat.

53. The gRNA of claim 51 or 52, wherein all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

54. The gRNA of claim 50, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

55. The gRNA of any one of claims 40-54, wherein the at least one BNA modification is not within a bulge of the gRNA.

56. The gRNA of any one of claims 40-55, wherein the at least one BNA modification is within the tail of the tracrRNA.

57. The gRNA of claim 56, wherein the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise BNA modifications.

58. The gRNA of claim 56, wherein the three terminal nucleotides at the 3’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

59. The gRNA of any one of claims 40-58, wherein at least three terminal nucleotides in the 3’ region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti-repeat comprise BNA modifications.

60. The gRNA of any one of claims 40-59, wherein all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the anti-repeat comprise BNA modifications.

61. The gRNA of any one of claims 40-60, wherein the at least one BNA modification is within the spacer.

62. The gRNA of claim 61, wherein three terminal nucleotides at the 5' region of the spacer comprise BNA modifications.

63. The gRNA of any one of claims 40-62, wherein the spacer is 18-30 nucleotides in length.

64. The gRNA of any one of claims 40-63, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

65. The gRNA of claim 64, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

66. The gRNA of claim 64 or 65, wherein the 2', 4' BNA is a LNA modification.

67. The gRNA of claim 64 or 65, wherein the 2', 4' BNA is a cEt modification.

68. The gRNA of any one of claims 40-67, wherein the gRNA further comprises at least one other modification.

69. The gRNA of claim 68, wherein the at least one other modification is within the crRNA.

70. The gRNA of claim 68 or 69, wherein the at least one other modification is within the 5' region or the 3' region of the crRNA.

71. The gRNA of claim 68 or 69, wherein the at least one other modification is within the 5' region and the 3' region of the crRNA.

72. The gRNA of any one of claims 68-71, wherein the at least one other chemical modification is within the crRNA repeat of the crRNA.

73. The gRNA of any one of claims 68-72, wherein the at least one other chemical modification is within the first stem of the crRNA repeat.

74. The gRNA of any one of claims 68-73, wherein the at least one other chemical modification is within the spacer of the crRNA.

75. The gRNA of claim 68, wherein the at least one other chemical modification is within the tracrRNA.

76. The gRNA of claim 75, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

77. The gRNA of claim 75 or 76, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

78. The gRNA of claim 75, wherein the at least one other chemical modification is within the tail of the tracrRNA.

79. The gRNA of any one of claims 68-78, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'- O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

80. The gRNA of claim 79, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications.

81. The gRNA of claim 79 or 80, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.

82. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

83. The gRNA of any one of claims 40-81 , wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

84. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of about 11 nucleotides.

85. The gRNA of any one of claims 40-81 , wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

86. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre -crRNA) or a GC-rich nucleotide sequence.

87. The gRNA of claim 86, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti-repeat at the 5 ’ region comprises a GC-rich nucleotide sequence, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

88. The gRNA of claim 79, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

89. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as:(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides;(d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;(e) SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides; or(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.

90. The gRNA of claim 89, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

91. The gRNA of claim 89 or 90, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

92. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides;(f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;(g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides;(h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides;(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;(j) SEQ ID NO: 360 orthat differs from SEQ ID NO: 360 by 1 or 2 nucleotides;(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;(l) SEQ ID NO: 389 orthat differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or(m) SEQ ID NO: 390 orthat differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

93. The gRNA of claim 92, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353- 355, 357-359, and 361-363.

94. The gRNA of claim 92 or 93, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

95. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:(a) SEQ ID NO: 324 orthat differs from SEQ ID NO: 324 by 1 or 2 nucleotides;(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;(c) SEQ ID NO: 332 orthat differs from SEQ ID NO: 332 by 1 or 2 nucleotides;(d) SEQ ID NO: 336 orthat differs from SEQ ID NO: 336 by 1 or 2 nucleotides;(e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;(f) SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides; and(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

96. The gRNA of claim 95, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

97. The gRNA of claim 95 or 96, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

98. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;(f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides;(g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides; and(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

99. The gRNA of claim 98, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

100. The gRNA of claim 98 or 99, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

101. The gRNA of any one of claims 40-100, wherein the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the anti -repeat.

102. The gRNA of claim 101, wherein the linker comprises an azide functional group or an alkyne functional group.

103. The gRNA of claim 101, wherein the linker is a polynucleotide.

104. The gRNA of claim 103, wherein the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG.

105. The gRNA of claim 103 or 104, wherein the linker has a nucleotide sequence set forth as AAAG.

106. The gRNA of any one of claims 103-105, wherein the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA.

107. The gRNA of claim 106, wherein the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

108. The gRNA of claim 106, wherein the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

109. The gRNA of any one of claims 40-108, wherein the gRNA is capable of binding to an RGN.

110. The gRNA of claim 109, wherein the RGN is a Type II RGN.

111. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

112. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

113. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93.

114. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

115. The gRNA of any one of claims 40-114, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

116. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:(a) a spacer; and(b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti-repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O- Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O- m ethyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

117. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:(a) a spacer; and(b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy- ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

118. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) of any one of claims 1-39; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

119. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) of claim 116 or 117; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

120. The RGN system of claim 118 or 119, wherein the crRNA and the tracrRNA form a guide RNA.

121. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA of any one of claims 40-115; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

122. The RGN system of any one of claims 118-121, wherein the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, orNGG.

123. The RGN system of any one of claims 118-122, wherein the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

124. The RGN system of any one of claims 118-123, wherein the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

125. The RGN system of any one of claims 118-124, wherein the RGN polypeptide and the gRNA are not found complexed to one another in nature.

126. The RGN system of any one of claims 118-125, wherein the RGN system binds a target sequence in a target nucleic acid molecule.

127. The RGN system of calim 126, wherein the target sequence is a eukaryotic target sequence.

128. The RGN system of claim 126 or 127, wherein the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

129. The RGN system of any one of claims 126-128, wherein the target sequence is within a cell.

130. The RGN system of any one of claims 126-129, wherein a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence.

131. The RGN system of claim 130, wherein the cleavage generates a double-stranded break.

132. The RGN system of claim 130, wherein the cleavage generates a single-stranded break.

133. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is nuclease inactive.

134. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is a nickase.

135. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is fused to a base-editing polypeptide.

136. The RGN system of claim 135, wherein the base-editing polypeptide comprises a deaminase.

137. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is fused to a prime editing polypeptide.

138. The RGN system of claim 137, wherein the prime editing polypeptide comprises a DNA polymerase.

139. The RGN system of claim 138, wherein the DNA polymerase comprises a reverse transcriptase.

140. The RGN system of any one of claims 137-139, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

141. The RGN system of any one of claims 118-140, wherein the RGN polypeptide is fused to a detectable label.

142. The RGN system of any one of claims 118-132, wherein the RGN system further comprises a donor polynucleotide.

143. The RGN system of any one of claims 118-142, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA.

144. The RGN system of any one of claims 118-142, wherein the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter.

145. The RGN system of any one of claims 118-142, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

146. A ribonucleoprotein (RNP) complex comprising the RGN system of any one of claims 118- 145.

147. A cell comprising the nucleic acid molecule comprising a tracrRNA of any one of claims 1- 39, the gRNA of any one of claims 40-115, the crRNA of claim 116 or 117, the RGN system of any one of claims 118-145, or the RNP complex of claim 146.

148. The cell of claim 147, wherein the cell comprises a target sequence capable of being bound by a formed crRNA / tracrRNA / RGN polypeptide or gRNA / RGN polypeptide complex of the RGN system of any one of claims 118-145, or by the RNP complex of claim 146.

149. The cell of claim 147 or 148, wherein the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

150. The cell of any one of claims 147-149, wherein the cell is a prokaryotic cell.

151. The cell of any one of claims 147-149, wherein the cell is a eukaryotic cell.

152. The cell of claim 151, wherein the eukaryotic cell is a primary cell.

153. The cell of claim 152, wherein the primary cell is a T cell.

154. The cell of claim 151, wherein the eukaryotic cell is a plant cell.

155. A plant comprising the cell of claim 154.

156. A seed comprising the cell of claim 154.

157. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tracrRNA of any one of claims 1-39, the gRNA of any one of claims 40-115, the crRNA of claim 116 or117, the RGN system of any one of claims 118-145, the RNP complex of claim 146, or the cell of any one of claims 147-153.

158. A method for binding a target sequence in a target nucleic acid molecule comprising delivering the RGN system of any one of claims 118-145, or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence.

159. The method of claim 158, wherein the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence.

160. The method of claim 158 or 159, wherein the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence.

161. A method for cleaving and / or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering the RGN system of any one of claims 118-145, or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

162. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of claims 1-39 and a CRISPR RNA (crRNA); and ii) a Type II RGN,thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

163. The method of claim 162, wherein the assembled RNP complex directs cleavage of the target sequence.

164. The method of any one of claims 158-162, wherein the RGN is fused to a prime editing polypeptide.

165. The method of claim 164, wherein the prime editing polypeptide comprises a DNA polymerase.

166. The method of claim 165, wherein the DNA polymerase comprises a reverse transcriptase.

167. The method of any one of claims 164-166, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

168. The method of any one of claims 158-162, wherein the RGN polypeptide is fused to a baseediting polypeptide.

169. The method of claim 168, wherein the base-editing polypeptide comprises a deaminase.

170. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of claims 1-39 and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

