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

Abstract

Compositions comprising engineered and / or chemically modified trans-activating 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 disclosed herein improve the gene editing (including base editing and prime editing) efficiency of the RNA-guided nuclease (RGN) system.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 in its entirety.

[0002] Reference to a sequence listing submitted electronically as an XML file This application contains a Sequence Listing that has been submitted in xml format via the USPTO Patent Center and is incorporated herein by reference in its entirety. The xml copy, created on August 24, 2023, is named L103438_1290WO_0235_5_SEQUENCE LISTING and is 2.18MB in size.

[0003] The present invention relates to the fields of molecular biology and gene editing. [Background technology]

[0004] Targeted genome editing or modification, along with genome modifications such as nucleic acid excision, deletion, and insertion; nucleotide substitution in nucleic acids; and many other possible modifications, is rapidly becoming an important tool for basic and applied research because it allows for the regulation of gene expression at specific locations within the genome. 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 cleaves nucleic acids, with a guide RNA. Hybridization of the guide RNA to a specific target sequence allows editing at a specific location within the genome. Therefore, genome editing systems using RNA-guided nucleases (RGNs) can be cost-effective and efficient for editing genome sequences, as the guide RNA is a programmable component of the RGN system, typically enabling genome editing of specific target sequences through simple design of the guide RNA. RGN genome editing systems have been adapted from many microorganisms, and these systems are classified into two classes, six types, and multiple subtypes.

[0005] In the microorganisms that are the source of type II and some type V RGN systems, guide RNAs (gRNAs) are expressed as a two-part RNA system consisting of a CRISPR-RNA (crRNA) containing a spacer sequence that recognizes target genomic sequences through Watson-Crick base pairing, and a scaffold (tracrRNA) that transactivates the crRNA. This two-part guide RNA requires base pairing between regions of the crRNA and tracrRNA molecules to form a dual guide RNA (dgRNA). For many applications, chimeric single guide RNA (sgRNA) molecules can be used, formed by physically linking the crRNA and tracrRNA with a short, flexible loop.

[0006] Elements of guide RNAs (e.g., phosphate backbone, ribose sugar, nucleobases) can be chemically modified, for example, to reduce degradation of the guide RNA. Many opportunities exist to delineate the type and / or degree of modification to improve guide RNA RGN systems, for example, to enhance stability, editing efficiency, and specificity for target sequences, and / or to reduce inflammatory responses associated with toxicity of the RGN system. Summary of the Invention

[0007] Provided herein are compositions comprising chemically modified trans-activating 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, the BNA modifications include 2',4' locked nucleic acid modifications of nucleotides 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'-O-Me), 2'-O-methyl 3' phosphorothioate (MS), and phosphorothioate (PS) modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure improve the gene editing efficiency of an RNA-guided nuclease (RGN) system compared to a reference RGN system comprising a tracrRNA, gRNA, and / or crRNA without a BNA modification. In some embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure enable the use of dual guide RNAs in applications where single guide RNAs would otherwise be required. In some embodiments, the present disclosure provides the use of BNA and / or other chemical modifications within the first stem of stem loop 1 of a dual guide RNA to enhance the performance of the RGN system in cells. In some embodiments, the use of BNA and / or other chemical modifications enables the use of shortened tracrRNAs, gRNAs, and / or crRNAs. In some embodiments, cells gene-edited with the RGN system of the present 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 for which the RGN system is applicable.

[0008] Methods are also provided for achieving RGN-based gene editing in cells using BNA-modified guide RNAs and increasing gene editing efficiency.

[0009] In one aspect, the present disclosure provides a nucleic acid molecule comprising a trans-activating 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 bridge nucleic acid (BNA) modification.

[0010] In some embodiments of the tracrRNA aspects, at least one BNA modification is within the anti-repeat. In some embodiments of the tracrRNA aspects, at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the tracrRNA aspects, the at least one BNA modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 BNA modifications on consecutive nucleotides or at least 2, 3, 4, 5, 6, or 7 BNA modifications on alternating nucleotides within the first stem of the anti-repeat. In some embodiments of the tracrRNA aspects, all nucleotides within the first stem of the anti-repeat comprise a BNA modification.

[0011] In some embodiments of the above tracrRNA aspects, at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above tracrRNA aspects, at least one BNA modification is not within the bulge of the tracrRNA. In some embodiments of the above tracrRNA aspects, the three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above tracrRNA aspects, the three terminal nucleotides of the tail of the tracrRNA comprise both BNA and phosphorothioate (PS) modifications.

[0012] In some embodiments of the above tracrRNA aspects, at least one BNA modification comprises a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is a locked nucleic acid (LNA) modification, a BNA NC

[0023] In some embodiments, the 2',4' BNA is an LNA modification. In some embodiments, the 2',4' BNA is a cEt modification.

[0013] In some embodiments of the above tracrRNA aspects, 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.

[0014] In some embodiments of the tracrRNA aspect, the at least one other chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; and a phosphorothioate (PS) modification. In some embodiments of the tracrRNA aspect, the three terminal nucleotides of the tail of the tracrRNA comprise MS modifications. In some embodiments of the tracrRNA aspect, the 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 modification is an LNA modification.

[0015] In some embodiments of the tracrRNA aspect above, the first stem of the anti-repeat comprises a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the tracrRNA aspect above, the first stem of the anti-repeat comprises a length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the tracrRNA aspect above, the first stem of the anti-repeat comprises a length of about 11 nucleotides. In some embodiments of the tracrRNA aspect above, the first stem of the anti-repeat comprises a length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

[0016] In some embodiments of the above tracrRNA aspects, the first stem of the anti-repeat comprises a nucleotide sequence from a natural precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence in its 5' region. In some embodiments of the above tracrRNA aspects, the first stem of the anti-repeat comprises a GC-rich nucleotide sequence in its 5' region, wherein the 5' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

[0017] In some embodiments of the above tracrRNA aspects, 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 greater than 180 nt.

[0018] In some embodiments of the above tracrRNA aspects, 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.

[0019] In some embodiments of the above tracrRNA aspects, 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.

[0020] In some embodiments of the above tracrRNA aspects, 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.

[0021] In some embodiments of the above tracrRNA aspects, 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.

[0022] In some embodiments of the tracrRNA aspects above, the tracrRNA is part of a gRNA that can bind to an RGN. In some embodiments, the RGN is a type II RGN.

[0023] In some embodiments of the above tracrRNA 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 SEQ ID NO:1.

[0024] In some embodiments of the above tracrRNA 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 SEQ ID NO:69.

[0025] In some embodiments of the above tracrRNA 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 SEQ ID NO:93.

[0026] In some embodiments of the above tracrRNA 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 SEQ ID NO:252.

[0027] In another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat comprising i) a spacer; and ii) a first stem and a second stem, and the tracrRNA comprises an anti-repeat comprising i) a tail; and ii) a first stem and a second stem, and at least one of the crRNA and the tracrRNA comprises at least one bridge nucleic acid (BNA) modification.

[0028] In some embodiments of the above gRNA aspects, 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 greater than 200 nt. In some embodiments of the above gRNA aspects, the gRNA is a dual guide RNA (dgRNA).

[0029] In some embodiments of the gRNA aspects, at least one BNA modification is within a crRNA repeat. In some embodiments of the gRNA aspects, at least one BNA modification is within the first stem of the crRNA repeat. In some embodiments of the gRNA aspects, 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 gRNA aspects, the three terminal nucleotides of the 3' region of the first stem of the crRNA repeat comprise a BNA modification. In some embodiments of the gRNA aspects, the three terminal nucleotides of the 3' region of the first stem of the crRNA repeat comprise a BNA modification and a phosphorothioate (PS) modification. In some embodiments of the gRNA aspects, at least one BNA modification is not within the second stem of the crRNA repeat.

[0030] In some embodiments of the above gRNA aspects, at least one BNA modification is within the anti-repeat. In some embodiments of the above gRNA aspects, at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above gRNA aspects, the at least one BNA modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 BNA modifications on consecutive nucleotides or at least 2, 3, 4, 5, 6, or 7 BNA modifications on alternating nucleotides within the first stem of the anti-repeat. In some embodiments of the above gRNA aspects, every nucleotide in the first stem of the anti-repeat comprises a BNA modification.

[0031] In some embodiments of the above gRNA aspects, at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above gRNA aspects, at least one BNA modification is not within the bulge of the gRNA. In some embodiments of the above gRNA aspects, at least one BNA modification is within the tail of the tracrRNA. In some embodiments of the above gRNA aspects, the three terminal nucleotides of the 3' region of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above gRNA aspects, the three terminal nucleotides of the 3' region of the tail of the tracrRNA comprise both BNA and phosphorothioate (PS) modifications.

[0032] In some embodiments of the above gRNA aspects, at least the 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. In some embodiments of the above gRNA aspects, all nucleotides in the first stem of the crRNA repeat lack a chemical modification and all nucleotides in the first stem of the anti-repeat comprise a BNA modification.

[0033] In some embodiments of the above gRNA aspects, at least one BNA modification is in the spacer. In some embodiments of the above gRNA aspects, the three terminal nucleotides in the 5' region of the spacer comprise BNA modifications. In some embodiments of the above gRNA aspects, the three terminal nucleotides in the 5' region of the spacer comprise both BNA and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspects, the spacer is 18-30 nucleotides in length.

[0034] In some embodiments of the above gRNA aspects, at least one BNA modification comprises a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is a locked nucleic acid (LNA) modification, a BNA NC

[0023] In some embodiments, the 2',4' BNA is an LNA modification. In some embodiments, the 2',4' BNA is a cEt modification.

[0035] In some embodiments of the above gRNA aspects, the gRNA further comprises at least one other modification. In some embodiments of the above gRNA aspects, the at least one other modification is in the crRNA. In some embodiments of the above gRNA aspects, the at least one other modification is in the 5' or 3' region of the crRNA. In some embodiments of the above gRNA aspects, the at least one other modification is in the 5' and 3' regions of the crRNA.

[0036] In some embodiments of the gRNA aspects, the at least one other chemical modification is within a crRNA repeat of the crRNA. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the first stem of the crRNA repeat. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the spacer of the crRNA. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the tracrRNA. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments of the gRNA aspects, the at least one other chemical modification is within the tail of the tracrRNA.

[0037] In some embodiments of the above gRNA aspects, the at least one other chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; and a phosphorothioate (PS) modification. In some embodiments of the above gRNA aspects, the three terminal nucleotides of both the 5' and 3' regions of the crRNA comprise MS modifications. In some embodiments of the above gRNA aspects, the three terminal nucleotides of both the 5' and 3' regions of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-O-Me modifications.

[0038] In some embodiments of the above gRNA aspects, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises an overall length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspects, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises an overall length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspects, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises an overall length of about 11 nucleotides. In some embodiments of the above gRNA aspects, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises an overall length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

[0039] In some embodiments of the above gRNA aspects, the first stem of the 3' crRNA repeat or the first stem of the 5' anti-repeat comprises a nucleotide sequence derived from a natural precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above gRNA aspects, the first stem of the 3' crRNA repeat or the first stem of the 5' anti-repeat comprises a GC-rich nucleotide sequence, and the first stem of the 3' crRNA repeat or the first stem of the 5' anti-repeat comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

[0040] In some embodiments of the above gRNA aspects, the three terminal nucleotides of both the 5' and 3' regions of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

[0041] In some embodiments of the above gRNA aspects, the crRNA repeat has a nucleotide sequence that is (a) set forth as SEQ ID NO: 39 or differs by one or two nucleotides from SEQ ID NO: 39, (b) set forth as SEQ ID NO: 384 or differs by one or two nucleotides from SEQ ID NO: 384, (c) set forth as SEQ ID NO: 385 or differs by one or two nucleotides from SEQ ID NO: 385, (d) set forth as SEQ ID NO: 386 or differs by one or two nucleotides from SEQ ID NO: 386, (e) set forth as SEQ ID NO: 387 or differs by one or two nucleotides from SEQ ID NO: 387, or (f) set forth as SEQ ID NO: 397 or differs by one or two nucleotides from SEQ ID NO: 397. In some embodiments of the above aspects, the crRNA has a nucleotide sequence that has 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 aspects, 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.

[0042] In some embodiments of the above gRNA aspects, the crRNA repeat is (a) set forth as SEQ ID NO:300 or differs by one or two nucleotides from SEQ ID NO:300; (b) set forth as SEQ ID NO:304 or differs by one or two nucleotides from SEQ ID NO:304; (c) set forth as SEQ ID NO:308 or differs by one or two nucleotides from SEQ ID NO:308; (d) set forth as SEQ ID NO:312 or differs by one or two nucleotides from SEQ ID NO:312; (e) set forth as SEQ ID NO:320 or differs by one or two nucleotides from SEQ ID NO:320; (f) set forth as SEQ ID NO:344 or differs by one or two nucleotides from SEQ ID NO:344; or (g) set forth as SEQ ID NO:348. (i) is set forth as SEQ ID NO: 356 or differs by one or two nucleotides from SEQ ID NO: 356; (j) is set forth as SEQ ID NO: 360 or differs by one or two nucleotides from SEQ ID NO: 360; (k) is set forth as SEQ ID NO: 388 or differs by one or two nucleotides from SEQ ID NO: 388; (l) is set forth as SEQ ID NO: 389 or differs by one or two nucleotides from SEQ ID NO: 389; or (m) is set forth as SEQ ID NO: 390 or differs by one or two nucleotides from SEQ ID NO: 390. In some embodiments of the above gRNA aspects, the crRNA has a nucleotide sequence with 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 aspects, the tracrRNA has a nucleotide sequence with 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.

[0043] In some embodiments of the above gRNA aspects, the crRNA repeat has a nucleotide sequence: (a) set forth as SEQ ID NO:324 or differing by one or two nucleotides from SEQ ID NO:324; (b) set forth as SEQ ID NO:328 or differing by one or two nucleotides from SEQ ID NO:328; (c) set forth as SEQ ID NO:332 or differing by one or two nucleotides from SEQ ID NO:332; (d) set forth as SEQ ID NO:336 or differing by one or two nucleotides from SEQ ID NO:336; (e) set forth as SEQ ID NO:391 or differing by one or two nucleotides from SEQ ID NO:391; (f) set forth as SEQ ID NO:392 or differing by one or two nucleotides from SEQ ID NO:392; or (g) set forth as SEQ ID NO:393 or differing by one or two nucleotides from SEQ ID NO:393. In some embodiments of the above gRNA aspects, 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 aspects, 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.

[0044] In some embodiments of the above gRNA aspects, the crRNA repeat has a nucleotide sequence: (a) set forth as SEQ ID NO:465 or differing by one or two nucleotides from SEQ ID NO:465; (b) set forth as SEQ ID NO:469 or differing by one or two nucleotides from SEQ ID NO:469; (c) set forth as SEQ ID NO:473 or differing by one or two nucleotides from SEQ ID NO:473; (d) set forth as SEQ ID NO:477 or differing by one or two nucleotides from SEQ ID NO:477; (e) set forth as SEQ ID NO:481 or differing by one or two nucleotides from SEQ ID NO:481; (f) set forth as SEQ ID NO:508 or differing by one or two nucleotides from SEQ ID NO:508; (g) set forth as SEQ ID NO:512 or differing by one or two nucleotides from SEQ ID NO:512; and (h) set forth as SEQ ID NO:516 or differing by one or two nucleotides from SEQ ID NO:516. In some embodiments of the above gRNA aspects, 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 aspects, 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.

[0045] In some embodiments of the gRNA aspect above, the crRNA and tracrRNA are linked by a linker between the 3'-terminal nucleotide of the crRNA repeat and the 5'-terminal nucleotide of the anti-repeat. In some embodiments, the linker comprises an azide or alkyne functional group. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker has a nucleotide sequence designated as AAAG, GAAA, ACUU, or CAAAGG. In some embodiments, the linker has a nucleotide sequence designated as AAAG.

[0046] In some embodiments of the above gRNA aspects, the gRNA is an sgRNA comprising a crRNA and a tracrRNA, wherein the sgRNA comprises a backbone and a spacer, and the sgRNA backbone comprises crRNA repeats, a linker, and a tracrRNA. In some embodiments of the above gRNA aspects, the sgRNA backbone 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.

[0047] In some embodiments of the gRNA aspects above, the 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.

[0048] In some embodiments of the gRNA aspects above, the gRNA is capable of binding to an RGN. In some embodiments, the RGN is a type II RGN.

[0049] In some embodiments of the above gRNA 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 SEQ ID NO:1.

[0050] In some embodiments of the above gRNA aspects, 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.

[0051] In some embodiments of the above gRNA 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 SEQ ID NO:93.

[0052] In some embodiments of the above gRNA 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 SEQ ID NO:252.

[0053] In some embodiments of the above gRNA aspects, the gRNA further comprises an extension that comprises an editing template for prime editing.

[0054] 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, which can hybridize 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, the at least one chemical modification being a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; 2'-Fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-Di-Cα-OMe modification; 2'-O-Methyl 3' Phosphorothioate (MS) modification; 2'-O-Methyl 3' Thiophosphonoacetate (MSP) modification; 2'-O-Methyl 3' Phosphorothioate (MP) modification; Phosphorothioate (PS) modification; and BNA modification, and at least one chemical modification is within the three terminal nucleotides of the 5' or 3' region of the crRNA.

[0055] In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; (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: a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA modification, and wherein the at least one chemical modification is within the three terminal nucleotides of the 5' or 3' region of the crRNA. In some embodiments, the gRNA containing the crRNA can bind to an RNA-guided nuclease (RGN) that requires the tracrRNA for activity.

