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

Abstract

The present invention provides compositions comprising an engineered and / or chemically modified trans-activated CRISPR RNA (tracrRNA), a guide RNA (gRNA), and / or a CRISPR RNA (crRNA). The chemically modified tracrRNA, gRNA and / or crRNA incorporates bridged nucleic acid (BNA) modifications and other chemical modifications. BNA modification includes 2 ', 4' locked nucleic acid (LNA) modification. Other 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 tracrRNAs, gRNAs, and / or crRNAs of the present disclosure increase the efficiency of gene editing, including base editing and leader editing, of RNA-guided nuclease (RGN) systems.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 373,498, filed on August 25, 2022; U.S. Provisional Application No. 63 / 385,887, filed on December 2, 2022; and U.S. Provisional Application No. 63 / 517,703, filed on August 4, 2023, each of which is hereby incorporated by reference in its entirety.

[0003] Reference to a sequence listing electronically submitted as an XML file

[0004] This application contains a sequence listing that has been submitted in xml format through the USPTO Patent Center, and this document is hereby incorporated by reference in its entirety. The xml copy was created on August 24, 2023, named L103438_1290WO_0235_5_Sequence Listing, and is 2.18MB in size. Technical field

[0005] The present invention relates to the fields of molecular biology and gene editing. Background art

[0006] Editing or modifying the targeted genome is rapidly becoming an important tool for basic and applied research because it allows for modification of the genome, such as: cleaving, deleting, and inserting nucleic acids; substituting nucleotides in nucleic acids; and regulating gene expression at specific locations in the genome, as well as many other possible modifications. Genome editing systems using 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 for editing at a specific location in the genome. Thus, genome editing systems using RNA - guided nucleases (RGNs) can edit genomic sequences economically and efficiently because the guide RNA is the programmable component of the RGN system, allowing for genome editing of specific target sequences by generally directly designing 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.

[0007] In microorganisms from which type II RGN systems and some type V RGN systems are derived, the guide RNA (gRNA) is expressed as a two-part RNA system: the CRISPR-RNA (crRNA), which contains a spacer sequence that recognizes the target genomic sequence through Watson-Crick base pairing, and the scaffold trans-activating crRNA (tracrRNA). This two-part guide RNA requires base pairing between the crRNA molecule and the tracrRNA molecule regions to form a dual guide RNA (dgRNA). For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

[0008] Elements of the guide RNA (such as the phosphate backbone, ribose, nucleobase) can be chemically modified, for example, to reduce degradation of the guide RNA. There are many opportunities to describe the type and / or degree of modification of the guide RNA to improve the RGN system, such as increasing the stability of the target sequence, editing efficiency and specificity and / or reducing the inflammatory response associated with the toxicity of the RGN system. Summary of the Invention

[0009] The compositions provided herein include 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, wherein the 2′ oxygen is covalently linked to the 4′ carbon via a methylene bridge. In some embodiments, the other modifications include 2′-O-methyl (2′-O-Me), 2′-O-methyl 3′-thiolphosphate (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 as compared to a reference RGN system comprising tracrRNA, gRNA, and / or crRNA that do not have BNA modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure permit the use of dual guide RNAs in applications where a single guide RNA was previously required. In some embodiments, the present disclosure provides for the use of BNA modifications 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 modifications and / or other chemical modifications permits the use of shortened tracrRNA, gRNA, and / or crRNA. In some embodiments, the cells in which gene editing is performed 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 in any model system, cell type, and target sequence in which an RGN system is applied.

[0010] Also provided are methods of achieving RGN-based gene editing and improving gene editing efficiency in cells using a BNA-modified guide RNA.

[0011] 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 sequence; (b) a tail; and (c) a stem-loop closest to the tail, wherein the anti-repeat sequence of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

[0012] In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification is located within the anti-repeat sequence. In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification is located within the first stem of the anti-repeat sequence. In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification includes at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides within the first stem of the anti-repeat sequence, or at least two, three, four, five, six, or seven BNA modifications on alternating nucleotides. In some embodiments in terms of the above-mentioned tracrRNA, all nucleotides within the first stem of the anti-repeat contain BNA modifications.

[0013] In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification is not within the second stem of the anti-repeat sequence. In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification is not within the bulge of the tracrRNA. In some embodiments in terms of the above-mentioned tracrRNA, the three terminal nucleotides of the tail of the tracrRNA contain BNA modifications. In some embodiments in terms of the above-mentioned tracrRNA, the three terminal nucleotides of the tail of the tracrRNA contain both BNA modifications and phosphorothioate (PS) modifications.

[0014] In some embodiments in terms of the above-mentioned tracrRNA, the at least one BNA modification includes 2′,4′-BNA modification. In some embodiments, the 2′,4′-BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2′,4′-BNA is an LNA modification. In some embodiments, the 2′,4′-BNA is a cEt modification.

[0015] In some embodiments in terms of the above-mentioned tracrRNA, the tracrRNA further comprises at least one other chemical modification. In some embodiments, the at least one other chemical modification is located within the anti-repeat sequence of the tracrRNA. In some embodiments, the at least one other chemical modification is located within the first stem of the anti-repeat sequence of the tracrRNA. In some embodiments, the at least one other chemical modification is located within the tail of the tracrRNA.

[0016] In some embodiments with respect to the above-mentioned tracrRNA, at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification. In some embodiments with respect to the above-mentioned tracrRNA, the three terminal nucleotides of the tail of the tracrRNA comprise MS modification. In some embodiments with respect to the above-mentioned tracrRNA, the three terminal nucleotides of the tail of the tracrRNA comprise MS modification, and all nucleotides of the first stem of the anti-repeat sequence comprise BNA modification. In some embodiments, the BNA modification is LNA modification.

[0017] In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence has a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides. In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence has a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides. In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence has a total length of about 11 nucleotides. In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence has a total length of 6-15 nucleotides, 8-13 nucleotides or 10-12 nucleotides.

[0018] In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence at the 5' region. In some embodiments with respect to the above-mentioned tracrRNA, the first stem of the anti-repeat sequence comprises a GC-rich nucleotide sequence at the 5' region, wherein the 5' region comprises at least 2, at least 3, at least 4 or at least 5 G or C.

[0019] In some embodiments with respect to the above-mentioned tracrRNA, the tracrRNA has 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.

[0020] In some embodiments regarding the above tracrRNA, 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 with any one of SEQ ID NO: 10, 12, 51 - 53, 294, 295, 383, and 709.

[0021] In some embodiments regarding the above tracrRNA, 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 with any one of SEQ ID NO: 80, 81, 364 - 367, 369, and 375 - 379.

[0022] In some embodiments regarding the above tracrRNA, 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 with any one of SEQ ID NO: 102, 103, 370 - 373, 710, and 711.

[0023] In some embodiments regarding the above tracrRNA, 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 with any one of SEQ ID NO: 499 - 501, 504, 505, 534, 535, and 537.

[0024] In some embodiments regarding the above tracrRNA, the tracrRNA is part of a gRNA capable of binding to an RGN. In some embodiments, the RGN is a type II RGN.

[0025] In some embodiments regarding the above tracrRNA, 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 with SEQ ID NO: 1.

[0026] In some embodiments regarding the above tracrRNA, 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 with SEQ ID NO: 69.

[0027] In some embodiments in terms of the above tracrRNA, 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 with SEQ ID NO:93.

[0028] In some embodiments in terms of the above tracrRNA, 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 with SEQ ID NO:252.

[0029] 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: i) a spacer; and ii) a crRNA repeat sequence comprising a first stem and a second stem, and wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat sequence comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

[0030] In some embodiments in terms of the above gRNA, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA has a total length of 100 - 120 nt, 120 - 140 nt, 140 - 160 nt, 160 - 180 nt, 180 - 200 nt or more than 200 nt. In some embodiments in terms of the above gRNA, the gRNA is a dual guide RNA (dgRNA).

[0031] In some embodiments in terms of the above gRNA, the at least one BNA modification is located within the crRNA repeat sequence. In some embodiments in terms of the above gRNA, the at least one BNA modification is located within the first stem of the crRNA repeat sequence. In some embodiments in terms of the above gRNA, the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat sequence. In some embodiments in terms of the above gRNA, the three terminal nucleotides at the 3' region of the first stem of the crRNA repeat sequence comprise BNA modifications. In some embodiments in terms of the above gRNA, the three terminal nucleotides at the 3' region of the first stem of the crRNA repeat sequence comprise both BNA modifications and phosphorothioate (PS) modifications. In some embodiments in terms of the above gRNA, the at least one BNA modification is not within the second stem of the crRNA repeat sequence.

[0032] In some embodiments of the above gRNA, the at least one BNA modification is located within the anti-repeat sequence. In some embodiments of the above gRNA, the at least one BNA modification is located within the first stem of the anti-repeat sequence. In some embodiments of the above gRNA, the at least one BNA modification includes at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides within the first stem of the anti-repeat sequence, or at least two, three, four, five, six, or seven BNA modifications on alternating nucleotides. In some embodiments of the above gRNA, all nucleotides within the first stem of the anti-repeat sequence contain BNA modifications.

[0033] In some embodiments of the above gRNA, the at least one BNA modification is not within the second stem of the anti-repeat sequence. In some embodiments of the above gRNA, the at least one BNA modification is not within the bulge of the gRNA. In some embodiments of the above gRNA, the at least one BNA modification is located within the tail of the tracrRNA. In some embodiments of the above gRNA, the three terminal nucleotides at the 3' region of the tail of the tracrRNA contain BNA modifications. In some embodiments of the above gRNA, the three terminal nucleotides at the 3' region of the tail of the tracrRNA contain both BNA modifications and phosphorothioate (PS) modifications.

[0034] In some embodiments of the above gRNA, at least three terminal nucleotides in the 3' region of the first stem of the crRNA repeat sequence and all nucleotides in the first stem of the anti-repeat sequence contain BNA modifications. In some embodiments of the above gRNA, all nucleotides in the first stem of the crRNA repeat sequence lack chemical modifications and all nucleotides in the first stem of the anti-repeat sequence contain BNA modifications.

[0035] In some embodiments of the above gRNA, the at least one BNA modification is located within the spacer region. In some embodiments of the above gRNA, the three terminal nucleotides at the 5' region of the spacer contain BNA modifications. In some embodiments of the above gRNA, the three terminal nucleotides at the 5' region of the spacer contain both BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA, the length of the spacer region is 18 - 30 nucleotides.

[0036] In some embodiments regarding the above gRNA, the at least one BNA modification includes 2′,4′ BNA modification. In some embodiments, the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2′,4′ BNA is LNA modification. In some embodiments, the 2′,4′ BNA is cEt modification.

[0037] In some embodiments regarding the above gRNA, the gRNA further comprises at least one other modification. In some embodiments regarding the above gRNA, the at least one other modification is located within the crRNA. In some embodiments regarding the above gRNA, the at least one other modification is located within the 5′ region or the 3′ region of the crRNA. In some embodiments regarding the above gRNA, the at least one other modification is located within both the 5′ region and the 3′ region of the crRNA.

[0038] In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the crRNA repeat sequence of the crRNA. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the first stem of the crRNA repeat sequence. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the spacer region of the crRNA. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the tracrRNA. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the anti-repeat sequence of the tracrRNA. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the first stem of the anti-repeat sequence of the tracrRNA. In some embodiments regarding the above gRNA, the at least one other chemical modification is located within the tail of the tracrRNA.

[0039] In some embodiments of the above gRNA, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments of the above gRNA, the three terminal nucleotides at the 5′ region and the 3′ region of the crRNA each comprise an MS modification. In some embodiments of the above gRNA, the three terminal nucleotides at the 5′ region and the 3′ region of the crRNA each comprise an MS modification, and the remaining nucleotides of the first stem of the crRNA repeat sequence comprise a 2'-O-Me modification.

[0040] In some embodiments of the above gRNA, the first stem of the crRNA repeat sequence or the first stem of the anti-repeat sequence has a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA, the first stem of the crRNA repeat sequence or the first stem of the anti-repeat sequence has a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA, the first stem of the crRNA repeat sequence or the first stem of the anti-repeat sequence has a total length of about 11 nucleotides. In some embodiments of the above gRNA, the first stem of the crRNA repeat sequence or the first stem of the anti-repeat sequence has a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

[0041] In some embodiments of the above gRNA, the 3′ region of the first stem of the crRNA repeat sequence or the 5′ region of the first stem of the anti-repeat sequence comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above gRNA, the 3′ region of the first stem of the crRNA repeat sequence or the 5′ region of the first stem of the anti-repeat sequence comprises a GC-rich nucleotide sequence, wherein the 3′ region of the first stem of the crRNA repeat sequence or the 5′ region of the first stem of the anti-repeat sequence comprises at least 2, at least 3, at least 4, or at least 5 G or C.

[0042] In some embodiments in terms of the above gRNA, the three terminal nucleotides at both the 5′ region and the 3′ region of the crRNA comprise MS modification, BNA modification or BNA+PS modification.

[0043] In some embodiments in terms of the above gRNA, the crRNA repeat sequence has a nucleotide sequence as shown below: (a) SEQ ID NO:39 or differs from SEQ ID NO:39 by 1 or 2 nucleotides; (b) SEQ ID NO:384 or differs from SEQ ID NO:384 by 1 or 2 nucleotides; (c) SEQ ID NO:385 or differs from SEQ ID NO:385 by 1 or 2 nucleotides; (d) SEQ ID NO:386 or differs from SEQ ID NO:386 by 1 or 2 nucleotides; (e) SEQ ID NO:387 or differs from SEQ ID NO:387 by 1 or 2 nucleotides; or (f) SEQ ID NO:397 or differs from SEQ ID NO:397 by 1 or 2 nucleotides. In some embodiments in terms of the above 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 with any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401 and 708. In some embodiments in terms of the above gRNA, 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 with any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383 and 709.

[0044] In some embodiments of the foregoing gRNA, the crRNA repeat sequence has the nucleotide sequence shown below: (a) SEQ ID NO: 300 or differs from SEQ ID NO: 300 by 1 or 2 nucleotides; (b) SEQ ID NO: 304 or differs from SEQ ID NO: 304 by 1 or 2 nucleotides; (c) SEQ ID NO: 308 or differs from SEQ ID NO: 308 by 1 or 2 nucleotides; (d) SEQ ID NO: 312 or differs from SEQ ID NO: 312 by 1 or 2 nucleotides; (e) SEQ ID NO: 320 or differs from SEQ ID NO: 320 by 1 or 2 nucleotides; (f) SEQ ID NO: 344 or differs from SEQ ID NO: 344 by 1 or 2 nucleotides; (g) SEQ ID NO: 348 or differs from SEQ ID NO: 348 by 1 or 2 nucleotides; (h) SEQ ID NO: 352 or differs from SEQ ID NO: 352 by 1 or 2 nucleotides; (i) SEQ ID NO: 356 or differs from SEQ ID NO: 356 by 1 or 2 nucleotides; (j) SEQ ID NO: 360 or differs from SEQ ID NO: 360 by 1 or 2 nucleotides; (k) SEQ ID NO: 388 or differs from SEQ ID NO: 388 by 1 or 2 nucleotides; (l) SEQ ID NO: 389 or differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or (m) SEQ ID NO: 390 or differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments of the foregoing gRNA, 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 with 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 foregoing gRNA, 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 with any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

[0045] In some embodiments in terms of the above-mentioned gRNA, the crRNA repeat sequence has a nucleotide sequence shown in any one of the following: (a) SEQ ID NO: 324 or having 1 or 2 nucleotides different from SEQ ID NO: 324; (b) SEQ ID NO: 328 or having 1 or 2 nucleotides different from SEQ ID NO: 328; (c) SEQ ID NO: 332 or having 1 or 2 nucleotides different from SEQ ID NO: 332; (d) SEQ ID NO: 336 or having 1 or 2 nucleotides different from SEQ ID NO: 336; (e) SEQ ID NO: 391 or having 1 or 2 nucleotides different from SEQ ID NO: 391; (f) SEQ ID NO: 392 or having 1 or 2 nucleotides different from SEQ ID NO: 392; and (g) SEQ ID NO: 393 or having 1 or 2 nucleotides different from SEQ ID NO: 393. In some embodiments in terms of the above-mentioned gRNA, 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 with any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335 and 337-339. In some embodiments in terms of the above-mentioned gRNA, 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 with any one of SEQ ID NOs: 102, 103, 370-373, 710 and 711.

[0046] In some embodiments of the above gRNA, the crRNA repeat sequence has a nucleotide sequence shown in any of the following: (a) SEQ ID NO: 465 or 1 or 2 nucleotides different from SEQ ID NO: 465; (b) SEQ ID NO: 469 or 1 or 2 nucleotides different from SEQ ID NO: 469; (c) SEQ ID NO: 473 or 1 or 2 nucleotides different from SEQ ID NO: 473; (d) SEQ ID NO: 477 or 1 or 2 nucleotides different from SEQ ID NO: 477; (e) SEQ ID NO: 481 or 1 or 2 nucleotides different from SEQ ID NO: 481; (f) SEQ ID NO: 508 or 1 or 2 nucleotides different from SEQ ID NO: 508; (g) SEQ ID NO: 512 or 1 or 2 nucleotides different from SEQ ID NO: 512; and (h) SEQ ID NO: 516 or 1 or 2 nucleotides different from SEQ ID NO: 516. In some embodiments of the above gRNA, 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 with 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, 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 with any one of SEQ ID NOs: 499 - 501, 504, 505, 534, 535 and 537.

[0047] In some embodiments of the above gRNA, the crRNA and the tracrRNA are linked by a linker between the 3'-terminal nucleotide of the crRNA repeat sequence and the 5'-terminal nucleotide of the anti-repeat sequence. In some embodiments, the linker contains an azide functional group or an alkyne functional group. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker has a nucleotide sequence shown in AAAG, GAAA, ACUU or CAAAGG. In some embodiments, the linker has a nucleotide sequence shown in AAAG.

