WO2024042489A1 - Modification chimique d'arn guides avec un acide nucléique verrouillé pour l'édition de gènes à médiation par nucléase guidée par l'arn - Google Patents

Modification chimique d'arn guides avec un acide nucléique verrouillé pour l'édition de gènes à médiation par nucléase guidée par l'arn Download PDF

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WO2024042489A1
WO2024042489A1 PCT/IB2023/058418 IB2023058418W WO2024042489A1 WO 2024042489 A1 WO2024042489 A1 WO 2024042489A1 IB 2023058418 W IB2023058418 W IB 2023058418W WO 2024042489 A1 WO2024042489 A1 WO 2024042489A1
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seq
crrna
modification
repeat
grna
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PCT/IB2023/058418
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English (en)
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Michael Coyle
Alexandra Briner CRAWLEY
Tedd D. Elich
Drew KELSO
Hui-Chia YU-KEMP
David Wiley
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LifeEDIT Therapeutics, Inc.
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Publication of WO2024042489A1 publication Critical patent/WO2024042489A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the present invention relates to the field of molecular biology and gene editing.
  • Genome editing systems that use RNA- guided nucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated (Cas) proteins of the CRISPR-Cas bacterial system, function by complexing a nuclease, an enzyme that cuts a nucleic acid, with a guide RNA. The hybridization of the guide RNA to a particular target sequence allows editing at a specific location in a genome.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • RGN RNA-guided nucleases
  • gRNAs guide RNAs
  • crRNA CRISPR-RNA
  • tracrRNA scaffold transactivating crRNA
  • This two-part guide RNA requires base pairing between regions of a crRNA molecule and a tracrRNA molecule to form a dual guide RNA (dgRNA).
  • dgRNA dual guide RNA
  • sgRNA chimeric single guide RNA
  • Elements of a guide RNA can be chemically modified to, for example, reduce degradation of the guide RNA.
  • a guide RNA e.g., the phosphate backbone, the ribose sugar, the nucleobase
  • compositions including chemically modified transactivating CRISPR RNA (tracrRNA), guide RNA (gRNA), and/or CRISPR RNA (crRNA).
  • the chemically modified tracrRNA, gRNA, and/or crRNA incorporate bridged nucleic acid (BNA) modifications and/or other chemical modifications.
  • BNA modifications include 2', 4' locked nucleic acid modifications of a nucleotide, in which the 2' oxygen is covalently linked to the 4' carbon via a methylene bridge.
  • additional modifications include 2'-O-methyl (2'-0-Me), 2'- O-methyl 3' phosphorothioate (MS), and phosphorothioate (PS) modifications.
  • the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure improves gene editing efficiency of an RNA-guided nuclease (RGN) system as compared to a reference RGN system comprising tracrRNA, gRNA, and/or crRNA having no BNA modifications.
  • RGN RNA-guided nuclease
  • the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure allows a dual guide RNA to be used in applications where otherwise a single guide RNA would be required.
  • the present disclosure provides for use of BNA modifications and/or other chemical modifications within the first stem of a stem loop 1 of a dual guide RNA to enhance the performance of an RGN system in a cell.
  • the use of BNA modifications and/or other chemical modifications allows the use of a shortened tracrRNA, gRNA, and/or crRNA.
  • the cells that are gene edited with an RGN system of the disclosure include primary cells.
  • the chemically modified tracrRNA, gRNA, and/or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.
  • the present disclosure provides a nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.
  • BNA bridged nucleic acid
  • the at least one BNA modification is within the anti-repeat.
  • the at least one BNA modification is within the first stem of the anti-repeat.
  • the at least one BNA modification comprises at least two three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive mucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat.
  • all nucleotides within the first stem of the anti -repeat comprise BNA modifications.
  • the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is not within a bulge of the tracrRNA. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.
  • PS phosphorothioate
  • the at least one BNA modification comprises a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNA NC [N-Me] modification 2'-O,4'-C-ethylene bridged nucleic acid
  • cEt S-constrained ethyl
  • the 2', 4' BNA is a LNA modification.
  • the 2', 4' BNA is a cEt modification.
  • the tracrRNA further comprises at least one other chemical modification.
  • the at least one other chemical modification is within the anti-repeat of the tracrRNA.
  • the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.
  • the at least one other chemical modification is within the tail of the tracrRNA.
  • the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3 'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.
  • 2'-O-methyl (2'-0-Me) modification 2'-O- methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • 2', 4'- di-Ca-OMe modification 2'-O-methyl 3 'phosphorothioate
  • three terminal nucleotides of the tail of the tracrRNA comprise MS modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the anti-repeat comprise BNA modifications. In some embodiments, the BNA modifications are LNA modifications.
  • the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of about 11 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.
  • the first stem of the anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above tracrRNA aspect, the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.
  • the tracrRNA comprises a total length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.
  • the tracrRNA is part of a gRNA that is capable of binding to an RGN.
  • the RGN is a Type II RGN.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.
  • the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BN A) modification.
  • gRNA guide RNA
  • crRNA CRISPR RNA
  • tracrRNA transactivating CRISPR RNA
  • the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem
  • the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt. In some embodiments of the above gRNA aspect, the gRNA is a dual guide RNA (dgRNA).
  • sgRNA single guide RNA
  • dgRNA dual guide RNA
  • the at least one BNA modification is within the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within the second stem of the crRNA repeat.
  • PS phosphorothioate
  • the at least one BNA modification is within the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, all nucleotides within the first stem of the anti-repeat comprises BNA modifications.
  • the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within a bulge of the gRNA. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the tail of the tracrRNA. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.
  • PS phosphorothioate
  • At least three terminal nucleotides in the 3' region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti-repeat comprise BNA modification.
  • all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the anti -repeat comprise BNA modifications.
  • the at least one BNA modification is within the spacer.
  • three terminal nucleotides at the 5' region of the spacer comprise BNA modifications.
  • the three terminal nucleotides at the 5' region of the spacer comprise both BNA modifications and phosphorothioate (PS) modifications.
  • the spacer is 18-30 nucleotides in length.
  • the at least one BNA modification comprises a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNA NC [N-Me] modification 2'-O,4'-C-ethylene bridged nucleic acid
  • cEt S-constrained ethyl
  • the 2', 4' BNA is a LNA modification.
  • the 2', 4' BNA is a cEt modification.
  • the gRNA further comprises at least one other modification.
  • the at least one other modification is within the crRNA.
  • the at least one other modification is within the 5' region or the 3' region of the crRNA.
  • the at least one other modification is within the 5' region and the 3' region of the crRNA.
  • the at least one other chemical modification is within the crRNA repeat of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the spacer of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tail of the tracrRNA.
  • the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.
  • 2'-O-methyl (2'-O-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • 2',4'-di-Ca-OMe modification 2'-O-methyl 3'phosphorothi
  • three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.
  • the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti -repeat comprises a total length of about 11 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.
  • the first stem of the crRNA repeat at the 3 ' region or the first stem of the anti -repeat at the 5 ’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.
  • the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5 ’ region comprises a GC-rich nucleotide sequence, wherein the first stem of the crRNA repeat at the 3 ’ region or the first stem of the anti -repeat at the 5 ’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.
  • three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.
  • the crRNA repeat has a nucleotide sequence set forth as: (a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides; (b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides; (c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides; (d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides; (e) SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides; or (f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.
  • the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.
  • the crRNA repeat has a nucleotide sequence set forth as (a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides; (b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides; (c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides; (d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides; (e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides; (f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides; (g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides; (h) SEQ
  • the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.
  • the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides; (b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides; (c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides; (d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides; (e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides; (f) SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides; and (g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleot
  • the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337- 339.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.
  • the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides; (b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides; (c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides; (d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides; (e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides; (f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides; (g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides
  • the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.
  • the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.
  • the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the antirepeat.
  • the linker comprises an azide functional group or an alkyne functional group.
  • the linker is a polynucleotide.
  • the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG. In some embodiments, the linker has a nucleotide sequence set forth as AAAG.
  • the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA.
  • the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.
  • the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.
  • the gRNA is capable of binding to an RGN.
  • the RGN is a Type II RGN.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl
  • the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is selected from the
  • the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) described hereinabove; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.
  • the tracrRNA and the crRNA form a gRNA.
  • the RGN system binds a target sequence in a target nucleic acid molecule.
  • the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA described hereinabove; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.
  • RGN RNA-guided nuclease
  • the RGN system binds a target sequence in a target nucleic acid molecule.
  • the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) described hereinabove; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.
  • the tracrRNA and the crRNA form a gRNA.
  • the RGN system binds a target sequence in a target nucleic acid molecule.
  • the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, or NGG.
  • PAM consensus protospacer adjacent motif
  • the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.
  • the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.
  • the RGN polypeptide and the gRNA are not found complexed to one another in nature.
  • the target sequence is a eukaryotic target sequence.
  • the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712. In some embodiments of the above RGN systems aspects, the target sequence is within a cell.
  • a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence.
  • the cleavage generates a double-stranded break.
  • the cleavage generates a single-stranded break.
  • the RGN polypeptide is nuclease inactive. In some embodiments of the above RGN systems aspects, the RGN polypeptide is a nickase.
  • the RGN polypeptide is fused to a base-editing polypeptide.
  • the base-editing polypeptide comprises a deaminase.
  • the RGN polypeptide is fused to a prime editing polypeptide.
  • the prime editing polypeptide comprises a DNA polymerase.
  • the DNA polymerase comprises a reverse transcriptase.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the RGN polypeptide is fused to a detectable label.
  • the RGN system further comprises a donor polynucleotide.
  • the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA. In some embodiments, the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter. In some embodiments, the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.
  • the present disclosure provides a ribonucleoprotein (RNP) complex comprising an RGN system as described hereinabove.
  • RNP ribonucleoprotein
  • the present disclosure provides a cell comprising a nucleic acid molecule comprising a tracrRNA, a gRNA, a crRNA, an RGN system, or an RNP complex as described hereinabove.
  • the cell comprises a target sequence capable of being bound by a formed gRNA/RGN polypeptide complex of an RGN system, or by an RNP complex, as described hereinabove.
  • the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.
  • 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.
  • the present disclosure provides a plant comprising a plant cell as described hereinabove.
  • the present disclosure provides a seed comprising a plant cell as described hereinabove.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tracrRNA, a gRNA, a crRNA, an RGN system, an RNP complex, or a cell as described hereinabove.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence.
  • the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence. In some embodiments of the above aspect, the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence. In some embodiments of the above aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the above aspect, the RGN polypeptide is fused to a base-editing polypeptide.
  • the present disclosure provides a method for cleaving and/or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.
  • RGN RNA-guided nuclease
  • the assembled RNP complex directs cleavage of the target sequence.