[0056] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, the RGN system comprising: a) the trans-activating crRNA (tracrRNA) described above; b) a crRNA; and c) a type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding a type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds to a target sequence in a target nucleic acid molecule.

[0057] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, the RGN system comprising: a) the gRNA described above; and b) a type II RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding a type II RGN polypeptide. In some embodiments, the RGN system binds to a target sequence in a target nucleic acid molecule.

[0058] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) a CRISPR RNA (crRNA) as described above; b) a tracrRNA; and c) a type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding a type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds to a target sequence in a target nucleic acid molecule.

[0059] In some embodiments of the RGN system aspects described above, the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence designated as NNNNCC, NNGRR, NNRYA, or NGG. In some embodiments of the RGN system aspects described above, the gRNA is an sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or greater than 200 nt. In some embodiments of the RGN system aspects described above, 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.

[0060] In some embodiments of the above RGN system aspects, the RGN polypeptide and gRNA are not naturally complexed to each other.

[0061] In some embodiments of the above RGN system aspects, the target sequence is a eukaryotic target sequence. In some embodiments, the target sequence has a nucleotide sequence set forth as any of SEQ ID NOs: 273-278 and 712. In some embodiments of the above RGN system aspects, the target sequence is intracellular.

[0062] In some embodiments of the above RGN system aspects, the complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence. In some embodiments, the cleavage results in a double-stranded break. In some embodiments, the cleavage results in a single-stranded break.

[0063] In some embodiments of the above RGN system aspects, the RGN polypeptide is nuclease inactive. In some embodiments of the above RGN system aspects, the RGN polypeptide is a nickase.

[0064] In some embodiments of the above RGN system aspects, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

[0065] In some embodiments of the above RGN system 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 system aspects, the gRNA further comprises an extension that comprises an editing template for prime editing.

[0066] In some embodiments of the above RGN system aspects, the RGN polypeptide is fused to a detectable label. In some embodiments of the above RGN system aspects, the RGN system further comprises a donor polynucleotide.

[0067] In some embodiments of the above RGN system aspects, the polynucleotide comprising a nucleotide sequence encoding RGN is 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 in a vector.

[0068] In another aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising an RGN system as described herein above.

[0069] In yet 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 herein above.

[0070] In some embodiments of the above aspects, the cell comprises a target sequence that can be bound by a formed gRNA / RGN polypeptide complex of the RGN system or by an RNP complex, as described herein above. In some embodiments, the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278 and 712.

[0071] 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.

[0072] In another aspect, the present disclosure provides a plant comprising the plant cell described above.

[0073] In another aspect, the present disclosure provides a seed comprising the plant cell described above.

[0074] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tracrRNA, gRNA, crRNA, RGN system, RNP complex, or cell as described herein above.

[0075] In another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule, the method comprising delivering an RGN system or an RNP complex as described herein above to the target sequence or a cell containing the target sequence.

[0076] In some embodiments of the above aspects, the RGN polypeptide or gRNA further comprises a detectable label, thereby enabling detection of the target sequence. In some embodiments of the above aspects, the RGN polypeptide or gRNA further comprises an expression modulator, thereby regulating expression of a target gene comprising the target sequence. In some embodiments of the above aspects, the RGN polypeptide is fused to a prime editing polypeptide. In some embodiments of the above aspects, the RGN polypeptide is fused to a base-editing polypeptide.

[0077] In yet another aspect, the present disclosure provides a method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, comprising delivering an RGN system or an RNP complex as described herein above to the target sequence or a cell comprising the target sequence, whereby cleavage or modification of the target nucleic acid molecule occurs.

[0078] In yet another aspect, the present disclosure provides a method for binding a target sequence within a target nucleic acid molecule with an RNA-guided nuclease (RGN), comprising: a) combining, under conditions suitable for the formation of a ribonucleoprotein (RNP) complex, i) a guide RNA (gRNA) comprising a trans-activating crRNA (tracrRNA) and a CRISPR RNA (crRNA) as described herein; 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 assembling 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 editing template for prime editing. In some embodiments of the method aspects, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

[0079] In yet another aspect, the disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), comprising contacting the target nucleic acid molecule or a cell containing the target nucleic acid molecule with: i) a guide RNA (gRNA) and a CRISPR RNA (crRNA) comprising a trans-activating crRNA (tracrRNA) as described herein above; and ii) a type II RGN or a polynucleotide encoding a type II RGN, thereby binding the target sequence to the RGN. In some embodiments of the method aspect, the formed complex between 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 editing 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 type II RGN is mRNA.

[0080] 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), comprising: a) combining, under conditions suitable for the formation of a ribonucleoprotein (RNP) complex, i) a guide RNA (gRNA) as described herein above; and ii) a type II RNA-guided nuclease (RGN), thereby assembling the RNP complex; and b) contacting the target nucleic acid molecule or a cell containing the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence to 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 that includes an editing template for prime editing.

[0081] In yet another aspect, the disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), comprising contacting the target nucleic acid molecule or a cell containing the target nucleic acid molecule with i) a guide RNA (gRNA) as described herein above; and ii) a type II RGN or a polynucleotide encoding a type II RGN, thereby binding the target sequence to the RGN. In some embodiments of the method aspect, the formed complex between 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 editing template for prime editing. In some embodiments of the method aspect, the polynucleotide encoding the type II RGN is mRNA.

[0082] 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), comprising: a) combining, under conditions suitable for the formation of a ribonucleoprotein (RNP) complex, i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described herein above; 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 assembling 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), 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 herein above and a tracrRNA; and ii) a type II RGN, or a polynucleotide encoding a type II RGN, thereby assembling the target sequence with the RGN.

[0083] In some embodiments of the above method aspects, the target sequence comprises a 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 for increasing the efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, comprising delivering an RGN system or RNP complex as described herein above to the target sequence or a cell comprising the target sequence, wherein the cleavage or modification of the nucleic acid molecule occurs with a higher efficiency compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering a reference RGN system or RNP complex to the target sequence or a cell comprising the target sequence, wherein the tracrRNA, gRNA, or 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 aspects, all nucleotides of the first stem of the anti-repeat of the tracrRNA or RNP complex of the RGN system as described herein above comprise BNA modifications. In some embodiments of the above aspects, at least the three terminal nucleotides of the 3' region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications. In some embodiments of the above aspects, the BNA modifications comprise LNA modifications. In some embodiments of the above aspects, the BNA modifications comprise cEt modifications. In some embodiments of the above aspects, the efficiency of cleaving and / or modifying the target sequence is increased by 15- to 30-fold. In some embodiments of the above aspects, the efficiency of cleaving and / or modifying the target sequence is determined by measuring the percentage of cells containing the target sequence that have altered expression of the target sequence or the polypeptide encoded by the target sequence. In some embodiments of the above aspects, 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 disclosure provides a method for engineering a gRNA, 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, and the 3' region of the crRNA repeat and the 5' region of the anti-repeat of the engineered gRNA comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has increased editing efficiency compared to the gRNA provided in step a).

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

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

[0089] In some embodiments of the above aspects, 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 a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA modification.

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

[0091] In some embodiments of the above aspects, 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 aspects, the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspects, the at least one chemical modification is on every nucleotide in the first stem of the anti-repeat. In some embodiments of the above aspects, the at least one chemical modification is on alternating nucleotides in the first stem of the anti-repeat.

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

[0093] In some embodiments of the above aspects, 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 a locked nucleic acid (LNA) modification, a BNANC[N-Me] modification, a 2'-O,4'-C-ethylene-bridged nucleic acid (2',4'-ENA) modification, and an 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.

[0094] In some embodiments of the above aspects, the editing efficiency of the engineered gRNA is increased by 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 aspects, the efficiency of cleavage and / or modification of the target sequence by the RGN system comprising the engineered gRNA is increased by 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 aspects, the efficiency is determined by measuring the percentage of cells comprising the target sequence or target sequence that have altered expression of the target sequence or polypeptide encoded by the target sequence. In some embodiments of the above aspects, 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 above method.