[0048] In some embodiments of the gRNA described above, the gRNA is an sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and a spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat sequence, a linker, and the tracrRNA. In some embodiments of the gRNA described above, 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 with any one of SEQ ID NOs: 35-37, 296, and 297.

[0049] In some embodiments of the gRNA described above, the sgRNA has a nucleotide sequence as shown in any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

[0050] In some embodiments of the gRNA described above, the gRNA is capable of binding to an RGN. In some embodiments, the RGN is a type II RGN.

[0051] In some embodiments of the gRNA described above, 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 with SEQ ID NO: 1.

[0052] In some embodiments of the gRNA described above, 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 with SEQ ID NO: 69.

[0053] In some embodiments of the gRNA described above, 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 with SEQ ID NO: 93.

[0054] In some embodiments of the gRNA described above, 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 with SEQ ID NO: 252.

[0055] In some embodiments of the gRNA described above, the gRNA further comprises an extension region, and the extension region comprises an editing template for prime editing.

[0056] In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA), the CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat sequence, wherein the crRNA repeat sequence is capable of hybridizing with the anti-repeat sequence of a tracrRNA to form a guide RNA (gRNA), the guide RNA comprising a stem-loop, the stem-loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat sequence and the anti-repeat sequence, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiolphosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA modification; and wherein the at least one chemical modification is located within three terminal nucleotides at the 5' region or 3' region of the crRNA.

[0057] In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA), the CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat sequence comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiolphosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA modification; and wherein the at least one chemical modification is located within three terminal nucleotides at the 5' region or 3' region of the crRNA. In some embodiments, the gRNA comprising the crRNA is capable of binding to an RNA-guided nuclease (RGN) that requires tracrRNA to be active.

[0058] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: 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 crRNA and the tracrRNA form a gRNA. In some embodiments, the RGN system binds to a target sequence in a target nucleic acid molecule.

[0059] In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: 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.

[0060] In still yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) 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 crRNA and the tracrRNA form a gRNA. In some embodiments, the RGN system binds to a target sequence in a target nucleic acid molecule.

[0061] In some embodiments of the above aspects of the RGN system, the RGN polypeptide recognizes a protospacer adjacent motif (PAM) having a nucleotide sequence shown as NNNNCC, NNGRR, NNRYA or NGG. In some embodiments of the above aspects of the RGN system, the gRNA is a sgRNA having a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt or more than 200 nt. In some embodiments of the above aspects of the RGN system, 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 with any one of SEQ ID NO: 1, 69, 93 or 252.

[0062] In some embodiments of the above aspects of the RGN system, the RGN polypeptide and the gRNA are not found to be complexed with each other in nature.

[0063] In some embodiments of the above-described RGN system, the target sequence is a eukaryotic target sequence. In some embodiments, the target sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 273-278 and 712. In some embodiments of the above-described RGN system, the target sequence is intracellular.

[0064] In some embodiments of the above-described RGN system, the complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence. In some embodiments, the cleavage produces a double-strand break. In some embodiments, the cleavage produces a single-strand break.

[0065] In some embodiments of the above-described RGN system, the RGN polypeptide is nuclease-inactivated. In some embodiments of the above-described RGN system, the RGN polypeptide is a nickase.

[0066] In some embodiments of the above-described RGN system, the RGN polypeptide is fused to a base editing polypeptide. In some embodiments, the base editing polypeptide includes a deaminase.

[0067] In some embodiments of the above-described RGN system, the RGN polypeptide is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide includes a DNA polymerase. In some embodiments, the DNA polymerase includes a reverse transcriptase. In some embodiments of the above-described RGN system, the gRNA further comprises an extension region that contains an editing template for prime editing.

[0068] In some embodiments of the above-described RGN system, the RGN polypeptide is fused to a detectable label. In some embodiments of the above-described RGN system, the RGN system further comprises a donor polynucleotide.

[0069] In some embodiments of the above-described RGN system, the polynucleotide comprising the nucleotide sequence encoding the 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 the nucleotide sequence encoding the RGN polypeptide is located in a vector.

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

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

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

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

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

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

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

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

[0078] In some embodiments of the above aspects, the RGN polypeptide or the gRNA further comprises a detectable tag, thereby allowing detection of the target sequence. In some embodiments of the above aspects, the RGN polypeptide or the gRNA further comprises an expression regulator, thereby regulating the expression of a target gene comprising the target sequence. In some embodiments of the above aspects, the RGN is fused to a prime editing polypeptide. In some embodiments of the above aspects, the RGN polypeptide is fused to a base editing polypeptide.

[0079] 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 the RGN system or RNP complex as described above to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

[0080] In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising: a) combining, under conditions suitable for forming a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a trans-activating crRNA (tracrRNA) and a CRISPR RNA (crRNA) as described 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 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 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 region that comprises 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.

[0081] In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) a guide RNA (gRNA) that comprises a trans-activating crRNA (tracrRNA) and a CRISPR RNA (crRNA) as described 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 complex formed by 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 region that comprises 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 the type II RGN is an mRNA.

[0082] In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising: a) combining, under conditions suitable for forming a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) as described above; and ii) a type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence 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 region that comprises an editing template for prime editing.

[0083] In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) a guide RNA (gRNA) as described 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 complex formed by 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 region that comprises an editing template for prime editing. In some embodiments of the method aspect, the polynucleotide encoding the type II RGN is an mRNA.

[0084] In a further aspect, the present disclosure provides a method of binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising: a) combining, under conditions suitable for forming a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA as described 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 binding the target sequence to the RGN. In a further aspect, the present disclosure provides a method of binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA as described above; and ii) a type II RGN, or a polynucleotide encoding a type II RGN, thereby binding the target sequence to the RGN.

[0085] In some embodiments of the above method aspects, the target sequence comprises a nucleotide sequence as set forth in 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 with any one of SEQ ID NO: 1, 69, 93 or 252.

[0086] In another aspect, the present disclosure provides a method of enhancing the efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system or an RNP complex as described above to the target sequence or a cell comprising the target sequence, wherein the nucleic acid molecule is cleaved or modified with a higher efficiency compared to cleaving or modifying the nucleic acid molecule by a method comprising delivering a reference RGN system or an 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 does not comprise any chemical modification.

[0087] In some embodiments of the above aspects, all nucleotides of the first stem of the anti-repeat sequence of the tracrRNA of the RGN system or RNP complex as described above include BNA modifications. In some embodiments of the above aspects, at least three terminal nucleotides at the 3' region of the first stem of the crRNA repeat sequence of the crRNA include BNA modifications. In some embodiments of the above aspects, the BNA modification includes LNA modification. In some embodiments of the above aspects, the BNA modification includes cEt modification. In some embodiments of the above aspects, the efficiency of cleaving and / or modifying the target sequence is increased by 15 to 30 times. In some embodiments of the above aspects, the efficiency of cleaving and / or modifying the target sequence is determined by measuring the percentage of target sequences or cells containing the target sequence in which the expression of the target sequence has changed or the expression of the polypeptide encoded by the target sequence has changed. In some embodiments of the above aspects, expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblotting, 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.

[0088] In another aspect, the present disclosure provides a method for engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat sequence and the tracrRNA comprises an anti-repeat sequence; and b) adding or replacing one or more nucleotides in the crRNA repeat sequence and one or more nucleotides in the anti-repeat sequence, wherein the one or more nucleotides added or replaced in the repeat sequence and the one or more nucleotides added or replaced in the anti-repeat sequence are capable of hybridizing to each other, wherein the 3' region of the crRNA repeat sequence and the 5' region of the anti-repeat sequence of the engineered gRNA comprise at least 2, at least 3, at least 4, or at least 5 G or C, and wherein the engineered gRNA has increased editing efficiency compared to the gRNA provided in step a).

[0089] 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 nucleotides added or replaced are located in the 3' region of the crRNA repeat sequence and the 5' region of the anti-repeat sequence, and wherein the 3' region of the crRNA repeat sequence and the 5' region of the anti-repeat sequence comprise at least 2, at least 3, at least 4, or at least 5 G or C.

[0090] In some embodiments of the above aspects, the gRNA is a dgRNA. In some embodiments of the above aspects, the gRNA is an sgRNA.

[0091] In some embodiments of the above aspects, it further comprises: c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA modification.

[0092] In some embodiments of the above aspects, the at least one chemical modification is located in the crRNA, the tracrRNA, or both. In some embodiments of the above aspects, the at least one chemical modification is located in: the crRNA repeat; the anti-repeat; the tail of the tracrRNA; the crRNA repeat and the anti-repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA. In some embodiments of the above aspects, the at least one chemical modification is located 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.

[0093] In some embodiments of the above aspects, the at least one chemical modification is located 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 located 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 located on all nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspects, the at least one chemical modification is located on alternating nucleotides in the first stem of the anti-repeat.

[0094] In some embodiments of the above aspects, the at least one chemical modification is located on all nucleotides in the first stem of the anti-repeat sequence and on three nucleotides at the 3' region of the tracrRNA tail. In some embodiments of the above aspects, the at least one chemical modification is located on all nucleotides in the first stem of the anti-repeat sequence and on at least one nucleotide in the first stem of the crRNA repeat sequence. In some embodiments of the above aspects, the at least one chemical modification is located on all nucleotides in the first stem of the anti-repeat sequence and on at least three terminal nucleotides at the 3' region of the first stem of the crRNA repeat sequence. In some embodiments of the above aspects, the at least one chemical modification is located on all nucleotides in the first stem of the anti-repeat sequence, on at least three terminal nucleotides at the 3' region of the first stem of the crRNA repeat sequence, and on three terminal nucleotides at the 3' region of the tracrRNA tail. In some embodiments of the above aspects, the at least one chemical modification is located on all nucleotides in the first stem of the anti-repeat sequence, on three terminal nucleotides at the 3' region of the tracrRNA tail, and on at least one nucleotide at the 3' region of the first stem of the crRNA repeat sequence.

[0095] In some embodiments of the above aspects, the at least one chemical modification comprises a BNA modification. In some embodiments, the BNA modification comprises a 2′,4′-BNA modification. In some embodiments, the 2′,4′-BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2′,4′-BNA is an LNA modification. In some embodiments, the 2′,4′-BNA is a cEt modification.

[0096] In some embodiments of the above aspects, compared to the gRNA provided in step a), 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. In some embodiments of the above aspects, compared to the RGN system comprising the gRNA provided in step a), the efficiency of the RGN system comprising the engineered gRNA to cleave and / or modify the target sequence 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. In some embodiments of the above aspects, the efficiency is determined by measuring the percentage of target sequences in which the expression of the target sequence has been altered or the percentage of cells comprising the target sequence in which the expression of the polypeptide encoded by the target sequence has been altered. In some embodiments of the above aspects, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblotting, 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.

[0097] In another aspect, the present disclosure provides an engineered gRNA produced by the method as described above.

[0098] 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 sequence, wherein the tracrRNA comprises an anti-repeat sequence, wherein the gRNA comprises a stem-loop containing a first stem and a second stem, wherein the first stem has a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0099] Still 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 sequence, wherein the tracrRNA comprises an anti-repeat sequence, wherein the gRNA comprises a stem-loop containing a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6 or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1Schematic diagrams of dual guide RNAs (dgRNAs) are provided, showing the pairing of crRNA and tracrRNA.

[0101] Figure 2A and 2B Schematic diagrams showing the various parts of the illustrated single guide (sgRNA) ( Figure 2A ) and dgRNA ( Figure 2B ). These parts include: the spacer region; stem-loop 1, including a first stem, a first bubble, and a second stem; stem-loop 2, including only the first stem; the inter-stem-loop region (ISR); stem-loop 3, including a loop, a first stem, a first bubble, a second stem, a second bubble, and a third stem; and the tail. Stem-loop 1 in sgRNA includes a loop, while stem-loop 1 in dgRNA does not include a loop. The CRISPR RNA (crRNA) repeat anneals with the anti-repeat of the trans-activating CRISPR RNA (tracrRNA) to form stem-loop 1.

[0102] Figure 3A and 3B Depictions of chemical modifications of dgRNAs for the APG07433.1 RNA-guided nuclease are provided. Figure 3A Shows tracrRNA modification schemes, from left to right: "MS-modified stem", where the first stem of stem-loop 1 contains 2'-O-methyl (2'-O-Me) modifications, 2'-O-methyl 3'-thiolphosphate (MS) modifications at the three terminal nucleotides in the 5' region, MS modifications at the three terminal nucleotides in the 3' region, and a 2'-O-Me modification on the nucleotide that is the fourth nucleotide from the 3' end of the tracrRNA molecule; "heavily MS-modified" – in addition to the modifications in "MS-modified stem", 2'-O-Me modifications are added to stem-loop 3 via the tail; "LNA-modified stem" – the first stem of stem-loop 1 contains all LNA modifications, the three terminal nucleotides in the 3' region have MS modifications, and the fourth nucleotide from the 3' end has a 2'-O-Me modification; "heavily LNA-modified" – in addition to the modifications in "LNA-modified stem", 2'-O-Me modifications are added to most of stem-loop 3, and a portion of the first and second stems of stem-loop 3 are LNA-modified. Figure 3B Shows a diagram of the crRNA modifications used in these experiments – all contain 5' MS modifications and 3' MS modifications, and the version shown on the left also has 2'-O-Me modifications on the first stem.

[0103] Figure 4shows the measurement of gene editing efficiency by knocking out the CD3 surface marker using flow cytometry in primary human T cells using different combinations of modified crRNA and modified tracrRNA. The guide RNA was designed to target the TRAC gene. These data indicate that modification at stem loop 3 abolishes editing activity, while modification of only stem loop 1 retains the editing ability of the dgRNA. Modifications were shown in the same scheme as Figure 3A and 3B . The tracrRNA is shown as depicted. The control sgRNA has MS modifications at three terminal nucleotides at both the 5' and 3' regions of the sgRNA. Mock indicates the case without RGN and gRNA, where cells were mixed with the nucleofection solution and subjected to the nucleofection process.

[0104] Figure 5 indicates that LNA modification enhances the editing efficiency of the dgRNA in the RNP complex with purified APG07433.1 protein, achieving an editing level similar to that of the sgRNA. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The control sgRNA and the control dgRNA each have MS modifications at three terminal nucleotides at both the 5' and 3' regions of the guide RNA.

[0105] Figure 6 shows that LNA-modified dgRNA promotes a high gene disruption rate, especially relative to dgRNA with only terminal modifications (MS modifications at three terminal nucleotides at both the 5' and 3' regions of the dgRNA) or dgRNA with an additional 2'-O'-Me modification (MS / PS modification) at stem loop 1, using the mRNA delivery method with two exemplary spacers. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The control sgRNA and the control dgRNA each have MS modifications at three terminal nucleotides at both the 5' and 3' regions of the guide RNA.

[0106] Figure 7 indicates that LNA-modified dgRNA is more potent in gene editing compared to the sgRNA, using two exemplary spacers. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The control sgRNA has MS modifications at three terminal nucleotides at both the 5' and 3' regions of the sgRNA.

[0107] Figure 8A and 8BThe results showed that LNA modification improved the editing efficiency of sgRNA for two different RNA-guided nucleases (RGNs), which were delivered in the form of a protein complexed with the guide RNA (RNP delivery) or an mRNA encoding the RGN (mRNA delivery).( Figure 8A ) APG07433.1 sgRNA. "Control RNP" and "Control mRNA" indicate the cases without RGN and gRNA for each delivery method, where the cells were mixed with the nucleofection solution but not subjected to the nucleofection process.( Figure 8B ) APG01604 sgRNA. "Control TRAC" and "Control_B2M" indicate the cases without RGN and gRNA, where the cells were mixed with the nucleofection solution but not subjected to the nucleofection process. The two different spacer names in the control indicate that antibodies against TRAC (2275) or B2M (1989) were used in flow cytometry to establish unedited reads. Two exemplary spacers were used for the sgRNA of each RGN. (-): Control sgRNA with MS modification at three terminal nucleotides at both the 5' region and 3' region of the sgRNA. (+): sgRNA with additional LNA modification, as shown in each schematic diagram. Each guide RNA schematic shows the LNA modification in the region of the inverted repeat forming the first stem and the MS modification at three terminal nucleotides at the 5' region and 3' region of the sgRNA. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker by editing of the TRAC target sequence or the B2M immunostaining by editing of the B2M target sequence using flow cytometry.

[0108] Figure 9A and 9B The results showed that MS modification of stem-loop 1 did not improve the editing efficiency. Figure 9A Eight cases of 2′-O-Me and / or MS modification in stem-loop 1 and / or stem-loop 3 of APG07433.1 sgRNA were described. Figure 9B The results showed that none of the tested sgRNAs with 2′-O-Me and / or MS modification enhanced sgRNA editing compared to the control sgRNA. Chemical modification at stem-loop 3 abolished sgRNA activity. The RGN was delivered in the form of a protein complexed with the guide RNA (RNP delivery) or an mRNA encoding the RGN (mRNA delivery). "Control" indicates the cases without RGN and gRNA for each delivery method, where the cells were mixed with the nucleofection solution but not subjected to the nucleofection process. The control sgRNA had MS modification at three terminal nucleotides at both the 5' region and 3' region of the sgRNA. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker using flow cytometry.