  • the RGN is fused to a prime editing polypeptide.
  • the prime editing polypeptide comprises a DNA polymerase.
  • the DNA polymerase comprises a reverse transcriptase.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the RGN polypeptide is fused to a base-editing polypeptide.
  • the base-editing polypeptide comprises a deaminase.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.
  • RGN RNA-guided nuclease
  • a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.
  • the RGN is fused to a prime editing polypeptide.
  • the prime editing polypeptide comprises a DNA polymerase.
  • the DNA polymerase comprises a reverse transcriptase.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the RGN polypeptide is fused to a base-editing polypeptide.
  • the base-editing polypeptide comprises a deaminase.
  • the polynucleotide encoding the Type II RGN is an mRNA.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) as described hereinabove; and ii) a Type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.
  • RNP ribonucleoprotein
  • gRNA guide RNA
  • RGN Type II RNA-guided nuclease
  • the assembled RNP complex directs cleavage of the target sequence.
  • the RGN polypeptide is fused to a base-editing polypeptide.
  • the base-editing polypeptide comprises a deaminase.
  • the RGN is fused to a prime editing polypeptide.
  • the prime editing polypeptide comprises a DNA polymerase.
  • the DNA polymerase comprises a reverse transcriptase.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) as described hereinabove; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.
  • a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.
  • the RGN polypeptide is fused to a base-editing polypeptide.
  • the base-editing polypeptide comprises a deaminase.
  • the RGN is fused to a prime editing polypeptide.
  • the prime editing polypeptide comprises a DNA polymerase.
  • the DNA polymerase comprises a reverse transcriptase.
  • the gRNA further comprises an extension comprising an edit template for prime editing.
  • the polynucleotide encoding the Type II RGN is an mRNA.
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.
  • RGN RNA-guided nuclease
  • the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a tracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.
  • gRNA guide RNA
  • crRNA CRISPR RNA
  • the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712.
  • the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.
  • the present disclosure provides a method of increasing efficiency of cleaving and/or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or any chemical modification.
  • BNA bridged nucleic acid
  • all nucleotides of the first stem of the anti-repeat of the tracrRNA of an RGN system or of an RNP complex as described hereinabove comprise BNA modifications.
  • at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications.
  • the BNA modifications comprise LNA modifications.
  • the BNA modifications comprise cEt modifications.
  • the efficiency of cleaving and/or modifying the target sequence is increased by 15-fold to 30-fold.
  • the efficiency of cleaving and/or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • the present disclosure provides a method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the crRNA repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the 3’ region of the crRNA repeat and the 5 ’ region of the anti-repeat of the engineered gRNA comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).
  • the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides.
  • the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.
  • the gRNA is a dgRNA. In some embodiments of the above aspect, the gRNA is a sgRNA.
  • the method further comprises c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.
  • 2'-O-methyl (2'-0-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • the at least one chemical modification is in the crRNA, the tracrRNA, or both. In some embodiments of the above aspect, the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti-repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA.
  • the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti-repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti-repeat, and the tail of the tracrRNA.
  • the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.
  • the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat.
  • the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3 ’ region of the first stem of the crRNA repeat.
  • the at least one chemical modification comprises a BNA modification.
  • the BNA modification comprises a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNANC[N-Me] modification 2'-O,4'-C-ethylene bridged nucleic acid
  • cEt S-constrained ethyl
  • the 2', 4' BNA is a LNA modification.
  • the 2', 4' BNA is a cEt modification.
  • the editing efficiency of the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the gRNA provided in step a).
  • the efficiency of cleaving and/or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the RGN system comprising the gRNA provided in step a).
  • the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • the present disclosure provides an engineered gRNA produced by the method described hereinabove.
  • the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.
  • gRNA guide RNA
  • crRNA CRISPR RNA
  • tracrRNA transactivating CRISPR RNA
  • the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BN A) modification.
  • gRNA guide RNA
  • crRNA CRISPR RNA
  • tracrRNA transactivating CRISPR RNA
  • the crRNA comprises a crRNA repeat
  • the tracrRNA comprises an anti-repeat
  • the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BN A)
  • FIG. 1 provides a schematic of a dual guide RNA (dgRNA) showing the pairing of the crRNA and the tracrRNA.
  • dgRNA dual guide RNA
  • FIGs. 2A and 2B show schematics illustrating the parts of a single guide (sgRNA) (FIG. 2A), and of a dgRNA (FIG. 2B).
  • the parts include: a spacer; a stem loop 1 including a first stem, a first bubble, and a second stem; a stem loop 2 comprising a first stem only; an inter stem loop region (ISR); a stem loop 3 including a loop, a first stem, a first bubble, a second stem, a second bubble, and a third stem; and a tail.
  • Stem loop 1 in the sgRNA includes a loop, while stem loop 1 in the dgRNA does not include a loop.
  • the CRISPR RNA (crRNA) repeat anneals to the anti -repeat of the transactivating CRISPR RNA (tracrRNA) to form stem loop 1.
  • FIGs. 3A and 3B provide a depiction of the chemical modifications of the dgRNA for APG07433.1 RNA guided nuclease.
  • FIG. 3A shows tracrRNA modification schemes, from left to right: “Stem MS modified”, in which the first stem of stem loop 1 comprises nucleotides with 2'-O- methyl (2'-0-Me) modifications, and with 2'-O-methyl 3'phosphorothioate (MS) modifications at the three terminal nucleotides at the 5' region and MS modifications at the three terminal nucleotides at the 3' region and a 2'-0-Me modification on the fourth nucleotide from the 3’ end of the tracrRNA molecule; “Heavily MS modified” - in addition to the modifications in “Stem MS modified”, adding 2’-0-Me modifications to stem loop 3 through the tail; “Stem LNA Modified” - the first stem of stem loop 1 contains all LNA modifications, and the three terminal nucleotides at the 3' region have
  • FIG. 4 shows the efficiency of gene editing, measured by knockout of the CD3 surface marker using flow cytometry, in primary human T cells using different combinations of the modified crRNA and modified tracrRNA.
  • Guide RNAs were designed to target the TRAC gene. These data demonstrate that modifications at stem loop 3 abolishes editing activity, while modifications at the stem loop 1 alone preserve editing ability of the dgRNA. The modifications are shown with the same scheme as in FIGs. 3A and 3B. TracrRNAs are shown diagrammed.
  • the control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. Mock indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process.
  • FIG. 5 demonstrates that LNA modification enhances the editing efficiency of dgRNA in a RNP complex with purified APG07433.1 protein, which reaches similar levels of editing as sgRNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.
  • FIG. 6 shows LNA-modified dgRNA facilitates a high rate of gene disruption, especially relative to dgRNA that is only end modified (the three terminal nucleotides at both the 5' region and 3' region of the dgRNA have MS modification) or dgRNA that has additional 2-0'-Me modifications (MS/PS mod) at stem loop 1, with an mRNA delivery method, using two exemplary spacers.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.
  • FIG. 7 demonstrates the higher potency of LNA-modified dgRNA in gene editing compared to sgRNA, using two exemplary spacers.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA.
  • FIGs. 8A and 8B show that LNA modification enhances the editing efficiency of sgRNAs for two different RNA-guided nucleases (RGNs), with the RGN being delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • RGNs RNA-guided nucleases
  • FIG. 8A APG07433.1 sgRNA.
  • ‘control_RNP’ and ‘control_mRNA’ indicate conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • FIG. 8B APG01604 sgRNA.
  • control TRAC and ‘control_B2M’ indicate conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • the two different spacer names in the controls indicate that antibodies against TRAC (2275) or B2M (1989) were used in the flow cytometry to establish a no-editing readout.
  • the sgRNAs for each RGN used two exemplary spacers.
  • (+) sgRNA with additional LNA modifications as depicted in each schematic.
  • Each guide RNA schematic shows the LNA modifications in a region of the antirepeat forming the first stem and the MS modifications of the 3 terminal nucleotides at the 5' and 3' regions of the sgRNAs.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker for editing of the TRAC target sequence or immunostaining B2M for editing of the B2M target sequence using flow cytometry.
  • FIGs. 9A and 9B show that MS modification of stem loop 1 does not enhance editing efficiency.
  • FIG. 9A depicts eight scenarios of 2'-0-Me and/or MS modifications in stem loop 1 and/or stem loop 3 for APG07433.1 sgRNA.
  • FIG. 9B shows that none of the tested sgRNAs with 2'-0-Me and/or MS modifications enhance sgRNA editing as compared to a control sgRNA. Chemical modification at stem loop 3 abolishes sgRNA activity.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control’ indicates conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • the control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • FIGs. 10A-10C show that the amount of LNA modifications correlates with guide RNA editing efficiency in primary human T cells as measured by knockout of the CD3 surface marker (for TRAC target sequence) or immunostaining B2M (for B2M target sequence) using flow cytometry.
  • FIG. 10A Gene editing efficiency of APG07433.1 dgRNA with 1, 3, 6, or 11 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1.
  • Schematics of the APG07433.1 dgRNA show: the ‘0 LNA @ stem loop 1’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified. The highest editing was achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified.
  • FIG. 10B Gene editing efficiency of APG01604 dgRNA with 3 or 7 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1.
  • Schematics of the APG01604 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA- modified.
  • FIG. 10C Gene editing efficiency of APG05586 dgRNA with 4 or 9 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1.
  • Schematics of the APG05586 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified.
  • Gene editing was improved for the LNA-modified APG01604 dgRNA and APG05586 dgRNA as compared to the ‘0 LNA’ dgRNA.
  • the dgRNAs for each RGN used two exemplary spacers. Each RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • ‘control_TRAC’ and ‘control_B2M’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process
  • FIGs. 11A-11C show that LNA modification maintains or increases gene editing efficiency for shortened APG07433.
  • 1 sgRNAs. Top: the full-length APG07433.
  • 1 sgRNA was shortened by a combination of truncations in various regions of the sgRNA: 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1 and 6 nt deleted from the tail (-10 first stem SL1, -6 tail); 5 nt pairs (10 nt) deleted from the first stem of stem loop 1, 4 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -4 tail, -2 first stem SL3); and 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1, 6 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, SL1,
  • shortened APG07433.1 sgRNAs have MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA and serve as controls to assess additional chemical modifications introduced into the first stem of stem loop 1.
  • FIG. 1 IB Gene editing efficiency for shortened APG07433.1 sgRNAs chemically modified as illustrated in FIG. 11A as compared to control full-length APG07433. 1 sgRNA or control shortened APG07433.1 sgRNA.
  • the control full-length and shortened sgRNAs each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not contain any chemical modifications at stem loop 1.
  • ‘Mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method.
  • the sgRNAs were used at a dilution factor of 1.
  • LNA modification increases editing potency for shortened APG07433.1 sgRNAs.
  • a serial dilution of the sgRNAs were conducted. Data were collected at day 4.