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

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

[0098] [Figure 1] Figure 1 provides a schematic diagram of dual guide RNA (dgRNA) showing pairing of crRNA and tracrRNA. [Figure 2] Figures 2A and 2B show schematic diagrams illustrating portions of a single-guide RNA (sgRNA) (Figure 2A) and a dgRNA (Figure 2B). The portions include a spacer; stem-loop 1, which includes the first stem, first bubble, and second stem; stem-loop 2, which includes only the first stem; an interstem-loop region (ISR); a loop; stem-loop 3, which includes the first stem, first bubble, second stem, second bubble, and third stem; and a tail. Stem-loop 1 in the sgRNA contains a loop, while stem-loop 1 in the dgRNA does not. The CRISPR RNA (crRNA) repeat anneals to the anti-repeat of the trans-activating CRISPR RNA (tracrRNA) to form stem-loop 1. [Figure 3]Figures 3A and 3B provide a depiction of the chemical modifications of the dgRNA of the APG07433.1 RNA-guided nuclease. Figure 3A shows the tracrRNA modification scheme from left to right: "Stem MS Modification," where the first stem of stem-loop 1 contains a nucleotide with a 2'-O-methyl (2'-O-Me) modification, and the three terminal nucleotides of the 5' region have 2'-O-methyl 3' phosphorothioate (MS) modifications, and the three terminal nucleotides of the 3' region have MS modifications, and the fourth nucleotide from the 3' end of the tracrRNA molecule has a 2'-O-Me modification; "Heavy MS Modification" - "Stem MS Modification" "Stem LNA modification" - the first stem of stem-loop 1 contains all LNA modifications, and the three terminal nucleotides of the 3' region have MS modifications, and the fourth nucleotide from the 3' end has a 2'-O-Me modification; "heavy LNA modification" - in addition to the modifications in "stem LNA modification", 2'-O-Me modifications are added throughout most of stem-loop 3, and portions of the first and second stems of stem-loop 3 are LNA modified. Figure 3B shows a diagram of the modifications of the crRNA used in these experiments, both containing 5' and 3' MS modifications, with the version shown on the left also having a 2'-O-Me modification on the first stem. [Figure 4] Figure 4 shows the efficiency of gene editing in primary human T cells using different combinations of modified crRNA and modified tracrRNA, as measured by knockout of the CD3 surface marker using flow cytometry. Guide RNAs were designed to target the TRAC gene. These data demonstrate that modifications in stem-loop 3 abolish editing activity, while modifications in stem-loop 1 alone preserve the editing ability of the dgRNA. These modifications are shown in the same scheme as in Figures 3A and 3B. A schematic representation of the tracrRNA is shown. The control sgRNA has MS modifications in the three terminal nucleotides of both the 5' and 3' regions of the sgRNA. Mock represents the condition in which cells are mixed with nucleofection solution and subjected to the nucleofection process without RGN or gRNA. [Figure 5] Figure 5 demonstrates that LNA modifications enhance the editing efficiency of dgRNA in RNP complexes with purified APG07433.1 protein, achieving 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 have MS modifications in the three terminal nucleotides of both the 5' and 3' regions of the guide RNA. [Figure 6] Figure 6 shows that LNA-modified dgRNAs promote a higher rate of gene disruption using two exemplary spacer mRNA delivery methods, particularly compared to terminally modified dgRNAs (MS modifications in the three terminal nucleotides of both the 5' and 3' regions of the dgRNA) or dgRNAs with an additional 2-O'-Me modification in stem-loop 1 (MS / PS modification). 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 have MS modifications in the three terminal nucleotides of both the 5' and 3' regions of the guide RNA. [Figure 7] Figure 7 demonstrates the higher efficacy of LNA-modified dgRNAs in gene editing compared to sgRNAs 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 in the three terminal nucleotides of both the 5' and 3' regions of the sgRNA. [Figure 8]Figures 8A and 8B show that LNA modification enhances the editing efficiency of sgRNAs for two different RNA-guided nucleases (RGNs). RGNs are delivered either as a protein complexed with the guide RNA (RNP delivery) or as mRNA encoding RGN (mRNA delivery). (Figure 8A) APG07433.1 sgRNA. "Control_RNP" and "Control_mRNA" indicate conditions without RGN or gRNA for each delivery method; cells are mixed with nucleofection solution but do not undergo the nucleofection process. (Figure 8B) APG01604 sgRNA. "Control_TRAC" and "Control_B2M" indicate conditions without RGN or gRNA; cells are mixed with nucleofection solution but do not undergo the nucleofection process. The two different spacer names in the controls indicate that no editing was detected using antibodies against TRAC (2275) or B2M (1989) in flow cytometry. Two exemplary spacers were used for each RGN sgRNA: (-): Control sgRNA with MS modifications in the three terminal nucleotides of both the 5' and 3' regions of the sgRNA; (+): sgRNA with additional LNA modifications as indicated in each schematic diagram. The schematic diagrams for each guide RNA show the LNA modification in the anti-repeat region forming the first stem and the MS modifications in the three terminal nucleotides of the 5' and 3' regions of the sgRNA. Gene editing efficiency in primary human T cells was measured by knockout of the CD3 surface marker for editing of TRAC target sequences or immunostaining of B2M for editing of B2M target sequences using flow cytometry. [Figure 9]Figures 9A and 9B show that MS modifications of stem-loop 1 do not enhance editing efficiency. Figure 9A shows eight scenarios of 2'-O-Me and / or MS modifications in stem-loop 1 and / or stem-loop 3 for the APG07433.1 sgRNA. Figure 9B shows that none of the test sgRNAs with 2'-O-Me and / or MS modifications enhanced sgRNA editing compared to the control sgRNA. Chemical modifications at stem-loop 3 abolished sgRNA activity. RGN was delivered as a protein complexed with a guide RNA (RNP delivery) or as an mRNA encoding RGN (mRNA delivery). "Control" indicates a condition without RGN or gRNA for each delivery method, in which cells were mixed with nucleofection solution but not passed through the nucleofection step. The control sgRNA had MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the sgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. [Figure 10]Figures 10A-10C show that the amount of LNA modification correlates with guide RNA editing efficiency in primary human T cells, as measured by flow cytometry knockout of the CD3 surface marker (for the TRAC target sequence) or immunostaining of B2M (for the B2M target sequence). (Figure 10A) Gene editing efficiency of APG07433.1 dgRNAs with 1, 3, 6, or 11 LNA-modified nucleotides within the anti-repeat region forming the first stem of stem-loop 1. A schematic diagram of the APG07433.1 dgRNA shows 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-modified" condition, which includes various numbers of LNA-modified nucleotides in addition to the "0 LNA" MS modifications. The highest editing was achieved when all nucleotides within the anti-repeat region forming the first stem of stem-loop 1 were LNA-modified. (Figure 10B) Gene editing efficiency of APG01604 dgRNA with 3 or 7 LNA-modified nucleotides in the region of the anti-repeat that forms the first stem of stem-loop 1. Schematic diagrams of 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-modified" condition, which includes various numbers of LNA-modified nucleotides in addition to the "0 LNA" MS modifications. (Figure 10C) Gene editing efficiency of APG05586 dgRNA with 4 or 9 LNA-modified nucleotides in the region of the anti-repeat that forms the first stem of stem-loop 1. Schematic diagrams of 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-modified" condition, which includes various numbers of LNA-modified nucleotides in addition to the "0 LNA" MS modifications. Gene editing was improved for the LNA-modified APG01604 dgRNA and APG05586 dgRNA compared to the "0 LNA" dgRNA. Each RGN dgRNA used two exemplary spacers. Each RGN was delivered either as a protein complexed with a guide RNA (RNP delivery) or as an mRNA encoding the RGN (mRNA delivery)."Control_TRAC" and "Control_B2M" indicate conditions without RGN and gRNA, in which cells are mixed with nucleofection solution but do not go through the nucleofection process, for two different spacers in the gRNA. [Figure 11]Figures 11A-11C show that LNA modifications maintain or increase the gene editing efficiency of truncated APG07433.1 sgRNAs. (Figure 11A) Top: The full-length APG07433.1 sgRNA was truncated by a combination of truncations in various regions of the sgRNA: a 5 nucleotide (nt) pair (10 nt) deleted from the first stem and 6 nt deleted from the tail of stem-loop 1 (-10 1st stem SL1, -6 tail); a 5 nt pair (10 nt) deleted from the first stem and 4 nt deleted from the tail of stem-loop 1, and a 1 nt pair (2 nt) deleted from the first stem of stem-loop 3 (-10 1st stem SL1, -4 tail, -2 1st stem SL3); and a 5 nucleotide (nt) pair (10 nt) deleted from the first stem of stem-loop 1 and 6 nt deleted from the tail, and a 1 nt pair (2 nt) deleted from the first stem of stem-loop 3 (-10 1st stem SL1, -6 tail, -2 1st stem SL3). These truncated APG07433.1 sgRNAs have MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the sgRNA and serve as controls to evaluate additional chemical modifications introduced into the first stem of stem-loop 1. Bottom: The truncated APG07433.1 sgRNAs shown above contain LNA and MS modifications in the first stem of stem-loop 1. (Figure 11B) Gene editing efficiency of the chemically modified truncated APG07433.1 sgRNAs shown in Figure 11A compared to the control full-length APG07433.1 sgRNA or the control truncated APG07433.1 sgRNA. The control full-length and truncated sgRNAs each have MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the sgRNA, but do not contain chemical modifications in stem-loop 1. "Mock" indicates a condition in which cells were mixed with nucleofection solution and subjected to nucleofection without RGN or gRNA for each delivery method. sgRNA was used at a dilution factor of 1. (Figure 11C) LNA modification increases the editing potency of the truncated APG07433.1 sgRNA. Serial dilutions of sgRNA were performed. Data were collected on day 4. The "3MS" full-length sgRNA and "3MS" truncated sgRNA are shown in Figure 11A (top).The modified truncated sgRNA is shown in Figure 11A (bottom). DF = dilution factor. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. RGN was delivered either as a protein complexed with a guide RNA (RNP delivery) or as mRNA encoding RGN (mRNA delivery). [Figure 12]Figures 12A and 12B show that LNA modifications maintain or increase the gene editing efficiency of truncated APG07433.1 dgRNAs via RNP delivery. The "M" truncation and chemical modification scheme works best for sgRNAs and dgRNAs. (Figure 12A) Chemical modification and truncation scheme for crRNA and tracrRNA. The crRNAs are truncated by the 5 terminal nt in the 3' region: left, MS modifications at the three terminal nucleotides of the 5' and 3' regions (O and Q represent two exemplary spacers used); right, MS modifications + 2'-O-Me modifications at the three terminal nucleotides of the 5' and 3' regions within the crRNA repeat that forms 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 the 5 terminal nt in the 5' region, all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modifications, the tail is shortened by 6 nt, and the three terminal nucleotides of the 3' region contain MS modifications; "tracr(M)", the anti-repeat forming the first stem of stem-loop 1 is shortened by the 5 terminal nt in the 5' region, and all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modifications. "tracr(N)", the anti-repeat forming the first stem of stem-loop 1 is shortened by 5 terminal nt in the 5' region and all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modifications; "tracr(N)", the tail is shortened by 6 nt, a 1 nt pair (2 nt) is deleted from the first stem of stem-loop 3 and the three terminal nucleotides in the 3' region contain MS modifications.(Figure 12B) Gene editing efficiency of a truncated APG07433.1 dgRNA chemically modified as shown in Figure 12A and a truncated APG07433.1 sgRNA chemically modified as shown in Figure 11A: gRNAs with untruncated backbones: 1, full-length sgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions but with an 1880 spacer with no chemical modifications elsewhere in the sgRNA; 2, dgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA but with an 1880 spacer with no chemical modifications elsewhere in the dgRNA; 3, dgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA but with an 1881 spacer with no chemical modifications elsewhere in the dgRNA; 4, crRNA cr(3) and LNA-modified tracrRNA dgRNA containing tracr(4) (see Figures 3A and 3B), with an 1880 spacer; and 5, dgRNA containing crRNA cr(3) and LNA-modified tracrRNA sgRNAs with shortened backbones (see Figure 11A) comprising tracr(4) (see Figures 3A and 3B) and having an 1881 spacer: 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 an additional chemical modification in the first stem of stem-loop 1; 10, shortened (M) sgRNA with an additional chemical modification in the first stem of stem-loop 1; and 11, shortened (N) sgRNA with an additional chemical modification 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 Figure 12A. "Control" indicates, for each delivery method, a condition in which cells are mixed with nucleofection solution but do not go through the nucleofection step, without RGN or gRNA."Mock" indicates a condition in which cells were mixed with nucleofection solution and subjected to nucleofection without RGN or gRNA for each delivery method. Two exemplary spacers were used for sgRNA and dgRNA. Gene editing efficiency in primary human T cells was measured by evaluating the knockout of CD3 surface marker using flow cytometry. RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding RGN (mRNA delivery). [Figure 13] Figure 13 shows a design for testing the gene editing efficiency of shortened ("M" backbone, see Figure 11A) chemically modified APG07433.1 gRNAs in sgRNA format. Top: The shortened "M" APG07433.1 sgRNA is modified with MS, LNA, or LNA+PS modifications at the three terminal nucleotides of the 5' and 3' regions of the sgRNA (3 MS, 3 LNA, 3 LNA / PS; 3 conditions) and has no additional chemical modifications in 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 of the 5' and 3' regions of the sgRNA (3 MS, 3 LNA, 3 LNA / PS; 3 conditions) and contains MS and / or LNA modifications in the first stem of stem-loop 1. [Figure 14] Figure 14 shows the gene editing efficiency of truncated "M" APG07433.1 sgRNAs chemically modified as shown in Figure 13 compared to a control truncated "M" APG07433.1 sgRNA without any chemical modifications. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. RGN was delivered as a protein complexed with a guide RNA (RNP delivery) or as an mRNA encoding RGN (mRNA delivery). "MS / LNA," "LNA," and "LNA" under the "Modification" bar indicate additional MS and / or LNA chemical modifications in the first stem of stem-loop 1 in truncated "M" APG07433.1 sgRNAs with 3 MS, 3 LNA, or 3 LNA / PS modifications, respectively. [Figure 15]Figure 15 shows the gene editing efficiency of chemically modified truncated "M" APG07433.1 sgRNAs as shown in Figure 13 compared to a control truncated "M" APG07433.1 sgRNA without any chemical modifications. Serial dilutions of the sgRNAs were performed. Data were collected on day 4. The "3MS," "3LNA," and "3LNA PS" truncated "M" APG07433.1 sgRNAs are shown in Figure 13. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. RGN was delivered as a protein complexed with a guide RNA (RNP delivery) or as mRNA encoding RGN (mRNA delivery). "MS / LNA," "LNA," and "LNA" under the "Modification" bar indicate additional MS and / or LNA chemical modifications at the first stem of stem-loop 1 in the truncated "M" APG07433.1 sgRNA, which has 3 MS, 3 LNA, or 3 LNA / PS modifications, respectively. [Figure 16]Figures 16A and 16B show the gene editing efficiency of dgRNAs with various chemical modifications at the three terminal nucleotides of the 5' and 3' regions. (Figure 16A, left) Design for testing the gene editing efficiency of wild-type (WT, full-length) chemically modified APG07433.1 gRNA in the dgRNA format. WT APG07433.1 gRNA is shown with possible chemical modifications: MS, LNA, or LNA+PS modifications at the three terminal nucleotides of the 5' and 3' regions 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 of the 3' region of the tracrRNA. (Figure 16A, right) Table showing the total 18 conditions tested, considering various combinations of 3MS, 3LNA, or 3LNA+PS in the crRNA, 3MS, 3LNA, or 3LNA+PS in the tracrRNA, and two delivery modes of RGN (RNP and mRNA). (Figure 16B) Gene editing efficiency of APG07433.1 dgRNAs with various combinations of chemical modifications shown in Figure 16A. All tested dgRNAs have LNA modifications at every nucleotide in the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used. "Control_TRAC" indicates the condition without RGN and dgRNA, in which cells are mixed with nucleofection solution but do not undergo the nucleofection step, for each delivery method. Control dgRNAs ("dg1880" and "dg1881") have MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA, but no chemical modifications elsewhere in the dgRNA. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered either as a protein complexed with a guide RNA (RNP delivery) or as mRNA encoding RGN (mRNA delivery). [Figure 17]Figures 17A and 17B show strategies for rescuing gene editing in the RGN system with dgRNAs of fewer than 11 nucleotide pairs in the first stem of stem-loop 1. Figure 17A shows a strategy involving extending the first stem at the distal end of the first bubble of stem-loop 1 of the APG05586 dgRNA (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA) by two nucleotide pairs using the native sequence of the APG05586 pre-crRNA and LNA modification of all nucleotides in the extended first stem of the anti-repeat. Figure 17B shows a strategy involving extending the first stem at the distal end of the first bubble of stem-loop 1 of the APG05586 dgRNA or the APG08167 dgRNA (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA) by two nucleotide pairs using the native sequence of the respective pre-crRNA. Figure 17B highlights the G:C-rich characteristics of the APG07433.1 nucleotide pair most distal to the first bubble in the first stem of stem-loop 1 (i.e., the nucleotide closest to the 3' region of the crRNA and the 5' region of the tracrRNA). WT APG07433.1 dgRNA, which has LNA modifications at all 11 nucleotides in the first stem of the anti-repeat, achieves the highest gene editing (see Figure 10A). Therefore, the nucleotide sequence from APG07433.1 most distal to the first bubble of stem-loop 1 is used to extend APG05586 and APG08167 as an alternative approach. [Figure 18]Figure 18 shows that gene editing is rescued for an RGN system with a WT (original) dgRNA with <11 nucleotide pairs in the first stem of stem-loop 1 by extending the first stem of stem-loop 1 distal to the first bubble of stem-loop 1 (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA) and modifying all nucleotides in the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiment, and there were two repeats per target gene. "Control" refers to a condition without RGN and dgRNA, in which cells are mixed with nucleofection solution but do not go through the nucleofection step. "Unmodified" refers to a dgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA (3MS), but no chemical modifications elsewhere in the dgRNA. "LNA" indicates a dgRNA with 3MS+LNA modifications at all nucleotides of the first stem of the anti-repeat. "Native sequence" indicates a dgRNA with the first stem of stem-loop 1 extended to the indicated nucleotide length using the native sequence from the respective pre-crRNA. "APG07433.1 seq" indicates a dgRNA with the first stem of stem-loop 1 extended to the indicated nucleotide length using the sequence from the APG07433.1 gRNA. (The nucleotide lengths shown are for the first stem of the anti-repeat, and the same nucleotide length is expected on the first stem of the crRNA repeat due to base pairing.) All nucleotides of the extended first stem of the anti-repeat are modified with LNA, and the first stem is extended at the end distal to the first bubble of stem-loop 1 (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA). Below the graph is a schematic of the APG01604 gRNA truncated in the first stem of stem-loop 1 (APG01604.1, 81 nt backbone length), showing the "unmodified" APG01604.1 gRNA.The nucleotide sequences above the data points indicate the sequence of the 5'-terminal four nucleotides of the original, unextended tracrRNA (for "unmodified" and "LNA") or the two terminal nucleotides added (the 5'-terminal two nucleotides of the extended tracrRNA). Flow cytometry was used to measure gene editing efficiency in primary human T cells by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). RGN was delivered as mRNA encoding RGN (mRNA delivery). [Figure 19]Figure 19 shows that extending the first stem distal to the first bubble of stem-loop 1 (i.e., the extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA) using nucleotide sequences from either the native pre-crRNA or the APG07433.1 gRNA and modifying all nucleotides of the first stem of the anti-repeat with LNAs rescues gene editing in the RGN system with a WT (original) dgRNA in the first stem of stem-loop 1 of <11 nucleotide pairs. The dgRNAs were extended to 11 or 13 nucleotide pairs in the first stem of stem-loop 1. "Native" indicates a dgRNA with the first stem of the anti-repeat extended to the indicated nucleotide length using the native sequence from the respective pre-crRNA. "APG07433.1" indicates a dgRNA in which the first stem of the anti-repeat was extended to the indicated nucleotide length using the sequence from the APG07433.1 gRNA. (The nucleotide lengths shown are for the first stem of the anti-repeat, and the same nucleotide length is expected on the first stem of the crRNA repeat due to base pairing.) All nucleotides of the extended first stem of the anti-repeat are modified with LNA, and the first stem is extended at the end distal to the first bubble of stem-loop 1 (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA). The nucleotide sequence above the data points indicates the sequence of the two added terminal nucleotides (the two nucleotides at the 5' end of the extended tracrRNA). Flow cytometry was used to measure gene editing efficiency in primary human T cells by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). RGN was delivered as an mRNA encoding RGN (mRNA delivery). [Figure 20]Figure 20 shows a strategy to improve gene editing efficiency by truncated dgRNA. Left: Schematic of WT APG07433.1 crRNA. Center: Schematic of truncated "M" APG07433.1 crRNA and tracrRNA. Right: The 3'3-terminal nucleotides of crRNA and the 5'2-terminal nucleotides of tracrRNA are replaced with C and G nucleotides (nucleotides marked with an asterisk), respectively. For both truncated (M) and engineered truncated (M), the first stem of the anti-repeat is LNA-modified. [Figure 21] Figure 21 shows that the gene editing efficiency of the truncated "M" APG07433.1 dgRNA is improved by substituting the nucleotides as shown in Figure 20. Two spacers were tested. "Original" indicates the truncated "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. RGN was delivered as mRNA encoding RGN (mRNA delivery). [Figure 22] Figures 22A and 22B show strategies for LNA modification of the anti-repeat-forming stem-loop 1 of tracrRNA in gRNAs. Figure 22A shows a modified APG07433.1 dgRNA that performs well in gene editing, with all 11 nucleotide LNAs modified in the first stem (FS) of the anti-repeat (Tracr(J); see Figure 10A). Figure 22B shows an APG07433.1 tracrRNA with modifications at all nucleotides in the FS and second stem (SS) of the anti-repeat (Tracr(Jb); FS+SS); all nucleotides in the SS of the anti-repeat (Tracr(Jc); SS); and all nucleotides in the anti-repeat, including nucleotides in the first stem, bubble, and second stem (Tracr(Jd); full stem-loop 1), modified. [Figure 23]Figure 23 shows that LNA modification of all nucleotides in the first stem of the anti-repeat in the gRNA tracrRNA is the most effective for gene editing compared to other LNA modification strategies for the anti-repeat. Gene editing efficiency is shown for the APG07433.1 dgRNA with LNA-modified tracrRNA (right side of the graph; Tracr(J), Tracr(Jb), Tracr(Jc), and Tracr(Jd)), as shown in Figures 22A and 22B. The crRNA had MS modifications in the three terminal nucleotides of both the 5' and 3' regions (3MS), as shown in Figure 22A. Two exemplary spacers (1880 and 1881) were used. "Control_TRAC" indicates a condition without RGN and dgRNA; cells are mixed with nucleofection solution but are not subjected to the nucleofection process. The control dgRNA on the left side of the graph has MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA, but no chemical modifications elsewhere in the dgRNA. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered as mRNA encoding RGN (mRNA delivery). [Figure 24]Figure 24 shows that LNA modification of all nucleotides in the first stem of the crRNA repeat in the gRNA crRNA is effective for gene editing, as long as all nucleotides in the first stem of the anti-repeat in the gRNA tracrRNA are also LNA modified (see upper graph, right). Having LNA modification of all nucleotides in the second stem of the crRNA repeat in the gRNA crRNA worsens the gene editing of gRNAs with LNA modification of all nucleotides in the first stem of the anti-repeat (see lower graph, right). APG07433.1 with LNA modification of all nucleotides in the first stem or second stem of the crRNA repeat Gene editing efficiency for dgRNA: (Top graph, upper left and left) shows a dgRNA with a crRNA with LNA modifications at every nucleotide in the first stem of the crRNA repeat and a tracrRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions (3MS); (Top graph, upper right and right) shows a dgRNA with a crRNA with LNA modifications at every nucleotide in the first stem of the crRNA repeat and a tracrRNA with LNA modifications at every nucleotide in the first stem of the anti-repeat; (Bottom graph, lower left and left) shows a dgRNA with a crRNA with LNA modifications at every nucleotide in the second stem of the crRNA repeat and a tracrRNA with 3MS; (Bottom graph, lower right and right) shows a crRNA with LNA modifications at every nucleotide in the second stem of the crRNA repeat and a tracrRNA with LNA modifications at every nucleotide in the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used. "Control" refers to a condition without RGN or dgRNA; cells are mixed with nucleofection solution but do not undergo the nucleofection process. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered as mRNA encoding RGN (mRNA delivery). [Figure 25]Figure 25 shows that LNA modification increases the editing efficacy of the APG05586 sgRNA. Serial dilutions of the sgRNA were performed. "Unmodified" refers to an sgRNA with MS modifications at the three terminal nucleotides of both the 5' and 3' regions and no other chemical modifications (3MS). "LNA@SL1" refers to an sgRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat, along with 3MS. "MS / LNA@SL1" refers to an sgRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat, an sgRNA with MS modifications at the three terminal nucleotides of the crRNA repeat closest 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. RGN was delivered either as a protein complexed with a guide RNA (RNP delivery) or as an mRNA encoding RGN (mRNA delivery). "Control (TRAC)" and "Control (B2M)" indicate conditions without RGN and gRNA, in which cells are mixed with nucleofection solution but do not undergo the nucleofection process, for two different spacers in the gRNA. [Figure 26] Figure 26 shows that the amount of LNA modification in the first stem of the anti-repeat correlates with the guide RNA editing efficiency and melting temperature (Tm) of the DNA / tracrRNA anti-repeat duplex. A schematic diagram of the APG07433.1 dgRNA shows 1, 3, 6, or 11 LNA-modified nucleotides in the region of the anti-repeat that forms the first stem of stem-loop 1. The dgRNA contains MS modifications at the 5' and 3' ends of the crRNA and tracrRNA. The highest editing and highest Tm were achieved when all nucleotides in the region of the anti-repeat that forms the first stem of stem-loop 1 were LNA-modified. The amount of LNA modification is shown on the x-axis, and Tm and gene editing efficiency are shown on the y-axis. 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 gene editing experiments. [Figure 27]Figure 27 shows that gene editing is rescued for the APG07991 RGN system (see schematic diagram on the left) with a WT (original) dgRNA with <11 (6) nucleotide pairs in the first stem of stem-loop 1 by extending the first stem distal to the first bubble of stem-loop 1 (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA) and modifying all nucleotides in the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiment, and there were two repeats per target gene. "Control" refers to conditions without RGN and dgRNA, in which cells are mixed with nucleofection solution but do not go through the nucleofection step. "Unmodified" refers to a dgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA (3MS), but no chemical modifications elsewhere in the dgRNA. "LNA" indicates a dgRNA with 3MS+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 extended to the indicated nucleotide length using the native sequence from the APG07991 pre-crRNA. "APG07433.1" indicates a dgRNA with the first stem of stem-loop 1 extended to the indicated nucleotide length using the sequence from the APG07433.1 gRNA. (The nucleotide lengths shown are for the first stem of the anti-repeat, and the same nucleotide length is expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the extended first stem of the anti-repeat are modified with LNA, and the first stem is extended at the end distal to the first bubble of stem-loop 1 (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA). The nucleotide sequences above the data points represent the sequence of the 5'-terminal six nucleotides of the original, unextended tracrRNA (in the case of "unmodified" and "LNA") or the two terminal nucleotides added (the two nucleotides at the 5'-end of the extended tracrRNA).Gene editing efficiency was measured in primary human T cells using flow cytometry by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). RGN was delivered as mRNA encoding RGN (mRNA delivery). [Figure 28]Figure 28 shows that the gene editing efficiency of the APG07991 dgRNA can be rescued by extending the first stem distal to the first bubble of stem-loop 1 by at least 11 nucleotide pairs (i.e., extension 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 a condition without RGN and dgRNA; cells are mixed with nucleofection solution but are not subjected to the nucleofection process. "sgRNA" indicates an APG07991 sgRNA control with an appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the sgRNA but no chemical modifications elsewhere in the sgRNA. 6, 8, 10, 11, 12, and 13 indicate the length of the first stem of stem-loop 1. "Native Sequence" indicates that the native sequence from the APG07991 pre-crRNA was used to extend the first stem of stem-loop 1 of the WT APG07991 dgRNA to the indicated nucleotide length. "APG07433.1 Seq" indicates that the sequence from the APG07433.1 gRNA was used to extend the first stem of stem-loop 1 of the WT APG07991 dgRNA to the indicated nucleotide length. "-" and "+" indicate whether all nucleotides in the first stem of the anti-repeat were modified with LNA. Flow cytometry was used to measure gene editing efficiency in primary human T cells by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). APG07991 RGN was delivered as mRNA encoding APG07991 RGN (mRNA delivery). Two exemplary spacers were used: TRAC and B2M. [Figure 29]Figure 29 shows that the gene editing efficiency of Streptococcus pyogenes Cas9 (SpyCas9) dgRNA, which cooperates with the APG07991 RGN for gene editing, can be rescued by extending the first stem distal to the first bubble of stem-loop 1 by at least 11 nucleotide pairs (i.e., extension 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 the WT (original) SpyCas9 dgRNA has 4 nucleotide pairs (see schematic diagram on the left), and shows very little gene editing with the APG07991 RGN. "Control" indicates a condition without RGN and dgRNA; cells are mixed with the nucleofection solution but are not subjected to 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 of both the 5' and 3' regions of the sgRNA, but no chemical modifications elsewhere in the sgRNA. Numbers 4, 8, 11, and 13 indicate the length of the first stem of stem-loop 1. "Native Sequence" indicates that the native sequence from SpyCas9 pre-crRNA was used to extend the first stem of stem-loop 1 of the WT SpyCas9 dgRNA to the indicated nucleotide length. "APG07433.1 Seq" indicates that the sequence from APG07433.1 gRNA was used to extend the first stem of stem-loop 1 of the WT SpyCas9 dgRNA to the indicated nucleotide length. Flow cytometry was used to measure gene editing efficiency in primary human T cells by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). APG07991 RGN was delivered as mRNA encoding APG07991 RGN (mRNA delivery). Two exemplary spacers (targeting the TRAC and B2M genes) were used. [Figure 30]Figure 30 shows that LNA modification of all nucleotides in the anti-repeat that forms the first stem of stem-loop 1 confers greater stability to the sgRNA. Schematic diagram on the left: (A) terminally modified sgRNA has MS modifications of the three terminal nucleotides in both the 5' and 3' regions of the sgRNA; (B) LNA-modified sgRNA has MS modifications of the three terminal nucleotides in both the 5' and 3' regions and LNA modifications of all nucleotides in the first stem of the anti-repeat; (C) MS / LNA-modified sgRNA has MS modifications of the three terminal nucleotides in both the 5' and 3' regions, MS modifications of the three terminal nucleotides in the 3' region of the crRNA repeat, and LNA modifications of all nucleotides in the first stem of the anti-repeat; and (D) MS / LNA-modified dgRNA has MS modifications of the three terminal nucleotides in both the 5' and 3' regions of the crRNA, LNA modifications of all nucleotides in the first stem of the anti-repeat, and MS modifications of the three terminal nucleotides in the 3' region of the tracrRNA. MS / LNA-modified dgRNAs enable efficient gene editing through simultaneous delivery of mRNA and gRNA components, but are not as stable as chemically modified sgRNAs, as demonstrated in the case of staggered delivery. [Figure 31] Figure 31 shows that using gRNAs with LNA modifications increases base editing efficiency. The gRNAs tested are as described in Figure 30. A, B, C, and D in the graph correspond to A, B, C, and D shown in the schematic diagram on the left. [Figure 32] Figure 32 shows that base editing efficiency increases using gRNAs with LNA modifications and truncated gRNAs with LNA modifications. No LNA, LNA, and MS / LNA in the graph correspond to those shown in the diagram on the left. "Shrt" indicates a truncated sgRNA. gRNAs with two exemplary spacers, SGN001880 and SGN001881, were used. [Figure 33]Figure 33 shows that the gene editing efficiency of a dgRNA chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to a level comparable to that of an LNA-modified dgRNA. The gene editing efficiency of the APG07433.1 dgRNA (shown in the schematic diagram at the top left, see also Figure 10A) with 11 LNA-modified nucleotides in the anti-repeat forming the first stem of stem-loop 1 was compared to that of the APG07433.1 dgRNA (shown in the schematic diagram at the bottom left) with 11 S-restrictive ethyl (cEt)-modified nucleotides in the anti-repeat forming the first stem of stem-loop 1. "Unmodified" indicates a dgRNA with MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the crRNA and tracrRNA (3MS), but no chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells using flow cytometry by evaluating knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). APG07433.1 RGN was delivered as mRNA encoding APG07433.1 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used. [Figure 34]Figure 34 shows that the gene editing efficiency of a dgRNA, cEt, extended at the first stem distal to the first bubble of stem-loop 1 and chemically modified with another bridging nucleic acid (BNA) is enhanced to a comparable level of gene editing efficiency of the same extended dgRNA modified with an LNA. The APG05586 dgRNA was extended at the first stem distal to the first bubble of stem-loop 1 by 11 nucleotide pairs (i.e., extension at the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA; see Figures 18 and 19), and all nucleotides in the first stem of the anti-repeat were modified with either LNA (shown in the top left schematic) or S-constrained ethyl (cEt) (shown in the bottom left schematic). "APG05586 Native Sequence" indicates that the native sequence from the APG05586 pre-crRNA was used to extend the first stem of stem-loop 1 of the WT APG05586 dgRNA to 11 nucleotides. "APG07433.1 Seq" indicates that the sequence from the APG07433.1 gRNA was used to extend the first stem of stem-loop 1 of the WT APG05586 dgRNA to 11 nucleotides. "Original" indicates the WT APG05586 dgRNA with 9 nucleotide pairs in the first stem of unextended stem-loop 1. "Unmodified" indicates a dgRNA with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the crRNA and tracrRNA (3MS), but no chemical modifications elsewhere in the dgRNA. Flow cytometry was used to measure gene editing efficiency in primary human T cells by assessing knockout of the CD3 surface marker (for editing of the TRAC target sequence) or immunostaining of B2M (for editing of the B2M target sequence). APG05586 RGN was delivered as mRNA encoding APG05586 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used. [Figure 35]Figure 35 shows that LNA modifications improved the gene editing efficiency of dgRNAs with extensions. The top part of Figure 35 shows a schematic diagram illustrating three chemically modified dgRNAs whose extensions are in the tail of the tracrRNA: (a) MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the crRNA (3' end), and MS modifications at the three terminal nucleotides of the 5' and 3' regions of the tracrRNA+ extension (3' end); (b) LNA modifications at the 3' end of the crRNA, all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides of the 3' region of the tracrRNA+ extension; (c) LNA modifications at the 3' end of the crRNA, all nucleotides of the first stem of the anti-repeat, and LNA modifications at the four nucleotides of the first stem of the stem-loop closest to the tail of the tracrRNA (i.e., the stem of the illustrated system). MS modifications at the three terminal nucleotides of the 3' region of the tracrRNA+ extension (3' MS end). The bar on the left side of the graph, labeled "Single," shows the gene editing efficiency of a control single guide RNA, corresponding to a dgRNA with no extension and MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the guide RNA (3' MS end), but no LNA modifications. The control single guide RNA serves as a benchmark for the gene editing efficiency of guide RNAs without extension. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered as an mRNA encoding RGN (mRNA delivery). SL1 = stem-loop 1. SL3 = stem-loop 3. [Figure 36]Figures 36A-C show schematics of guide RNAs with extensions and details of the engineering of truncated guide RNAs. Figure 36A: The unengineered guide RNA + extension has a 46-nt crRNA length (spacer + 21-nt crRNA repeats) and a 79-nt tracrRNA length (85-nt WT length minus 6 nt from the tail). Figure 36B: The truncated guide RNA + extension has a shortened crRNA / tracrRNA backbone and a 41-nt crRNA length (spacer + 16-nt truncated crRNA repeats) and a 72-nt tracrRNA length. Figure 36C: Schematic diagrams of the engineered truncated crRNA and tracrRNA (without extension) showing that the 3'3-terminal nucleotides of the crRNA and the 5'2-terminal nucleotides of the tracrRNA are replaced with C and G nucleotides (nucleotides marked with an asterisk). For both dgRNA + extensions shown in Figures 36A and 36B, the first stem of the anti-repeat is LNA-modified. [Figure 37]Figure 37 shows that LNA modifications improved the gene editing efficiency of an engineered shortened dgRNA+ extension using nucleotide substitutions at the 3' end of the crRNA and the 5' end of the tracrRNA, a dgRNA+ extension with an engineered shortened backbone and MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the crRNA (3MS termini), and MS modifications at the three terminal nucleotides of the 5' and 3' regions of the tracrRNA+ extension (3MS termini). a, dgRNA+ extension with an engineered shortened backbone and 3MS termini to the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides in the 3' region of the tracrRNA+ extension. The bar on the left side of the graph, labeled "Single," shows the gene editing efficiency of a control single-guide RNA without extended or shortened backbone or nucleotide engineering, with MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the guide RNA (3MS termini), and without LNA modifications. The control single-guide RNA serves as a benchmark for the gene editing efficiency of guide RNAs without extended or shortened backbone or nucleotide engineering. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered as an mRNA encoding RGN (mRNA delivery). SL1 = stem-loop 1. SL3 = stem-loop 3. [Figure 38]Figure 38 shows that LNA modification in the first stem of the anti-repeat is key to the editing efficiency of dgRNA+ extension. (a) MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the crRNA (3MS termini), and at the three terminal nucleotides of the 5' and 3' regions of the tracrRNA+ extension (3MS termini); (b) LNA modifications at the 3MS termini of the crRNA, at all nucleotides of the first stem of the anti-repeat, and at the three terminal nucleotides of the 3' region of the tracrRNA+ extension; (c) LNA modifications at the 3MS termini of the crRNA, at all nucleotides of the first stem of the anti-repeat, at the four nucleotides of the first stem of the stem-loop closest to the tail of the tracrRNA (stem-loop 3 in this system), and at the three terminal nucleotides of the 3' region of the tracrRNA+ extension; (d) MS modifications at the three terminal nucleotides of the 5' region of the crRNA, at all nucleotides of the first stem of the crRNA repeat, and at the tracrRNA+ extension. (e) MS modifications at the three terminal nucleotides of 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 of the 3' region of the tracrRNA+ extension; (f) MS modifications at the three terminal nucleotides of 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 the fourth nucleotide of the first stem of stem-loop 3, and MS modifications at the three terminal nucleotides of the 3' region of the tracrRNA+ extension; (g) MS modifications at the three terminal nucleotides of the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3 MS modifications at the tracrRNA+ extension;(h) MS modifications at the three terminal nucleotides of 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 of the 3' region of the tracrRNA+ extension; (g) and (h) the dgRNA+ extension has a shortened backbone engineered as described in Figure 36C. The bar on the left side of the graph labeled "Single" shows the gene editing efficiency of a control single guide RNA without an extended or shortened backbone or nucleotide engineering, with MS modifications at the three terminal nucleotides of both the 5' and 3' regions of the guide RNA (3MS), but without LNA modifications. The control single guide RNA serves as a benchmark for the gene editing efficiency of guide RNAs without an extended or shortened backbone or nucleotide engineering. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. RGN was delivered as mRNA encoding RGN (mRNA delivery). SL1 = stem-loop 1. SL3 = stem-loop 3. ; DETAILED DESCRIPTION OF THE INVENTION