[0109] Figure 10A-10CIt is shown that the amount of LNA modification correlates with the guide RNA editing efficiency in primary human T cells, which is measured by flow cytometry through the knockout of the CD3 surface marker (for the TRAC target sequence) or immunostaining of B2M (for the B2M target sequence).( Figure 10A )The gene editing efficiency of APG07433.1 dgRNA, which has 1, 3, 6, or 11 LNA-modified nucleotides in the inverted repeat region of the first stem forming stem-loop 1. The schematic diagram of APG07433.1 dgRNA shows the "0LNA@stem-loop 1" case, including MS modifications at the 5' and 3' ends of the tracrRNA and crRNA; and the "LNA-modified" case, including the MS modification of "0LNA" plus different numbers of LNA-modified nucleotides. The highest editing was achieved when all nucleotides in the inverted repeat region of the first stem forming stem-loop 1 were LNA-modified.( Figure 10B )The gene editing efficiency of APG01604 dgRNA, which has 3 or 7 LNA-modified nucleotides in the inverted repeat region of the first stem forming stem-loop 1. The schematic diagram of APG01604 dgRNA shows the "0LNA, -" case, including MS modifications at the 5' and 3' ends of the tracrRNA and crRNA; and the "LNA-modified" case, including the MS modification of "0LNA" plus different numbers of LNA-modified nucleotides.( Figure 10C )The gene editing efficiency of APG05586 dgRNA, which has 4 or 9 LNA-modified nucleotides in the inverted repeat region of the first stem forming stem-loop 1. The schematic diagram of APG05586 dgRNA shows the "0LNA, -" case, including MS modifications at the 5' and 3' ends of the tracrRNA and crRNA; and the "LNA-modified" case, including the MS modification of "0LNA" plus different numbers of LNA-modified nucleotides. The gene editing of LNA-modified APG01604 dgRNA and APG05586 dgRNA was improved compared to the "0LNA" dgRNA. The dgRNA of each RGN uses two exemplary spacers. Each RGN is delivered in the form of a protein complexed with the guide RNA (RNP delivery) or mRNA encoding the RGN (mRNA delivery). "Control_TRAC" and "Control_B2M" indicate that for two different spacers in the gRNA, in the absence of RGN and gRNA, the cells were mixed with the nucleofection solution but not subjected to the nucleofection process.

[0110] Figure 11A-11CThis suggests that LNA modification maintains or improves the gene editing efficiency of the shortened APG07433.1 sgRNA. Figure 11A ) Top: The full-length APG07433.1 sgRNA was shortened by a combination of truncations of various regions of the sgRNA: deletion of 5 nucleotide (nt) pairs (10nt) from the first stem of stem-loop 1 and 6 nt from the tail (-10 first stem SL1, -6 tail); deletion of 5 nt pairs (10nt) from the first stem of stem-loop 1, 4 nt from the tail, and 1 nt pair (2nt) from the first stem of stem-loop 3 (-10 first stem SL1, -4 tail, -2 first stem SL3); and deletion of 5 nucleotide (nt) pairs (10nt) from the first stem of stem-loop 1, 6 nt from the tail, and 1 nt pair (2nt) from the first stem of stem-loop 3 (-10 first stem SL1, -6 tail, -2 first stem SL3). These shortened APG07433.1 sgRNAs have MS modifications at the three terminal nucleotides in both the 5' and 3' regions of the sgRNA and serve as controls for evaluating other chemical modifications introduced into the first stem of stem-loop 1. Bottom panel: shortened APG07433.1 sgRNA as in the top panel, but including LNA and MS modifications in the first stem of stem-loop 1. ( Figure 11B ) compared with the control full-length APG07433.1 sgRNA or the control shortened APG07433.1 sgRNA, as Figure 11A Gene editing efficiency of the chemically modified shortened APG07433.1 sgRNA shown. The control full-length sgRNA and shortened sgRNA each had MS modifications at the three terminal nucleotides at both the 5' and 3' regions of the sgRNA, but did not contain any chemical modification at stem-loop 1. "Mock" represents the case without RGN and gRNA, where for each delivery method, cells were mixed with nucleofection solution and underwent nucleofection. sgRNA was used at a dilution factor of 1. ( Figure 11C )LNA modification increases the editing efficacy of the shortened APG07433.1 sgRNA. The sgRNAs were serially diluted. Data were collected on day 4. The “3MS” full-length and “3MS” shortened sgRNAs were shown in Figure 11A As shown in the figure above. The modified shortened sgRNA is Figure 11A Shown at the bottom. DF = dilution factor. Gene editing efficiency in primary human T cells was measured by assessing knockdown of CD3 surface markers using flow cytometry. RGNs were delivered as proteins complexed with guide RNA (RNP delivery) or mRNA encoding RGNs (mRNA delivery).

[0111] Figure 12A and 12BIt is shown that the LNA modification maintains or improves the gene editing efficiency of the shortened APG07433.1 dgRNA delivered using RNP. The "M" shortening and chemical modification scheme perform best for sgRNA and dgRNA.( Figure 12A )Chemical modification and shortening schemes for crRNA and tracrRNA. The crRNA was shortened by 5 terminal nt in the 3' region: left panel, MS modification was performed at the three terminal nucleotides in the 5' and 3' regions (O and Q represent two exemplary spacers used); right panel, MS modification of the three terminal nucleotides in the 5' and 3' regions plus 2'-O-Me modification within the crRNA repeat forming the first stem of stem-loop 1 (P and R represent two exemplary spacers used). tracrRNA: 'tracr(L)', the anti-repeat forming the first stem of stem-loop 1 was shortened by 5 terminal nt in the 5' region, all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modification, the tail was shortened by 6 nt, and the three terminal nucleotides in the 3' region contain MS modification; tracrRNA: 'tracr(M)', the anti-repeat forming the first stem of stem-loop 1 was shortened by 5 terminal nt in the 5' region, all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modification, the tail was shortened by 4 nt, 1 nt pair (2 nt) was deleted from the first stem of stem-loop 3, and the three terminal nucleotides in the 3' region contain MS modification; 'tracr(N)', the anti-repeat forming the first stem of stem-loop 1 was shortened by 5 terminal nt in the 5' region, all nucleotides of the anti-repeat forming the first stem of stem-loop 1 contain LNA modification, the tail was shortened by 6 nt, 1 nt pair (2 nt) was deleted from the first stem of stem-loop 3, and the three terminal nucleotides in the 3' region contain MS modification.( Figure 12B )As Figure 12A shown, the gene editing efficiency of the chemically modified shortened APG07433.1 dgRNA, as Figure 11AGene editing efficiency of the chemically modified shortened APG07433.1 sgRNA. gRNAs with an unshortened backbone: 1, full-length sgRNA with 1880 spacer has MS modification at the three terminal nucleotides at both the 5'-region and 3'-region, but no chemical modification at other positions in the sgRNA; 2, dgRNA with 1880 spacer has MS modification at the three terminal nucleotides at both the 5'-region and 3'-region of tracrRNA and crRNA, but no chemical modification at other positions in the dgRNA; 3, dgRNA with 1881 spacer has MS modification at the three terminal nucleotides at both the 5'-region and 3'-region of tracrRNA and crRNA, but no chemical modification at other positions in the dgRNA; 4, dgRNA containing crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see Figure 3A and 3B ), with 1880 spacer; and 5, dgRNA containing crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see Figure 3A and 3B ), with 1881 spacer. sgRNAs with a shortened backbone (see Figure 11A ): 6, shortened (L) sgRNA without chemical modification in the first stem of stem-loop 1; 7, shortened (M) sgRNA without chemical modification in the first stem of stem-loop 1; 8, shortened (N) sgRNA without chemical modification in the first stem of stem-loop 1; 9, shortened (L) sgRNA with additional chemical modification in the first stem of stem-loop 1; 10, shortened (M) sgRNA with additional chemical modification in the first stem of stem-loop 1; and 11, shortened (N) sgRNA with additional chemical modification in the first stem of stem-loop 1. dgRNAs with a shortened backbone: shortened and chemically modified crRNAs (O, Q, P, and R) and tracrRNAs (L, M, N) are as Figure 12A shown. "Control" indicates the case without RGN and gRNA, where for each delivery method, cells are mixed with the nucleofection solution but do not undergo the nucleofection process. "Mock" indicates the case without RGN and gRNA, where for each delivery method, cells are mixed with the nucleofection solution and undergo the nucleofection process. Two exemplary spacers were used for sgRNAs and dgRNAs. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. RGN was delivered in the form of a protein complexed with the guide RNA (RNP delivery) or mRNA encoding RGN (mRNA delivery).

[0112] Figure 13Shows the design for testing the gene editing efficiency of shortened (“M” backbone, see Figure 11A ) and chemically modified APG07433.1 gRNAs. Upper panel: The shortened “M” APG07433.1 sgRNA was modified as follows: MS, LNA, or LNA+PS modifications (3MS, 3LNA, 3LNA / PS; 3 cases) at the three terminal nucleotides at the 5' and 3' regions of the sgRNA, and no additional chemical modification at the first stem of stem-loop 1. Lower panel: The shortened “M” APG07433.1 sgRNA was modified as follows: MS, LNA, or LNA+PS modifications (3MS, 3LNA, 3LNA / PS; 3 cases) at the three terminal nucleotides at the 5' and 3' regions of the sgRNA, and included MS and / or LNA modifications at the first stem of stem-loop 1.

[0113] Figure 14 Shows the gene editing efficiency of the chemically modified shortened “M” APG07433.1 sgRNA as shown Figure 13 compared to the control shortened “M” APG07433.1 sgRNA without any chemical modification. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The RGN was delivered in the form of a protein complexed with the guide RNA (RNP delivery) or mRNA encoding the RGN (mRNA delivery). “MS / LNA”, “LNA”, and “LNA” below the “Modification” bars indicate additional MS and / or LNA chemical modifications in the first stem of stem-loop 1 in the shortened “M” APG07433.1 sgRNA with 3MS, 3LNA, or 3LNA / PS modifications, respectively.

[0114] Figure 15 Shows the gene editing efficiency of the chemically modified shortened “M” APG07433.1 sgRNA as shown Figure 13 compared to the control shortened “M” APG07433.1 sgRNA without any chemical modification. The sgRNA was serially diluted. Data were collected on day 4. The “3MS”, “3LNA”, “3LNA PS” shortened “M” APG07433.1 sgRNAs as shown Figure 13As shown. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The RGN was delivered in the form of a protein complexed with the guide RNA (RNP delivery) or mRNA encoding the RGN (mRNA delivery). "MS / LNA", "LNA", and "LNA" under the "Modified" bar indicate additional MS and / or LNA chemical modifications at the first stem of stem-loop 1 in the shortened "M" APG07433.1 sgRNA with 3MS, 3LNA, or 3LNA / PS modifications, respectively.

[0115] Figure 16A and 16B . Gene editing efficiency of dgRNAs with various chemical modifications at the three terminal nucleotides at 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 form of dgRNA. The WT APG07433.1 gRNA is shown with possible chemical modifications: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' 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 at the 3' region of the tracrRNA. ( Figure 16A , right) Table showing all 18 cases tested under various given combinations of 3MS, 3LNA, or 3LNA+PS in the crRNA, 3MS, 3LNA, or 3LNA+PS in the tracrRNA, and two delivery modes (RNP and mRNA) of the RGN. Figure 16B ) Gene editing efficiency of APG07433.1 dgRNAs with various combinations of chemical modifications as Figure 16A shown. All tested dgRNAs had LNA modifications at all nucleotides of the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used. "Control_TRAC" indicates the case without RGN and dgRNA, where for each delivery method, the cells were mixed with the nucleofection solution but not subjected to the nucleofection process. Control dgRNAs ("dg1880" and "dg1881") had MS modifications at the three terminal nucleotides at both the 5' and 3' regions of the tracrRNA and crRNA, but no chemical modifications at other positions of the dgRNA. Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. The RGN was delivered in the form of a protein complexed with the guide RNA (RNP delivery) or mRNA encoding the RGN (mRNA delivery).

[0116] Figure 17A and 17B shows strategies to rescue gene editing of the RGN system with dgRNAs that have fewer than 11 nucleotide pairs in the first stem of stem-loop 1. Figure 17A shows a strategy that includes using the native sequence of APG05586 pre-crRNA and LNA modification of all nucleotides in the extended first stem of the anti-repeat, extending the first stem at the first bubble distal end of stem-loop 1 of APG05586 dgRNA by 2 nucleotide pairs (i.e., at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA). Figure 17B The strategy shown uses the native sequence of the corresponding pre-crRNA to extend the first stem at the first bubble distal end of stem-loop 1 of APG05586 dgRNA or APG08167 dgRNA by 2 nucleotide pairs (i.e., at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA). Figure 17B Highlights the G:C-rich nature of the APG07433.1 nucleotide pair that is farthest from the first bubble in the first stem of stem-loop 1 (i.e., the nucleotide closest to the 3' region of crRNA and the 5' region of tracrRNA). WT APG07433.1 dgRNA with LNA modification on all 11 nucleotides of the first stem of the anti-repeat achieved the highest gene editing (see Figure 10A ). Thus, the nucleotide sequence from APG07433.1 that is farthest from the first bubble of stem-loop 1 will be used to extend APG05586 and APG08167 as an alternative method.

[0117] Figure 18It was shown that gene editing was rescued in the RGN system of WT (original) dgRNA with fewer than 11 nucleotide pairs in the first stem of stem-loop 1 by extending the first stem at the distal end of the first bulge of stem-loop 1 (i.e., extending at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. In the experiment, two genes were targeted for editing, with two replicates for each target gene. "Control" indicates the situation without RGN and dgRNA, where cells were mixed with the nucleofection solution but not subjected to the nucleofection process. "Unmodified" indicates a dgRNA with MS modification (3MS) at the three terminal nucleotides at both the 5'-region and 3'-region of tracrRNA and crRNA, but no chemical modification at other positions of the dgRNA. "LNA" indicates a dgRNA with 3MS plus LNA modification on all nucleotides of the first stem of the anti-repeat. "Native sequence" indicates a dgRNA that extends the first stem of stem-loop 1 to the indicated nucleotide length using the native sequence from the corresponding pre-crRNA. "APG07433.1 seq" indicates a dgRNA that extends the first stem of stem-loop 1 to the indicated nucleotide length using the sequence from APG07433.1 gRNA. (The indicated nucleotide length is for the first stem of the anti-repeat, and the first stem of the crRNA repeat is expected to have the same nucleotide length 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 distal end of the first bulge of stem-loop 1 (i.e., extended at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA). A schematic diagram of the shortened APG01604 gRNA (APG01604.1, 81 nt backbone length) in the first stem of stem-loop 1 below the figure illustrates the "unmodified" APG01604.1 gRNA. The nucleotide sequence above the data points represents the sequence of 4 nucleotides at the 5'-terminal of the original unextended tracrRNA (for "unmodified" and "LNA") or the sequence of 2 added terminal nucleotides (2 nucleotides at the 5'-terminal of the extended tracrRNA). Gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining for B2M (for editing the B2M target sequence) using flow cytometry. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery).

[0118] Figure 19It shows that extending the first stem at the distal end of the first bubble of stem-loop 1 using the nucleotide sequence from natural pre-crRNA or APG07433.1 gRNA (i.e., extending at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA), and modifying all nucleotides of the first stem of the anti-repeat with LNA, rescued gene editing of the RGN system with WT (original) dgRNA, which had fewer than 11 nucleotide pairs in the first stem of stem-loop 1. The dgRNA was extended to 11 nucleotide pairs or 13 nucleotide pairs in the first stem of stem-loop 1. "Natural" indicates a dgRNA that extends the first stem of the anti-repeat to the indicated nucleotide length using the natural sequence from the corresponding pre-crRNA. "APG07433.1" indicates a dgRNA that extends the first stem of the anti-repeat to the indicated nucleotide length using the sequence from APG07433.1 gRNA. (The indicated nucleotide length is for the first stem of the anti-repeat, and the first stem of the crRNA repeat is expected to have the same nucleotide length for base pairing.) All nucleotides of the extended first stem of the anti-repeat were modified with LNA, and the first stem was extended at the distal end of the first bubble of stem-loop 1 (i.e., extending at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA). The nucleotide sequence above the data points represents the sequence of the 2 added terminal nucleotides (2 nucleotides at the 5'-end of the extended tracrRNA). Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining for B2M (for editing the B2M target sequence) using flow cytometry. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery).

[0119] Figure 20 It shows a strategy to improve gene editing efficiency with shortened dgRNA. Left panel: Schematic of WT APG07433.1 crRNA. Middle panel: Schematic of shortened "M" APG07433.1 crRNA and tracrRNA. Right panel: The 3 terminal nucleotides at the 3' of crRNA and the 2 terminal nucleotides at the 5' of tracrRNA were replaced with C and G nucleotides, respectively (nucleotides with asterisks). For both the shortened (M) and engineered shortened (M), the first stem of the anti-repeat was LNA-modified.

[0120] Figure 21 It shows that by replacing Figure 20For the nucleotides shown in , the gene editing efficiency of the shortened "M" APG07433.1 dgRNA was improved. Two spacers were tested. "Original" refers to the shortened "M" APG07433.1 dgRNA without nucleotide substitutions. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

[0121] Figure 22A and 22B shows the LNA modification strategy for the inverted repeat of stem - loop 1 forming tracrRNA in the gRNA. Figure 22A shows the APG07433.1 dgRNA with good performance modification in gene editing, in which all 11 nucleotides in the first stem (FS) of the inverted repeat were modified with LNA (Tracr(J); see Figure 10A ). Figure 22B shows the APG07433.1 tracrRNA modified at all nucleotides in the FS and the second stem (SS) of the inverted repeat (Tracr(Jb); FS + SS); modified at all nucleotides in the SS of the inverted repeat (Tracr(Jc); SS); and modified at all nucleotides in the inverted repeat (including nucleotides in the first stem, bubble, and second stem) (Tracr(Jd); full stem - loop 1).

[0122] Figure 23 shows that having LNA modification at all nucleotides of the first stem of the inverted repeat in the tracrRNA of the gRNA is the most effective for gene editing compared to other LNA modification strategies for the inverted repeat. The gene editing efficiency of the APG07433.1 dgRNA with LNA - modified tracrRNA (right side of the figure; Tracr(J), Tracr(Jb), Tracr(Jc), and Tracr(Jd)) is shown, as Figure 22A and 22B shown. The crRNA has MS modification at three terminal nucleotides at both the 5' region and the 3' region (3MS), as Figure 22AAs shown. Two exemplary spacers (1880 and 1881) were used. "Control_TRAC" represents the case without RGN and dgRNA, where cells were mixed with the nucleofection solution but not subjected to the nucleofection process. The control dgRNA on the left side of the figure has MS modifications at three terminal nucleotides at both the 5' and 3' regions of the tracrRNA and crRNA, but no chemical modifications at other positions of the dgRNA. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery).