  • the ‘3MS’ full-length and ‘3MS’ shortened sgRNAs are as depicted in FIG. 11A, top.
  • the modified shortened sgRNAs are as depicted in FIG. 11A, bottom.
  • DF dilution factor.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • FIGs. 12A and 12B show that LNA modification maintains or increases gene editing efficiency for shortened APG07433.1 dgRNAs with RNP delivery.
  • the ‘M’ shortening and chemical modification scheme performs the best for sgRNA and dgRNA.
  • FIG. 12A Chemical modification and shortening schemes for crRNA and tracrRNA.
  • the crRNA is shortened by 5 terminal nt at the 3' region: left, MS modifications at the three terminal nucleotides at the 5' and 3' regions (O and Q represent two exemplary spacers used); right, MS modifications at the three terminal nucleotides at the 5' and 3' regions plus 2’-O-Me modifications within the crRNA repeat forming the first stem of stem loop 1 (P and R represent two exemplary spacers used).
  • tracrRNA ‘tracr(L)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 6 nt, and the three terminal nucleotides at the 3' region comprise MS modifications; ‘tracr(M)’, the anti -repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 4 nt, 1 nt pair (2 nt) is deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3’ region comprise MS modifications; ‘tracr(N)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucle
  • FIG. 12B Gene editing efficiency for shortened APG07433.1 dgRNAs chemically modified as illustrated in FIG. 12A and for shortened APG07433.
  • gRNAs with non-shortened backbones 1, full-length sgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region but not having any chemical modifications elsewhere in the sgRNA; 2, dgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 3, dgRNA with 1881 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 4, dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4)
  • dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see FIGs. 3A and 3B), with 1881 spacer.
  • sgRNAs with shortened backbones see FIG.
  • control indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process, for each delivery method
  • ‘mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method.
  • the sgRNAs and dgRNAs used two exemplary spacers.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • FIG. 13 shows the designs for testing gene editing efficiency of a shortened (‘M’ backbone, see FIG. 11A), chemically modified APG07433.1 gRNA in a sgRNA format.
  • the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA/PS; 3 conditions) and has no additional chemical modifications at the first stem of stem loop 1.
  • the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA/PS; 3 conditions) and includes MS and/or LNA modifications at the first stem of stem loop 1.
  • FIG. 14 shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • the ‘MS/LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and/or LNA chemical modifications in the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA/PS modifications, respectively.
  • FIG. 15. shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications.
  • a serial dilution of the sgRNAs were conducted. Data were collected at day 4.
  • the ‘3MS’, ‘3LNA’, ‘3LNA PS’ shortened ‘M’ APG07433.1 sgRNAs are as depicted in FIG. 13.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • the ‘MS/LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and/or LNA chemical modifications at the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA/PS modifications, respectively.
  • FIGs. 16A and 16B Gene editing efficiencies of a dgRNA with various chemical modifications at the three terminal nucleotides at the 5' region and 3' region.
  • FIG. 16A, left A design for testing gene editing efficiency of a wild-type (WT, full-length), chemically modified APG07433. 1 gRNA in a dgRNA format.
  • the WT APG07433.1 gRNA is shown with possible chemical modifications: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the crRNA; LNA modifications at all nucleotides of the first stem of the anti-repeat; and MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 3' region of the tracrRNA.
  • FIG. 16A, right a table showing the 18 total conditions tested given various combinations of 3MS, 3LNA, or 3LNA+PS in the crRNA, 3MS, 3LNA, or 3LNA+PS in the tracrRNA, and two delivery modes of the RGN (RNP and mRNA).
  • FIG. 16B Gene editing efficiency for APG07433.
  • the control dgRNA (‘dgl880’ and ‘dgl881’) has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA.
  • RGN Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).
  • FIGs. 17A and 17B show strategies for rescuing gene editing of RGN systems having dgRNAs with ⁇ 11 nucleotide pairs in the first stem of stem loop 1.
  • FIG. 17A shows a strategy that includes lengthening the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of APG05586 pre-crRNA and LNA modification of all nucleotides in the lengthened first stem of the anti -repeat.
  • FIG. 17A shows a strategy that includes lengthening the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2
  • FIG. 17B show strategies to lengthen the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA or APG08167 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of the respective pre-crRNAs.
  • FIG. 17B highlights the G:C rich characteristic of the APG07433.1 nucleotide pairs most distal to the first bubble in the first stem of stem loop 1 (i.e. the nucleotides most proximal to the 3’ region of the crRNA and the 5’ region of the tracrRNA).
  • WT APG07433.1 dgRNA having LNA modifications at all 11 nucleotides of the first stem of the anti-repeat achieves the highest gene editing (see FIG. 10A). Therefore, nucleotide sequence from APG07433. 1 most distal to the first bubble of stem loop 1 will be used to lenghthen APG05586 and APG08167 as an alternative approach.
  • FIG. 18 shows that gene editing is rescued for RGN systems having WT (original) dgRNAs with ⁇ 11 nucleotide pairs in the first stem of stem loop 1 by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA.
  • Two genes were targeted for editing in the experiments, and there were two repeats per target gene.
  • the ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • ‘Unmod’ indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA.
  • ‘LNA’ indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the antirepeat.
  • ‘Native seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA.
  • ‘APG07433.1 seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA.
  • nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.
  • All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA).
  • a schematic of a APG01604 gRNA shortened in the first stem of stem loop 1 (APG01604.1, 81 nt backbone length) below the graph illustrates the ‘unmod’ APG01604.1 gRNA.
  • the nucleotide sequences above the data points indicate the sequence of the 4 nucleotides at the 5’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA).
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIG. 19 shows that lengthening the first stem at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) using either nucleotide sequences from a native pre-crRNA or from APG07433.
  • 1 gRNA and modifying all nucleotides of the first stem of the anti-repeat with LNA rescues gene editing for RGN systems having WT (original) dgRNAs with ⁇ 11 nucleotide pairs in the first stem of stem loop 1.
  • the dgRNAs were lengthened to 11 nucleotide pairs or 13 nucleotide pairs in the first stem of stem loop 1.
  • ‘native’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA.
  • ‘APG07433.1’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA.
  • nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.
  • All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA).
  • the nucleotide sequences above the data points indicate the sequence of the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of the lengthened tracrRNA).
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIG. 20 shows a strategy to improve gene editing efficiency by a shortened dgRNA.
  • Left schematic of a WT APG07433.1 crRNA.
  • Center schematic of a shortened ‘M’ APG07433.1 crRNA and tracrRNA.
  • the first stem of the anti-repeat is LNA modified.
  • FIG. 21 shows that gene editing efficiency is improved for a shortened ‘M’ APG07433. 1 dgRNA by substituting nucleotides as shown in FIG. 20. Two spacers were tested. ‘Original’ indicates the shortened ‘M’ APG07433.1 dgRNA without nucleotide substitutions. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIGs. 22A and 22B show strategies for LNA modification of the anti-repeat forming stem loop 1 of the tracrRNA in a gRNA.
  • FIG. 22A shows a modified APG07433.1 dgRNA that performs well in gene editing, having all 11 nucleotides LNA modified in the first stem (FS) of the anti-repeat (Tracr(J); see FIG. 10A).
  • FIG. FS first stem
  • 22B shows APG07433.1 tracrRNA modified at all nucleotides in the FS and in the second stem (SS) of the anti-repeat (Tracr(Jb); FS+SS); modified at all nucleotides in the SS of the anti-repeat (Tracr(Jc); SS); and modified at all nucleotides in the anti-repeat, including nucleotides of the first stem, the bubble, and the second stem (Tracr(Jd); full stem loop 1).
  • FIG. 23 shows that having LNA modification of all nucleotides of the first stem of the antirepeat in the tracrRNA of a gRNA is most effective for gene editing as compared to other LNA modification strategies for the anti-repeat.
  • Gene editing efficiencies are shown for APG07433.1 dgRNAs having LNA-modified tracrRNA (right-hand side of the graph; Tracr(J), Tracr(Jb), Tracr(Jc), and Tracr(Jd)), as shown in FIGs. 22A and 22B.
  • the crRNA had MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS), as shown in FIG. 22A.
  • Two exemplary spacers (1880 and 1881) were used.
  • control_TRAC indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • the control dgRNAs on the left-hand side of the graph has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIG. 24 shows that LNA modification of all nucleotides of the first stem of the crRNA repeat in the crRNA of a gRNA is effective for gene editing as long as all nucleotides of the first stem of the anti -repeat in the tracrRNA of the gRNA are also LNA-modified (see top graph, right side). Having LNA modification of all nucleotides of the second stem of the crRNA repeat in the crRNA of a gRNA worsens gene editing for a gRNA having LNA modification of all nucleotides of the first stem of the anti-repeat (see bottom graph, right side).
  • dgRNAs having LNA modifications at all nucleotides of the first stem or the second stem of the crRNA repeat upper left and left side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS); upper right and right side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat; lower left and left side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with 3MS; and lower right and right side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with 3MS; and lower right and
  • control indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIG. 25 shows that LNA modification increases editing potency for APG05586 sgRNAs.
  • a serial dilution of the sgRNAs were conducted.
  • the ‘unmod’ indicates sgRNAs having MS modifications at the three terminal nucleotides at both the 5' region and 3' region and no other chemical modifications (3MS).
  • the ‘LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, along with 3MS.
  • the ‘MS/LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, MS modifications at the three terminal nucleotides of the crRNA repeat most proximal to the loop of stem loop 1, and 3MS.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.
  • the RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control (TRAC)’ and ‘control (B2M)’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • FIG. 26 shows that the amount of LNA modification at the first stem of the anti -repeat correlates with guide RNA editing efficiency and melting temperature (Tm) of a DNA/tracrRNA antirepeat heteroduplex.
  • Tm melting temperature
  • the amount of LNA modification is shown on the x-axis, and the Tm and gene editing efficiency are shown on the vertical axes.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Two spacers (1062 and 1881) were tested in the gene editing experiments.
  • FIG. 27 shows that gene editing is rescued for an APG07991 RGN system having WT (original) dgRNA with ⁇ 11 (6) nucleotide pairs in the first stem of stem loop 1 (see left schematic) by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA.
  • Two genes were targeted for editing in the experiments, and there were two repeats per target gene.
  • control indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • Unmod indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA.
  • LNA indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the anti-repeat.
  • Nearest indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the APG07991pre-crRNA.
  • ‘APG07433.1’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA.
  • the indicated nucleotide lengths are for the first stem of the anti -repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.
  • All nucleotides of the lengthened first stem of the anti-repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA).
  • the nucleotide sequences above the data points indicate the sequence of the 6 nucleotides at the 5 ’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA).
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • FIG. 28 shows that gene editing efficiency for APG07991 dgRNA can be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA.
  • ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • sgRNA indicates an APG07991 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA.