[0099] Many modifications and other embodiments of the inventions described 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. It is to be understood, therefore, that the invention is not 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.

[0100] I. Overview The present disclosure provides, inter alia, compositions and methods relating to modified guide RNAs (gRNAs) for use in RNA-guided nuclease (RGN) systems, as well as related systems and methods. In various embodiments, the inclusion of one or more bridged nucleic acids, e.g., locked nucleic acids, within specific regions of the gRNA (in some embodiments, but not within other regions) improves the editing efficiency of RGN systems. Furthermore, such modifications open up the possibility of editing in mRNA-based dual-guide systems that was previously impossible with unmodified dual-guide RNAs available in the prior art. This is a significant advance in the art, as new editing modes, 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 on a large scale in a single-guide format using current manufacturing processes. Thus, the lack of manufacturability in the art significantly limits the commercial viability of these mRNA-based therapeutics. These and other advances are presented in the present disclosure.

[0101] The RGN system allows targeted manipulation of specific sites within the genome and is useful in the context of gene targeting for therapeutic and research applications. In various organisms, including mammals, the RGN system has been used, for example, to create single- or double-strand breaks in polynucleotides, modify polynucleotides, detect specific sites within polynucleotides, or modify the expression of specific genes. The RGN system comprises a complex of RGN and gRNA. Hybridization of the gRNA to a specific target sequence allows the guide RNA / RGN complex to be targeted to a specific location within the genome for editing.

[0102] In the RGN system, gRNA can exist as a two-part gRNA or a single gRNA. Two-part gRNA systems include a CRISPR RNA (crRNA) containing a spacer sequence that recognizes the target genomic sequence through Watson-Crick base pairing, and a scaffold (tracrRNA) that transactivates the crRNA. The crRNA hybridizes to the tracrRNA to form a dual guide RNA (dgRNA) held together as a duplex at the crRNA:tracrRNA annealing region. For many applications, chimeric single guide RNA (sgRNA) molecules formed by physically linking the crRNA and tracrRNA with a short, flexible loop can be used.

[0103] Both dgRNAs and sgRNAs have advantages and disadvantages. sgRNAs generally provide relatively good editing efficiency. However, while somewhat convenient as a single chemical species, sgRNAs are relatively long, generally 100 nt or longer. A common method for producing gRNAs is by solid-phase oligonucleotide synthesis, a sequential synthesis pathway. As length increases, despite high coupling efficiency of each individual step, the yield and purity of the full-length product are low (Reese, Org Biomol Chem, 2005; Beaucage & Reese, Curr Protoc Nucleic Acid Chem, 2009; Beaucage, Curr Opin Drug Di De, 2008; Shiba, Nucleic Acids, 2007; 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 synthesized. Another advantage of using dgRNAs is that because tracrRNAs pair with different crRNAs containing various spacer sequences, tracrRNA scaffolds can be generated in large batches and paired with individual crRNAs for specific genomic targets.

[0104] However, dgRNA has relatively low gene editing efficiency, especially in the delivery method of introducing mRNA encoding RGN into cells for RGN expression. The additional exposed 5' and 3' ends of crRNA and tracrRNA, and the possibly weaker duplex strength compared to sgRNA (intramolecular components hybridize), may reduce RNA stability and therefore performance. In some embodiments, the effectiveness of dgRNA can be enhanced and rescued by strengthening the duplex.

[0105] Several chemical modifications, including 2'-fluoro-ribose (2'-F), 2'-O-methyl (2'-O-Me), 3' phosphorothioate (PS), and 2'-O-Me 3' phosphorothioate (MS), along with other modifications, have been used to improve gRNA stability and thus enhance editing efficiency in cells ex vivo and in vivo. However, these methods cannot enable effective editing when transfecting mRNA encoding RGN with dgRNA. Instead, they require the production of long sgRNAs. Newer techniques, such as prime editing using additional guide extensions, may not be able to produce sgRNAs on a large scale and with high levels of purity. Furthermore, while the above-mentioned modifications protect RNA from nuclease degradation, other mechanisms may induce degradation or inactivation, particularly disruption of secondary structure. Bridged nucleic acids (BNAs) contain nucleotide analogs with restricted conformations due to intramolecular bonds or crosslinks. Locked nucleic acids (LNAs), a type of BNA, have been used in PCR probe design, for example, to shorten probe length and increase hybridization strength. LNAs contain a covalent bond between the 2' oxygen and 4' carbon on the ribose sugar of a nucleotide and have been shown to contribute to highly efficient complementary pairing for improved mismatch discrimination and nuclease resistance (You et al. Nucleic Acids Res., 2006; Vester & Wengel, J Biochemistry, 2004). Provided herein is a method for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications. The data in this application demonstrate that such guide RNAs modified with BNA modifications are an improvement over prior art methods, significantly enhancing the efficacy of guide RNAs and enabling the use of dual guide RNAs in applications that would otherwise be inappropriate (e.g., when gRNAs are cotransfected with RGN-encoding mRNA).Without being limited by speculation, the inventors believe that these advantages are at least due to the BNA modification stabilizing the stem-loop formed by hybridization between the crRNA repeat and the tracrRNA anti-repeat. In some instances, the BNA is an LNA. In some instances, the BNA is an S-restrictive ethyl (cEt). In some examples, 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 BNAs (e.g., LNAs and / or cEt) or other chemical modifications into the 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, both the crRNA and the tracrRNA include BNA (e.g., LNAs and / or cEt) modifications. In certain embodiments, either the crRNA or the tracrRNA includes BNA (e.g., LNAs and / or cEt) modifications. In some embodiments, the tracrRNA contains BNA modifications, but the crRNA does not contain BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, the tracrRNA contains BNA modifications and the crRNA contains MS modifications. In embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure improve the gene editing efficiency of an RGN system compared to a reference RGN system having a tracrRNA, gRNA, and / or crRNA that contains only MS modifications in the three terminal nucleotides of the 5' and 3' regions. In embodiments, the modified tracrRNA, gRNA, and / or crRNA of the present disclosure enable the use of dgRNA in applications where sgRNA would otherwise be more desirable. In some embodiments, the present disclosure provides the use of BNA (e.g., LNA and / or cEt) modifications within the crRNA:tracrRNA annealing region of the 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 allow for shortened crRNA:tracrRNA annealing regions. In some embodiments, BNA (e.g., LNA) modifications combined with engineering the crRNA:tracrRNA annealing region of the dgRNA enhance the performance of the RGN system in cells. In some embodiments, the gene-edited cells comprise 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 in which the RGN system is applicable.