[0123] Figure 24 It is shown that the LNA modification of all nucleotides in the first stem of the crRNA repeat sequence in the gRNA's crRNA is effective for gene editing as long as all nucleotides in the first stem of the anti-repeat sequence in the tracrRNA of the gRNA are also LNA-modified (see the figure above, right). The LNA modification of all nucleotides in the second stem of the crRNA repeat sequence in the gRNA's crRNA deteriorates the gene editing of the gRNA with LNA modification at all nucleotides in the first stem of the anti-repeat sequence (see the figure below, right). The gene editing efficiency of the APG07433.1 dgRNA with LNA modification at all nucleotides in the first or second stem of the crRNA repeat sequence is shown: upper left and left side of the upper figure, the dgRNA with crRNA having LNA modification at all nucleotides in the first stem of the crRNA repeat sequence and tracrRNA having MS modification (3MS) at three terminal nucleotides at both the 5' and 3' regions; upper right and right side of the upper figure, the dgRNA with crRNA having LNA modification at all nucleotides in the first stem of the crRNA repeat sequence and tracrRNA having LNA modification at all nucleotides in the first stem of the anti-repeat sequence; lower left and left side of the lower figure, the dgRNA with crRNA having LNA modification at all nucleotides in the second stem of the crRNA repeat sequence and tracrRNA having 3MS; lower right and right side of the lower figure, the dgRNA with crRNA having LNA modification at all nucleotides in the second stem of the crRNA repeat sequence and tracrRNA having LNA modification at all nucleotides in the first stem of the anti-repeat sequence. Two exemplary spacers (1880 and 1881) were used. "Control" represents the case without RGN and dgRNA, where cells were mixed with the nucleofection solution but not subjected to the nucleofection process. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery).

[0124] Figure 25 Shown is that LNA modification increases the editing potency of APG05586 sgRNA. The sgRNA was serially diluted. "Unmodified" means that the sgRNA has MS modification at three terminal nucleotides at both the 5'-region and 3'-region and no other chemical modification (3MS). "LNA@SL1" means the sgRNA with LNA modification at all nucleotides of the first stem of the antisense repeat and 3MS. "MS / LNA@SL1" means the sgRNA with LNA modification at all nucleotides of the first stem of the antisense repeat, MS modification at three terminal nucleotides of the crRNA repeat of the loop closest to stem-loop 1, and 3MS. The gene editing efficiency in primary human T cells was measured by assessing the knockout of CD3 surface marker using flow cytometry. The RGN was delivered in the form of protein complexed with guide RNA (RNP delivery) or mRNA encoding the RGN (mRNA delivery). "Control (TRAC)" and "Control (B2M)" mean that for two different spacers in the gRNA, in the absence of RGN and gRNA, the cells were mixed with the nucleofection solution but not subjected to the nucleofection process.

[0125] Figure 26 Shown is that the amount of LNA modification at the first stem of the antisense repeat is related to the guide RNA editing efficiency and melting temperature (Tm) of the DNA / tracrRNA antisense repeat heteroduplex. The schematic of APG07433.1 dgRNA shows 1, 3, 6, or 11 LNA-modified nucleotides within the antisense repeat region of the first stem forming stem-loop 1. The dgRNA includes MS modification at the 5'- and 3'-termini of tracrRNA and crRNA. The highest editing and highest Tm were achieved when all nucleotides within the antisense repeat region of the first stem forming 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. The gene editing efficiency in primary human T cells was measured by assessing the knockout of CD3 surface marker using flow cytometry. Two spacers (1062 and 1881) were tested in the gene editing experiment.

[0126] Figure 27Shows gene editing rescue of the APG07991 RGN system with WT (original) dgRNA, which has fewer than 11 (6) nucleotide pairs in the first stem of stem-loop 1 (see left schematic), by extending the first stem at the distal end of the first bubble of stem-loop 1 (i.e., extending at the 3’-terminal nucleotide of crRNA and the 5’-terminal nucleotide of tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiment, with two replicates for each target gene. “Control” indicates the situation without RGN and dgRNA, where cells were mixed with the nucleofection solution but not subjected to the nucleofection process. “Unmodified” indicates a dgRNA with MS modification (3MS) at the three terminal nucleotides at both the 5′ and 3′ regions of tracrRNA and crRNA, but no chemical modification at other positions of the dgRNA. “LNA” indicates a dgRNA with 3MS plus LNA modification at all nucleotides of the first stem of the anti-repeat. “Native” indicates a dgRNA that extends the first stem of stem-loop 1 to the indicated nucleotide length using the native sequence from APG07991 pre-crRNA. “APG07433.1” indicates a dgRNA that extends the first stem of stem-loop 1 to the indicated nucleotide length using the sequence from APG07433.1 gRNA. (The indicated nucleotide length is 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 distal end of the first bubble of stem-loop 1 (i.e., extending at the 3'-terminal nucleotide of crRNA and the 5'-terminal nucleotide of tracrRNA). The nucleotide sequence above the data points represents the sequence of 6 nucleotides at the 5' end of the original, unextended tracrRNA (for “Unmodified” and “LNA”) or the sequence of 2 added terminal nucleotides (2 nucleotides at the 5' end of the extended tracrRNA). Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining for B2M (for editing the B2M target sequence) using flow cytometry. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery).

[0127] Figure 28It shows that the gene editing efficiency of APG07991 dgRNA can be rescued by extending the first stem of stem-loop 1 at the distal end of the first bubble to at least 11 nucleotide pairs (i.e., extending 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 sequence with LNA. "Control" indicates the case without RGN and dgRNA, where cells are mixed with the nucleofection solution but not subjected to the nucleofection process. "sgRNA" indicates the APG07991 sgRNA control with an appropriate spacer (TRAC or B2M), which has MS modifications at the three terminal nucleotides at both the 5'-region and 3'-region of the sgRNA, but no chemical modifications at other positions of the sgRNA. The numbers 6, 8, 10, 11, 12, and 13 represent the lengths of the first stem of stem-loop 1. "Native sequence" indicates the native sequence from APG07991 pre-crRNA used to extend the first stem of stem-loop 1 of WT APG07991 dgRNA to the specified nucleotide length. "APG07433.1 Seq" indicates the sequence from APG07433.1 gRNA used to extend the first stem of stem-loop 1 of WT APG07991 dgRNA to the specified nucleotide length. "-" and "+" indicate whether all nucleotides of the first stem of the anti-repeat sequence are modified with LNA. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining for B2M (for editing the B2M target sequence) using flow cytometry. APG07991 RGN was delivered in the form of mRNA encoding APG07991 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0128] Figure 29It is also shown that the gene editing efficiency of Streptococcus pyogenes Cas9 (SpyCas9) dgRNA, which is used for gene editing together with APG07991 RGN, can be rescued by extending the first stem of stem-loop 1 at the distal end of the first bubble to at least 11 nucleotide pairs (i.e., extending 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 sequence with LNA. The first stem of stem-loop 1 of the WT (original) SpyCas9 dgRNA has 4 nucleotide pairs (see the left figure), and the gene editing using APG07991 RGN is very low. "Control" indicates the situation without RGN and dgRNA, where cells are mixed with the nucleofection solution but not subjected to the nucleofection process. "sgRNA" indicates the SpyCas9 sgRNA control with appropriate spacers (TRAC or B2M), which has MS modifications at the three terminal nucleotides at both the 5'-region and 3'-region of the sgRNA, but no chemical modifications at other positions in the sgRNA. The numbers 4, 8, 11, and 13 represent the lengths of the first stem of stem-loop 1. "Native sequence" indicates the native sequence from SpyCas9 pre-crRNA used to extend the first stem of stem-loop 1 of the WT SpyCas9 dgRNA to the specified nucleotide length. "APG07433.1 Seq" indicates the sequence from APG07433.1 gRNA used to extend the first stem of stem-loop 1 of the WT SpyCas9 dgRNA to the specified nucleotide length. The gene editing efficiency in primary human T cells was measured by evaluating the knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining for B2M (for editing the B2M target sequence) using flow cytometry. The APG07991 RGN was delivered in the form of mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (targeting the TRAC and B2M genes) were used.

[0129] Figure 30LNA modification of all nucleotides showing the inverted repeats forming the first stem of stem - loop 1 confers higher stability to the sgRNA. Left schematic: (A) The end - modified sgRNA has MS modification at three terminal nucleotides at both the 5' region and 3' region of the sgRNA; (B) The LNA - modified sgRNA has MS modification at three terminal nucleotides at both the 5' region and 3' region, and has LNA modification at all nucleotides of the first stem of the inverted repeats; (C) The MS / LNA - modified sgRNA has MS modification at three terminal nucleotides at both the 5' region and 3' region, has MS modification at three terminal nucleotides at the 3' region of the crRNA repeat, and has LNA modification at all nucleotides of the first stem of the inverted repeats; and (D) The MS / LNA - modified dgRNA has MS modification at three terminal nucleotides at both the 5' region and 3' region of the crRNA, has LNA modification at all nucleotides of the first stem of the inverted repeats, and has MS modification at three terminal nucleotides at the 3' region of the tracrRNA. The MS / LNA - modified dgRNA can perform efficient gene editing by co - delivery of the mRNA and gRNA components, but is less stable than the chemically modified sgRNA, as demonstrated in the case of staggered delivery.

[0130] Figure 31 Base editing efficiency was shown to be improved using gRNAs with LNA modifications. The gRNAs tested were as Figure 30 shown. Panels A, B, C, and D of the figure correspond to A, B, C, and D described in the left schematic.

[0131] Figure 32 Base editing efficiency was shown to be improved using both gRNAs with LNA modifications and shortened gRNAs with LNA modifications. The no - LNA, LNA, and MS / LNA of the figure correspond to what is depicted in the left schematic. "shrt" indicates a shortened sgRNA. gRNAs with two exemplary spacers SGN001880 and SGN001881 were used.

[0132] Figure 33 The gene editing efficiency of dgRNAs chemically modified with another bridged nucleic acid (BNA) cEt was shown to be improved to a level comparable to that of LNA - modified dgRNAs. The APG07433.1 dgRNA with 11 LNA - modified nucleotides within the inverted repeats forming the first stem of stem - loop 1 (depicted in the upper - left schematic, see also Figure 10A) The gene editing efficiency was compared with that of the APG07433.1 dgRNA having 11 S-constrained ethyl (cEt) modified nucleotides within the inverted repeat of the first stem forming stem-loop 1 (depicted in the lower left schematic). "Unmodified" refers to a dgRNA having MS modifications (3MS) at the three terminal nucleotides at both the 5' and 3' regions of the tracrRNA and crRNA, but no chemical modifications at other positions of the dgRNA. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining of B2M (for editing the B2M target sequence) using flow cytometry. The APG07433.1 RGN was delivered in the form of mRNA encoding the APG07433.1 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0133] Figure 34 It was shown that the gene editing efficiency of the dgRNA extended on the first stem distal to the first bubble of stem-loop 1 and chemically modified with another bridged nucleic acid (BNA) cEt was increased to a level comparable to that of the dgRNA of the same extension modified with LNA. The APG05586 dgRNA was extended to 11 nucleotide pairs at the first stem distal to the first bubble of stem-loop 1 (i.e., extended at the 3' terminal nucleotide of the crRNA and the 5' terminal nucleotide of the tracrRNA; see Figure 18 and 19), and all nucleotides of the first stem of the anti-repeat sequence are modified with LNA (depicted in the upper left schematic) or S-constrained ethyl (cEt) (depicted in the lower left schematic). "Native sequence of APG05586" refers to the native sequence from APG05586 pre-crRNA used to extend the first stem of stem-loop 1 of WT APG05586 dgRNA to 11 nucleotides. "APG07433.1 Seq" refers to the sequence from APG07433.1 gRNA used to extend the first stem of stem-loop 1 of WT APG05586 dgRNA to 11 nucleotides. "Original" means that WT APG05586 dgRNA has 9 nucleotide pairs in the first stem of stem-loop 1 and has not been extended. "Unmodified" means a dgRNA that has MS modifications (3MS) at the three terminal nucleotides at both the 5' and 3' regions of the tracrRNA and crRNA but no chemical modifications at other positions of the dgRNA. Gene editing efficiency in primary human T cells was measured by assessing knockout of the CD3 surface marker (for editing the TRAC target sequence) or immunostaining of B2M (for editing the B2M target sequence) using flow cytometry. APG05586 RGN was delivered in the form of mRNA encoding APG05586 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

[0134] Figure 35 It was shown that LNA modification improved the gene editing efficiency of dgRNAs with an extended region. Figure 35The top shows schematic diagrams of three chemically modified dgRNAs, where the extension region is located at the tail of the tracrRNA: (a) with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA (3MS termini), and at the three terminal nucleotides at the 5' region and the three terminal nucleotides at the 3' region of the tracrRNA + extension region (3MS termini); (b) the 3MS termini of the crRNA, with LNA modifications at all nucleotides of the first stem of the anti-repeat sequence, and with MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension region; (c) at the 3MS termini of the crRNA, with LNA modifications at all nucleotides of the first stem of the anti-repeat sequence, with LNA modifications at 4 nucleotides of the first stem of the stem-loop closest to the tracrRNA tail (i.e., stem-loop 3 of the depicted system), and with MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension region. The left bar labeled "single" in the figure represents the gene editing efficiency of the control single guide RNA corresponding to the dgRNA without the extension region, which has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS termini) of the guide RNA and no LNA modifications. The control single guide RNA serves as a benchmark for the gene editing efficiency of the guide RNA without the extension region. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery). SL1 = stem-loop 1. SL3 = stem-loop 3.

[0135] Figure 36A-36C Shows a schematic diagram of the guide RNA with the extension region and the engineering details of the shortened guide RNA. Figure 36A The non-engineered guide RNA + extension region has: a crRNA length of 46 nt (spacer + 21 nt crRNA repeat); and a tracrRNA length of 79 nt (85 nt WT length minus 6 nt at the tail). Figure 36B The shortened guide RNA + extension region has a shortened crRNA / tracrRNA backbone and has: a crRNA length of 41 nt (spacer + 16 nt shortened crRNA repeat); and a tracrRNA length of 72 nt. Figure 36C Schematic diagrams of the engineered shortened crRNA and tracrRNA (except without the extension region), showing that the 3 terminal nucleotides at the 3′ of the crRNA and the 2 terminal nucleotides at the 5′ of the tracrRNA are replaced by C and G nucleotides (asterisk nucleotides). For Figure 36A and 36BOf the two dgRNA + extension regions shown, the first stem of the anti-repeat sequence is LNA-modified.

[0136] Figure 37 It is shown that LNA modification improves the gene editing efficiency of the shortened dgRNA + extension region, which is engineered with nucleotide substitutions at the 3' end of the crRNA and the 5' end of the tracrRNA. a, A dgRNA + extension region with an engineered shortened backbone and MS modifications (3MS ends) at the three terminal nucleotides at both the 5' region and the 3' region of the crRNA, and MS modifications (3MS ends) at the three terminal nucleotides at the 5' region and the three terminal nucleotides at the 3' region of the tracrRNA + extension region; and b, A dgRNA + extension region with an engineered shortened backbone and 3MS ends of the crRNA, with LNA modifications at all nucleotides of the first stem of the anti-repeat sequence, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension region. The left bar labeled "single" in the figure represents the gene editing efficiency of a control single-guide RNA without an extension region or a shortened backbone or nucleotide engineering, which has MS modifications at the three terminal nucleotides at both the 5' region and the 3' region (3MS ends) of the guide RNA and no LNA modification. The control single-guide RNA serves as a benchmark for the gene editing efficiency of guide RNAs without an extension region or a shortened backbone or nucleotide engineering. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery). SL1 = stem-loop 1. SL3 = stem-loop 3.