  • the numbers 6, 8, 10, 11, 12, and 13 indicate the length of the first stem of stem loop 1.
  • Near Seq indicates that native sequence from the APG07991 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length.
  • ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length. ‘-’ and ‘+’ indicate whether all nucleotides of the first stem of the anti-repeat are modified with LNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.
  • FIG. 29 shows that gene editing efficiency for Streptococcus pyogenes Cas9 (SpyCas9) dgRNA, which works with APG07991 RGN for gene editing, can also be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA.
  • SpyCas9 dgRNA Streptococcus pyogenes Cas9
  • the first stem of stem loop 1 of WT (original) SpyCas9 dgRNA has 4 nucleotide pairs (see left schematic) and has very low gene editing with APG07991 RGN.
  • control indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.
  • sgRNA indicates a SpyCas9 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA.
  • the numbers 4, 8, 11, and 13 indicate the length of the first stem of stem loop 1.
  • ‘Native Seq’ indicates that native sequence from the SpyCas9 pre- crRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length.
  • ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (targeting the TRAC and B2M genes) were used.
  • FIG. 30 shows that LNA modifications of all nucleotides of the anti-repeat forming the first stem of stem loop 1 confer more stability to an sgRNA.
  • End modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the sgRNA
  • LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions and LNA modifications at all nucleotides of the first stem of the anti-repeat
  • C MS/LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions, MS modifications of the three terminal nucleotides at the 3 ’ region of the crRNA repeat, and LNA modifications at all nucleotides of the first stem of the anti-repeat
  • D MS/LNA modified dgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the crRNA
  • FIG. 31 shows that base editing efficiency is increased using gRNAs having LNA modifications.
  • the tested gRNAs are as described in FIG. 30.
  • the A, B, C, and D of the graphs correspond to the A, B, C, and D depicted in the left schematic.
  • FIG. 32 shows that base editing efficiency is increased using gRNAs having LNA modifications and also shortened gRNAs having LNA modifications.
  • the No LNA, LNA, and MS/LNA of the graphs correspond to what is depicted in the left schematic, ‘shrt’ indicates a shortened sgRNA.
  • FIG. 33 shows that gene editing efficiency for a dgRNA chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of a LNA-modified dgRNA.
  • BNA bridged nucleic acid
  • ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the APG07433.1 RGN was delivered as mRNA encoding the APG07433.1 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.
  • FIG. 34 shows that gene editing efficiency for a dgRNA lengthened at the first stem distal to the first bubble of stem loop 1 and chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of the same lengthened dgRNA that has been LNA-modified.
  • BNA bridged nucleic acid
  • nucleotides of the first stem of the anti-repeat were modified with either LNA (depicted in upper left schematic) or with S- constrained ethyl (cEt) (depicted in lower left schematic).
  • LNA depicted in upper left schematic
  • cEt S- constrained ethyl
  • ‘Native seq for APG05586’ indicates that native sequence from the APG05586 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides.
  • APG07433.1 Seq indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides.
  • ‘Original’ indicates WT APG05586 dgRNA having 9 nucleotide pairs in the first stem of stem loop 1 that has not been lengthened.
  • ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry.
  • the APG05586 RGN was delivered as mRNA encoding the APG05586 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.
  • FIG. 35 shows that LNA modification improved gene editing efficiency of a dgRNA having an extension.
  • the top of FIG. 35 shows schematics illustrating three chemically modified dgRNAs where the extensions are at the tail of the tracrRNA: (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat , and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the
  • the left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA corresponding to a dgRNA that does not have the extension, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications.
  • the control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without the extension.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • SL1 stem loop 1.
  • SL3 stem loop 3.
  • FIGs. 36A-36C show schematics of guide RNAs having extensions and details of the engineering of a shortened guide RNA.
  • a non-engineered guide RNA + extension has: a crRNA length of 46 nt (a spacer + 21 nt crRNA repeat); and a tracrRNA length of 79 nt (85 nt WT length minus 6 nt from the tail).
  • FIG. 36B a shortened guide RNA + extension has a shortened crRNA/tracrRNA backbone and has: a crRNA length of 41 nt (a spacer + 16 nt shortened crRNA repeat); and a tracrRNA length of 72 nt.
  • FIG. 36A a non-engineered guide RNA + extension has: a crRNA length of 46 nt (a spacer + 21 nt crRNA repeat); and a tracrRNA length of 79 nt (85 nt WT length minus 6 nt from the tail).
  • FIG. 36B a
  • FIG. 37 shows that LNA modification improved gene editing efficiency of shortened dgRNAs + extensions that are engineered with nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA.
  • a dgRNA + extension with engineered shortened backbone and having MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends);
  • b dgRNA + extension with engineered shortened backbone and having 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension.
  • the left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications.
  • the control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • SL1 stem loop 1.
  • SL3 stem loop 3.
  • FIG. 38 shows that LNA modification at the first stem of the anti-repeat is the key for editing efficiency of dgRNA + extension, (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti -repeat, and MS modifications at the three terminal nucleotides at the 3 ' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail of the tracrRNA (stem loop 3 in this system), and MS modifications at the three terminal nucle
  • the left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS) of the guide RNA, and has no LNA modifications.
  • the control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering.
  • Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4.
  • the RGN was delivered as mRNA encoding the RGN (mRNA delivery).
  • SL1 stem loop 1.
  • SL3 stem loop 3.
  • the present disclosure provides, inter alia, compositions and methods related to modified guide RNA (gRNA) for use in RNA-guided nuclease (RGN) systems, and systems and methods related thereto.
  • gRNA modified guide RNA
  • RGN RNA-guided nuclease
  • inclusion of one or more bridged nucleic acid, such as, e.g., locked nucleic acid, within certain regions of the gRNA (but in some embodiments, not within other regions) improves editing efficiency of RGN systems.
  • bridged nucleic acid such as, e.g., locked nucleic acid
  • An RGN system allows for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications.
  • RGN systems have been used for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene, for example.
  • An RGN system involves a complex of an RGN with a gRNA. The hybridization of the gRNA to a particular target sequence allows targeting of the guide RNA/RGN complex to a specific location in a genome for editing.
  • gRNAs can exist as a two-part gRNA or as a single gRNA.
  • the two-part gRNA system includes a CRISPR RNA (crRNA) containing a spacer sequence which recognizes the target genomic sequence via Watson-Crick base pairing, and a scaffold transactivating crRNA (tracrRNA).
  • the crRNA hybridizes to the tracrRNA to form a dual guide RNA (dgRNA) held together as a duplex at a crRNA IracrRNA annealing region.
  • dgRNA dual guide RNA
  • sgRNA chimeric single guide RNA
  • sgRNAs generally provide relatively good editing efficiency.
  • a sgRNA while somewhat convenient as a single chemical species, is relatively long - generally at or above 100 nt.
  • a common method to produce gRNAs is through solid phase oligonucleotide synthesis, which is a serial synthetic route.
  • a dgRNA has relatively low gene editing efficiency, especially in a delivery method where mRNA encoding an RGN is introduced to a cell for expression of the RGN.
  • the additional exposed 5' and 3' ends of the crRNA and tracrRNA and likely weaker duplex strength relative to sgRNA (in which intramolecular components are hybridizing) may result in lower RNA stability and hence poor performance.
  • strengthening the duplex can enhance and rescue the effectiveness of dgRNA.
  • a bridged nucleic acid includes a nucleotide analog having a restricted conformation due to an intramolecular bond or crosslink.
  • a type of BNA a locked nucleic acid (LNA) has been used, for example, in the design of PCR probes to shorten the length of the probes and to increase hybridization strength.
  • An LNA includes a covalent linkage between the 2' oxygen to the 4' carbon on the ribose sugar of a nucleotide and has been shown to contribute to highly efficient complementary pairing to improve mismatch discrimination, as well as to improve nuclease resistance (Y on et al. Nucleic Acids Res., 2006; Vester & Wengel, J Biochemistry, 2004).
  • the BNA is LNA.
  • the BNA is S- constrained ethyl (cEt).
  • methods are provided herein for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications and/or other chemical modifications.
  • the present disclosure incorporates BNA (e.g., LNA and/or cEt) or other chemical modifications in tracrRNA, gRNA, and/or crRNA to generate chemically modified tracrRNA, gRNA, and/or crRNA for use in RGN-based gene editing systems.
  • the crRNA and tracrRNA both comprise BNA (e.g., LNA and/or cEt) modifications.
  • either the crRNA or the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications.
  • the tracrRNA comprises BNA modifications whereas the crRNA does not comprise BNA (e.g., LNA and/or cEt) modifications.
  • the tracrRNA comprises BNA modifications and the crRNA comprises MS modifications.
  • the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure improves gene editing efficiency of an RGN system as compared to a reference RGN system with tracrRNA, gRNA, and/or crRNA comprising only MS modifications at the three terminal nucleotides at the 5 ’ and 3’ regions.
  • the modified tracrRNA, gRNA, and/or crRNA of the disclosure allows a dgRNA to be used in applications where otherwise a sgRNA would be more desirable.
  • the present disclosure provides for use of BNA (e.g., LNA and/or cEt) modifications within the crRNA: tracrRNA annealing region of a dgRNA to enhance the performance (e.g., editing efficiency) of an RGN system in a cell.
  • BNA e.g., LNA and/or cEt
  • BNA e.g., LNA and/or cEt modifications allows a shortened crRNA IracrRNA annealing region.
  • BNA e.g., LNA
  • BNA e.g., LNA
  • the cells that are gene edited include primary cells.
  • the modified tracrRNA, gRNA, and/or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.
  • the present disclosure provides guide RNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification.
  • BNA bridged nucleic acid
  • the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA.
  • the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA.
  • guide RNA is known in the art and generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a RNA-guided nuclease (RGN) and aid in targeting the RGN to a specific location within a target polynucleotide (e.g., a DNA or an mRNA molecule) such as, e.g., a genomic locus.
  • RGN RNA-guided nuclease
  • the guide RNA comprises a nucleotide sequence (i.e., a spacer) having sufficient complementarity with a target strand nucleotide sequence to hybridize with the target strand and direct sequence -specific binding of an RGN to the target nucleotide sequence.
  • the target nucleotide sequence when the target nucleotide sequence is double-stranded as is the case with DNA, the target nucleotide sequence comprises a non-target strand (which comprises the PAM sequence) and the target strand, which hybridizes with the spacer of the guide RNA.
  • the guide RNA has sufficient complementarity with the target strand of a double-stranded target sequence (e.g., target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequencespecific binding of an associated RGN to the target sequence (e.g., target DNA sequence). Therefore, in some embodiments, a guide RNA includes a spacer that is identical to the sequence of the nontarget strand except that uracil (U) replaces thymidine (T) in the guide RNA.
  • An RGN’s respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target strand and/or the non-target strand.