[0106] Without being bound by any theory, chemical modifications to nucleotides in the gRNA may increase stability by preventing degradation of the gRNA by endogenous nucleases and / or stabilize RNA-RNA interactions in the chemically modified region.

[0107] II. Guide RNA 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 group of RNA molecules) that can bind to an RNA-guided nuclease (RGN) and help target the RGN to a specific location within a target polynucleotide (e.g., a DNA or mRNA molecule), such as a genomic locus. In some embodiments, the guide RNA comprises a nucleotide sequence (i.e., a spacer) that has sufficient complementarity with the target strand nucleotide sequence to hybridize with the target strand and direct sequence-specific binding of the RGN to the target nucleotide sequence. In some embodiments, when the target nucleotide sequence is double-stranded, such as in the case of DNA, the target nucleotide sequence comprises a non-target strand (including a PAM sequence) and a target strand that hybridizes with a spacer of the guide RNA. In these embodiments, the guide RNA has sufficient complementarity with the target strand of the double-stranded target sequence (e.g., a target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequence-specific binding of the associated RGN to the target sequence (e.g., the target DNA sequence). Thus, in some embodiments, the guide RNA comprises a spacer that is identical to the sequence of the non-target strand, except that uracil (U) replaces thymidine (T) in the guide RNA.

[0108] Each guide RNA of an RGN is one or more RNA molecules (generally one or two) that can bind to the RGN and guide the RGN to bind to a specific target sequence, and in embodiments where the RGN has nickase or nuclease activity, also cleaves the target strand and / or non-target strand. Generally, guide RNAs include CRISPR RNAs (crRNAs) and trans-activating CRISPR RNAs (tracrRNAs).

[0109] The term "guide RNA" also collectively encompasses a group of two or more RNA molecules in which the crRNA and tracrRNA segments are located within separate RNA molecules. Natural guide RNAs, including both crRNA and tracrRNA, generally comprise two separate RNA molecules that hybridize to each other via 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 type of chemical linkage that covalently links two molecules together, such as a linkage formed by click chemistry or any other chemical reaction, a polynucleotide, a polymer, or any entity capable of linking two molecules. In some embodiments, the linker connecting the crRNA and tracrRNA comprises a multinucleotide linker (e.g., a 4-nucleotide linker) to form a single guide RNA molecule, and the crRNA and tracrRNA hybridize to each other via the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. Guide RNA therefore encompasses single guide RNAs (sgRNAs) in which the crRNA segment and the tracrRNA segment are located on the same RNA molecule or strand.

[0110] The guide RNAs crRNA and tracrRNA can be linked by organic molecules, groups, polymers, or chemical moieties. In some embodiments, the guide RNAs crRNA and tracrRNA are linked by click chemistry. Click chemistry involves the rapid generation of compounds by linking small units together via heteroatom linkages (CXC). The primary goal of click chemistry is to develop a set of powerful, selective, modular "building blocks" useful for small- and large-scale applications. Click chemistry reactions are fast, modular, efficient, often do not produce toxic waste, can be performed using water as a solvent, and can be configured to be stereospecific.

[0111] Click chemistry is a versatile reaction that can be used to synthesize a variety of conjugates. Virtually any biomolecule can participate, and labeling with small molecules such as fluorescent dyes, biotin, and other groups can be easily achieved. The click chemistry reaction occurs between two components: an azide functional group and an alkyne functional group. The azide is represented by the formula N3 - and structure - N=N + =N - It is a linear polyatomic anion with the formula RN3. It is the conjugate base of hydrazoic acid HN3. Organic azides are organic compounds with the formula RN3 that contain an azide functional group. Alkynes are unsaturated hydrocarbons that contain 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, have the general chemical formula C n H 2n-2 A terminal alkyne has the formula RC2H. An example is methylacetylene (propyne using IUPAC nomenclature). Both the azide group and the alkyne group are rarely encountered in natural biomolecules. Therefore, the reaction can occur within biological systems without interfering with other cellular processes (i.e., highly bioorthogonal) and specifically.

[0112] A well-known click reaction is the Huisgen 1,3-dipolar cycloaddition of azides and alkynes. This reaction, which produces triazoles, has become the gold standard in click chemistry due to its reliability, specificity, and biocompatibility. Such cycloadditions have high activation energies (ΔG) when the reaction involves simpler alkenes or azides. ‡ The Cu(I) catalyst promotes the reaction of terminal alkynes and azides to give 1,4-disubstituted-1,2,3 triazoles. This reaction is an ideal "click" reaction and has been widely adopted in materials science, medicinal chemistry, and chemical biology.

[0113] However, the cytotoxicity of transition metals used as catalysts for click reactions precludes their use for in vivo applications. Alternative approaches with lower activation barriers and copper-free reactions have been established. Such reactions are called "copper-free click chemistry." Instead of using copper to activate alkynes, the alkynes are instead introduced into strained difluorooctynes ​​(DIFOs), and the electron-withdrawing propargylic gem fluorine, along with the ring strain, significantly destabilizes the alkynes (Agard et al. (2006) ACS Chem. Biol. 1(10):644-648). This destabilization increases the driving force and the desire to relieve the ring strain of the cycloalkyne. Copper-free click chemistry proceeds as a concerted [3 + 2] cycloaddition, with the same mechanism as the Huisgen 1,3-dipolar cycloaddition. Cycloctynes ​​can also tolerate non-fluorine substituents, such as benzene rings.

[0114] The reactive groups in click chemistry (e.g., dibenzocyclooctyne (DBCO)) can be introduced into any form of nucleic acid molecule and can be introduced enzymatically or chemically. Alkyne- and azide-modified oligonucleotides can be ordered from an oligo synthesis facility or company. Azide-modified nucleotides can be introduced into a first RNA molecule during RNA synthesis, and alkyne-modified nucleotides can be introduced into a second RNA molecule during RNA synthesis. The resulting click-functionalized nucleic acid molecules can be isolated and purified to remove unreacted reagents or by-products that may 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 contain a copper catalyst to promote the reaction between the azide and the alkyne, or it can be copper-free. The azide and alkyne functional groups react to form a covalent bond, linking the two nucleic acid molecules together. The linked nucleic acid molecules can be further purified, and analytical techniques such as gel electrophoresis or mass spectrometry can be used to verify the success of the linkage of the nucleic acid molecules and evaluate the purity of the product.

[0115] Click chemistry is further described in, for example, 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; U.S. Patent No. 7,375,234; U.S. Patent No. 7,070,941; and U.S. Patent Application Publication No. 2013 / 0046084, the contents of each of which are incorporated herein by reference in their entireties.

[0116] Ligation of crRNA and tracrRNA can proceed as follows: By solid-phase synthesis, crRNA contains a 3' amino modifier, and tracrRNA contains a 5' amino modifier. After synthesis, deprotection, and purification of the RNA oligonucleotides, an azide group is introduced at the 5' region of tracrRNA using the NHS ester of azidobutyrate (4-azidobutanoic acid N-hydroxysuccinimide ester; available, for example, from Glen Research, catalog number 50-1904-24), and a cyclooctyne group is introduced at the 3' region of crRNA using the NHS ester of DBCO (dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester; available, for example, from Sigma-Aldrich, catalog number 764019). Assembly in aqueous medium (optionally optimizing ionic strength, pH, and reagent concentrations) allows for the strain-promoted azide-alkyne Huisgen cycloaddition ("copper-free click chemistry") to proceed.

[0117] In some embodiments, the crRNA and tracrRNA linked by click chemistry are linked by a chemical moiety. In some embodiments, the crRNA and tracrRNA linked by a chemical moiety include an azide group at one or more nucleotides in the anti-repeat of the tracrRNA and an alkyne group at one or more nucleotides in the crRNA repeat of the crRNA. In some embodiments, the crRNA and tracrRNA linked by a chemical moiety include an azide group at one or more nucleotides in the crRNA repeat of the crRNA and an alkyne group at one or more nucleotides in the anti-repeat of the tracrRNA. One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be within the stem, 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 the stem, bubble, or both of the anti-repeat of the guide RNA. In some embodiments, the crRNA and tracrRNA linked by the chemical moiety are single guide RNAs and contain azide- or alkyne-modified nucleotides at one or more nucleotides in the nucleotide loop connecting the crRNA repeat and anti-repeat.

[0118] A variety of additional chemical reactions and corresponding modifications are available to those skilled in the art for covalently linking nucleic acid molecules (e.g., crRNA and tracrRNA) to one another. These modifications include various crosslinkers, such as thiol modifications, such as thioctic acid N-hydroxysuccinimide (NHS) ester, which are chemical groups that react with primary amines (-NH). These primary amines are positively charged at physiological pH and nucleophilic, making them easy to target for conjugation with several reactive groups. Numerous synthetic chemical groups exist that form chemical bonds with primary amines. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfo-NHS esters containing a sulfonate (-SO3) group, such as bis(sulfosuccinimidyl)suberate (BS3), sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), anhydrides, and fluorophenyl esters.

[0119] As described herein, the guide RNA can include a crRNA and a tracrRNA, where the crRNA includes: (i) a spacer; (ii) a crRNA repeat including a first stem and a second stem; and the tracrRNA includes: (i) a tail; and (ii) an anti-repeat including a first stem and a second stem, and at least one of the crRNA and the tracrRNA includes at least one BNA modification. In some embodiments, the anti-repeat can hybridize to the crRNA repeat to form a stem-loop including the first stem and the second stem.

[0120] As described herein, the present disclosure also provides a nucleic acid molecule comprising a tracrRNA, the tracrRNA comprising (a) an anti-repeat; (b) a tail; and (c) a stem-loop proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and the tracrRNA comprises at least one BNA modification. In some embodiments, the anti-repeat of the tracrRNA can hybridize to a crRNA repeat of the crRNA to form a stem-loop comprising the first stem and the second stem. In some embodiments, the gRNA comprising the tracrRNA can bind to an RGN.

[0121] As described herein, the present disclosure also provides a nucleic acid molecule 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, the at least one chemical modification being selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA modification; wherein the at least one chemical modification is within the three terminal nucleotides of the 5' or 3' region of the crRNA. In some embodiments, the crRNA repeat can hybridize to the anti-repeat of the tracrRNA to form a stem-loop comprising a first stem and a second stem. In some embodiments, the gRNA comprising the crRNA can bind to an RNA-guided nuclease (RGN) that requires the tracrRNA for activity.

[0122] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, the tracrRNA comprises an anti-repeat, the gRNA comprises a stem-loop comprising a first stem and a second stem, the first stem comprising an overall length of about 11 base pairs, and the first stem comprises at least one bridge nucleic acid (BNA) modification.

[0123] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, the tracrRNA comprises an anti-repeat, the gRNA comprises a stem-loop comprising a first stem and a second stem, the first stem comprising at least 3, 4, 5, 6, or 7 GC base pairs, and the first stem comprises at least one bridge nucleic acid (BNA) modification.

[0124] The present invention provides, inter alia, CRISPR RNA (crRNA) or a polynucleotide encoding a CRISPR RNA containing 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 a nucleotide sequence that directly hybridizes to the target strand of a target sequence, and a CRISPR repeat, which includes a nucleotide sequence that, by itself or in cooperation with a hybridized tracrRNA, forms a structure recognized by an RGN molecule. As used herein, the term "tracrRNA" or "transactivating crRNA" refers to an RNA molecule that includes an anti-repeat sequence that is sufficiently complementary to hybridize to at least a portion of the CRISPR repeat of the crRNA to form a structure recognized by an RGN molecule. In some embodiments, an additional secondary structure (e.g., a stem-loop) within the tracrRNA molecule is required for binding to RGN.

[0125] In some embodiments, the crRNA comprises at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a 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 a locked nucleic acid (LNA) modification, BNA NC The 2',4' BNA is selected from the group consisting of an [N-Me] modification, a 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and an S-restricted 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, at least one chemical modification is a 2'-O-Me modification. In certain embodiments, at least one chemical modification is an MS modification.

[0126] The crRNA comprises a spacer and CRISPR repeats. A "spacer" is a nucleotide sequence that directly hybridizes to the target strand of a target sequence of interest (e.g., a target DNA sequence). The spacer is engineered to have full or partial complementarity to the target strand of the 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 the spacer and the target strand of a target sequence (e.g., a target DNA sequence), when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to, 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 the spacer and the target strand of the target sequence (e.g., the target DNA sequence) 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 when optimally aligned using a suitable alignment algorithm. In some embodiments, the spacer can be identical in sequence to the non-target strand of the target sequence.In some embodiments where the target sequence is a target DNA sequence, the spacer may be identical in sequence to the non-target strand of the target DNA sequence, except that thymidines (Ts) in the non-target strand are replaced by uracils (Us) in the spacer. In embodiments, the spacer does not contain 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 (e.g., Gruber et al. (2008) Cell 106(1):23-24).

[0127] In some embodiments, the spacer of the present disclosure comprises a chemical modification to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the spacer. In certain embodiments, the spacer of the present disclosure comprises at least one chemical modification. In some embodiments, the at least one modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, the spacer of the present disclosure comprises at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the spacer of the present disclosure comprises at least a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is a locked nucleic acid (LNA) modification, BNA NCThe spacer is selected from the group consisting of an [N-Me] modification, a 2'-O,4'-C-ethylene-bridged nucleic acid (2',4'-ENA) modification, and an S-constrained ethyl (cEt) modification. In some embodiments, a spacer of the present disclosure comprises at least one LNA modification. In some embodiments, a spacer of the present disclosure comprises at least one 2'-O-Me modification. In some embodiments, a spacer of the present disclosure comprises at least one MS modification. In some embodiments, a spacer of the present disclosure comprises at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, a spacer of the present disclosure comprises 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 spacer of the present disclosure comprises at least one BNA (e.g., LNA and / or cEt) modification and at least one PS modification. In some embodiments, the spacer, in the absence of chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14 or differs from SEQ ID NO: 14 by one or two nucleotides. In some embodiments, the spacer, in the absence of any chemical modification, has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 14. In some embodiments, the spacer, in the absence of any chemical modification, has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 14. In some embodiments, the spacer, in the absence of any chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 14.

[0128] For clarity, as used herein, when a nucleotide sequence "differs by a certain number of nucleotides from a SEQ ID NO" or "has a certain percentage identity to a SEQ ID NO," the difference occurs only in the nucleotide sequence; the chemical modification, or lack thereof, remains the same.

[0129] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO: 16.

[0130] In some embodiments, the spacer, absent any chemical modification, has the nucleotide sequence set forth as SEQ ID NO: 15, or differs by one or two nucleotides from SEQ ID NO: 15. In some embodiments, the spacer, absent any chemical modification, has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 15. In some embodiments, the spacer, absent any chemical modification, has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 15. In some embodiments, the spacer, absent any chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 15.

[0131] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO: 17.

[0132] In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89, or differs by one or two nucleotides from SEQ ID NO: 89. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 89. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 89. In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 89.

[0133] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO:91 or that differs from SEQ ID NO:91 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:91 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:91 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:91 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:91 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:91 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO:91.

[0134] In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90, or differs by one or two nucleotides from SEQ ID NO: 90. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 90. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 90. In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 90.

[0135] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO:92 or that differs from SEQ ID NO:92 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:92 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:92 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:92 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:92 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:92 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO:92.

[0136] In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111, or differs by one or two nucleotides from SEQ ID NO: 111. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by ... one nucleotide from SEQ ID NO: 111. In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 111.

[0137] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO:113 or that differs from SEQ ID NO:113 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:113 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:113 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:113 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:113 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:113 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO:113.

[0138] In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112, or differs by one or two nucleotides from SEQ ID NO: 112. In some embodiments, the spacer, in the absence of any chemical modifications, has a nucleotide sequence that differs by ... one nucleotide from SEQ ID NO: 112. In some embodiments, the spacer, in the absence of any chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 112.

[0139] In some embodiments, the chemically modified spacer has a nucleotide sequence set forth as SEQ ID NO:114 or that differs from SEQ ID NO:114 by 1-5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:114 by 5 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:114 by 4 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:114 by 3 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:114 by 2 nucleotides. In some embodiments, the chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO:114 by 1 nucleotide. In some embodiments, the chemically modified spacer has the nucleotide sequence set forth in SEQ ID NO:114.

[0140] The crRNA further comprises a CRISPR RNA (crRNA) repeat, along with a spacer. The crRNA repeat comprises a nucleotide sequence that, by itself or in conjunction with a hybridized tracrRNA, forms a structure recognized by an 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, 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 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 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 certain embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat 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 when optimally aligned using a suitable alignment algorithm.