[0137] Figure 38Shows that LNA modification at the first stem of the anti-repeat sequence is crucial for the editing efficiency of dgRNA + extension region. (a) MS modification at the three terminal nucleotides at both the 5′ region and 3′ region of crRNA (3MS termini), and MS modification at the three terminal nucleotides at the 5′ region and the three terminal nucleotides at the 3′ region of tracrRNA + extension region (3MS termini); (b) 3MS termini of crRNA, LNA modification at all nucleotides of the first stem of the anti-repeat sequence, and MS modification at the three terminal nucleotides at the 3′ region of tracrRNA + extension region; (c) 3MS termini of crRNA, LNA modification at all nucleotides of the first stem of the anti-repeat sequence, LNA modification at 4 nucleotides of the first stem of the stem-loop closest to the tracrRNA tail (stem-loop 3 in this system), and MS modification at the three terminal nucleotides at the 3′ region of tracrRNA + extension region; (d) MS modification at the three terminal nucleotides at the 5′ region of crRNA, LNA modification at all nucleotides of the first stem of the crRNA repeat sequence, and 3MS termini of tracrRNA + extension region; (e) MS modification at the three terminal nucleotides at the 5′ region of crRNA, LNA modification at all nucleotides of the first stem of the crRNA repeat sequence, LNA modification at all nucleotides of the first stem of the anti-repeat sequence, and MS modification at the three terminal nucleotides at the 3′ region of tracrRNA + extension region; (f) MS modification at the three terminal nucleotides of the 5′ region of crRNA, LNA modification at all nucleotides of the first stem of the crRNA repeat sequence, LNA modification at all nucleotides of the first stem of the anti-repeat sequence, LNA modification at 4 nucleotides of the first stem of stem-loop 3, and MS modification at the three terminal nucleotides at the 3′ region of tracrRNA + extension region; (g) MS modification at the three terminal nucleotides at the 5′ region of crRNA, LNA modification at all nucleotides of the first stem of the crRNA repeat sequence, and 3MS termini of tracrRNA + extension region; (h) MS modification at the three terminal nucleotides at the 5′ region of crRNA, LNA modification at all nucleotides of the first stem of the crRNA repeat sequence, LNA modification at all nucleotides of the first stem of the anti-repeat sequence, and MS modification at the three terminal nucleotides at the 3′ region of tracrRNA + extension region. (g) and (h) The dgRNA + extension region has as Figure 36CThe engineered shortened backbone. The left bar labeled "single" in the figure represents the gene editing efficiency of a control single guide RNA without an extended region or a shortened backbone or nucleotide engineering, which has MS modifications (3MS termini) at three terminal nucleotides at both the 5' and 3' regions of the guide RNA and no LNA modifications. The control single guide RNA serves as a benchmark for the gene editing efficiency of guide RNAs without an extended region or a shortened backbone or nucleotide engineering. The gene editing efficiency in primary human T cells was measured by assessing the knockout of the CD3 surface marker using flow cytometry. Data were collected on day 4. The RGN was delivered in the form of mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3. DETAILED DESCRIPTION OF THE INVENTION

[0139] Benefiting from the teachings presented in the foregoing description and the related drawings, many modifications and other embodiments of the inventions described herein will come to mind to those of ordinary skill in the art to which these inventions pertain. Accordingly, it is to be understood that the inventions are not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0140] I. Overview

[0141] Among other things, the present disclosure provides compositions and methods related to modified guide RNAs (gRNAs) for RNA-guided nuclease (RGN) systems, as well as related systems and methods. In various embodiments, the inclusion of one or more bridging nucleic acids (such as locked nucleic acids) within certain regions (but not in some embodiments, other regions) of the gRNA increases the editing efficiency of the RGN system. Additionally, such modifications unlock the editing potential in mRNA-based dual-guide systems, which was not achievable with unmodified dual-guide RNAs available in the prior art. This is a key advancement in the art because new editing modalities, such as prime editing (also known as reverse transcriptase editing or RT editing), require very long gRNA templates that are difficult or even impossible to synthesize on a large scale in single-guide form using current manufacturing processes. Thus, the lack of production feasibility in the art significantly limits the commercial potential of these mRNA-based therapies. These and other advancements are presented in the present disclosure.

[0142] The RGN system allows for targeted manipulation of specific loci within the genome and is highly useful in gene targeting scenarios for therapeutic and research applications. For example, in various organisms including mammals, the RGN system has been used to generate single-stranded or double-stranded breaks in polynucleotides, modify polynucleotides, detect specific sites within polynucleotides, or modify the expression of specific genes. The RGN system involves a complex of RGN and gRNA. Hybridization of the gRNA with a specific target sequence allows the guide RNA / RGN complex to be targeted to a specific location in the genome for editing.

[0143] In the RGN system, the gRNA can exist as a two-part gRNA or a single gRNA. The two-part gRNA system contains a CRISPR RNA (crRNA) that contains a spacer sequence that recognizes the target genomic sequence through Watson-Crick base pairing, and a scaffold trans-activating crRNA (tracrRNA). The crRNA hybridizes with the tracrRNA to form a dual guide RNA (dgRNA) that binds together as a duplex in the crRNA:tracrRNA annealing region. For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

[0144] Both dgRNA and sgRNA have advantages and disadvantages. sgRNA generally provides relatively good editing efficiency. However, although sgRNA is somewhat convenient as a single chemical species, it is relatively long - typically 100 nt or higher. A common method for generating gRNA is through solid-phase oligonucleotide synthesis, which is a sequential synthesis route. As the length increases, although the coupling efficiency of each individual step is high, 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 DiDe, 2008; Shiba, Nucleic Acids, 2007; LeProust, Nuc. Acids Res. 2010). Therefore, the ability to use shorter gRNA and / or dgRNA has some utility in reducing the maximum length of the oligonucleotide to be synthesized. Another advantage of using dgRNA is that the tracrRNA pairs with different crRNAs containing different spacer sequences, so the tracrRNA scaffold can be prepared in large batches and paired with individual crRNAs targeting specific genomic targets.

[0145] However, the gene editing efficiency of dgRNA is relatively low, especially in delivery methods where the mRNA encoding the RGN is introduced into cells to express the RGN. The additional exposed 5' and 3' ends of the tracrRNA and crRNA and the relatively weak duplex strength relative to sgRNA (where intramolecular components hybridize) may result in lower RNA stability and thus poor performance. In some embodiments, strengthening the duplex can enhance and rescue the effectiveness of dgRNA.

[0146] Many chemical modifications, including 2'-fluoro ribose (2′-F), 2'-O-methyl (2′-O-Me), 3'-thio phosphate (PS), 2'-O-Me 3'-thio phosphate (MS), and other modifications, have been used to improve gRNA stability and thus enhance editing efficiency in vitro and in vivo. However, when transfecting the mRNA encoding RGN with dgRNA, these methods fail to achieve efficient editing. Instead, they require the production of long sgRNAs. With the emergence of new technologies, such as prime editing using additional guide extensions, sgRNAs may not be produced on a large scale and with high purity. In addition, while the above modifications protect RNA from nuclease degradation, other mechanisms may lead to guide degradation or inactivation, particularly disruption of secondary structure. Bridged nucleic acids (BNAs) include nucleotide analogs with conformationally restricted backbones due to intramolecular bonds or crosslinks. For example, in the design of PCR probes, a BNA, namely locked nucleic acid (LNA), has been used to shorten the probe length and enhance hybridization strength. LNA includes a covalent bond between the 2'-oxygen and 4'-carbon on the ribose of the nucleotide and has been shown to facilitate efficient complementary pairing to improve mismatch discrimination and enhance nuclease resistance (You et al., Nucleic Acids Res., 2006; Vester & Wengel, J. Biochemistry, 2004). This disclosure provides methods for achieving RGN-based gene editing in cells using BNA-modified guide RNAs. The data in this application demonstrate that such guide RNAs modified with BNA modifications are improved compared to prior art methods, significantly enhancing the potency of the guide RNA and enabling the use of dual-guide RNAs in applications where they were previously not applicable, such as when gRNA is co-transfected with the mRNA encoding RGN. Without being bound by conjecture, we believe these benefits are at least due to the BNA modification stabilizing the stem-loop formed by hybridization between the crRNA repeat sequence and the tracrRNA anti-repeat sequence. In some cases, the BNA is LNA. In some cases, the BNA is S-constrained ethyl (cEt). In some cases, this disclosure provides methods for achieving RGN-based gene editing in cells using BNA-modified and / or other chemically modified guide RNAs. In various embodiments, the present disclosure incorporates BNA (e.g., LNA and / or cEt) or other chemical modifications into tracrRNA, gRNA, and / or crRNA to produce chemically modified tracrRNA, gRNA, and / or crRNA for use in RGN-based gene editing systems. In some embodiments, both crRNA and tracrRNA contain BNA (e.g., LNA and / or cEt) modifications. In certain embodiments, either crRNA or tracrRNA contains BNA (e.g., LNA and / or cEt) modifications.In some embodiments, the tracrRNA comprises a BNA modification, while the crRNA does not comprise a BNA (e.g., LNA and / or cEt) modification. In certain embodiments, the tracrRNA comprises a BNA modification and the crRNA comprises an MS modification. In embodiments, the chemically modified tracrRNA, gRNA, and / or crRNA of the present disclosure improve the gene editing efficiency of the RGN system as compared to a reference RGN system having a tracrRNA, gRNA, and / or crRNA that comprises only MS modifications at three terminal nucleotides at the 5' region and the 3' region. In embodiments, the modified tracrRNA, gRNA, and / or crRNA of the present disclosure allow for the use of dgRNA in applications where an sgRNA would otherwise be more desirable. In some embodiments, the present disclosure provides for 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 the RGN system in cells. In some embodiments, the BNA (e.g., LNA and / or cEt) modifications allow for a shortened crRNA:tracrRNA annealing region. In some embodiments, the BNA (e.g., LNA) modification in combination with engineering of the crRNA:tracrRNA annealing region of the dgRNA enhances the performance of the RGN system in cells. In some embodiments, the gene-edited cells include primary cells. The modified tracrRNA, gRNA, and / or crRNA of the present disclosure can be used with any model system, cell type, and target sequence that employs an RGN system.

[0147] Without being bound by any one theory, chemical modification of nucleotides in the gRNA can enhance stability and / or stabilize RNA-RNA interactions in the chemically modified region by interfering with degradation of the gRNA by endogenous nucleases.

[0148] II. Guide RNA

[0149] The present disclosure provides guide RNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and / or cEt) modification. In some embodiments, at least one BNA (e.g., LNA and / or cEt) modification is located in the first stem of the anti-repeat sequence 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 located in the first stem of the anti-repeat sequence of the tracrRNA. The term "guide RNA" is known in the art and generally refers to an RNA molecule (or set of RNA molecules) that can bind to an RNA-guided nuclease (RGN) and facilitate targeting of the RGN to a specific location (such as, for example, a genomic locus) within a target polynucleotide (e.g., a DNA or mRNA molecule). In some embodiments, the guide RNA comprises a nucleotide sequence (i.e., a spacer) that has sufficient complementarity to a target strand nucleotide sequence to hybridize to 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 DNA, the target nucleotide sequence comprises a non-target strand (including the PAM sequence) and a target strand, and the target strand hybridizes to the spacer of the guide RNA. In these embodiments, the guide RNA has sufficient complementarity to the target strand of the double-stranded target sequence (e.g., the target DNA sequence) such that the guide RNA hybridizes to 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 non-target strand sequence, except that uracil (U) replaces thymidine (T) in the guide RNA.

[0150] The guide RNA for each RGN is one or more RNA molecules (usually one or two) that can bind to the RGN and direct the RGN to bind to a specific target sequence, and also cleave the target strand and / or the non-target strand in those embodiments where the RGN has nickase or nuclease activity. Generally, the guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).

[0151] The term "guide RNA" generally also encompasses a set of two or more RNA molecules, where the crRNA fragment and the tracrRNA fragment are located in separate RNA molecules. A native guide RNA containing both crRNA and tracrRNA typically consists of two separate RNA molecules that hybridize to each other through the repeat sequence of crRNA and the anti-repeat sequence of 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 connects two molecules, e.g., a linkage formed by click chemistry or any other chemical reaction, a polynucleotide, a polymer, or any entity that can connect two molecules. In some embodiments, the linker that connects crRNA and tracrRNA includes a polynucleotide linker (e.g., a tetranucleotide linker) to form a single guide RNA molecule, where the crRNA and tracrRNA hybridize to each other through the repeat sequence of crRNA and the anti-repeat sequence of tracrRNA. Thus, guide RNA encompasses single guide RNA (sgRNA), where the crRNA fragment and the tracrRNA fragment are located in the same RNA molecule or strand.

[0152] The crRNA and tracrRNA of the guide RNA can be linked by an organic molecule, group, polymer, or chemical moiety. In some embodiments, the crRNA and tracrRNA of the guide RNA are linked by click chemistry. Click chemistry involves joining small units together through heteroatom links (C-X-C) to rapidly generate compounds. The main goal of click chemistry is to develop a set of powerful, selective, and modular "parts" that are suitable for small-scale and large-scale applications. Click chemical reactions are fast, modular, efficient, generally do not produce toxic waste, can be accomplished using water as a solvent, and can be made stereospecific.

[0153] Click chemistry is a versatile reaction that can be used to synthesize a variety of conjugates. Almost any biomolecule can participate, and it is easy to achieve the labeling of small molecules such as fluorescent dyes, biotin, and other groups. Click chemical reactions occur between two components: an azide functional group and an alkyne functional group. An azide is a linear polyatomic anion with the molecular formula N3 - , with the structure - N=N + =N - . It is the conjugate base of hydrazoic acid HN3. An organic azide is an organic compound with the molecular formula RN3 that contains an azide functional group. An alkyne is an unsaturated hydrocarbon containing at least one carbon-carbon triple bond (-C≡C-; e.g., terminal acetylene). The simplest acyclic alkyne has only one triple bond and no other functional groups, forming a homologous series with the general chemical formula C n H2n-2 。The molecular formula of terminal alkynes is RC2H. An example is methylacetylene (propyne using IUPAC nomenclature). Neither azide nor alkyne groups occur almost ever in natural biomolecules. Thus, the reaction can occur within biological systems without interfering with other cellular processes (i.e., highly bioorthogonal) and specifically.

[0154] A well-known click reaction is the Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This reaction produces triazoles and has become the gold standard of click chemistry due to its reliability, specificity, and biocompatibility. When the reaction involves simpler alkenes or azides, this cycloaddition reaction requires high temperature or high pressure because the activation energy is very high. Cu(I) catalysts facilitate the reaction of terminal alkynes and azides to give 1,4-disubstituted-1,2,3-triazoles. This reaction is an ideal click reaction and is widely used in materials science, medicinal chemistry, and chemical biology.

[0155] However, the cytotoxic nature of transition metals used as click reaction catalysts renders them unsuitable for in vivo applications. Alternative methods 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, alkynes are introduced into strained difluorooctyne (DIFO), where electron-withdrawing, propargylic, gemfluorines act together with ring strain to greatly disrupt the stability of the alkyne (Agard et al. (2006) ACS Chem. Biol. 1(10):644-648). This instability increases the reaction driving force, as well as the desire of cycloalkynes to relieve their ring strain. Copper-free click chemistry proceeds via a concerted [3+2] cycloaddition reaction, the mechanism of which is the same as that of the Huisgen 1,3-dipolar cycloaddition reaction. Substituents other than fluorine, such as benzene rings, are also allowed on cyclooctynes.

[0156] Reactive groups in click chemistry (e.g., azides, terminal alkynes, strained alkynes (e.g., dibenzocyclooctyne (DBCO))) can be introduced into nucleic acid molecules in any form and can be introduced enzymatically or chemically. Alkyne-modified and azide-modified oligonucleotides can be ordered from oligonucleotide synthesis facilities or companies. During RNA synthesis, an azide-modified version of a nucleotide can be introduced into a first RNA molecule, and an alkyne-modified version of a nucleotide 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 any unreacted reagents or by-products that may interfere with subsequent click reactions. The purified click-functionalized nucleic acid molecules can be mixed together in a reaction buffer that supports the click reaction. This can include a copper catalyst to facilitate the reaction between the azide and the alkyne and can also be copper-free. The azide functional group and the alkyne functional group react to form a covalent bond that links 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 successful ligation of the nucleic acid molecules and to assess the purity of the product.

[0157] Click chemistry is further described in, e.g.: Kumar et al., (2007) J. Am. Chem. Soc, 129:6859-6864; El-Sagheer and Brown (2010) Chem. Soc. Rev. 39:1388-1405; Haque and Peng (2014) Sci. China Chem. 57:215-231; Wittig and Krebs 1961 Chem. Ber. 1961, 94, 3260–3275; US 7,375,234; US 7,070,941; US2013 / 0046084, the content of each of which is incorporated herein by reference in its entirety.

[0158] The ligation of crRNA and tracrRNA can be carried out as follows. By solid-phase synthesis, the crRNA contains a 3'-amino modifier and the tracrRNA contains a 5'-amino modifier. After synthesizing, deprotecting, and purifying the RNA oligonucleotides, an azide group is installed at the 5'-region of the tracrRNA using the NHS ester of azidobutyric acid (4-azido-1-butyric acid N-hydroxysuccinimide ester; e.g., available from Glen Research, catalog number 50-1904-24), and a cyclooctyne group is installed at the 3'-region of the crRNA using the NHS ester of DBCO (dibenzocyclooctyne-PEG4-N-hydroxysuccinimide ester; e.g., available from Sigma Aldrich, catalog number 764019). Assembly in an aqueous medium (optionally optimizing the ionic strength, pH, and reagent concentration) allows the strain-promoted azide-alkyne Huisgen cycloaddition reaction ("copper-free click chemistry") to proceed.

[0159] 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 contain an azide group at one or more nucleotides at the anti-repeat sequence of the tracrRNA and an alkyne group at one or more nucleotides at the crRNA repeat sequence of the crRNA. In some embodiments, the crRNA and tracrRNA linked by a chemical moiety contain an azide group at one or more nucleotides at the crRNA repeat sequence of the crRNA and an alkyne group at one or more nucleotides at the anti-repeat sequence of the tracrRNA. One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be located within the stem, the loop, or both of the crRNA repeat sequence of the guide RNA. One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be located within the stem, the loop, or both of the anti-repeat sequence of the guide RNA. In some embodiments, the crRNA and tracrRNA linked by a chemical moiety are a single guide RNA and contain an azide-modified nucleotide or an alkyne-modified nucleotide at one or more nucleotides within the nucleotide loop that connects the crRNA repeat sequence and the anti-repeat sequence.

[0160] Those skilled in the art can utilize various further chemical reactions and corresponding modifications to covalently link nucleic acid molecules (e.g., crRNA and tracrRNA) to each other. These modifications include various crosslinking agents, such as thiol modifications, such as lipoic acid N-hydroxysuccinimide (NHS) ester, chemical groups that react with primary amines (-NH2). These primary amines are positively charged and nucleophilic at physiological pH; this makes them easy to target and conjugate with multiple reactive groups. There are many synthetic chemical groups 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, ethylene oxides, carbonates, aryl halides, imidates, carbodiimides, such as, for example, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), acid anhydrides, and fluorophenyl esters.

[0161] As described herein, the guide RNA can comprise a crRNA and a tracrRNA, wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat sequence comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat sequence comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one BNA modification. In some embodiments, the anti-repeat sequence is capable of hybridizing with the crRNA repeat sequence to form a stem-loop comprising a first stem and a second stem.