  • a guide RNA comprises a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA).
  • guide RNA also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules.
  • Native guide RNAs that comprise both a crRNA and a tracrRNA generally comprise two separate RNA molecules that hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA.
  • the crRNA and tracrRNA are linked together by a linker.
  • a "linker" can be any kind of chemical linkage that covalently connects two molecules together, for example, a linkage formed by click chemistry or any other chemical reactions, a polynucleotide, a polymer, or any entity that can link two molecules.
  • a linker connecting a crRNA and a tracrRNA comprises a multi-nucleotide linker (e.g., a four-nucleotide linker) to form a single guide RNA molecule, wherein the crRNA and the tracrRNA hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA.
  • a guide RNA encompasses a single-guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule or strand.
  • the crRNA and tracrRNA of a guide RNA can be linked by an organic molecule, group, polymer, or chemical moiety.
  • the crRNA and tracrRNA of a guide RNA is linked by click chemistry.
  • Click chemistry involves the rapid generation of compounds by joining small units together via heteroatom links (C-X-C).
  • the main objective of click chemistry is to develop a set of powerful, selective, and modular “blocks” that are useful for small- and large-scale applications. Click chemistry reactions are fast, modular, efficient, often do not produce toxic waste products, can be done with water as a solvent, and can be set up to be stereospecific.
  • Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecules can be involved, and labeling with small molecules, such as fluorescent dyes, biotin, and other groups can be readily achieved. Click chemistry reaction takes place between two components: an azide functional group and an alkyne functional group.
  • Organic azides are organic compounds with the formula RN3, containing the azide functional group.
  • a well-known click reaction is the Huisgen 1,3-dipolar cycloaddition of azides and alkynes.
  • This reaction yielding triazoles, has become the gold standard of click chemistry for its reliability, specificity, and biocompatibility.
  • Such cycloadditions need high temperatures or pressures when the reaction involves simpler alkene or azides, since the activation energies are high (AG t »+26 kcal/mol).
  • Cu(I) catalysts expedite the reaction of terminal alkynes and azides, thereby affording 1,4- disubstituted-I,2,3-triazoles.
  • This reaction is an ideal click reaction and is widely employed in material science, medicinal chemistry, and chemical biology.
  • the alkyne is instead introduced in a strained difluorooctyne (DIFO), in which the electron-withdrawing, propargylic, gemfluorines act together with the ring strain to greatly destabilize the alkyne (Agard et al. (2006) ACS Chem. Biol. 1(10): 644-648).
  • DIFO strained difluorooctyne
  • Copper-free click chemistry proceeds as a concerted [3+2] cycloaddition in the same mechanism as the Huisgen 1,3-dipolar cycloaddition.
  • Substituents other than fluorines, such as benzene rings, are also allowed on the cyclooctyne.
  • the reactive groups in click chemistry can be introduced into any form of nucleic acid molecule and can be introduced enzymatically or chemically.
  • Alkyne-modified and azide-modified oligonucleotides can be ordered from an oligo-synthesizing facility or company.
  • An azide-modified version of a nucleotide can be introduced during RNA synthesis into a first RNA molecule, and an alkyne-modified version of a nucleotide can be introduced during RNA synthesis into a second RNA molecule.
  • the resulting Click-functionalized nucleic acid molecules can be isolated and purified to remove any unreacted reagents or byproducts that might interfere with the subsequent click reaction.
  • the purified Click- functionalized nucleic acid molecules can be mixed together in a reaction buffer that supports the click reaction. This can include a copper catalyst to facilitate the reaction between the azide and alkyne or can be copper-free.
  • the azide and alkyne functional groups react to form a covalent bond, linking the two nucleic acid molecules together.
  • Linked nucleic acid molecules can be further purified, and analytical techniques such as gel electrophoresis or mass spectrometry can be used to verify the successful linking of the nucleic acid molecules and assess the purity of the product.
  • Linking of a crRNA and a tracrRNA can proceed in the following manner.
  • a 3' amino modifer is included on the crRNA and a 5' amino modifier is included on the tracrRNA by solid phase synthesis.
  • an NHS ester of azidobutyrate (4-azido-butan-l-oic acid A-hydroxysuccinimide ester; available from, e.g., Glen Research cat no.
  • DBCO dibenzocyclooctyne-PEG4-JV-hydroxysuccinimidyl ester; available, for example, from Sigma Aldrich cat. no. 764019
  • Assembly in aqueous media allows the strain promoted azide-alkyne Huisgen cycloaddition ("copper free click chemistry") to proceed.
  • a crRNA and tracrRNA linked by click chemistry are linked by chemical moieties.
  • a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the anti-repeat of the tracrRNA and an alkyne group at one or more nucleotides at the crRNA repeat of the crRNA.
  • a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the crRNA repeat of the crRNA and an alkyne group at one or more nucleotides at the anti-repeat of the tracrRNA.
  • One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the crRNA repeat of the guide RNA.
  • One or more azide- modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the anti-repeat of the guide RNA.
  • a crRNA and tracrRNA linked by chemical moieties is a single guide RNA and comprises an azide-modified nucleotide or an alkyne- modified nucleotide at one or more nucleotides in the nucleotide loop connecting the crRNA repeat and the anti-repeat.
  • nucleic acid molecules e.g., crRNA and tracrRNA
  • modifications include a variety of crosslinkers, such as thiol modifications, like a thioctic acid N-hydroxy succinimide (NHS) ester, chemical groups that react with primary amines ( — NH2). These primary amines are positively charged at physiologic pH and nucleophilic; this makes them easy to target for conjugation with several reactive groups.
  • crosslinkers such as thiol modifications, like a thioctic acid N-hydroxy succinimide (NHS) ester, chemical groups that react with primary amines ( — NH2).
  • NHS thioctic acid N-hydroxy succinimide
  • isothiocyanates include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfo-NHS esters containing a sulfonate ( — SO3) group, for example, bis(sulfosuccinimidyl)suberate (BS3), sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, such as, for example l-ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), anhydrides, and fluorophenyl esters.
  • — SO3 sulfonate
  • EDC dimethylaminopropyljcarbodiimide
  • DCC dicyclohexylcarbodiimide
  • a guide RNA can comprise a crRNA and a tracrRNA, wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one BNA modification.
  • the anti -repeat is capable of hybridizing to the crRNA repeat to form a stem loop comprising a first stem and a second stem.
  • the present disclosure also provides a nucleic acid molecule comprising a tracrRNA, wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one BNA modification.
  • the antirepeat of the tracrRNA is capable of hybridizing to a crRNA repeat of a crRNA to form a stem loop comprising a first stem and a second stem.
  • a gRNA comprising the tracrRNA is capable of binding to an RGN.
  • the present disclosure also provides a nucleic acid molecule comprising a crRNA comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides
  • the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a stem loop comprising a first stem and a second stem.
  • a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.
  • RGN RNA guided nuclease
  • the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.
  • BNA bridged nucleic acid
  • the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.
  • BNA bridged nucleic acid
  • the present invention provides, inter alia, CRISPR RNAs (crRNAs) or polynucleotides encoding CRISPR RNAs that comprise at least one BNA (e.g., LNA and/or cEt) modification.
  • crRNA refers to an RNA molecule or portion thereof that includes a spacer, which is the nucleotide sequence that directly hybridizes with the target strand of a target sequence, and a CRISPR repeat that comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule.
  • tracrRNA or “transactivating crRNA” refers to an RNA molecule that comprises an anti-repeat sequence that has sufficient complementarity to hybridize to at least a portion of the CRISPR repeat of a crRNA to form a structure that is recognized by an RGN molecule.
  • additional secondary structure(s) e.g., stem-loops
  • stem-loops within the tracrRNA molecule is required for binding to an RGN.
  • the crRNAs comprise at least one other chemical modification.
  • the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification.
  • BNA e.g., LNA and/or cEt
  • the at least one modification is a BNA (e.g., LNA and/or cEt) modification.
  • the BNA modification comprises a 2', 4' BNA modification.
  • 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.
  • the 2', 4' BNA is an LNA modification.
  • the 2', 4' BNA is a cEt modification.
  • the at least one chemical modification is a 2'-0-Me modification. In certain embodiments, the at least one chemical modification is an MS modification.
  • a crRNA comprises a spacer and a CRISPR repeat.
  • the “spacer” is a nucleotide sequence that directly hybridizes with the target strand of a target sequence (e.g., target DNA sequence) of interest.
  • the spacer is engineered to have full or partial complementarity with the target strand of a target sequence of interest.
  • the spacer can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the spacer can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In some embodiments, the spacer is about 30 nucleotides in length. In some embodiments, the spacer is 30 nucleotides in length.
  • the degree of complementarity between a spacer and the target strand of a target sequence is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a spacer and the target strand of a target 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.
  • the spacer can be identical in sequence to the non-target strand of a target sequence.
  • the spacer can be identical in sequence to the non-target strand of the target DNA sequence, with the exception of the thymidines (Ts) in the nontarget strand are replaced by uracils (Us) in the spacer.
  • the spacer is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(l):23-24).
  • a spacer of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the spacer. In certain embodiments, a spacer of the disclosure includes at least one chemical modification.
  • the at least one modification is selected from the group consisting of: 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification.
  • 2'-O- methyl (2'-0-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification.
  • a spacer of the disclosure includes at least a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNA NC [N-Me] modification BNA NC [N-Me] modification
  • 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification
  • S-constrained ethyl (cEt) modification S-constrained ethyl
  • a spacer of the disclosure includes at least one 2'-0-Me modification and at least one MS modification.
  • a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2'-0-Me or MS).
  • a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one PS modification.
  • a spacer, absent any chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 14 or that differs from SEQ ID NO: 14 by 1 or 2 nucleotides.
  • a spacer absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14.
  • nucleotide sequence “differs from a SEQ ID NO by a certain number of nucleotides” or “has a certain percentage identity to a SEQ ID NO”, the difference only occurs in the nucleotide sequence, and chemical modifications or lack thereof remain the same.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15 or differs from SEQ ID NO: 15 by 1 or 2 nucleotides.
  • a spacer, absent any chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 15 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 15 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17.
  • a spacer, absent any chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 89 or that differs from SEQ ID NO: 89 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91 or that differs from SEQ ID NO: 91 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91.
  • a spacer, absent any chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 90 or that differs from SEQ ID NO: 90 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92 or that differs from SEQ ID NO: 92 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92.
  • a spacer absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111 or that differs from SEQ ID NO: 111 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113 or that differs from SEQ ID NO: 113 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113.
  • a spacer, absent any chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 112 or that differs from SEQ ID NO: 112 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112.
  • a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 3 nucleotides.
  • a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114.
  • a crRNA further comprises a CRISPR RNA (crRNA) repeat.
  • the crRNA repeat comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule.