[0141] In some embodiments, the crRNA repeat of the present disclosure comprises a chemical modification to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the crRNA repeat. In certain embodiments, the crRNA repeat of the present disclosure comprises at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, the crRNA repeats of the present disclosure comprise at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the crRNA repeats of the present disclosure comprise at least a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is a locked nucleic acid (LNA) modification, a BNA NCThe crRNA repeat of the present disclosure is selected from the group consisting of an [N-Me] modification, a 2'-O,4'-C-ethylene-bridged nucleic acid (2',4'-ENA) modification, and an S-restricted ethyl (cEt) modification. In certain embodiments, the crRNA repeat of the present disclosure comprises at least one LNA modification. In certain embodiments, the crRNA repeat of the present disclosure comprises at least one cEt modification. In some embodiments, the crRNA repeat of the present disclosure comprises at least one 2'-O-Me modification. In some embodiments, the crRNA repeat of the present disclosure comprises at least one MS modification. In some embodiments, the crRNA repeat of the present disclosure comprises at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, the crRNA repeat of the present disclosure comprises 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, the crRNA repeats of the present disclosure comprise at least one BNA (e.g., LNA; in certain embodiments, the crRNA repeats of the present disclosure comprise at least one LNA modification) and at least one PS modification.

[0142] 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, which, when contained within a guide RNA, can direct sequence-specific binding of the associated RNA-guided nuclease provided herein to the presently disclosed target sequences. In some embodiments, the 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 the 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, the 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 consecutive nucleotides of the 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, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth as SEQ ID NO: 2 or differs from SEQ ID NO: 2 by one or two nucleotides. In some embodiments, the crRNA repeat, in the absence of chemical modification, has a nucleotide sequence differing from SEQ ID NO: 2 by two nucleotides. In some embodiments, the crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO:2 in the absence of chemical modifications.In some embodiments, the crRNA repeat without chemical modifications has the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70 or differs from SEQ ID NO: 70 by one or two nucleotides. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 70 by two nucleotides. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 70 by one nucleotide. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 70. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 94 or differs from SEQ ID NO: 94 by one or two nucleotides. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 94 by ... one nucleotide. In some embodiments, the crRNA repeat, in the absence of chemical modifications, has the nucleotide sequence set forth in SEQ ID NO: 94 by one nucleotide. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth as SEQ ID NO: 241 or differs by one or two nucleotides from SEQ ID NO: 241. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 241 or differs by one or two nucleotides from SEQ ID NO: 241. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 241. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 253 or differs by one or two nucleotides from SEQ ID NO: 253.In some embodiments, the crRNA repeat, in the absence of chemical modification, has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 253. In some embodiments, the crRNA repeat, in the absence of chemical modification, has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 253. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 253. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 538 or differs by one or two nucleotides from SEQ ID NO: 538. In some embodiments, the crRNA repeat, in the absence of chemical modification, has a nucleotide sequence that differs by ... one nucleotide from SEQ ID NO: 538. In some embodiments, the crRNA repeat, in the absence of chemical modification, has the nucleotide sequence set forth in SEQ ID NO: 538.

[0143] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs by one or two nucleotides from SEQ ID NO: 39. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 39. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 39. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 39.

[0144] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384 or that differs by one or two nucleotides from SEQ ID NO: 384. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 384. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 384. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 384.

[0145] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385 or that differs by one or two nucleotides from SEQ ID NO: 385. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 385. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 385. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 385.

[0146] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as (c) SEQ ID NO: 386 or that differs by one or two nucleotides from SEQ ID NO: 386. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 386. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 386. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 386.

[0147] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387 or that differs by one or two nucleotides from SEQ ID NO: 387. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 387. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 387. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 387.

[0148] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs by one or two nucleotides from SEQ ID NO: 300. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 300. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 300. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 300.

[0149] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs by one or two nucleotides from SEQ ID NO: 304. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 304. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 304. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 304.

[0150] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs by one or two nucleotides from SEQ ID NO: 308. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 308. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 308. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 308.

[0151] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs by one or two nucleotides from SEQ ID NO: 312. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 312. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 312. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 312.

[0152] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs by one or two nucleotides from SEQ ID NO: 320. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 320. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 320. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 320.

[0153] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs by one or two nucleotides from SEQ ID NO: 344. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 344. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 344. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 344.

[0154] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs by one or two nucleotides from SEQ ID NO: 348. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 348. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 348. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 348.

[0155] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs by one or two nucleotides from SEQ ID NO: 352. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 352. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 352. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 352.

[0156] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs by one or two nucleotides from SEQ ID NO: 356. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence or that differs by two nucleotides from SEQ ID NO: 356. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 356. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 356.

[0157] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs by one or two nucleotides from SEQ ID NO: 360. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence or that differs by two nucleotides from SEQ ID NO: 360. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 360. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 360.

[0158] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388 or that differs by one or two nucleotides from SEQ ID NO: 388. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 388. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 388. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 388.

[0159] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389 or that differs by one or two nucleotides from SEQ ID NO: 389. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 389. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 389. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 389.

[0160] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390 or that differs by one or two nucleotides from SEQ ID NO: 390. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 390. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 390. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 390.

[0161] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs by one or two nucleotides from SEQ ID NO: 324. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 324. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 324. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 324.

[0162] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs by one or two nucleotides from SEQ ID NO: 328. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 328. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 328. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 328.

[0163] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs by one or two nucleotides from SEQ ID NO: 332. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 332. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 332. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 332.

[0164] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs by one or two nucleotides from SEQ ID NO: 336. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 336. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 336. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 336.

[0165] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391 or that differs by one or two nucleotides from SEQ ID NO: 391. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 391. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 391. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 391.

[0166] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392 or that differs by one or two nucleotides from SEQ ID NO: 392. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 392. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 392. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 392.

[0167] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393 or that differs by one or two nucleotides from SEQ ID NO: 393. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 393. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 393. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 393.

[0168] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397 or that differs by one or two nucleotides from SEQ ID NO: 397. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 397. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 397. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 397.

[0169] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs by one or two nucleotides from SEQ ID NO: 465. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 465. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 465. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 465.

[0170] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs by one or two nucleotides from SEQ ID NO: 469. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 469. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 469. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 469.

[0171] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs by one or two nucleotides from SEQ ID NO: 473. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 473. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 473. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 473.

[0172] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs by one or two nucleotides from SEQ ID NO: 477. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 477. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 477. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 477.

[0173] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs by one or two nucleotides from SEQ ID NO: 481. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 481. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 481. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 481.

[0174] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs by one or two nucleotides from SEQ ID NO: 508. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO: 508. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO: 508. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO: 508.

[0175] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO:512 or that differs by one or two nucleotides from SEQ ID NO:512. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO:512. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO:512. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO:512.

[0176] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO:516 or that differs by one or two nucleotides from SEQ ID NO:516. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by two nucleotides from SEQ ID NO:516. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs by one nucleotide from SEQ ID NO:516. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence set forth in SEQ ID NO:516.

[0177] In some embodiments, the crRNA does not occur in nature. In some embodiments, the particular crRNA repeat sequence is not linked to an 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 does not occur in nature.

[0178] In some embodiments, the crRNA of the present disclosure comprises a chemical modification to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the crRNA. In certain embodiments, the crRNA of the present disclosure comprises at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, the crRNA of the present disclosure comprises at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the crRNA of the present disclosure comprises at least a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is a locked nucleic acid (LNA) modification, a BNA NCThe modification is selected from the group consisting of an [N-Me] modification, a 2'-O,4'-C-ethylene-bridged nucleic acid (2',4'-ENA) modification, and an S-restricted ethyl (cEt) modification. In certain embodiments, the crRNA of this disclosure contains at least one LNA modification. In some embodiments, the crRNA of this disclosure contains at least one 2'-O-Me modification. In some embodiments, the crRNA of this disclosure contains at least one MS modification. In some embodiments, the crRNA of this disclosure contains at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, the crRNA of this disclosure contains 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, the crRNA of this disclosure contains at least one BNA (e.g., LNA and / or cEt) modification and at least one PS modification. In some embodiments, the chemically modified crRNA has a 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, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 18.In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 19. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the crRNA, in the absence of chemical modifications, has at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, the crRNA, in the absence of chemical modifications, has at least 95% sequence identity to SEQ ID NO: 71. In some embodiments, the crRNA, in the absence of chemical modifications, has at least 100% sequence identity to SEQ ID NO: 71. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 74. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 72. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 95. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 95.In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 95. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 95. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 96. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, the crRNA, in the absence of chemical modifications, has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 96.

[0179] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 4.

[0180] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 5.

[0181] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 6.

[0182] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 7.

[0183] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 8.

[0184] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 9.

[0185] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 708. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 708. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 708. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 708.

[0186] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 292. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 292. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 292. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth as SEQ ID NO: 292, herein referred to as SEQ ID NO: 292.

[0187] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 293.

[0188] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 73.

[0189] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 74.

[0190] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 75.

[0191] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 301.

[0192] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 302.

[0193] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 303.

[0194] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 305.

[0195] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 306.

[0196] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 307.

[0197] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 309.

[0198] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 310.

[0199] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 311.

[0200] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 313.

[0201] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 314.

[0202] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 315.

[0203] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 321.

[0204] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 322.

[0205] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 323.

[0206] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 345.

[0207] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 346.

[0208] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 347.

[0209] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 349.

[0210] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 350.

[0211] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 351.

[0212] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 353.

[0213] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 354.

[0214] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 355.

[0215] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 357.

[0216] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 358.

[0217] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 359.

[0218] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 361.

[0219] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 362.

[0220] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 363.

[0221] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 97.

[0222] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 98.

[0223] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 99.

[0224] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 325.

[0225] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 326.

[0226] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 327.

[0227] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 329.

[0228] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 330.

[0229] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 331.

[0230] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 333.

[0231] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 334.

[0232] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 335.

[0233] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 337.

[0234] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 338.

[0235] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 339.

[0236] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 42.

[0237] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 43.

[0238] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 44.

[0239] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 380.

[0240] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 381.

[0241] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 382.

[0242] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 399. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 399. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 399. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 399.

[0243] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 400. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 400. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 400. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 400.

[0244] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 401. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 401. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 401. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 401.

[0245] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 466.

[0246] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 467.

[0247] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 468.

[0248] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 470.

[0249] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 471.

[0250] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 472.

[0251] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 474.

[0252] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 475.

[0253] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 476.

[0254] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 478.

[0255] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 479.

[0256] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 480.

[0257] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 482.

[0258] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 483.

[0259] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 484.

[0260] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 509.

[0261] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 510.

[0262] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 511.

[0263] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 513.

[0264] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 514.

[0265] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 515.

[0266] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 517.

[0267] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 518.

[0268] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, the chemically modified crRNA has the nucleotide sequence set forth in SEQ ID NO: 519.

[0269] Presently disclosed guide RNAs include crRNA and trans-activating CRISPR RNA (tracrRNA). A tracrRNA molecule comprises a nucleotide sequence containing a region, referred to herein as an anti-repeat, that is sufficiently complementary to hybridize to a crRNA repeat. In embodiments, a tracrRNA molecule further comprises a region having a secondary structure (e.g., a stem-loop). In some embodiments, the secondary structure comprises nucleotides that are in one of two states: paired or unpaired, and the nucleotides or base pairs include base-base hydrogen bonding interactions between two complementary nucleic acid strands to form a helix (e.g., adenine (A) pairs with uracil (U), and cytosine (C) pairs with guanine (G)). In some embodiments, a combination of one or more helical elements interspersed with unpaired single-stranded nucleotides constitutes the RNA structure.

[0270] As used herein, "stem-loop" refers to a secondary structural form found in polynucleotides that contains at least one "stem" and at least one "loop," "bulge," or "bubble." Stem-loops can form intramolecularly (within a single molecule, e.g., within a tracrRNA or sgRNA) or intermolecularly (e.g., between two different nucleic acids in a dgRNA, e.g., between a crRNA repeat of a crRNA and an anti-repeat of a tracrRNA). A stem-loop is formed when at least some complementarity exists between two nucleic acid sequences to form a paired double helix. The paired double helix region, which has perfect complementarity or sometimes contains a G:U wobble base pair (or I:U, I:A, or I:C, where I refers to inosine), is referred to as the "stem." The terms "loop," "bulge," or "bubble" refer to a single-stranded region within a "stem-loop" structure where there is no complementarity between nucleotides, except for G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine). Thus, "loops," "bulges," and "bubbles" comprise unpaired nucleotides. In some embodiments, a "loop" is distinguished from a "bulge" or "bubble" by being located at one end of the "stem-loop" structure, and a "bulge" or "bubble" is located between the two "stems" of the "stem-loop" structure.

[0271] In certain embodiments, the stem-loop structure comprises a stem and a loop at one end of the stem. In some embodiments, the stem-loop structure comprises a first stem and a second stem with a bubble between the stems. In some embodiments, the stem-loop structure comprises a loop, multiple stems, and multiple bubbles between the stems. In this context, the bubble closest to the loop is referred to as the "first bubble," "second bubble," "third bubble," etc., and the stem closest to the loop is referred to as the "first stem," "second stem," "third stem," etc. In dgRNA embodiments, the stem-loop formed by the crRNA repeats of the crRNA and the anti-repeats of the tracrRNA does not contain a loop, and therefore the bubbles closer to the 5' end of the tracrRNA (or the 3' end of the crRNA) are referred to as the "first bubble," "second bubble," "third bubble," etc., and the stems closer to the 5' end of the tracrRNA (or the 3' end of the crRNA) are referred to as the "first stem," "second stem," "third stem," etc.

[0272] The terms "first stem of the crRNA repeat of a crRNA," "first stem of the crRNA repeat," or "first stem of the crRNA" refer to the region within the crRNA repeat of a crRNA that forms the first stem of a stem-loop structure when hybridized with the anti-repeat of a tracrRNA. The terms "second stem of the crRNA repeat of a crRNA," "second stem of the crRNA repeat," or "second stem of the crRNA" refer to the region within the crRNA repeat of a crRNA that forms the second stem of a stem-loop structure when hybridized with the anti-repeat of a tracrRNA. Similarly, the terms "first stem of the anti-repeat of a tracrRNA," "first stem of the anti-repeat," or "first stem of the tracrRNA" refer to the region within the anti-repeat of a tracrRNA that forms the first stem of a stem-loop structure when hybridized with the crRNA repeat of a crRNA. The term "second stem of the anti-repeat of tracrRNA," "second stem of the anti-repeat," or "second stem of the tracrRNA" refers to a region within the anti-repeat of tracrRNA that forms the second stem of a stem-loop structure when hybridized with a crRNA repeat of the crRNA.

[0273] In some embodiments, the stem loop formed in the molecule is a hairpin stem loop. Base pairing occurs in the stem portion of the stem loop, typically involving guanine-cytosine and adenine-uracil (thymidine) pairing, although guanine-uracil pairing is also possible. Base-stacking interactions promote helix formation. The loop portion of the stem loop contains unpaired bases. In some embodiments, the loop is the point at which the nucleic acid strand turns back on itself due to nucleotide pairing to create the stem. In some embodiments, loops less than three bases long are sterically impossible and will not form. In some embodiments, the optimal loop length is about 4-8 bases. Common loops with four nucleotide sequences, such as GAAA, AAAG, ACUU, or UUCG, are known as "tetraloops" and are particularly stable due to the base-stacking interactions of their constituent nucleotides.

[0274] In some embodiments, the region of the tracrRNA that is fully or partially complementary to the crRNA repeats is at the 5' end of the molecule, and the 3' end of the tracrRNA contains secondary structure. This region of secondary structure generally contains several hairpin structures, including the nexus hairpin found adjacent to the anti-repeat. The nexus forms the core of interaction between the guide RNA and RGN and is located at the intersection between the guide RNA, RGN, and target sequence. The nexus hairpin often has a conserved nucleotide sequence at the base of the hairpin stem, with the motif UNANNC being found in many nexus hairpins in tracrRNA. In embodiments, the guide RNA or RGN system of the present disclosure uses tracrRNAs that contain non-canonical sequences at the base of the hairpin stem of their nexus hairpins, including UNANNG and CNANNC. In some embodiments, the guide RNA or RGN system of the present disclosure uses tracrRNAs that contain the non-canonical sequence UNANNG at the base of the nexus hairpin stem. In some embodiments, the guide RNA or RGN system of the present disclosure uses a tracrRNA that contains the non-canonical sequence CNANNC at the base of the hairpin stem of the nexus. The 3' end of the tracrRNA often contains a terminal hairpin that can vary in structure and number, but often contains a GC-rich Rho-independent transcription terminator hairpin followed by a string of U's at the 3' end. See, e.g., 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. Patent Application Publication No. 2017 / 0275648, each of which is incorporated by reference in its entirety.