[0162] As described herein, the present disclosure also provides a nucleic acid molecule comprising a tracrRNA, wherein the tracrRNA comprises: (a) an anti-repeat sequence; (b) a tail; and (c) a stem-loop closest to the tail, wherein the anti-repeat sequence of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one BNA modification. In some embodiments, the anti-repeat sequence of the tracrRNA is capable of hybridizing with the crRNA repeat sequence of the crRNA to form a stem-loop comprising a first stem and a second stem. In some embodiments, the gRNA comprising the tracrRNA is capable of binding to the RGN.

[0163] As described herein, the present disclosure also provides a nucleic acid molecule comprising a crRNA, which comprises: (a) a spacer; and (b) a crRNA repeat sequence comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, and wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiolphosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5' region or 3' region of the crRNA. In some embodiments, the crRNA repeat sequence is capable of hybridizing with the anti-repeat sequence of a tracrRNA to form a stem-loop comprising a first stem and a second stem. In some embodiments, a gRNA comprising a crRNA is capable of binding to an RNA-guided nuclease (RGN) that requires a tracrRNA for activity.

[0164] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat sequence, wherein the tracrRNA comprises an anti-repeat sequence, wherein the gRNA comprises a stem-loop comprising a first stem and a second stem, wherein the first stem has a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0165] As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat sequence, wherein the tracrRNA comprises an anti-repeat sequence, wherein the gRNA comprises a stem-loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

[0166] The present invention provides, among other things, a CRISPR RNA (crRNA) or a polynucleotide encoding a CRISPR RNA, which comprises at least one BNA (e.g., LNA and / or cEt) modification. As used herein, the term "crRNA" refers to an RNA molecule or a portion thereof, which includes a spacer, which is a nucleotide sequence that directly hybridizes to the target strand of a target sequence, and a CRISPR repeat sequence, which contains a nucleotide sequence that forms a structure recognized by an RGN molecule, alone or in cooperation with a hybridized tracrRNA. As used herein, the term "tracrRNA" or "trans-activating crRNA" refers to an RNA molecule that contains an anti-repeat sequence, which has sufficient complementarity to hybridize to at least a portion of the CRISPR repeat sequence of a crRNA to form a structure that can be recognized by an RGN molecule. In some embodiments, additional secondary structures (e.g., stem-loops) within the tracrRNA molecule are required for binding to the RGN.

[0167] 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: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiolphosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and 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 includes 2′,4′ BNA modification. In certain embodiments, the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2′,4′ BNA is an LNA modification. In some embodiments, the 2′,4′ BNA is a cEt modification. In some embodiments, the at least one chemical modification is a 2′-O-Me modification. In certain embodiments, the at least one chemical modification is an MS modification.

[0168] The crRNA comprises a spacer region and CRISPR repeat sequences. The "spacer region" 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 region is engineered to have complete or partial complementarity with the target strand of the target sequence of interest. In some embodiments, the spacer region can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the spacer region 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 some embodiments, the length of the spacer region 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 some embodiments, the length of the spacer region is from about 10 to about 26 nucleotides, or from about 12 to about 30 nucleotides. In some embodiments, the length of the spacer region is about 30 nucleotides. In some embodiments, the length of the spacer region is 30 nucleotides. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the degree of complementarity between the spacer region and the target strand of the target sequence (e.g., a target DNA sequence) is 50% to 99% or higher, including but not limited to, about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the degree of complementarity between the spacer region and the target strand of the target sequence (e.g., a 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. In some embodiments, the sequence of the spacer region can be the same as the non-target strand of the target sequence. In some of those embodiments in which the target sequence is a target DNA sequence, the sequence of the spacer region can be the same as the non-target strand of the target DNA sequence, except that thymidine (T) in the non-target strand is replaced by uracil (U) in the spacer region.In embodiments, the spacer has no secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al., (2008) Cell 106(1):23-24).

[0169] In some embodiments, the spacer of the present disclosure includes chemical modifications 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 includes at least one chemical modification. In some embodiments, the at least one modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiolphosphate (MS) modification; 2'-O-methyl 3'-thiolphosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA (e.g., LNA) modification. In certain embodiments, the spacer of the present disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the spacer of the present disclosure includes at least 2′,4′ BNA modification. In some embodiments, the 2′,4′ BNA modification is selected from the group consisting of locked nucleic acid (LNA) modification, BNA NC[N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the spacer of the present disclosure comprises at least one LNA modification. In some embodiments, the spacer of the present disclosure comprises at least one 2′-O-Me modification. In some embodiments, the spacer of the present disclosure comprises at least one MS modification. In some embodiments, the spacer of the present disclosure comprises at least one 2′-O-Me modification and at least one MS modification. In certain embodiments, the 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, the 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 without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:14 or that differs from SEQ ID NO:14 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:14 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:14 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:14.

[0170] For clarity, as described herein, when a nucleotide sequence "differs from SEQ ID NO by a certain number of nucleotides" or "has a certain percentage identity to SEQ ID NO", the differences occur only in the nucleotide sequence, and chemical modifications or lack thereof remain unchanged.

[0171] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in SEQ ID NO:16 or that differs from SEQ ID NO:16 by 1 to 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 as set forth in SEQ ID NO:16.

[0172] In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:15 or that differs from SEQ ID NO:15 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:15 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:15 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has the nucleotide sequence as set forth in SEQ ID NO:15.

[0173] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in SEQ ID NO:17 or that differs from SEQ ID NO:17 by 1 to 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 as set forth in SEQ ID NO:17.

[0174] In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:89 or that differs from SEQ ID NO:89 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:89 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:89 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has the nucleotide sequence as set forth in SEQ ID NO:89.

[0175] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in 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 as set forth in SEQ ID NO:91.

[0176] In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:90 or that differs from SEQ ID NO:90 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:90 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:90 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has the nucleotide sequence as set forth in SEQ ID NO:90.

[0177] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in 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 as set forth in SEQ ID NO:92.

[0178] In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO:111 or that differs from SEQ ID NO:111 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:111 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO:111 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has the nucleotide sequence as set forth in SEQ ID NO:111.

[0179] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in 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 as set forth in SEQ ID NO:113.

[0180] In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO: 112 or that differs from SEQ ID NO: 112 by 1 or 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO: 112 by 2 nucleotides. In some embodiments, the spacer without any chemical modification has a nucleotide sequence that differs from SEQ ID NO: 112 by 1 nucleotide. In some embodiments, the spacer without any chemical modification has a nucleotide sequence as set forth in SEQ ID NO: 112.

[0181] In some embodiments, the chemically modified spacer has a nucleotide sequence as set forth in 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 a nucleotide sequence as set forth in SEQ ID NO: 114.

[0182] Together with the spacer, the crRNA also includes a CRISPR RNA (crRNA) repeat sequence. The crRNA repeat sequence contains a nucleotide sequence that forms a structure recognized by the RGN molecule, acting alone or in concert with a hybridized tracrRNA. In embodiments, the crRNA repeat sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the crRNA repeat sequence 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 embodiments, the length of the crRNA repeat sequence 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 embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the crRNA repeat sequence and its corresponding tracrRNA anti-repeat sequence is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more. In a specific embodiment, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the crRNA repeat sequence and its corresponding tracrRNA anti-repeat 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.

[0183] In some embodiments, the crRNA repeats of the present disclosure include chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the crRNA repeats. In certain embodiments, the crRNA repeats of the present disclosure include at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA (e.g., LNA and / or cEt) modification. In certain embodiments, the crRNA repeats of the present disclosure include at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the crRNA repeats of the present disclosure include at least 2′,4′ BNA modification. In some embodiments, the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, the crRNA repeats of the present disclosure include at least one LNA modification. In certain embodiments, the crRNA repeats of the present disclosure include at least one cEt modification. In some embodiments, the disclosed crRNA repeats include at least one 2'-O-Me modification. In some embodiments, the crRNA repeats of the present disclosure include at least one MS modification. In some embodiments, the crRNA repeats of the present disclosure include at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, the crRNA repeats of the present disclosure include 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 include at least one BNA (e.g., LNA. In certain embodiments, the crRNA repeats of the present disclosure include at least one LNA modification.) modification and at least one PS modification.

[0184] In some embodiments, the crRNA repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NO: 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 included in a guide RNA, is capable of directing sequence - specific binding of the relevant RNA - guided nuclease provided herein to a target sequence of the present disclosure. In some embodiments, an active crRNA repeat 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 the nucleotide sequence shown in any one of SEQ ID NO: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384 - 393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. In some embodiments, an active crRNA repeat sequence 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 shown in any one of SEQ ID NO: 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 sequence without chemical modification has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence that differs from SEQ ID NO: 2 by 1 or 2 nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO: 2 by 2 nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO: 2 by 1 nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO: 2. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence as shown in SEQ ID NO: 70 or a nucleotide sequence that differs from SEQ ID NO: 70 by 1 or 2 nucleotides.In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:70 by two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:70 by one nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:70. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:94 or a nucleotide sequence that differs from SEQ ID NO:94 by one or two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:94 by two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:94 by one nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:94. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:241 or a nucleotide sequence that differs from SEQ ID NO:241 by one or two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:241 by two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:241 by one nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:241. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:253 or a nucleotide sequence that differs from SEQ ID NO:253 by one or two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:253 by two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:253 by one nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:253. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as shown in SEQ ID NO:538 or a nucleotide sequence that differs from SEQ ID NO:538 by one or two nucleotides. In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO:538 by two nucleotides.In some embodiments, the crRNA repeat sequence without chemical modification has a nucleotide sequence that differs from SEQ ID NO: 538 by 1 nucleotide. In some embodiments, the crRNA repeat sequence without chemical modification has the nucleotide sequence as set forth in SEQ ID NO: 538.

[0185] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 39.

[0186] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 384.

[0187] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 385.

[0188] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in (c)SEQ ID NO:386 or that differs from SEQ ID NO:386 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:386 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:386 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO:386.

[0189] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO:387 or that differs from SEQ ID NO:387 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:387 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:387 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO:387.

[0190] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO:300 or that differs from SEQ ID NO:300 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:300 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:300 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO:300.

[0191] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO:304 or that differs from SEQ ID NO:304 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:304 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO:304 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO:304.

[0192] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 308.

[0193] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 312.

[0194] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 320.

[0195] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 344 or a nucleotide sequence that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 344.

[0196] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 348 or a nucleotide sequence that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 348.

[0197] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 352 or a nucleotide sequence that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 352.

[0198] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 356 or a nucleotide sequence that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 356.

[0199] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 360.

[0200] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 388.

[0201] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 389.

[0202] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 390.

[0203] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 324.

[0204] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 328.

[0205] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 332.

[0206] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 336.

[0207] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 391.

[0208] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 392.

[0209] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 393.

[0210] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 397.

[0211] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 465.

[0212] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO: 469.

[0213] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 473 or a nucleotide sequence that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 473.

[0214] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 477 or a nucleotide sequence that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 477.

[0215] In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence as set forth in SEQ ID NO: 481 or a nucleotide sequence that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat sequence has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat sequence has the nucleotide sequence as set forth in SEQ ID NO: 481.

[0216] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO:508 or that differs from SEQ ID NO:508 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:508 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:508 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO:508.

[0217] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO:512 or that differs from SEQ ID NO:512 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:512 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:512 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO:512.

[0218] In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence as set forth in SEQ ID NO:516 or that differs from SEQ ID NO:516 by 1 or 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:516 by 2 nucleotides. In some embodiments, the chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO:516 by 1 nucleotide. In some embodiments, the chemically modified crRNA repeat has the nucleotide sequence as set forth in SEQ ID NO:516.

[0219] In some embodiments, the crRNA is not naturally occurring. In some embodiments, a particular crRNA repeat is not linked to an engineered spacer sequence in nature, and the crRNA repeat is considered heterologous to the spacer sequence. In some embodiments, the spacer sequence is a non-naturally occurring engineered sequence.

[0220] In some embodiments, the crRNAs of the present disclosure include chemical modifications 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 crRNAs of the present disclosure include at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; thiophosphate (PS) modification; and BNA (e.g., LNA) modification. In certain embodiments, the crRNAs of the present disclosure include at least one BNA (e.g., LNA and / or cEt) modification. In some embodiments, the crRNAs of the present disclosure include at least 2′,4′ BNA modification. In some embodiments, the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC[N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, the crRNAs of the present disclosure include at least one LNA modification. In some embodiments, the disclosed crRNAs include at least one 2'-O-Me modification. In some embodiments, the crRNAs of the present disclosure include at least one MS modification. In some embodiments, the crRNAs of the present disclosure include at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, the crRNAs of the present disclosure include 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 crRNAs of the present disclosure include at least one BNA (e.g., LNA and / or cEt) modification and at least one PS modification. In some embodiments, the chemically modified crRNAs have a nucleotide sequence as shown in 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 unmodified crRNAs have a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, the unmodified crRNAs have a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, the unmodified crRNAs have a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, the unmodified crRNAs have a nucleotide sequence having 100% sequence identity to SEQ ID NO: 18. In some embodiments, the unmodified crRNAs have a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, the unmodified crRNAs have a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the unmodified crRNAs have a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19.In some embodiments, the crRNA without chemical modification has a nucleotide sequence with 100% sequence identity to SEQ ID NO:19. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:71. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:71. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:71. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with 100% sequence identity to SEQ ID NO:71. In some embodiments, the chemically modified crRNA has the nucleotide sequence as shown in SEQ ID NO:74. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:72. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:72. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:72. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with 100% sequence identity to SEQ ID NO:72. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:95. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:95. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:95. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with 100% sequence identity to SEQ ID NO:95. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:96. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:96. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:96. In some embodiments, the crRNA without chemical modification has a nucleotide sequence with 100% sequence identity to SEQ ID NO:96.

[0221] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:4. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:4. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:4.

[0222] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:5. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:5. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:5.

[0223] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:6. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:6. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:6.

[0224] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:7. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:7. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:7. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:7.

[0225] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:8. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:8. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:8. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:8.

[0226] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:9. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:9. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:9. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:9.

[0227] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:708. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:708. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:708. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:708.

[0228] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:292. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:292. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:292. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:292, and wherein reference is made to SEQ ID NO:292.

[0229] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 293. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 293.

[0230] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 73. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 73.

[0231] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 74. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 74.

[0232] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 75. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 75.

[0233] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:301. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:301. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:301. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:301.

[0234] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:302. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:302. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:302. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:302.

[0235] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:303. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:303. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:303. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:303.

[0236] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:305. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:305. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:305. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:305.

[0237] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 306. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 306.

[0238] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 307. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 307.

[0239] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 309. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 309.

[0240] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 310. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 310.

[0241] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 311. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 311.

[0242] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 313. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 313.

[0243] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 314. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 314.

[0244] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 315. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 315.

[0245] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 321. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 321.

[0246] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 322. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 322.

[0247] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 323. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 323.

[0248] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 345. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 345.

[0249] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:346. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:346. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:346. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:346.

[0250] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:347. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:347. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:347. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:347.

[0251] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:349. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:349. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:349. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:349.

[0252] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:350. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:350. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:350. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:350.

[0253] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:351. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:351. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:351. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:351.

[0254] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:353. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:353. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:353. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:353.

[0255] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:354. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:354. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:354. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:354.

[0256] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:355. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:355. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:355. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:355.

[0257] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:357. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:357. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:357. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:357.

[0258] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:358. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:358. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:358. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:358.

[0259] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:359. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:359. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:359. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:359.

[0260] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:361. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:361. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:361. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:361.

[0261] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 362. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 362.

[0262] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 363. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 363.

[0263] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 97. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQID NO: 97.

[0264] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 98. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQID NO: 98.

[0265] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:99. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:99. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:99. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:99.

[0266] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:325. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:325. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:325. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:325.

[0267] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:326. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:326. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:326. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:326.

[0268] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:327. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:327. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:327. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:327.

[0269] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 329. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 329.

[0270] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 330. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 330.

[0271] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 331. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 331.

[0272] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 333. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 333.

[0273] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 334. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 334.

[0274] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 335. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 335.

[0275] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 337. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 337.

[0276] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 338. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 338.

[0277] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 339. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 339.

[0278] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 42. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQID NO: 42.

[0279] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 43. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQID NO: 43.

[0280] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 44. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQID NO: 44.

[0281] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:380. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:380. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:380. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:380.

[0282] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:381. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:381. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:381. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:381.

[0283] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:382. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:382. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:382. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:382.

[0284] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:399. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:399. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:399. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:399.

[0285] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:400. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:400. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:400. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:400.

[0286] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:401. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:401. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:401. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:401.

[0287] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:466. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:466. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:466. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:466.

[0288] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:467. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:467. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:467. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:467.

[0289] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 468. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 468.

[0290] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 470. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 470.

[0291] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 471. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 471.

[0292] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 472. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 472.

[0293] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 474. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 474.

[0294] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 475. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 475.

[0295] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 476. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 476.

[0296] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 478. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 478.

[0297] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:479. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:479. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:479. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:479.

[0298] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:480. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:480. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:480. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:480.

[0299] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:482. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:482. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:482. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:482.

[0300] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:483. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:483. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:483. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:483.

[0301] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 484. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 484.

[0302] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 509. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 509.

[0303] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 510. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 510.

[0304] In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 511. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO: 511.

[0305] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:513. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:513. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:513. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:513.

[0306] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:514. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:514. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:514. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:514.

[0307] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:515. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:515. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:515. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:515.

[0308] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:517. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:517. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:517. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:517.

[0309] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:518. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:518. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:518. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:518.

[0310] In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:519. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:519. In some embodiments, the chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:519. In some embodiments, the chemically modified crRNA has the nucleotide sequence as set forth in SEQ ID NO:519.

[0311] The guide RNAs of the present disclosure include a crRNA and a trans-activating CRISPR RNA (tracrRNA). The tracrRNA molecule includes a nucleotide sequence containing a region herein referred to as an anti-repeat sequence, which has sufficient complementarity to hybridize with the crRNA repeat sequence. In an embodiment, the tracrRNA molecule further includes a region having a secondary structure (e.g., a stem-loop). In some embodiments, the secondary structure includes nucleotides in one of two states, paired or unpaired, wherein nucleotide or base pairing includes base-base hydrogen bonding interactions between two complementary nucleic acid strands (e.g., adenine (A) pairs with uracil (U), cytosine (C) pairs with guanine (G)) to form a helix. In some embodiments, a combination of unpaired single-stranded nucleotides interspersed with one or more helical elements constitutes an RNA structure.