  • the crRNA repeat can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the crRNA repeat is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.
  • the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • a crRNA repeat of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the crRNA repeat. In certain embodiments, a crRNA repeat of the disclosure includes at least one chemical modification.
  • the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification.
  • 2'-O-methyl (2'-O-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification.
  • a crRNA repeat of the disclosure includes at least a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNA NC [N-Me] modification BNA NC [N-Me] modification
  • 2'-O,4’-C-ethylene bridged nucleic acid 2'-O,4’-C-ethylene bridged nucleic acid
  • cEt S-constrained ethyl
  • a crRNA repeat of the disclosure includes at least one cEt modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification. In some embodiments, a crRNA repeat of the disclosure includes at least one MS modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification and at least one MS modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2’- O-Me or MS). In some embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA In certain embodiments, a crRNA repeat of the disclosure includes at least one LNA modification.) modification and at least one PS modification.
  • BNA e.g., LNA
  • a crRNA repeat of the disclosure includes at least one LNA modification.
  • the crRNA repeat comprises the nucleotide sequence of any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or an active variant or fragment thereof, that when comprised within a guide RNA, is capable of directing the sequence-specific binding of an associated RNA-guided nuclease provided herein to a presently disclosed target sequence.
  • an active crRNA repeat variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516.
  • an active crRNA repeat fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516.
  • a crRNA repeat, absent chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 1 or 2 nucleotides.
  • a crRNA repeat, absent chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 2 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 2 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 2. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70 or differs from SEQ ID NO: 70 by 1 or 2 nucleotides.
  • a crRNA repeat, absent chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 70 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 70 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94 or differs from SEQ ID NO: 94 by 1 or 2 nucleotides.
  • a crRNA repeat, absent chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 94 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 94 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241 or differs from SEQ ID NO: 241 by 1 or 2 nucleotides.
  • a crRNA repeat, absent chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 241 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 241 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253 or differs from SEQ ID NO: 253 by 1 or 2 nucleotides.
  • a crRNA repeat, absent chemical modifications has a nucleotide sequence that differs from SEQ ID NO: 253 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 253 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538 or differs from SEQ ID NO: 538 by 1 or 2 nucleotides.
  • a crRNA repeat absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as (c) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 386.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512.
  • a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516.
  • the crRNA is not naturally-occurring.
  • the specific crRNA repeat sequence is not linked to the engineered spacer sequence in nature and the crRNA repeat sequence is considered heterologous to the spacer sequence.
  • the spacer sequence is an engineered sequence that is not naturally occurring.
  • a crRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the crRNA. In certain embodiments, a crRNA of the disclosure includes at least one chemical modification.
  • the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'- fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'- O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification.
  • 2'-O-methyl (2'-O-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'- fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification.
  • a crRNA of the disclosure includes at least a 2', 4' BNA modification.
  • 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.
  • LNA locked nucleic acid
  • BNA NC [N-Me] modification BNA NC [N-Me] modification
  • 2'- O,4’-C-ethylene bridged nucleic acid 2',4'-ENA
  • cEt S-constrained ethyl
  • a crRNA of the disclosure includes at least one 2'-O-Me modification. In some embodiments, a crRNA of the disclosure includes at least one MS modification. In some embodiments, a crRNA of the disclosure includes at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2'-O-Me or MS). In some embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one PS modification.
  • BNA e.g., LNA and/or cEt
  • a chemically modified crRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337- 339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.
  • a crRNA, absent chemical modifications has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19.
  • a crRNA, absent chemical modifications has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 71.
  • a crRNA, absent chemical modifications has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 71. In some embodiments, a chemically modified crRNA has the nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 72.
  • a crRNA, absent chemical modifications has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 95.
  • a crRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 96.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 4.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 5.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 6.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 7.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 9.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 708.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 292, and wherein with reference to SEQ ID NO: 292.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 293.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 73.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 75.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 75.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 301.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 302.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 303.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 305.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 307.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 309.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 310.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 311.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 313.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 315.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 321.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 322.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 323.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 345.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 347.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 349.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 350.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 351.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 353.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 355.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 357.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 358.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 359.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 361.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 363.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 97.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 98.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 99.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 325.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 327.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 329.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 330.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 331.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 333.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 335.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 337.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 338.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 339.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 42.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 44.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 380.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 381.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 382.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 399.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 401.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 401.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 466.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 467.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 468.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 470.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 472.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 474.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 475.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 476.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 478.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 480.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 482.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 483.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 484.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 509.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 511.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 513.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 514.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 515.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 517.
  • a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519.
  • a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 519.
  • tracrRNAs comprise a crRNA and a trans-activating CRISPR RNA (tracrRNA).
  • a tracrRNA molecule comprises a nucleotide sequence comprising a region, referred to herein as the anti-repeat, that has sufficient complementarity to hybridize to a crRNA repeat.
  • the tracrRNA molecule further comprises a region with secondary structure (e.g., stemloop).
  • secondary structure includes nucleotides that are in one of two states, paired or unpaired, where nucleotide or base pairing includes base-base hydrogen bonding interactions (e.g., adenine (A) pairs with uracil (U), cytosine (C) pairs with guanine (G)) between two complementary nucleic acid strands to form a helix.
  • nucleotide or base pairing includes base-base hydrogen bonding interactions (e.g., adenine (A) pairs with uracil (U), cytosine (C) pairs with guanine (G)) between two complementary nucleic acid strands to form a helix.
  • the combination of one or more helical elements interspersed with unpaired, single-stranded nucleotides constitutes an RNA structure.
  • a “stem loop” as used herein refers to a form of secondary structure comprising at least one “stem” and at least one “loop”, “bulge”, or “bubble” found in polynucleotides.
  • a stem loop can form intramolecularly (within one molecule, e.g., within a tracrRNA or a sgRNA) or intermolecularly (between two distinct nucleic acids, e.g., in a dgRNA by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA).
  • Stem loops are created when there is at least some complementarity between two nucleic acid sequences to form a paired double helix.
  • the paired double helix region with full complementarity or sometimes including a G:U wobble base pair (or I:U, I:A, or I:C, where I refers to inosine) is referred to as a “stem”.
  • the term “loop”, “bulge”, or “bubble” refers to a single stranded region within the “stem loop” structure where there is no complementarity between nucleotides, excluding G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine).
  • “loops”, “bulges” and “bubbles” include nucleotides that are not paired.
  • a “loop” is distinguished from a “bulge” or “bubble” by being located at one end of the “stem loop” structure, while a “bulge” or a “bubble” is located between two “stems” in the “stem loop” structure.
  • a stem loop structure comprises a stem and a loop at one end of the stem.
  • a stem loop structure comprises a first stem and a second stem with a bubble in between the stems.
  • a stem loop structure comprises a loop, multiple stems and multiple bubbles in between the stems.
  • the bubbles in the order of closeness to the loop are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc.
  • the stems in the order of closeness to the loop are referred to as a “first stem”, a “second stem”, a “third stem”, etc.
  • the stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA does not include a loop, and thus the bubbles in the order of closeness to the 5’ end of the tracrRNA (or 3’ end of the crRNA) are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc., and the stems in the order of closeness to the 5’ end of the tracrRNA (or 3 ’ end of the crRNA) are referred to as a “first stem”, a “second stem”, a “third stem”, etc.
  • first stem of a crRNA repeat of a crRNA means the region in the crRNA repeat of the crRNA that forms the first stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA.
  • second stem of a crRNA repeat of a crRNA means the region in the crRNA repeat of the crRNA that forms the second stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA.
  • first stem of an anti-repeat of a tracrRNA means the region in the anti-repeat of the tracrRNA that forms the first stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA.
  • second stem of an anti-repeat of a tracrRNA means the region in the anti-repeat of the tracrRNA that forms the second stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA.
  • a stem loop formed intramolecularly is a hairpin stem loop.
  • Base pairings occur in the stem part of a stem loop and typically involve guanine-cytosine base pairing and adenine-uracil(thymidine) base pairing, although guanine -uracil base pairing is possible.
  • Base stacking interactions promote helix formation.
  • the loop part of a stem loop includes bases that are not paired.
  • a loop is the point at which a nucleic acid strand turns back on itself for nucleotide pairing to create a stem.
  • loops that are less than three bases long are sterically impossible and do not form.
  • optimal loop length is about 4-8 bases long.
  • Common loops with four nucleotide sequences such as GAAA, AAAG, ACUU, or UUCG are known as the "tetraloop" and are particularly stable due to the base-stacking interactions of its component nucleotides.
  • the region of the tracrRNA that is fully or partially complementary to a crRNA repeat is at the 5' end of the molecule and the 3' end of the tracrRNA comprises secondary structure.
  • This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat.
  • the nexus forms the core of the interactions between the guide RNA and the RGN, and is at the intersection between the guide RNA, the RGN, and the target sequence.
  • the nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs.
  • guide RNAs or RGN systems of the disclosure use tracrRNAs that comprise non- canonical sequences in the base of the hairpin stem of their nexus hairpins, including UNANNG and CNANNC.
  • a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of UNANNG.
  • a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of CNANNC.
  • terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc, doi: 10. 1101/pdb.top090902, and U.S. Publication No. 2017/0275648, each of which is herein incorporated by reference in its entirety.
  • a tracrRNA of the disclosure includes additional hairpin or stem loop structures in addition to the nexus hairpin.
  • a tracrRNA includes at least one stem loop.
  • a tracrRNA includes at least one stem loop proximal to the antirepeat and at least one stem loop proximal to the 3’ end of the tracrRNA.
  • Proximal refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides of a region or an end of a nucleic acid molecule.
  • proximal refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides of a region or an end of a nucleic acid molecule.
  • “Most proximal” refers to being the nearest to a region or to an end of a nucleic acid molecule.
  • a stem loop most proximal to the tail of a tracrRNA is the first stem loop nearest the tail of the tracrRNA.
  • “Distal” refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a region or an end of a nucleic acid molecule.
  • distal refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a structure of a nucleic acid molecule (e.g., bubble, loop).
  • a nucleic acid molecule e.g., bubble, loop
  • the first stem of the anti -repeat of a dual guide RNA lengthened at the end distal to the first bubble of the stem loop is lengthened from the 3 ’ terminal nucleotide of the crRNA and from the 5’ terminal nucleotide of the tracrRNA.
  • a tracrRNA also forms secondary structure upon hybridizing with its corresponding crRNA.
  • the anti-repeat region of a tracrRNA is fully or partially complementary to the crRNA repeat of a crRNA.
  • a portion of the anti-repeat of a tracrRNA and a portion of a crRNA repeat hybridize and form a stem.
  • the crRNA:tracrRNA stem includes at least one nucleotide pair (i.e.