[0275] In some embodiments, the tracrRNA of the present disclosure comprises an additional hairpin or stem-loop structure in addition to the nexus hairpin. In some embodiments, the tracrRNA comprises at least one stem-loop. In some embodiments, the tracrRNA comprises 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, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a region or end of a nucleic acid molecule. In certain embodiments, "proximal" refers to being within 1, 2, 3, 4, 5, or 6 nucleotides of a region or end of a nucleic acid molecule. "Most proximal" refers to being closest to a region or end of a nucleic acid molecule. For example, the stem-loop closest to the tail of the tracrRNA is the first stem-loop closest to the tail of the tracrRNA. "Distal" refers to being at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from a region or end of a nucleic acid molecule. In some embodiments, "distal" refers to being at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides away from a structure of a nucleic acid molecule (e.g., a bubble or loop). For example, the first stem of the anti-repeat of the dual guide RNA, extended at the distal end of the first bubble of the stem-loop, extends from the 3'-terminal nucleotide of the crRNA and the 5'-terminal nucleotide of the tracrRNA. The tracrRNA also forms a secondary structure when hybridized with its corresponding crRNA. The anti-repeat region of the tracrRNA is fully or partially complementary to the crRNA repeat of the crRNA.In some embodiments, a portion of the tracrRNA anti-repeat and a portion of the crRNA repeat hybridize and form a stem. In some embodiments, the crRNA:tracrRNA stem comprises 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, the portion of the tracrRNA anti-repeat that forms the first stem is the first stem of the anti-repeat, the portion of the tracrRNA anti-repeat that forms the second stem is the second stem of the anti-repeat, the portion of the tracrRNA anti-repeat that forms the third stem is the third stem of the anti-repeat, etc. As described elsewhere herein, the portion of the crRNA repeat of the crRNA that forms the first stem is the first stem of the crRNA repeat, the portion of the crRNA repeat of the second stem is the second stem of the crRNA repeat, the portion of the crRNA repeat of the third stem is the third stem of the crRNA repeat, etc. In some embodiments, the portion of the anti-repeat of the tracrRNA and the portion of the crRNA repeat are not complementary to each other and therefore do not hybridize to form base pairs. In some embodiments, the non-complementary region between the anti-repeat and the crRNA repeat forms a bulge or bubble. In some embodiments, hybridization of the anti-repeat of the tracrRNA with the crRNA repeat of the crRNA forms a secondary structure comprising at least one stem. In some embodiments, hybridization of the anti-repeat of the tracrRNA with the crRNA repeat of the crRNA forms a secondary structure comprising at least one bubble. In some embodiments, hybridization of the anti-repeats of the tracrRNA with the crRNA repeats of the crRNA forms a secondary structure comprising at least one stem and at least one bubble.In some embodiments, hybridization of the anti-repeat of the tracrRNA with the crRNA repeat of the crRNA forms a secondary structure comprising two stems with a bubble between them.

[0276] In certain embodiments, the stem-loop in the gRNA formed by only a portion of the tracrRNA does not contain a BNA (e.g., LNA and / or cEt) modification. In some embodiments, the stem-loop in the gRNA formed by only a portion of the tracrRNA does not contain a chemical modification.

[0277] In certain embodiments, the nucleotides in the loop, bulge, or bubble do not comprise a BNA (e.g., LNA) modification. In some embodiments, the nucleotides in the loop, bulge, or bubble do not comprise a chemical modification.

[0278] In some embodiments, the anti-repeat of the tracrRNA, which is fully or partially complementary to the crRNA repeat, comprises from about 8 nucleotides to about 30 or more nucleotides. For example, the stem formed by the tracrRNA anti-repeat and crRNA repeat can be about 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 stem formed by the tracrRNA anti-repeat and 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 is 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 when optimally aligned using a suitable alignment algorithm. In some embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat 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 when optimally aligned using a suitable alignment algorithm.

[0279] 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 60-80 nucleotides, 80-100 nucleotides, 100-120 nucleotides, 120-140 nucleotides, 140-160 nucleotides, 160-180 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 between 70 and 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.

[0280] In some embodiments, a tracrRNA of the present disclosure comprises a chemical modification 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 present disclosure comprises at least one BNA (e.g., LNA and / or cEt) modification. In certain embodiments, a tracrRNA of the present disclosure comprises 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 a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; and a 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 a locked nucleic acid (LNA) modification, a BNA NCThe BNA modification is selected from the group consisting of an [N-Me] modification, a 2'-O,4'-C-ethylene-bridged nucleic acid (2',4'-ENA) modification, and an S-restricted 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 a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; and a 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 a 2'-O-methyl (2'-O-Me) modification, a 2'-O-methoxyethyl (2'MOE) modification, a 2'-fluoro (2'-F) modification, a 2'F-4'Cα-OMe modification, a 2',4'-di-Cα-OMe modification, a 2'-O-methyl 3' phosphorothioate (MS) modification, a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification, a 2'-O-methyl 3' phosphonoacetate (MP) modification, and a phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one PS modification.

[0281] 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, which, when contained within a guide RNA, can direct sequence-specific binding of the associated RNA-guided nuclease provided herein to the presently disclosed target sequences. 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, the 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.

[0282] In some embodiments, the tracrRNA contains at least one chemical modification in its 5' region or its 3' region. In some embodiments, the tracrRNA contains at least one chemical modification in both its 5' region and 3' region. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 100. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 100. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 242.In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 242. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 242. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 242. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 254. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 254. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 254. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 254. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 539. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 539. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 539. In some embodiments, the tracrRNA without chemical modifications has a nucleotide sequence that has at least 100% sequence identity to SEQ ID NO: 539.

[0283] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 10.

[0284] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 12.

[0285] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 709. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 709. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 709. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 709.

[0286] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 713. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 713. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 713. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 713.

[0287] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 294. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 294. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 294. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 294.

[0288] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 295. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 295. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 295. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 295.

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

[0290] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 81. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 81. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 81. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 81.

[0291] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 364.

[0292] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 365.

[0293] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 366.

[0294] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 367.

[0295] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 369.

[0296] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 375.

[0297] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 376.

[0298] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 377.

[0299] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 378.

[0300] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 379.

[0301] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 102. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 102. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 102.

[0302] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 103. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 103. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 103.

[0303] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 370.

[0304] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 371.

[0305] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 372.

[0306] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 373.

[0307] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 710.

[0308] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 711.

[0309] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 51.

[0310] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 52.

[0311] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 53.

[0312] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 383.

[0313] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 499.

[0314] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 500.

[0315] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 501.

[0316] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 504.

[0317] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 505.

[0318] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 534.

[0319] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 535.

[0320] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence set forth in SEQ ID NO: 537.

[0321] The term "derived from," as used herein in reference to a polynucleotide molecule, refers to a molecule generated or synthesized using information from a parent molecule or its parent molecule. For example, a tracrRNA, gRNA, or crRNA of the present disclosure that includes at least one BNA (e.g., LNA) modification is derived from its respective unmodified tracrRNA, gRNA, or crRNA by modifying at least one of its nucleotides with a BNA (e.g., LNA) modification. In some embodiments, a tracrRNA, gRNA, or crRNA that includes 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.

[0322] Two polynucleotide sequences can be considered substantially complementary if they hybridize to each other under stringent conditions. Similarly, an RGN is considered to bind to a particular target sequence in a sequence-specific manner if a guide RNA bound to the RGN binds to the target sequence under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which two polynucleotide sequences hybridize to each other to a detectably greater extent than other sequences (e.g., at least twice background). Stringent conditions are sequence-dependent and will vary in different circumstances. Typically, stringent conditions are conditions in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salt), 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., more than 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate) at 37°C, followed by washing in 1X-2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50-55°C. Exemplary medium stringency conditions include hybridization with 40-45% formamide, 1.0 M NaCl, and 1% SDS at 37°C, followed by washing in 0.5X-1X SSC at 55-60°C. Exemplary high stringency conditions include hybridization with 50% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by washing in 0.1X SSC at 60-65°C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash period is at least long enough to reach equilibrium.

[0323] 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, Tm can be approximated by the formula of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% GC) - 500 / L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, % GC is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a specific sequence and its complement at a defined ionic strength and pH. However, highly stringent conditions can utilize hybridization and / or washing at 1, 2, 3, or 4° C. below the thermal melting point (Tm). Moderately stringent conditions can utilize hybridization and / or washing at 6, 7, 8, 9, or 10° C. below the thermal melting point (Tm). Low stringency conditions can utilize hybridization and / or washing at 11, 12, 13, 14, 15, or 20° C. below the thermal melting point (Tm). Using the formula, hybridization and washing composition, and desired Tm, one of skill in the art will understand that variations in stringency of hybridization and / or washing solutions are essentially described. Extensive guides to nucleic acid hybridization are 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 (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0324] The term "sequence-specific" can also refer to binding of an RGN polypeptide to a target sequence with higher affinity than the binding to a randomized background sequence.

[0325] The guide RNA can be a single guide RNA (sgRNA) or a dual guide RNA (dgRNA). An sgRNA comprises a crRNA and a tracrRNA on a single molecule of RNA, while a dgRNA comprises a crRNA and a tracrRNA present on two different RNA molecules hybridized to each other via at least a portion of the crRNA repeat of the crRNA and at least a portion of the anti-repeat of the tracrRNA, which may be fully or partially complementary to each other. Hybridization of the anti-repeat of the tracrRNA with the crRNA repeat of the crRNA forms a stem-loop comprising the anti-repeat and the crRNA repeat. In some embodiments, the stem-loop comprises one or more stems formed by the anti-repeat and the crRNA repeat. In some embodiments, the guide RNA is an sgRNA, and the crRNA and tracrRNA are separated by a linker nucleotide sequence. Generally, the linker nucleotide sequence does not contain bases complementary to itself or to other portions of the sgRNA to avoid the formation of secondary structures within or involving the nucleotides of the linker nucleotide sequence. In certain embodiments, the linker forms a loop at one end of the first stem in a stem-loop structure comprising the crRNA repeat and anti-repeat. In some embodiments, the linker nucleotide sequence between the crRNA and the 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 the sgRNA is at least 4 nucleotides in length. In certain embodiments, the linker nucleotide sequence comprises a nucleotide sequence represented as any of AAAG, GAAA, ACUU, and CAAAGG.

[0326] The total length of the guide RNA may be 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 the 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, or more. t, 123nt, 124nt, 125nt, 126nt, 127nt, 128nt, 129nt, 130nt, 131nt, 132nt, 133nt, 134nt, 135nt, 136nt, 137nt, 138nt, 139nt, 140nt, 141nt, 142nt, 143nt, 144nt, 145nt, 146nt, 147nt, 148nt, 14 9nt, 150nt, 151nt, 152nt, 153nt, 154nt, 155nt, 156nt, 157nt, 158nt, 159nt, 160nt, 161nt, 162n t, 163nt, 164nt, 165nt, 166nt, 167nt, 168nt, 169nt, 170nt, 171nt, 172nt, 173nt, 174nt, 175nt, 176nt, 177nt, 178nt, 179nt, 180nt, 181nt, 182nt, 183nt, 184nt, 185nt, 186nt, 187nt, 188nt, 189nt, 190nt, 191nt, 192nt, 193nt, 194nt, 195nt, 196nt, 197nt, 198nt, 199nt, 200nt or more.

[0327] In some embodiments, the 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.

[0328] sgRNA or dgRNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between RGN and guide RNA are known in the art and include, but are not limited to, in vitro binding assays between expressed RGN and guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in pull-down detection assays in which the guide RNA:RGN complex is captured via a detectable label (e.g., streptavidin beads). A control guide RNA with a sequence or structure unrelated to the guide RNA can be used as a negative control for non-specific binding of RGN to RNA.

[0329] In some embodiments, the guide RNA can be introduced into the target cell, organelle, or embryo as an RNA molecule. The guide RNA can be chemically synthesized.

[0330] In embodiments, the guide RNA may 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.

[0331] The guide RNA directs the associated RGN to a specific 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 the RGN in vitro or in cells. The target sequence can comprise DNA, RNA, or a combination of both, and can be single-stranded or double-stranded. In some embodiments, the 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 embodiments where the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plastid, or mitochondrial chromosomal sequence. In the compositions and methods of the present disclosure, 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 includes a target strand and a non-target strand, and the target sequence (i.e., the sequence on the non-target strand) has a nucleotide sequence set forth as any of SEQ ID NOs: 273-278 and 712.

[0332] The target sequence is adjacent to a protospacer adjacent motif (PAM), and the non-target strand of the target sequence is the strand containing the PAM. The PAM is immediately adjacent to the target sequence and often includes Ns, which represent any nucleotide. In some embodiments, the PAM includes 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, the PAM includes 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 the non-target strand. In some embodiments, the PAM is 3' of the target sequence on the non-target strand of the guide RNA and RGN systems of the present disclosure. 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.

[0333] In some embodiments, the PAM sequence adjacent to the presently disclosed target sequence on the non-target strand comprises a consensus sequence set forth as any one of the PAM sequences in Table 1. In some embodiments, the PAM sequence adjacent to the presently disclosed target sequence on the non-target strand comprises a consensus sequence set forth as any one of NNNNCC, NNGRR, NNRYA, and NGG. In some embodiments, the PAM sequence is 3' to the target sequence on the non-target strand.

[0334] It is well known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (e.g., Karvelis et al. (2015) Genome Biol 16:253), which can be modified by modifying the promoter used to express RGN or the amount of ribonucleoprotein complex delivered to a cell, organelle, or embryo.

[0335] Upon recognizing its corresponding PAM sequence, an RGN can cleave one or both strands of a target sequence at a specific cleavage site. As used herein, a cleavage site is comprised of two specific nucleotides within a target sequence between which the RGN cleaves the target strand, the non-target strand, or both strands of the target sequence. The cleavage site may include the first and second, second and third, third and fourth, fourth and fifth, fifth and sixth, seventh and eighth, or eighth and ninth nucleotides from the PAM in either the 5' or 3' direction. In embodiments, the cleavage site may be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction. Because an RGN can cleave a target sequence and shift its terminus, in embodiments, the cleavage site is defined based on a distance of two nucleotides from the PAM on the non-target strand of the target sequence and a distance of two nucleotides from the complement of the PAM on the target strand.

[0336] III. Chemical and length modifications to guide RNA Nucleotides of a crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA of the present disclosure may, in some embodiments, comprise at least one BNA (e.g., LNA) modification. In some embodiments, 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 that comprises at least one BNA (e.g., LNA) modification in the first stem of the anti-repeat of the tracrRNA. Nucleotides of a crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA of the present disclosure may, in some embodiments, comprise a modification in a ribose (e.g., sugar) group, a phosphate group, a nucleobase, or any combination thereof. The term "chemical modification" in the context of an oligonucleotide or polynucleotide includes, but is not limited to, (a) terminal modifications, such as 5'-end modifications or 3'-end modifications; (b) nucleobase (or "base") modifications, including base substitution or removal; (c) sugar modifications, including modifications at the 2', 3', and / or 4' positions; and (d) backbone modifications, including modification or replacement of a phosphodiester bond. The term "modified nucleotide" generally refers to a nucleotide having modifications to one or more chemical structures of the phosphodiester bond or backbone moiety, including the base, sugar, and nucleotide phosphate. The terms "modification" and "chemical modification" are used interchangeably herein.

[0337] In some embodiments, the modified nucleotide comprises a sugar modification, non-limiting examples of which include 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotides (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy 2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine-5'-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine-5'-triphosphate, 2'-aruridine-5'-triphosphate), azidotriphosphates (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate), and combinations thereof.

[0338] In some embodiments, a modified molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the present disclosure comprises one or more 2'-fluoro, 2'-amino, and / or 2'-thio modifications. In some embodiments, the modifications are 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, ribothymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and / or 5-fluoro-uridine.

[0339] There are over 96 naturally occurring nucleoside modifications found in mammalian RNA. See, for example, Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994). A nucleoside comprises a purine or pyrimidine base linked to a sugar (i.e., a nucleotide without a phosphate group). The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art and is described, for example, in U.S. Pat. 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. Many modified nucleosides and modified nucleotides suitable for use in the present disclosure are commercially available. The nucleoside can be an analog of a naturally occurring nucleoside. In some embodiments, the nucleoside analog includes dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxycytidine, and deoxyuridine.

[0340] In some cases, modified molecules or regions (e.g., crRNA, crRNA repeats, spacers, tracrRNA, anti-repeats, or guide RNAs) of the present disclosure comprise nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase in place of a naturally occurring nucleobase. Non-limiting examples of modified nucleobases that can be incorporated into modified nucleosides and nucleotides include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine), m2A (2-methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2-methylthio-N6-methyladenosine), i6A ( N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycinylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine) m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxynorvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine), Ar(p) (2'-O-ribosyladenosine (phosphate)), I (inosine), mIl (1-methylinosine), m'Im (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2T-O- Methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), f5C (5-phenylcytidine), m5Cm (5,2-O-dimethylcytidine), ac4Cm (N4-acetyl-2TOmethylcytidine), k2C (lysidine), m1G (1-methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-O-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* (unmodified hydroxywybutosine), imG (wybutosine), mimG (methylguanosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deoxy-queuosine), azaguanosine), 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-hydroxyacetic acid), mcmo5U (uridine 5-hydroxyacetic acid methylester) ester), chm5U (5-(carboxyhydroxymethyl)uridine)), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5s2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5 se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine), 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 (N6,0-dimethyladenosine), rn62Am (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'-O-methylcytidine), mlGm (l,2'-O-dimethylguanosine), m'Am (1,2-O-dimethyladenosine), tm5s2U (S-taurinomethyl-2-thiouridine), imG-14 (4-demethylguanosine), i mG2 (isoguanosine), or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6) -Alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Ci-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N, 2 -dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)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-deazapurines, 7-deaza-7-substituted purines, 7-deaza-8-substituted purines, and combinations thereof.

[0341] In some embodiments, modified molecules or regions (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the present disclosure comprise one or more modifications to the phosphate backbone. Modifications can include one or more of phosphorothioate, phosphorodithioate, phosphoramidate (e.g., N3'-P5'-phosphoramidate (NP)), and / or methylphosphonate linkages. In some embodiments, backbone modifications include neutral backbone modifications, 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.