[0312] As used herein, "stem-loop" refers to a secondary structure form found in polynucleotides that contains at least one "stem" and at least one "loop", "bulge", or "bubble". Stem-loops can form intramolecularly (within one molecule, e.g., within a tracrRNA or sgRNA) or intermolecularly (between two different nucleic acids, e.g., in a dgRNA, formed by the crRNA repeat sequence of crRNA and the anti-repeat sequence of tracrRNA). Stem-loops are generated when there is at least some complementarity between two nucleic acid sequences to form a paired double helix. The paired double helix region with perfect complementarity or sometimes including G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine) is called the "stem". The terms "loop", "bulge", or "bubble" refer to single-stranded regions in the "stem-loop" structure where the nucleotides are not complementary, excluding G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine). Thus, "loops", "bulges", and "bubbles" include unpaired nucleotides. In some embodiments, the difference between a "loop" and a "bulge" or "bubble" is that the "loop" is located at one end of the "stem-loop" structure, while the "bulge" or "bubble" is located between two "stems" in the "stem-loop" structure.

[0313] In certain embodiments, the stem-loop structure includes a stem and a loop located at one end of the stem. In certain embodiments, the stem-loop structure includes a first stem and a second stem with a bubble between the stems. In some embodiments, the stem-loop structure includes a loop, multiple stems, and multiple bubbles located between the stems. At this time, in the order close to the loop, the bubbles are sequentially called the "first bubble", "second bubble", "third bubble", etc., and in the order close to the loop, the stems are sequentially called the "first stem", "second stem", "third stem", etc. In the embodiment of dgRNA, the stem-loop formed by the crRNA repeat sequence of crRNA and the anti-repeat sequence of tracrRNA does not include a loop. Therefore, in the order close to the 5' end of tracrRNA (or the 3' end of crRNA), the bubbles are sequentially called the "first bubble", "second bubble", "third bubble", etc., and in the order close to the 5' end of tracrRNA (or the 3' end of crRNA), the stems are sequentially called the "first stem", "second stem", "third stem", etc.

[0314] The term "first stem of the crRNA repeat of crRNA", "first stem of the crRNA repeat", or "first stem of crRNA" refers to the region of the first stem of the crRNA repeat of crRNA that forms a stem-loop structure when hybridized to the anti-repeat sequence of tracrRNA. The term "second stem of the crRNA repeat of crRNA", "second stem of the crRNA repeat", or "second stem of crRNA" refers to the region of the second stem of the crRNA repeat of crRNA that forms a stem-loop structure when hybridized to the anti-repeat sequence of tracrRNA. Similarly, the term "first stem of the anti-repeat sequence of tracrRNA", "first stem of the anti-repeat sequence", or "first stem of tracrRNA" refers to the region of the first stem of the anti-repeat sequence of tracrRNA that forms a stem-loop structure when hybridized to the crRNA repeat of crRNA. The term "second stem of the anti-repeat sequence of tracrRNA", "second stem of the anti-repeat sequence", or "second stem of tracrRNA" refers to the region of the second stem of the anti-repeat sequence of tracrRNA that forms a stem-loop structure when hybridized to the crRNA repeat of crRNA.

[0315] In some embodiments, the stem-loop formed intramolecularly is a hairpin-like stem-loop. Base pairing occurs in the stem portion of the stem-loop and typically includes guanine-cytosine base pairing and adenine-uracil (thymidine) base pairing, although guanine-uracil base pairing is also possible. Base stacking interactions facilitate helix formation. The loop portion of the stem-loop includes unpaired bases. In some embodiments, the loop is the point at which the nucleic acid strand folds back on itself to effect nucleotide pairing to form the stem. In some embodiments, a loop of less than three bases is sterically impossible and no loop is formed. In some embodiments, the optimal loop length is about 4-8 bases. Common loops with a four-nucleotide sequence, such as GAAA, AAAG, ACUU, or UUCG, are referred to as "tetraloops" and are particularly stable due to base stacking interactions of their component nucleotides.

[0316] In some embodiments, the region of the tracrRNA that is fully or partially complementary to the crRNA repeat is located at the 5’ end of the molecule, while the 3’ end of the tracrRNA contains a secondary structure. This region of the secondary structure typically contains several hairpin structures, including a nexus hairpin structure near the anti-repeat sequence. The nexus forms the core of the interaction between the guide RNA and the RGN and is located at the intersection of the guide RNA, the RGN, and the target sequence. The nexus hairpin typically has a conserved nucleotide sequence at the base of the hairpin stem, and the motif UNANNC is found in many nexus hairpins in the tracrRNA. In embodiments, the tracrRNA used in the guide RNA or RGN system of the present disclosure contains non-classical sequences at the base of the hairpin stem of its nexus hairpin, including UNANNG and CNANNC. In some embodiments, the tracrRNA used in the guide RNA or RGN system of the present disclosure contains the non-classical sequence UNANNG at the base of the nexus hairpin stem. In some embodiments, the tracrRNA used in the guide RNA or RGN system of the present disclosure contains the non-classical sequence CNANNC at the base of the nexus hairpin stem. A terminal hairpin is typically present at the 3’ end of the tracrRNA, and its structure and number can vary, but it typically contains a GC-rich Rho-independent transcriptional 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 Publication No. 2017 / 0275648, each of which is incorporated herein by reference in its entirety.

[0317] In some embodiments, the tracrRNA of the present disclosure includes additional hairpins or stem-loop structures in addition to the nexus hairpin. In some embodiments, the tracrRNA includes at least one stem-loop. In some embodiments, the tracrRNA includes at least one stem-loop proximal to the anti-repeat sequence and at least one stem-loop proximal to the 3' end of the tracrRNA. "Proximal" means within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides of a nucleic acid molecule region or end. In certain embodiments, "proximal" means within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides of a nucleic acid molecule region or end. "Most proximal" means closest to the 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" means at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more nucleotides away from a nucleic acid molecule region or end. In some embodiments, "distal" means at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more nucleotides away from a structure (e.g., bubble, loop) of a nucleic acid molecule. For example, the first stem of the anti-repeat sequence of the dual-guide RNA extended at one end away from the first bubble of the stem-loop extends from the 3' terminal nucleotide of the crRNA and the 5' terminal nucleotide of the tracrRNA. A secondary structure is also formed when the tracrRNA hybridizes with its corresponding crRNA. The anti-repeat sequence region of the tracrRNA is fully or partially complementary to the crRNA repeat sequence of the crRNA. In some embodiments, a portion of the anti-repeat sequence of the tracrRNA hybridizes with a portion of the crRNA repeat sequence and forms a stem. In some embodiments, the crRNA:tracrRNA stem includes at least one nucleotide pair (i.e., base pair) because these portions of the anti-repeat sequence and the crRNA repeat sequence are complementary. As described elsewhere herein, the portion of the anti-repeat sequence of the tracrRNA that forms the first stem is the first stem of the anti-repeat sequence, the portion of the anti-repeat sequence of the tracrRNA that forms the second stem is the second stem of the anti-repeat sequence, the portion of the anti-repeat sequence of the tracrRNA that forms the third stem is the third stem of the anti-repeat sequence, and so on.As described elsewhere herein, the crRNA repeat sequence portion of the crRNA that forms the first stem is the first stem of the crRNA repeat sequence, the crRNA repeat sequence portion of the crRNA that forms the second stem is the second stem of the crRNA repeat sequence, the crRNA repeat sequence portion of the crRNA that forms the third stem is the third stem of the crRNA repeat sequence, and so on. In some embodiments, the anti-repeat sequence portion of the tracrRNA and the crRNA repeat sequence portion are not complementary to each other and thus do not hybridize to form base pairs. In some embodiments, the non-complementary region between the anti-repeat sequence and the crRNA repeat sequence forms a bulge or a bubble. In some embodiments, the hybridization of the anti-repeat sequence of the tracrRNA with the crRNA repeat sequence of the crRNA forms a secondary structure comprising at least one stem. In some embodiments, the hybridization of the anti-repeat sequence of the tracrRNA with the crRNA repeat sequence of the crRNA forms a secondary structure comprising at least one bubble. In some embodiments, the hybridization of the anti-repeat sequence of the tracrRNA with the crRNA repeat sequence of the crRNA forms a secondary structure comprising at least one stem and at least one bubble. In some embodiments, the hybridization of the anti-repeat sequence of the tracrRNA with the crRNA repeat sequence of the crRNA forms a secondary structure comprising two stems and a bubble therebetween.

[0318] In certain embodiments, the stem-loop in the gRNA formed solely by a portion of the tracrRNA does not contain BNA (e.g., LNA and / or cEt) modifications. In some embodiments, the stem-loop in the gRNA formed solely by a portion of the tracrRNA does not contain any chemical modifications.

[0319] In certain embodiments, the nucleotides in the loop, bulge, or bubble do not contain BNA (e.g., LNA) modifications. In certain embodiments, the nucleotides in the loop, bulge, or bubble do not contain any chemical modifications.

[0320] In some embodiments, the anti-repeat sequence of the tracrRNA that is fully or partially complementary to the crRNA repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the stem formed by the tracrRNA anti-repeat sequence and the crRNA repeat sequence 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 some embodiments, the length of the stem formed by the tracrRNA anti-repeat sequence and the crRNA repeat sequence 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 some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the crRNA repeat sequence and its corresponding tracrRNA anti-repeat sequence is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the crRNA repeat sequence and its corresponding tracrRNA anti-repeat 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 higher.

[0321] In some embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. In some embodiments, the total length of the tracrRNA comprises 60 to 80 nucleotides, 80 to 100 nucleotides, 100 to 120 nucleotides, 120 to 140 nucleotides, 140 to 160 nucleotides, 160 to 180 nucleotides, or more than 180 nucleotides. For example, the length of 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 an embodiment, the length of 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 some embodiments, the length of the tracrRNA is from about 70 to about 105 nucleotides, including lengths of 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 some embodiments, the length of the tracrRNA is 70 to 105 nucleotides, including lengths of 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 some embodiments, the length of the tracrRNA is from about 90 to about 125 nucleotides, including lengths of 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 some embodiments, the tracrRNA has a length of 90 to 125 nucleotides, including lengths of 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.

[0322] In some embodiments, the tracrRNA of the present disclosure includes chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and / or the phosphate backbone of the tracrRNA. In certain embodiments, the tracrRNA of the present disclosure includes at least one BNA (e.g., LNA and / or cEt) modification. In certain embodiments, the tracrRNA of the present disclosure includes at least one BNA (e.g., LNA and / or cEt) modification and at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'-MOE) modification; 2'-fluoro (2'-F) modification; 2'-F-4'-Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiol phosphate (MS) modification; 2'-O-methyl 3'-thiol phosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification. In certain embodiments, the BNA modification includes 2′,4′ BNA modification. In some embodiments, the 2′,4′ BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC[N-Me] modification, 2′-O,4′-C-ethylene-bridged nucleic acid (2′,4′-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the BNA modification is an LNA modification. Thus, in some embodiments, the tracrRNA comprises at least one LNA modification. In some embodiments, the BNA modification is a cEt modification. Thus, in some embodiments, the tracrRNA comprises at least one cEt modification. In certain embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification, and the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (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, and the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Cα-OMe modification; 2',4'-di-Cα-OMe modification; 2'-O-methyl 3'-thiophosphate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; and thiophosphate (PS) modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one PS modification.

[0323] In some embodiments, the tracrRNA comprises a nucleotide sequence set forth in any of SEQ ID NO: 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 included in a guide RNA, is capable of directing the sequence - specific binding of a related RNA - guided nuclease provided herein to a target sequence of the present disclosure. 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 of the nucleotide sequences set forth in SEQ ID NO: 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 an embodiment, an active tracrRNA sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of any of the nucleotide sequences set forth in SEQ ID NO: 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.

[0324] In some embodiments, the tracrRNA comprises at least one chemical modification in its 5' region or in its 3' region. In some embodiments, the tracrRNA comprises at least one chemical modification in both its 5' region and 3' region. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 100% sequence identity to SEQ ID NO:3. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 100% sequence identity to SEQ ID NO:76 or 77. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:100. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:100. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:100. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 100% sequence identity to SEQ ID NO:100. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:242. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 90% sequence identity to SEQ ID NO:242. In some embodiments, the tracrRNA without chemical modification has a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:242.In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 100% sequence identity with SEQ ID NO:242. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:254. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:254. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:254. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 100% sequence identity with SEQ ID NO:254. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:539. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:539. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:539. In some embodiments, the unmodified tracrRNA has a nucleotide sequence having at least 100% sequence identity with SEQ ID NO:539.

[0325] In some embodiments, the modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:10. In some embodiments, the modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:10. In some embodiments, the modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:10. In some embodiments, the modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:10.

[0326] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:12. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:12. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:12. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:12.

[0327] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:709. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:709. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:709. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:709.

[0328] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:713. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:713. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:713. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:713.

[0329] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:294. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:294. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:294. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:294.

[0330] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:295. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:295. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:295. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:295.

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

[0332] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:81. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:81. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:81. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:81.

[0333] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:364. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:364. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:364. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:364.

[0334] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 365. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 365.

[0335] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 366. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 366.

[0336] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 367. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 367.

[0337] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 369. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 369.

[0338] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:375. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:375. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:375. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:375.

[0339] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:376. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:376. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:376. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:376.

[0340] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:377. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:377. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:377. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:377.

[0341] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:378. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:378. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:378. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:378.

[0342] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:379. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:379. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:379. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:379.

[0343] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:102. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:102. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:102. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:102.

[0344] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:103. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:103. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:103. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:103.

[0345] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:370. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:370. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:370. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:370.

[0346] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 371. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 371.

[0347] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 372. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 372.

[0348] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 373. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 373.

[0349] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 710. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 710.

[0350] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:711. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:711. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:711. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:711.

[0351] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:51. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:51. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:51. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:51.

[0352] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:52. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:52. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:52. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:52.

[0353] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:53. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:53. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:53. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:53.

[0354] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 383. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 383.

[0355] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 499. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 499.

[0356] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 500. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 500.

[0357] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 501. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO: 501.

[0358] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:504. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:504. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:504. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:504.

[0359] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:505. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:505. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:505. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:505.

[0360] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:534. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:534. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:534. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:534.

[0361] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:535. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:535. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:535. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:535.

[0362] In some embodiments, the chemically modified tracrRNA has a nucleotide sequence that has at least 80% sequence identity with SEQ ID NO:537. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:537. In some embodiments, the chemically modified tracrRNA has a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:537. In some embodiments, the chemically modified tracrRNA has the nucleotide sequence as set forth in SEQ ID NO:537.

[0363] As used herein, the term "derived from" in the context of a polynucleotide molecule refers to a molecule generated or synthesized using a parental molecule or information from that parental molecule. For example, a tracrRNA, gRNA, or crRNA of the present disclosure comprising at least one BNA (e.g., LNA) modification is derived from its respective unmodified tracrRNA, gRNA, or crRNA by modifying at least one nucleotide thereof with a BNA (e.g., LNA) modification. In some embodiments, a tracrRNA, gRNA, or crRNA comprising at least one BNA (e.g., LNA) modification derived from its respective unmodified parental tracrRNA, gRNA, or crRNA has the same polynucleotide sequence as the parental molecule. In the context of a crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA, the term "unmodified" 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.

[0364] Two polynucleotide sequences are considered substantially complementary when they hybridize to each other under stringent conditions. Similarly, if a guide RNA that binds to an RGN binds to a target sequence under stringent conditions, the RGN is considered to bind to the specific target sequence in a sequence-specific manner. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which two polynucleotide sequences hybridize to each other, and the degree of such hybridization is detectably higher than hybridization to other sequences (e.g., at least 2-fold above background). Stringent conditions are sequence-dependent and will vary in different circumstances. Typically, stringent conditions are salt concentrations less than about 1.5 M Na ions, usually about 0.01 to 1.0 M Na ion concentration (or other salts), pH 7.0 to 8.3, and for short sequences (e.g., 10 to 50 nucleotides) the temperature is at least about 30 °C, and for long sequences (e.g., greater than 50 nucleotides) at least about 60 °C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low-stringency conditions include hybridization in a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C, and washing in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55 °C. Exemplary medium-stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 °C, and washing in 0.5X to 1X SSC at 55 to 60 °C. Exemplary high-stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 °C, and washing in 0.1X SSC at 60 to 65 °C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The hybridization duration is typically less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time is at least long enough to reach equilibrium.

[0365] Tm is the temperature at which 50% of the complementary target sequence hybridizes to a perfectly matched sequence (at a specified ionic strength and pH). For DNA-DNA hybrids, Tm can be approximated using the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (%form) - 500 / L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Typically, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a particular sequence and its complement at a specified ionic strength and pH. However, highly stringent conditions can be utilized at a temperature 1, 2, 3, or 4°C lower than the thermal melting point (Tm) for hybridization and / or washing; moderately stringent conditions can be utilized at a temperature 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm) for hybridization and / or washing; and low stringency conditions can be utilized at a temperature 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm) for hybridization and / or washing. Using this equation, hybridization and wash compositions, and the desired Tm, one of ordinary skill in the art will appreciate that the stringency of the hybridization and / or wash solution can be varied. Exhaustive guidelines for nucleic acid hybridization can be 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).

[0366] The term "sequence specific" can also refer to the binding affinity of an RGN polypeptide for a target sequence being greater than its binding affinity for a random background sequence.