  • a portion of the anti-repeat of a tracrRNA forming a first stem is the first stem of the anti-repeat
  • a portion of the anti-repeat of a tracrRNA forming a second stem is the second stem of the anti-repeat
  • a portion of the anti-repeat of a tracrRNA forming a third stem is the third stem of the anti-repeat, etc.
  • a portion of the crRNA repeat of a crRNA forming a first stem is the first stem of the crRNA repeat
  • a portion of the crRNA repeat of a crRNA forming a second stem is the second stem of the crRNA repeat
  • a portion of the crRNA repeat of a crRNA forming a third stem is the third stem of the crRNA repeat
  • a portion of the anti-repeat of a tracrRNA and a portion of the crRNA repeat are not complementary with each other and thus do not hybridize to form base pairs.
  • the region of non-complementarity between the anti -repeat and the crRNA repeat forms a bulge or a bubble.
  • hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem. In some embodiments, hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem and at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes two stems and one bubble in between.
  • a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include BNA (e.g., LNA and/or cEt) modifications. In some embodiments, a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include any chemical modifications.
  • BNA e.g., LNA and/or cEt
  • the nucleotides in a loop, bulge, or bubble do not include BNA (e.g., LNA) modifications. In some embodiments, the nucleotides in a loop, bulge, or bubble do not include any chemical modifications.
  • BNA e.g., LNA
  • the anti-repeat of the tracrRNA that is fully or partially complementary to the crRNA repeat comprises from about 8 nucleotides to about 30 nucleotides, or more.
  • the stem formed by the tracrRNA anti-repeat and the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the stem formed by the tracrRNA anti-repeat and the crRNA repeat is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.
  • the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. In some embodiments, the tracrRNA comprises a total length of 60 to 80 nucleotides, 80 to 100 nucleotides, 100 to 120 nucleotides, 120 to 140 nucleotides, 140 to 160 nucleotides, 160 to 180 nucleotides, or more than 180 nucleotides.
  • the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, or more nucleotides in length.
  • the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides in length.
  • the tracrRNA is about 70 to about 105 nucleotides in length, including about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, and about 105 nucleotides in length.
  • the tracrRNA is 70 to 105 nucleotides in length, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105 nucleotides in length.
  • the tracrRNA is about 90 to about 125 nucleotides in length, including about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, and about 125 nucleotides in length.
  • the tracrRNA is 90 to 125 nucleotides in length, including 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and 125 nucleotides in length.
  • a tracrRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or the phosphate backbone of the tracrRNA.
  • a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification.
  • a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification.
  • the at least one other chemical modification is selected from the group consisting of: 2'- O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.
  • the BNA modification comprises a 2', 4' BNA modification.
  • 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.
  • the BNA modification is an LNA modification.
  • the tracrRNA comprises at least one LNA modification.
  • the BNA modification is a cEt modification.
  • the tracrRNA comprises at least one cEt modification.
  • the tracrRNA comprises at least one LNA modification and at least one other chemical modification.
  • the tracrRNA comprises at least one LNA modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'- O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.
  • 2'-O-methyl (2'-0-Me) modification 2'- O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • 2',4'-di-Ca-OMe modification 2'-
  • the tracrRNA comprises at least one LNA modification and at least one PS modification. In certain embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one
  • the tracrRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or an active variant or fragment thereof, that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA- guided nuclease provided herein to a presently disclosed target sequence.
  • an active tracrRNA sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51 - 53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713.
  • an active tracrRNA sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713.
  • a tracrRNA comprises at least one chemical modification at its 5' region or at its 3' region. In some embodiments, a tracrRNA comprises at least one chemical modification at both its 5' region and 3' region. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3.
  • a tracrRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 76 or 77.
  • a tracrRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 100.
  • a tracrRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 242.
  • a tracrRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 254.
  • a tracrRNA, absent chemical modifications has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 539.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity toSEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 10.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 12.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 709.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 713.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 294. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 294.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 295. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 295.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 80.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 81.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 364.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 365.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 366.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 367.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 369.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 375.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 376.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 377.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 378.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 379.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 102.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 103.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 370.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 371.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 372.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 373.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 710.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 711.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 51.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 52.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 53.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 383.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 499.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 500.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 501.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 504.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 505.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 534.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 535.
  • a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 537.
  • a tracrRNA, gRNA, or crRNA of the disclosure comprising at least one BNA (e.g., LNA) modification is derived from its respective unmodified tracrRNA, gRNA, or crRNA by having at least one of its nucleotides modified with a BNA (e.g., LNA) modification.
  • BNA e.g., LNA
  • the tracrRNA, gRNA, or crRNA comprising at least one BNA (e.g., LNA) modification derived from its respective unmodified parent tracrRNA, gRNA, or crRNA has the same polynucleotide sequence as the parent molecule.
  • BNA e.g., LNA
  • the term “unmodified” in the context of a crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA refers to a conventional crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA that does not include any modified nucleotides, BNA modifications, modified sugars, modified nucleobases, and/or modified phosphate backbones, or any chemical modifications.
  • Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to a target sequence under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g. , greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm).
  • sequence specific can also refer to the binding of a RGN polypeptide to a target sequence at a greater affinity than binding to a randomized background sequence.
  • the guide RNA can be a single guide RNA (sgRNA) or a dual -guide RNA (dgRNA).
  • sgRNA comprises the crRNA and tracrRNA on a single molecule of RNA
  • dgRNA comprises a crRNA and a tracrRNA present on two distinct RNA molecules, hybridized to one another through at least a portion of the crRNA repeat of the crRNA and at least a portion of the antirepeat of the tracrRNA, which may be fully or partially complementary to each other.
  • Hybridization of the anti-repeat of a tracrRNA to the crRNA repeat of a crRNA forms a stem loop comprising the anti-repeat and the crRNA repeat.
  • the stem loop includes one or more stems formed by the anti-repeat and the crRNA repeat.
  • the crRNA and tracrRNA are separated by a linker nucleotide sequence.
  • the linker nucleotide sequence is one that does not include bases complementary within itself or to other parts of the sgRNA in order to avoid the formation of secondary structure within or comprising nucleotides of the linker nucleotide sequence.
  • the linker forms a loop at one end of the first stem in the stem loop structure comprising the crRNA repeat and the anti-repeat.
  • the linker nucleotide sequence between the crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length.
  • the linker nucleotide sequence of a sgRNA is at least 4 nucleotides in length.
  • the linker nucleotide sequence includes a nucleotide sequence set forth as any of AAAG, GAAA, ACUU, and CAAAGG.
  • the total length of a guide RNA can comprise about 100 nt to 120 nt, about 120 nt to 140 nt, about 140 nt to about 160 nt, about 160 nt to about 180 nt, about 180 nt to about 200 nt, or more.
  • the total length of a guide RNA is 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt,
  • a chemically modified sgRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.
  • the sgRNA or dgRNA can be synthesized chemically or via in vitro transcription.
  • Assays for determining sequence -specific binding between an RGN and a guide RNA are known in the art and include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads).
  • a control guide RNA with an unrelated sequence or structure to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA.
  • the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule.
  • the guide RNA can be chemically synthesized.
  • 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.
  • the guide RNA directs an associated RGN to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target sequence of interest.
  • the target sequence can be bound (and in some embodiments, cleaved) by an RGN in vitro or in a cell.
  • a target sequence can comprise DNA, RNA, or a combination of both and can be single -stranded or doublestranded.
  • a target sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, episomal DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA).
  • the chromosomal sequence can be a nuclear, plastid or mitochondrial chromosomal sequence.
  • the target sequence is within a target nucleic acid molecule that is double-stranded (e.g., a target DNA sequence).
  • the target sequence is unique in the target genome.
  • the target sequence comprises a target strand and a non-target strand, and the target sequence (i.e., the sequence on the non-target strand) has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) and the non-target strand of the target sequence is the strand that comprises the PAM.
  • the PAM is immediately adjacent to the target sequence and often comprise Ns, which represent any nucleotide.
  • the PAM comprises about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns.
  • a PAM comprises 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns.
  • the PAM can be 5' or 3' of the target sequence on its non-target strand.
  • the PAM is 3' of the target sequence on its non-target strand for the presently disclosed guide RNAs and RGN systems.
  • the PAM is a consensus sequence of about 3-4 nucleotides, but in some embodiments, it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.
  • a PAM sequence adjacent to a presently disclosed target sequence on its non-target strand comprises the consensus sequence set forth as any one of the PAM sequences in Table 1.
  • a PAM sequence adjacent to the presently disclosed target sequence on its non-target strand includes the consensus sequence set forth as any one of NNNNCC, NNGRR, NNRYA, and NGG.
  • the PAM sequence is 3' of the target sequence on its non- target strand.
  • PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015) Genome Biol 16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo.
  • the RGN can cleave one or both strands of a target sequence at a specific cleavage site.
  • a cleavage site is made up of the two particular nucleotides within a target sequence between which the target strand, non-target strand, or both strands of a target sequence are cleaved by an RGN.
  • the cleavage site can comprise the 1 st and 2 nd , 2 nd and 3 rd , 3 rd and 4 th , 4 th and 5 th , 5 th and 6 th , 7 th and 8 th , or 8 th and 9 th nucleotides from the PAM in either the 5' or 3' direction.
  • the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction.
  • the cleavage site is defined based on the distance of the two nucleotides from the PAM on the non-target strand of the target sequence, and for the target strand, the distance of the two nucleotides from the complement of the PAM.
  • Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA of the disclosure can in some embodiments comprise at least one BNA (e.g., LNA) modification.
  • the at least one BNA (e.g., LNA) modification is in the first stem of the anti-repeat of the tracrRNA.
  • the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA) modification in the first stem of the anti -repeat of the tracrRNA.
  • Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA of the disclosure can in some embodiments include a modification in the ribose (e.g., sugar) group, phosphate group, nucleobase, or any combination thereof.
  • ribose e.g., sugar
  • modified nucleotide in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5' end modifications or 3' end modifications, (b) nucleobase (or “base”) modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2', 3', and/or 4' positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • modified nucleotide generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates.
  • modification and “chemical modification” are used interchangeably herein.
  • a modified nucleotide includes a sugar modification.
  • sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'- deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5 '-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'- deoxyuridine-5'- triphosphate), 2'-O-alkyl oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2 '-O- methylcytidine-5'-triphosphate, 2'-methyluridine-5 '-triphosphate), 2'-C-alkyl oligoribonucleotide, and isomers thereof (2'-aracytidine-5
  • a modified molecule or region of the disclosure e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA
  • a modified molecule or region of the disclosure includes one or more 2'-fluoro, 2'-amino and/or 2'-thio modifications.
  • the modification is a 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'- amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, 5 -amino-allyl- uridine, 5- bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl- pyrene -uridine, 5-fluoro-cytidine, and/or 5 -fluoro-uridine.
  • a nucleoside includes a purine or pyrimidine base linked to a sugar (i.e., nucleotides without a phosphate group).