[0342] In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) comprise a modification 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, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) comprise a 2'-O-methyl (2'-O-Me) modification ("B" represents "base" in the chemical structures herein): [ka]

[0343] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a 2'-O-methoxy-ethyl (2'-MOE) modification: [ka]

[0344] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a 2'-fluoro (2'-F) modification: [ka]

[0345] In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprise modifications at the 2' and 4' positions of the ribose sugar. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprise a 2'F-4'Cα-OMe modification: [ka]

[0346] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a 2',4'-diCα-OMe modification: [ka]

[0347] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a phosphorothioate (PS) modification (e.g., in the backbone): [ka]

[0348] In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) comprise a modification at the 2' position of the ribose sugar and phosphate backbone. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) comprise a 2'-O-methyl 3'-phosphorothioate (MS) modification: [ka]

[0349] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a 2'-O-methyl 3' thiophosphonoacetate (MSP; 2'-O-methyl 3' thioPACE) modification: [ka]

[0350] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the disclosure include a 2'-O-methyl 3' phosphonoacetate (MP) modification: [ka]

[0351] In certain embodiments, one or more modifications of a molecule or region (e.g., crRNA, crRNA repeat, spacer, tracrRNA, anti-repeat, or guide RNA) of the present disclosure include a 2'-O-methyl (2'-O-Me) modification, a 2'-O-methoxyethyl (2'MOE) modification, a 2'-fluoro (2'-F) modification, a 2'F-4'Cα-OMe modification, a 2',4'-di-Cα-OMe modification, a 2'-O-methyl 3' phosphorothioate (MS) modification, a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification, a 2'-O-methyl 3' phosphonoacetate (MP) modification, a phosphorothioate (PS) modification, and a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, a modified molecule or region of the present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises 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 present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises 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 present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises 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 present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises 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 present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises one or more MS modifications and one or more LNA modifications. In some embodiments, a modified molecule or region of the present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises one or more MS modifications and one or more cEt modifications.In certain embodiments, a modified molecule or region of the present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises one or more PS modifications and one or more LNA modifications. In certain embodiments, a modified molecule or region of the present disclosure (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) comprises one or more PS modifications and one or more cEt modifications.

[0352] In some embodiments, the modification comprises a bridged nucleic acid (BNA) modification. The term "bridged nucleic acid" refers to a nucleic acid having a structure in which the degrees of freedom of the nucleic acid are restricted by an intramolecular bond or bridge. In certain embodiments, the BNA modification comprises a 2',4' BNA modification. In some embodiments, the 2' oxygen and 4' carbon of the ribose are linked via a "bridge."

[0353] First generation BNA modifications include locked nucleic acid (LNA) modifications, which 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 A-type helix preference (You et al. (2006) Nucleic Acids Res 34(8):e60, shown as follows: [ka]

[0354] LNAs exhibit 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(1):439-44...

Claims

1. A nucleic acid molecule comprising a trans-activating CRISPR RNA (tracrRNA), wherein the tracrRNA is (a) anti-repeat; (b) a tail; and (c) the stem loop most proximal to the tail Including, A nucleic acid molecule, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and the tracrRNA comprises at least one bridge 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. 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. 4. The nucleic acid molecule of claim 3, wherein the at least one BNA modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 BNA modifications on consecutive nucleotides or at least 2, 3, 4, 5, 6, or 7 BNA modifications on alternating nucleotides within the first stem of the anti-repeat.

5. 5. The nucleic acid molecule of claim 3 or 4, wherein all nucleotides in the first stem of the anti-repeat contain a BNA modification.

6. 3. 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. 7. The nucleic acid molecule of claim 1, wherein the at least one BNA modification is not within the bulge of the tracrRNA.

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

9. 8. The nucleic acid molecule of claim 1, wherein the three terminal nucleotides of the tail of the tracrRNA comprise both BNA and phosphorothioate (PS) modifications.

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

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

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

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

14. The nucleic acid molecule of any one of claims 1 to 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. 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. 18. The nucleic acid molecule of any one of claims 14 to 17, wherein the at least one other chemical modification is selected from the group consisting of a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a 2'-fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; and a phosphorothioate (PS) modification.

19. 19. The nucleic acid molecule of Claim 18, wherein the three terminal nucleotides of the tail of the tracrRNA comprise an MS modification.

20. 19. The nucleic acid molecule of Claim 18, wherein the 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. 21. The nucleic acid molecule of claim 20, wherein the BNA modification comprises an LNA modification.

22. 21. The nucleic acid molecule of claim 20, wherein the BNA modification comprises a cEt modification.

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

24. 23. The nucleic acid molecule of any one of claims 1 to 22, wherein 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.

25. 23. The nucleic acid molecule of any one of claims 1 to 22, wherein the first stem of the anti-repeat comprises a total length of about 11 nucleotides.

26. 23. The nucleic acid molecule of any one of claims 1 to 22, wherein the first stem of the anti-repeat comprises an overall length of 6 to 15 nucleotides, 8 to 13 nucleotides, or 10 to 12 nucleotides.

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

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

29. 29. The nucleic acid molecule of any one of claims 1 to 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. 30. The nucleic acid molecule of any one of claims 1 to 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. 30. The nucleic acid molecule of any one of claims 1 to 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. 30. The nucleic acid molecule of any one of claims 1 to 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. 30. The nucleic acid molecule of any one of claims 1 to 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. 34. The nucleic acid molecule of any one of claims 1 to 33, wherein the tracrRNA is part of a gRNA that can bind to an RGN.

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

36. 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. 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:

69.

38. 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:

93.

39. 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:

252.

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

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

42. 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. 41. The gRNA of Claim 40, wherein the gRNA is a dual guide RNA (dgRNA).

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

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

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

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

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

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

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

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

52. 52. The gRNA of Claim 51, wherein the at least one BNA modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 BNA modifications on consecutive nucleotides or at least 2, 3, 4, 5, 6, or 7 BNA modifications on alternating nucleotides in the first stem of the anti-repeat.

53. 53. The gRNA of Claim 51 or 52, wherein all nucleotides in the first stem of the anti-repeat comprise a BNA modification.

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

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

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

57. 57. The gRNA of Claim 56, wherein the three terminal nucleotides of the 3' region of the tail of the tracrRNA comprise BNA modifications.

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

59. 59. The gRNA of any one of claims 40-58, wherein at least the 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. 60. The gRNA of any one of claims 40-59, wherein all nucleotides in the first stem of the crRNA repeat lack a chemical modification and all nucleotides in the first stem of the anti-repeat comprise a BNA modification.

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

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

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

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

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

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

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

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

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

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

71. 70. The gRNA of Claim 68 or 69, wherein the at least one other modification is in the 5' and 3' regions of the crRNA.

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

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

75. 69. The gRNA of Claim 68, wherein the at least one other chemical modification is in the tracrRNA.

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

77. 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. 76. The gRNA of Claim 75, wherein the at least one other chemical modification is in the tail of the tracrRNA.

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

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

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

82. 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 an overall length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

83. 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 an overall length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

84. 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 about 11 nucleotides.

85. 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 an overall length of 6 to 15 nucleotides, 8 to 13 nucleotides, or 10 to 12 nucleotides.

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

87. 87. The gRNA of Claim 86, wherein the first stem of the crRNA repeat of the 3' region or the first stem of the anti-repeat of the 5' region comprises a GC-rich nucleotide sequence, and wherein the first stem of the crRNA repeat of the 3' region or the first stem of the anti-repeat of the 5' region comprises at least 2, at least 3, at least 4, or at least 5 G or C.

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

89. the crRNA repeats are (a) set forth as SEQ ID NO: 39, or which differs by one or two nucleotides from SEQ ID NO: 39; (b) set forth as SEQ ID NO: 384, or differing by one or two nucleotides from SEQ ID NO: 384; (c) set forth as SEQ ID NO: 385, or differing by one or two nucleotides from SEQ ID NO: 385; (d) set forth as SEQ ID NO: 386, or differs by one or two nucleotides from SEQ ID NO: 386; (e) set forth as SEQ ID NO: 387, or differing by one or two nucleotides from SEQ ID NO: 387; or (f) set forth as SEQ ID NO: 397, or differing by one or two nucleotides from SEQ ID NO: 397; 89. The gRNA of any one of claims 40 to 88, having a nucleotide sequence.

90. 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. 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 crRNA repeats are (a) set forth as SEQ ID NO: 300, or which differs by one or two nucleotides from SEQ ID NO: 300; (b) set forth as SEQ ID NO: 304, or differing by one or two nucleotides from SEQ ID NO: 304; (c) set forth as SEQ ID NO: 308, or differing by one or two nucleotides from SEQ ID NO: 308; (d) set forth as SEQ ID NO: 312, or differing by one or two nucleotides from SEQ ID NO: 312; (e) set forth as SEQ ID NO: 320, or differing by one or two nucleotides from SEQ ID NO: 320; (f) set forth as SEQ ID NO: 344, or differing by one or two nucleotides from SEQ ID NO: 344; (g) set forth as SEQ ID NO: 348, or differing by one or two nucleotides from SEQ ID NO: 348; (h) set forth as SEQ ID NO: 352, or differing by one or two nucleotides from SEQ ID NO: 352; (i) set forth as SEQ ID NO: 356, or differing by one or two nucleotides from SEQ ID NO: 356; (j) set forth as SEQ ID NO: 360, or differing by one or two nucleotides from SEQ ID NO: 360; (k) set forth as SEQ ID NO: 388, or differing by one or two nucleotides from SEQ ID NO: 388; (l) set forth as SEQ ID NO: 389, or differing by one or two nucleotides from SEQ ID NO: 389; or (m) set forth as SEQ ID NO: 390, or differing by one or two nucleotides from SEQ ID NO: 390; 89. The gRNA of any one of claims 40 to 88, having a nucleotide sequence.

93. 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. 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 crRNA repeats are (a) set forth as SEQ ID NO: 324, or which differs by one or two nucleotides from SEQ ID NO: 324; (b) set forth as SEQ ID NO: 328, or differing by one or two nucleotides from SEQ ID NO: 328; (c) set forth as SEQ ID NO: 332, or differing by one or two nucleotides from SEQ ID NO: 332; (d) set forth as SEQ ID NO: 336, or differs by one or two nucleotides from SEQ ID NO: 336; (e) set forth as SEQ ID NO: 391, or differing by one or two nucleotides from SEQ ID NO: 391; (f) set forth as SEQ ID NO: 392 or differing by one or two nucleotides from SEQ ID NO: 392; and (g) set forth as SEQ ID NO: 393, or differing by one or two nucleotides from SEQ ID NO: 393; The gRNA of any one of claims 40 to 88, having any one of the nucleotide sequences:

96. 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. 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 crRNA repeats are (a) set forth as SEQ ID NO: 465, or which differs by one or two nucleotides from SEQ ID NO: 465; (b) set forth as SEQ ID NO: 469, or differing by one or two nucleotides from SEQ ID NO: 469; (c) set forth as SEQ ID NO: 473, or differing by one or two nucleotides from SEQ ID NO: 473; (d) set forth as SEQ ID NO: 477, or differs by one or two nucleotides from SEQ ID NO: 477; (e) set forth as SEQ ID NO: 481, or differing by one or two nucleotides from SEQ ID NO: 481; (f) set forth as SEQ ID NO: 508, or differing by one or two nucleotides from SEQ ID NO: 508; (g) set forth as SEQ ID NO: 512 or differing by one or two nucleotides from SEQ ID NO: 512; and (h) set forth as SEQ ID NO: 516, or differing by one or two nucleotides from SEQ ID NO: 516; The gRNA of any one of claims 40 to 88, having any one of the nucleotide sequences:

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

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

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

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

105. 105. The gRNA of Claim 103 or 104, wherein the linker has a nucleotide sequence shown as AAAG.

106. The gRNA of any one of claims 103 to 105, wherein the gRNA is an sgRNA comprising the crRNA and the tracrRNA, the sgRNA comprising a backbone and the spacer, and the backbone of the sgRNA comprising the crRNA repeats, the linker, and the tracrRNA.

107. 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. 107. The gRNA of claim 106, wherein the 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.

109. The gRNA of any one of claims 40 to 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. 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. 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:

69.

113. 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:

93.

114. 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:

252.

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

116. (a) a spacer; and (b) crRNA repeat A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: The crRNA repeat can hybridize 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 the crRNA comprises at least one chemical modification, the at least one chemical modification being a 2'-O-methyl (2'-O-Me) modification; a 2'-O-methoxyethyl (2'MOE) modification; a fluoro (2'-F) modification; a 2'F-4'Cα-OMe modification; a 2',4'-di-Cα-OMe modification; a 2'-O-methyl 3' phosphorothioate (MS) modification; a 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; a 2'-O-methyl 3' phosphonoacetate (MP) modification; a phosphorothioate (PS) modification; and a BNA modification, and the at least one chemical modification is within the three terminal nucleotides of the 5' region or the 3' region of the crRNA. Nucleic acid molecule.

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

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

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

120. 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, the RGN system comprising: a) a gRNA according to any one of claims 40 to 115; and b) a type II RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding said type II RGN polypeptide An RNA-guided nuclease (RGN) system comprising:

122. The RGN system of any one of claims 118 to 121, wherein the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence designated as NNNNCC, NNGRR, NNRYA, or NGG.

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

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

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

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

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

129. The RGN system of any one of claims 126 to 128, wherein the target sequence is intracellular.

130. The RGN system of any one of claims 126 to 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-strand break.

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

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

135. The RGN system of any one of claims 118 to 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 to 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 to 139, wherein the gRNA further comprises an extension comprising an editing template for prime editing.

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

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

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

144. The RGN system of any one of claims 118 to 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 to 142, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is in a vector.

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

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

148. 148. The cell of claim 147, comprising a target sequence that can be bound by the formed crRNA / tracrRNA / RGN polypeptide or gRNA / RGN polypeptide complex of the RGN system of any one of claims 118 to 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 to 149, wherein the cell is a prokaryotic cell.

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

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

155. 155. A plant comprising the cell of claim 154.

156. 155. A seed comprising the cell of claim 154.

157. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tracrRNA according to any one of claims 1 to 39, a gRNA according to any one of claims 40 to 115, a crRNA according to claims 116 or 117, an RGN system according to any one of claims 118 to 145, an RNP complex according to claim 146, or a cell according to any one of claims 147 to 153.

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

159. 159. The method of claim 158, wherein the RGN polypeptide or the gRNA further comprises a detectable label, thereby enabling 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 regulating expression of a target gene comprising the target sequence.

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

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

163. 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 to 162, wherein the RGN is fused to a prime editing polypeptide.

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

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

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

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

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

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

171. 171. The method of Claim 170, wherein the formed complex of the gRNA and the type II RGN directs cleavage of the target sequence.

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

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

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

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

176. 171. 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. 171. The method of claim 170, wherein the polynucleotide encoding the type II RGN is mRNA.

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

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

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

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

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

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

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

188. 188. The method of claim 187, wherein the formed complex of the 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. 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. 192. The method of Claim 191, wherein the prime editing polypeptide comprises a DNA polymerase.

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

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

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

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

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

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

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

200. 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. 147. A method for increasing the efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system described in any one of claims 118 to 145 or an RNP complex described in claim 146 to the target sequence or a cell comprising the target sequence, wherein the cleavage or modification of the nucleic acid molecule occurs with a higher efficiency compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering a reference RGN system or RNP complex to the target sequence or a cell comprising the target sequence, and wherein the tracrRNA, gRNA, or 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 to 145 or the RNP complex of claim 146 comprises a BNA modification.

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

204. 202. The method of claim 201, wherein the BNA modification comprises an LNA modification.

205. 202. The method of claim 201, wherein the BNA modification comprises a cEt modification.

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

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

208. 208. The method of claim 207, wherein said 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.

209. 1. A method for manipulating gRNA, 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; 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, the one or more added or substituted nucleotides comprise at least 2, at least 3, at least 4, or at least 5 G or C, and the engineered gRNA has increased editing efficiency compared to the gRNA provided in step a); A method comprising:

210. 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. 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 the 5' region of the anti-repeat, and the 3' region of the crRNA repeat and the 5' region of the anti-repeat contain at least 2, at least 3, at least 4, or at least 5 G or C.

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

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

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

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

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

217. 216. The method of claim 215, wherein the at least one chemical modification is in the first stem of the crRNA repeat; the first stem of the anti-repeat; the 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. 218. The method of claim 217, wherein the at least one chemical modification is in the first stem of the anti-repeat.

219. 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. 219. 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. 219. The method of claim 218, wherein said at least one chemical modification is on alternating nucleotides in said first stem of said anti-repeat.

222. 219. The method of claim 218, wherein said at least one chemical modification is on every nucleotide in said first stem of said anti-repeat.

223. 223. The method of Claim 222, wherein the at least one chemical modification is on every nucleotide of the first stem of the anti-repeat and on the three terminal nucleotides of the 3' region of the tail of the tracrRNA.

224. 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 at least one nucleotide in the first stem of the crRNA repeat.

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

226. 223. The method of Claim 222, wherein the at least one chemical modification is on every nucleotide of the first stem of the anti-repeat, on at least the three terminal nucleotides of the 3' region of the first stem of the crRNA repeat, and on the three terminal nucleotides of the 3' region of the tail of the tracrRNA.

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

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

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

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

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

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

233. 233. The method of any one of claims 209 to 232, wherein the efficiency of cleaving and / or modifying a target sequence by an RGN system comprising said engineered gRNA is increased by 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).

234. 234. The method of claim 233, wherein the efficiency is determined by measuring the percentage of cells containing the target sequence or the target sequence in which expression of the target sequence or the polypeptide encoded by the target sequence is altered.

235. 235. The method of claim 234, wherein said 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.

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

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

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

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

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