[0367] The guide RNA can be a single guide RNA (sgRNA) or a dual guide RNA (dgRNA). The sgRNA comprises a crRNA and a tracrRNA on a single RNA molecule, while the dgRNA comprises a crRNA and a tracrRNA on two different RNA molecules, which hybridize to each other through at least a portion of the crRNA repeat sequence of the crRNA and at least a portion of the anti-repeat sequence of the tracrRNA, and they can be completely or partially complementary to each other. The anti-repeat sequence of the tracrRNA hybridizes with the crRNA repeat sequence of the crRNA to form a stem-loop containing the anti-repeat sequence and the crRNA repeat sequence. In some embodiments, the stem-loop comprises one or more stems formed by the anti-repeat sequence and the crRNA repeat sequence. In some embodiments where the guide RNA is an sgRNA, the crRNA and the tracrRNA are separated by a linker nucleotide sequence. Generally, the linker nucleotide sequence does not include base complementarity within itself or with other parts of the sgRNA to avoid forming a secondary structure within the nucleotides of the linker nucleotide sequence or forming a secondary structure containing the nucleotides of the linker nucleotide sequence. In certain embodiments, the linker forms a loop at one end of the first stem in the stem-loop structure containing the crRNA repeat sequence and the anti-repeat sequence. In some embodiments, the length of 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 certain embodiments, the length of the linker nucleotide sequence of the sgRNA is at least 4 nucleotides. In certain embodiments, the linker nucleotide sequence comprises a nucleotide sequence as shown by any one of AAAG, GAAA, ACUU and CAAAGG.

[0368] The total length of the guide RNA can include 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, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 171 nt, 172 nt, 173 nt, 174 nt, 175 nt, 176 nt, 177 nt, 178 nt, 179 nt, 180 nt, 181 nt, 182 nt, 183 nt, 184 nt, 185 nt, 186 nt, 187 nt, 188 nt, 189 nt, 190 nt, 191 nt, 192 nt, 193 nt, 194 nt, 195 nt, 196 nt, 197 nt, 198 nt, 199 nt, 200 nt or more.

[0369] In some embodiments, the chemically modified sgRNA has a nucleotide sequence as shown in any one of SEQ ID NOs: 25 - 30, 60 - 68, 86 - 88, 108 - 110, 298, 299, and 405 - 407.

[0370] sgRNA or dgRNA can be synthesized by chemical synthesis or in vitro transcription. Assays for determining sequence-specific binding between the RGN and the guide RNA are known in the art and include, but are not limited to, in vitro binding assays between the expressed RGN and the guide RNA, which can be labeled with a detectable label (e.g., biotin) and used in a pull-down assay, wherein the guide RNA:RGN complex is captured by the detectable label (e.g., using streptavidin beads). A control guide RNA having a sequence or structure unrelated to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA.

[0371] In some embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be synthesized by chemical synthesis.

[0372] In embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex as described herein, wherein the guide RNA is bound to an RGN polypeptide.

[0373] The guide RNA directs the associated RGN to a specific target nucleotide sequence of interest by 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 intracellularly. The target sequence can include 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 those 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 located within a double-stranded target nucleic acid molecule (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 as shown in any of SEQ ID NOs: 273-278 and 712.

[0374] The target sequence is adjacent to the protospacer adjacent motif (PAM), and the non-target strand of the target sequence is the strand containing the PAM. The PAM is adjacent to the target sequence and typically contains N, which represents any nucleotide. In some embodiments, the PAM contains from 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 contains 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. The PAM can be located 5' or 3' of the target sequence on its non-target strand. In some embodiments, for the guide RNAs and RGN systems of the present disclosure, the PAM is located 3' of the target sequence on its non-target strand. Typically, the PAM is a consensus sequence of about 3-4 nucleotides, but in some embodiments, its length can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides.

[0375] In some embodiments, the PAM sequence adjacent to the target sequence of the present disclosure on its non-target strand comprises a consensus sequence as shown by any one of the PAM sequences in Table 1. In some embodiments, the PAM sequence adjacent to the target sequence of the present disclosure on its non-target strand comprises a consensus sequence as shown by any one of NNNNCC, NNGRR, NNRYA, and NGG. In some embodiments, the PAM sequence is located 3’ of the target sequence on its non-target strand.

[0376] As is well known in the art, the PAM sequence specific to a given nuclease is affected by the enzyme concentration (see, e.g., Karvelis et al., (2015) Genome Biol 16:253), and the enzyme concentration can be altered by changing the promoter used to express the RGN or the amount of ribonucleoprotein complex delivered to cells, organelles, or embryos.

[0377] After recognizing its corresponding PAM sequence, the RGN can cleave one or both strands of the target sequence at a specific cleavage site. As used herein, the cleavage site consists of two specific nucleotides within the target sequence, and the RGN cleaves the target strand, non-target strand, or both strands of the target sequence between these two specific nucleotides. The cleavage site can include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides from the PAM in the 5' or 3' direction. In embodiments, the cleavage site can be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in the 5' or 3' direction. Since the RGN can cleave the target sequence, resulting in staggered ends, in embodiments, the cleavage site is defined based on the distance of two nucleotides from the PAM on the non-target strand of the target sequence, and for the target strand, it is defined based on the distance of two nucleotides from the complementary sequence of the PAM.

[0378] III. Chemical Modification and Length Modification of Guide RNA

[0379] In some embodiments, the nucleotides of the crRNA, crRNA repeat sequence, spacer, tracrRNA, anti-repeat sequence, or guide RNA of the present disclosure may comprise at least one BNA (e.g., LNA) modification. In some embodiments, the at least one BNA (e.g., LNA) modification is located in the first stem of the anti-repeat sequence of 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 sequence of tracrRNA. In some embodiments, the nucleotides of the crRNA, crRNA repeat sequence, spacer, tracrRNA, anti-repeat sequence, or guide RNA of the present disclosure may comprise modifications in the ribose (e.g., sugar) group, phosphate group, nucleobase, or any combination thereof. In the context of oligonucleotides or polynucleotides, the term "chemical modification" includes, but is not limited to: (a) terminal modifications, such as 5'-terminal or 3'-terminal modifications, (b) nucleobase (or "base") modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2', 3', and / or 4' positions, and (d) backbone modifications, including modification or replacement of phosphodiester bonds. The term "modified nucleotide" generally refers to a nucleotide in which the chemical structure of one or more of the base, sugar, and phosphodiester bond or backbone moiety (including nucleotide phosphates) is modified. The terms "modification" and "chemical modification" are used interchangeably herein.

[0380] In some embodiments, the modified nucleotide comprises a sugar modification. Non-limiting examples of sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamino-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'-arachidonoyl cytidine-5'-triphosphate, 2'-arachidonoyl uridine-5'-triphosphate), azidotriphosphates (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate), and combinations thereof.

[0381] In some embodiments, the modified molecules or regions of the disclosure (e.g., crRNA, crRNA repeat sequences, spacers, tracrRNA, anti-repeat sequences, or guide RNAs) include one or more 2'-fluoro, 2'-amino, and / or 2'-thio modifications. In some embodiments, the modifications are 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, 5-allylaminouridine, 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2'-aminobutyrylpyrene uridine, 5-fluorocytidine, and / or 5-fluorouridine.

[0382] Over 96 naturally occurring nucleoside modifications have been found on mammalian RNAs. See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994). Nucleosides include purine or pyrimidine bases linked to a sugar (i.e., nucleotides without a phosphate group). The preparation of nucleotides, modified nucleotides, and nucleosides is well known in the art and is described, e.g., in U.S. Patent Nos. 4,373,071; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319; 5,262,530; and 5,700,642. A variety of modified nucleosides and modified nucleotides suitable for the disclosure are commercially available. Nucleosides can be analogs of naturally occurring nucleosides. In some embodiments, nucleoside analogs include dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxycytidine, and deoxythymidine.

[0383] In some cases, the modifying molecules or regions of the present disclosure (e.g., crRNA, crRNA repeat sequences, spacers, tracrRNA, anti-repeat sequences, or guide RNAs) include ribonucleotides with modified nucleobases, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Non-limiting examples of modified nucleobases that can be incorporated into modified nucleosides and modified nucleotides include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine), m2A (2-methyladenosine), Am (2'-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-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), Ar(p) (2'-O-riboadenosine (phosphate)), I (inosine), mIl (1-methylinosine), m'Im (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), f5C (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (2-methylimidazoline), 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-riboguanosine (phosphate)), yW (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW* (unmodified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (Q nucleoside), oQ (epoxy-Q nucleoside), galQ (galactosyl-Q nucleoside), manQ (mannosyl-Q nucleoside), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G (archaeosine), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-hydroxyacetate), mcmo5U (methyl uridine 5-hydroxyacetate), chm5U (5-(carboxyhydroxymethyl)uridine)), mchm5U (methyl 5-(carboxyhydroxymethyl)uridine), 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), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cnmm5Um (5-carboxymethylaminomethyl-2-L-O-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,T-O-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 (1,2'-O-dimethylguanosine), m'Am (1,2-O-dimethyladenosine), iminomethyluridine), tm5s2U (S-taurine methyl-2-thiouridine)), imG-14 (4-demethylguanosine), imG2 (isoguanosine) or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, 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-(C1-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-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine and combinations thereof.

[0384] In some embodiments, the modified molecules or regions of the present disclosure (e.g., crRNA, crRNA repeat sequences, spacers, tracrRNA, anti-repeat sequences or guide RNAs) include one or more modifications in the phosphate backbone. The modifications can include phosphorothioates, dithiophosphates, aminoph...

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

21. The nucleic acid molecule according to claim 20, wherein the BNA modification comprises LNA modification.

22. The nucleic acid molecule according to claim 20, wherein the BNA modification comprises cEt modification.

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

24. The nucleic acid molecule according to any one of claims 1-22, wherein the first stem of the anti-repeat sequence comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides.

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

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

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

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

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

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

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

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

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

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

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

36. The nucleic acid molecule according to 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 with SEQ ID NO:

1.

37. The nucleic acid molecule according to 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 with SEQ ID NO:

69.

38. The nucleic acid molecule according to 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 with SEQ ID NO:

93.

39. The nucleic acid molecule according to 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 with SEQ ID NO:

252.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

66. The gRNA according to claim 64 or 65, wherein the 2′,4′-BNA is an LNA modification.

67. The gRNA according to claim 64 or 65, wherein the 2′,4′-BNA is a cEt modification.

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

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

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

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

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

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

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

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

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

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

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

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

80. The gRNA according to claim 79, wherein the three terminal nucleotides at the 5′ region and the 3′ region of the crRNA each comprise an MS modification.

81. The gRNA according to claim 79 or 80, wherein the three terminal nucleotides at the 5′ region and the 3′ region of the crRNA all comprise MS modification, and the remaining nucleotides of the first stem of the crRNA repeat sequence comprise 2′-O-Me modification.

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

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

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

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

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

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

88. The gRNA according to claim 79, wherein the three terminal nucleotides at the 5′ region and the 3′ region of the crRNA all comprise MS modification, BNA modification or BNA+PS modification.

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

90. The gRNA according to 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 with any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401 and 708.

91. The gRNA according to 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 with any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709 and 713.

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

93. The gRNA according to 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 with any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359 and 361-363.

94. The gRNA according to 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 with any one of SEQ ID NOs: 80, 81, 364-367, 369 and 375-379.

95. The gRNA according to any one of claims 40-88, wherein the crRNA repeat sequence has a nucleotide sequence as shown in any of the following: (a) SEQ ID NO: 324 or 1 or 2 nucleotides different from SEQ ID NO: 324; (b) SEQ ID NO: 328 or 1 or 2 nucleotides different from SEQ ID NO: 328; (c) SEQ ID NO: 332 or 1 or 2 nucleotides different from SEQ ID NO: 332; (d) SEQ ID NO: 336 or 1 or 2 nucleotides different from SEQ ID NO: 336; (e) SEQ ID NO: 391 or 1 or 2 nucleotides different from SEQ ID NO: 391; (f) SEQ ID NO: 392 or 1 or 2 nucleotides different from SEQ ID NO: 392; and (g) SEQ ID NO: 393 or 1 or 2 nucleotides different from SEQ ID NO:

393.

96. The gRNA according to 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 with any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335 and 337-339.

97. The gRNA according to 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 with any one of SEQ ID NOs: 102, 103, 370-373, 710 and 711.

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

99. The gRNA according to 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 with any one of SEQ ID NOs: 466 - 468, 470 - 472, 474 - 476, 478 - 480, 482 - 484, 509 - 511, 513 - 515 and 517 - 519.

100. The gRNA according to 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 with any one of SEQ ID NOs: 499 - 501, 504, 505, 534, 535 and 537.

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

102. The gRNA according to claim 101, wherein the linker contains an azide functional group or an alkyne functional group.

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

104. The gRNA according to claim 103, wherein the linker has a nucleotide sequence as shown by AAAG, GAAA, ACUU or CAAAGG.

105. The gRNA according to claim 103 or 104, wherein the linker has a nucleotide sequence as shown by AAAG.

106. The gRNA according to any one of claims 103-105, wherein the gRNA is a single guide RNA (sgRNA) comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and a spacer region, and wherein the backbone of the sgRNA comprises the crRNA repeat sequence, a linker, and the tracrRNA.

107. The gRNA according to 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 with any one of SEQ ID NOs: 35-37, 296, and 297.

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

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

110. The gRNA according to claim 109, wherein the RGN is a type II RGN.

111. The gRNA according to 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 with SEQ ID NO:

1.

112. The gRNA according to 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 with SEQ ID NO:

69.

113. The gRNA according to 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 with SEQ ID NO:

93.

114. The gRNA according to 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 with SEQ ID NO:

252.

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

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

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

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

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

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

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

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

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

124. The RGN system according to 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 with any one of SEQ ID NO: 1, 69, 93 or 252.

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

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

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

128. The RGN system according to claim 126 or 127, wherein the target sequence has a nucleotide sequence as shown by any one of SEQ ID NO: 273-278 and 712.

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

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

131. The RGN system according to claim 130, wherein the cleavage produces a double-strand break.

132. The RGN system according to claim 130, wherein the cleavage produces a single-strand break.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

149. The cell according to claim 147 or 148, wherein the target sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO: 273 - 278 and 712.

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

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

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

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

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

155. A plant comprising the cell according to claim 154.

156. A seed comprising the cell according to claim 154.

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

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

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

160. The method according to claim 158 or 159, wherein the RGN polypeptide or the gRNA further comprises an expression regulator, thereby regulating the 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 according to any one of claims 118-145 or an RNP complex according to claim 146 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

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

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

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

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

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

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

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

169. The method according to claim 168, wherein the base editing polypeptide comprises a deaminase.

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

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

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

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

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

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

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

177. The method according to claim 176, wherein the base editing polypeptide comprises a deaminase.

178. The method according to claim 170, wherein the polynucleotide encoding the type II RGN is an mRNA.

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

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

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

182. The method according to claim 181, wherein the base editing polypeptide comprises a deaminase.

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

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

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

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

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

188. The method according to claim 187, wherein the complex formed by the gRNA and the type II RGN directs cleavage of the target sequence.

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

190. The method according to claim 189, wherein the base editing polypeptide comprises a deaminase.

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

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

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

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

195. The method according to claim 187, wherein the polynucleotide encoding the type II RGN is an mRNA.

196. A method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising: a) combining, under conditions suitable for forming a ribonucleoprotein (RNP) complex: 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, so as to assemble 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, so as to bind the target sequence to the RGN.

197. A method for binding a target sequence in a target nucleic acid molecule to an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) 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. The method according to claim 197, wherein the polynucleotide encoding the type II RGN is an mRNA.

199. The method according to any one of claims 158 - 198, wherein the target sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO: 273 - 278 and 712.

200. The method according to 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 with any one of SEQ ID NO: 1, 69, 93 or 252.

201. A method for enhancing the efficiency of cleaving and / or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system according to any one of claims 118 - 145 or an RNP complex according to claim 146 to the target sequence or a cell comprising the target sequence, wherein the nucleic acid molecule is cleaved or modified with a higher efficiency compared to cleaving or modifying 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 does not comprise any chemical modification.

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

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

204. The method according to claim 201, wherein the BNA modification comprises a locked nucleic acid (LNA) modification.

205. The method according to claim 201, wherein the BNA modification comprises a 2'-O,4'-C-ethylene-bridged nucleic acid (cEt) modification.

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

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

208. The method according to claim 207, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblotting, 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. A method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat sequence and the tracrRNA comprises an anti-repeat sequence; and b) adding or replacing one or more nucleotides in the crRNA repeat sequence and one or more nucleotides in the anti-repeat sequence, wherein the one or more nucleotides added or replaced in the repeat sequence and the one or more nucleotides added or replaced in the anti-repeat sequence are capable of hybridizing to each other, wherein the one or more nucleotides added or replaced comprise at least 2, at least 3, at least 4, or at least 5 G or C, and wherein the engineered gRNA has an increased editing efficiency compared to the gRNA provided in step a).

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

211. The method according to claim 209 or 210, wherein the one or more nucleotides added or replaced are located in the 3' region of the crRNA repeat sequence and the 5' region of the anti-repeat sequence, and wherein the 3' region of the crRNA repeat sequence and the 5' region of the anti-repeat sequence comprise at least 2, at least 3, at least 4, or at least 5 G or C.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

229. The method according to claim 228, wherein the BNA modification comprises a 2′,4′-BNA modification.

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

231. The method according to claim 230, wherein the 2′,4′-BNA is an LNA modification.

232. The method according to claim 230, wherein the 2′,4′-BNA is a cEt modification.

233. The method according to any one of claims 209 - 232, wherein the efficiency of the RGN system comprising the engineered gRNA to cleave and / or modify the target sequence 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. The method according to claim 233, wherein the efficiency is determined by measuring the percentage of target sequences or cells comprising the target sequence in which the expression of the target sequence has been altered or the expression of the polypeptide encoded by the target sequence has been altered.

235. The method according to claim 234, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblotting, 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. The method according to any one of claims 209-235, wherein the engineered gRNA further comprises an extension region, and the extension region comprises an editing template for prime editing.

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

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

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

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