171. The method of claim 170, wherein a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.

172. The method of claim 170, wherein the RGN is fused to a prime editing polypeptide.

173. The method of claim 172, wherein the prime editing polypeptide comprises a DNA polymerase.

174. The method of claim 173, wherein the DNA polymerase comprises a reverse transcriptase.

175. The method of any one of claims 172-174, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

176. The method of claim 170, wherein the RGN polypeptide is fused to a base-editing polypeptide.

177. The method of claim 176, wherein the base-editing polypeptide comprises a deaminase.

178. The method of claim 170, wherein the polynucleotide encoding the Type II RGN is an mRNA.

179. A method for binding a target sequence in a target nucleic acid molecule with RNA-guided nuclease (RGN), the method comprising:a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) the guide RNA (gRNA) of any one of claims 40-115; and ii) a Type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

180. The method of claim 179, wherein the assembled RNP complex directs cleavage of the target sequence.

181. The method of claim 179, wherein the RGN polypeptide is fused to a base-editing polypeptide.

182. The method of claim 181, wherein the base-editing polypeptide comprises a deaminase.

183. The method of claim 179, wherein the RGN is fused to a prime editing polypeptide.

184. The method of claim 183, wherein the prime editing polypeptide comprises a DNA polymerase.

185. The method of claim 184, wherein the DNA polymerase comprises a reverse transcriptase.

186. The method of any one of claims 183-185, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

187. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) of any one of claims 40-115; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

188. The method of claim 187, wherein a formed complex ofthe gRNA and the Type II RGN directs cleavage of the target sequence.

189. The method of claim 187, wherein the RGN polypeptide is fused to a base-editing polypeptide.

190. The method of claim 189, wherein the base-editing polypeptide comprises a deaminase.

191. The method of claim 187, wherein the RGN is fused to a prime editing polypeptide.

192. The method of claim 191, wherein the prime editing polypeptide comprises a DNA polymerase.

193. The method of claim 192, wherein the DNA polymerase comprises a reverse transcriptase.

194. The method of any one of claims 191-193, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

195. The method of claim 187, wherein the polynucleotide encoding the Type II RGN is an mRNA.

196. A method for binding a target sequence in a target nucleic acid molecule with RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of claim 116 or 117 and a tracrRNA; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

197. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of claim 116 or 117 and a tracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

198. The method of claim 197, wherein the polynucleotide encoding the Type II RGN is an mRNA.

199. The method of any one of claims 158-198, wherein the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712.

200. The method of any one of claims 162-199, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

201. A method of increasing efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering the RGN system of any one of claims 118- 145 or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or does not comprise any chemical modification.

202. The method of claim 201, wherein all nucleotides of the first stem of the anti-repeat of the tracrRNA of the RGN system of any one of claims 118-145 or the RNP complex of claim 146 comprise BNA modifications.

203. The method of claim 202, wherein at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications.

204. The method of claim 201, wherein the BNA modifications comprise LNA modifications.

205. The method of claim 201, wherein the BNA modifications comprise cEt modifications.

206. The method of any one of claims 201-205, wherein the efficiency of cleaving and / or modifying the target sequence is increased by 15-fold to 30-fold.

207. The method of claim 206, wherein the efficiency of cleaving and / or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

208. The method of claim 207, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immuno staining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), mass spectrometry, or a combination thereof.

209. A method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the added or substituted one or more nucleotides comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).

210. The method of claim 209, wherein the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides.

211. The method of claim 209 or 210, wherein the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

212. The method of any one of claims 209-211, wherein the gRNA is a dgRNA.

213. The method of any one of claims 209-211, wherein the gRNA is a sgRNA.

214. The method of any one of claims 209-213, further comprising: c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2’-O-methyl (2’-0-Me) modification; 2’-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'- O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.

215. The method of claim 214, wherein the at least one chemical modification is in the crRNA, the tracrRNA, or both.

216. The method of claim 215, wherein the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti -repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA.

217. The method of claim 215, wherein the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti -repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti -repeat, and the tail of the tracrRNA.

218. The method of claim 217, wherein the at least one chemical modification is in the first stem of the anti-repeat.

219. The method of claim 218, wherein the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat.

220. The method of claim 218, wherein the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat.

221. The method of claim 218, wherein the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.

222. The method of claim 218, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat.

223. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA.

224. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat.

225. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat.

226. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA.

227. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3’ region of the first stem of the crRNA repeat.

228. The method of any one of claims 214-227, wherein the at least one chemical modification comprises a BNA modification.

229. The method of claim 228, wherein the BNA modification comprises a 2', 4' BNA modification.

230. The method of claim 229, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C -ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

231. The method of claim 230, wherein the 2', 4' BNA is a LNA modification.

232. The method of claim 230, wherein the 2', 4' BNA is a cEt modification.

233. The method of any one of claims 209-232, wherein efficiency of cleaving and / or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or more compared to the RGN system comprising the gRNA provided in step a).

234. The method of claim 233, wherein the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

235. The method of claim 234, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immuno staining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), mass spectrometry, or a combination thereof.

236. The method of any one of claims 209-235, wherein the engineered gRNA further comprises an extension comprising an edit template for prime editing.

237. An engineered gRNA produced by the method of any one of the claims 209-236.

238. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

239. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

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