  • the preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art and described in, e.g., U.S. Patent Nos.
  • nucleoside can be an analogue of a naturally occurring nucleoside.
  • a nucleoside analogue includes dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxy cytodine, and deoxyuridine.
  • a modified molecule or region of the disclosure includes a nucleobase-modified ribonucleotide, i.e., a ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.
  • Non-limiting examples of modified nucleobases which can be incorporated into modified nucleosides and modified nucleotides include m5C (5 -methylcytidine), m5U (5 - methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1- methyl adenosine), m2A (2- methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2- methylthio-N6-methyladenosine), i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio- N6isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2- methylthio- N6-(cis-hydroxyisopenten
  • a modified molecule or region of the disclosure includes one or more modifications in the phosphate backbone.
  • the modification can include one or more of phosphorothioate, phosphorodithioate, phosphoramidate (e.g., N3'- P5'-phosphoramidate (NP)), and/or methylphosphonate linkages.
  • a backbone modification includes a neutral backbone modification including: phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA) modifications. In some embodiments, all stereoisomers of these backbone modifications are useful in the present disclosure.
  • one or more of the modified nucleotides of a molecule or region of the disclosure include modifications at the 2’ position of the ribose sugar.
  • one or more modifications of a molecule or region of the disclosure include a 2'-O-methyl (2'-0-Me) modification (“B” indicates “base” in chemical structures herein):
  • one or more modifications of a molecule or region of the disclosure include a 2'-O- methoxy-ethyl (2'-M0E) modification:
  • one or more modifications of a molecule or region of the disclosure include a 2'-fluoro (2'- F) modification:
  • one or more of the modified nucleotides of a molecule or region of the disclosure include modifications at the 2' position and 4' position of the ribose sugar.
  • one or more modifications of a molecule or region of the disclosure include a 2'F-4'Ca-OMe modification:
  • one or more modifications of a molecule or region of the disclosure include a 2',4'-di-Ca- OMe modification:
  • one or more modifications of a molecule or region of the disclosure include a phosphorothioate (PS) modification (e.g., in the backbone):
  • PS phosphorothioate
  • one or more of the modified nucleotides of a molecule or region of the disclosure include modifications at the 2' position of the ribose sugar and the phosphate backbone.
  • one or more modifications of a molecule or region of the disclosure include a 2'-O-methyl 3'- phosphorothioate (MS) modification:
  • one or more modifications of a molecule or region of the disclosure include a 2'-O-methyl 3'thiophosphonoacetate (MSP; 2'-O-methyl 3'thioPACE) modification:
  • one or more modifications of a molecule or region of the disclosure include a 2'-O-methyl 3'phosphonoacetate (MP) modification:
  • one or more modifications of a molecule or region of the disclosure include 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2’-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3 ’thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification.
  • 2'-O-methyl (2'-0-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification 2'F-4'Ca-OMe modification
  • a modified molecule or region of the disclosure includes one or more MS modifications and one or more BNA (e.g., LNA and/or cEt) modifications.
  • a modified molecule or region of the disclosure includes one or more PS modifications and one or more BNA (e.g., LNA and/or cEt) modifications.
  • a modified molecule or region of the disclosure includes one or more MS modifications and one or more 2’,4’-BNA (e.g., LNA and/or cEt) modifications.
  • a modified molecule or region of the disclosure includes one or more PS modifications and one or more 2’,4’-BNA (e.g., LNA and/or cEt) modifications.
  • a modified molecule or region of the disclosure includes one or more MS modifications and one or more LNA modifications.
  • a modified molecule or region of the disclosure includes one or more MS modifications and one or more cEt modifications.
  • a modified molecule or region of the disclosure includes one or more PS modifications and one or more LNA modifications.
  • a modified molecule or region of the disclosure includes one or more PS modifications and one or more cEt modifications.
  • a modification includes a bridged nucleic acid (BNA) modification.
  • BNA bridged nucleic acid
  • the term "bridged nucleic acid” refers to a nucleic acid having a structure wherein the degree of freedom of the nucleic acid is restricted through an intramolecular bond or crosslink.
  • a BNA modification includes a 2', 4' BNA modification.
  • the 2' oxygen and 4' carbon of the ribose are linked through a “bridge”.
  • First generation BNA modifications include locked nucleic acid (LNA) modifications.
  • LNA nucleotides comprise conformationally-restricted RNA nucleotides in which the 2' oxygen in the ribose forms a covalent bond to the 4' carbon, inducing N-type (C3'-endo) sugar puckering and preference for an A-form helix (Y ou et al. (2006) Nucleic Acids Res 34(8):e60), depicted as follows:
  • LNAs display improved base stacking and thermal stability compared to RNA, resulting in highly efficient binding to complementary nucleic acids and improved mismatch discrimination, as well as nuclease resistance (You et al. (2006) Nucleic Acids Res 34(8):e60; Vester & Wengel (2004) Biochemistry 43(42): 13233-13241). They have been successfully used in numerous applications ranging from SNP detection assays to siRNA (Vester & Wengel (2004) Biochemistry 43(42): 13233- 13241; Elmen et al. (2005) Nucleic Acids Res 33(l):439-447).
  • BNA NC [N-Me] N-methyl substituted bridged nucleic acids
  • BNAs include: 2'-O,4'-C- ethylene BNA (2',3'-ENA); 2'-O,4'-C-methylenecytidine; 2'-O,4'-C-methyleneuridine; 2',4'-BNA-l- isoquinolone; 2',4'-BNA-2-pyridone; 2',4'-BNA-TeNA; 2',4'-BNA-TrNA; 2',4'-BNA C0C ; 2', 4'- BNA NC [NBn]; 2',4'-BNA NC [NH]; 2',4'-BNA NC [NMe]; 3'-amino-2',4'-BNA; AmNA; DpNA (3,4- dihydro-2H-pyran bridge moiety; EoNA; Gu
  • one or more modifications of a molecule or region of the disclosure include a BNA NC [N-Me] modification:
  • one or more modifications of a molecule or region of the disclosure include a 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification:
  • one or more modifications of a molecule or region of the disclosure include a S- constrained ethyl (cEt) modification:
  • one or more modifications of a molecule or region of the disclosure include a combination of chemical modifications disclosed herein.
  • One or more nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA of the disclosure can be a modified nucleotide.
  • a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all nucleotides being chemically modified.
  • a crRNA repeat of a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified.
  • a spacer of a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified.
  • a tracrRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all nucleotides being chemically modified.
  • an anti-repeat of a tracrRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified.
  • a guide RNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all chemically nucleotides being modified.
  • the modified nucleotides can be located at any nucleotide position of the RNA sequence, including the first nucleotide position, the last nucleotide position, and/or any nucleotide position between the first and last nucleotide positions.
  • the one or more modified nucleotides can be located at nucleotide position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, and/or position 20 from the 5’ end of the guide RNA.
  • from about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 30%, about 20% to about 30%, or about 25% to about 30% of an RNA molecule of the disclosure can comprise modified nucleotides.
  • RNA molecule or region of the disclosure can comprise modified nucleotides.
  • the chemical modifications are within one or more stems in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).
  • the chemical modifications are within the one or more stems of one stem loop.
  • the chemical modifications are within the one or more stems of multiple stem loops.
  • the chemical modifications are within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are present on all nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem but not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within the first stem and one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are present on all nucleotides within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).
  • the chemical modifications are within one stem of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within one stem of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are present on all nucleotides within one stem of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are within two stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within each of the two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).
  • the chemical modifications are present on all nucleotides within two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are within three stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within each of the three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are present on one or more nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are present on all nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of a crRNA and a tracrRNA. In some embodiments, a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of crRNA repeat of a crRNA and anti-repeat of a tracrRNA.
  • the chemical modifications are within the “tail” in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the term “tail” as used herein refers to the non-complementary region closest to the 3' end (e.g., within twelve, eleven, ten, nine, eight, seven, six, five nucleotides from the 3' end) of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • a tail of a tracrRNA includes 1-12, 1-8, or 1-6 nucleotides from the 3' end of the tracrRNA. In some embodiments, a tail of a tracrRNA includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides from the 3' end of the tracrRNA. In some embodiments, the chemical modifications are within one or more stems and within the tail in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).
  • the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within the first stem and no chemical modifications are within the second stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA, and chemical modifications are present within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In certain embodiments, the chemical modifications are within one or more stems but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within one or more stems and within the tail but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within the first stem and not within any other stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).
  • the chemical modifications are within one or more stems and present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within one or more stems and within the tail as well as present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).
  • the chemical modifications are within a first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within a first and second stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within a first, second, and third stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).
  • a first stem is formed by hybridization between the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA.
  • the crRNA and the tracrRNA are two separate molecules, which together form a dgRNA.
  • the first stem is the stem closest to the 3' end of the crRNA and closest to the 5' end of the tracrRNA.
  • the crRNA and the tracrRNA are within one molecule of a sgRNA.
  • the first stem is the stem immediately adjacent to the loop in a stem loop formed by hybridization between the crRNA repeat of the crRNA and the anti-repeat of the tracrRNA.
  • At least one BNA (e.g., LNA and/or cEt) modification is within a crRNA repeat.
  • at least one BNA modification (e.g., LNA and/or cEt) is within the first stem of a crRNA repeat.
  • the first stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the first stem of a stem loop by hybridization with nucleotides of an anti-repeat of a tracrRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least two consecutive nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the second stem of a crRNA repeat.
  • the second stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the second stem of a stem loop by hybridization with nucleotides of an antirepeat of a tracrRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least two consecutive nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • no chemical modifications are within the second stem of a crRNA repeat.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the first and second stems of a crRNA repeat.
  • the terminal 5' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 3' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 5' nucleotides and the terminal 3' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • BNA e.g., LNA and/or cEt
  • at least two consecutive nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least one BNA (e.g., LNA and/or cEt) modification is within the first stem and no chemical modifications are within the second stem of a crRNA repeat.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the third stem of a crRNA repeat.
  • the third stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the third stem of a stem loop by hybridization with nucleotides of an anti-repeat of a tracrRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least two consecutive nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 5' nucleotide of each stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of each stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • At least two consecutive nucleotides of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, only the first stem of a crRNA repeat comprises BNA (e.g., LNA and/or cEt) modifications.
  • nucleotides of a crRNA repeat within a loop, bulge, or bubble in a stem loop formed by hybridization of the crRNA repeat and the anti-repeat do not comprise BNA (e.g., LNA and/or cEt) modifications.
  • a crRNA repeat does not comprise BNA (e.g., LNA and/or cEt) modifications but hybridizes to the anti-repeat of a tracrRNA comprising at least one BNA (e.g., LNA and/or cEt) modification.
  • the crRNA repeat lacking BNA (e.g., LNA and/or cEt) modifications hybridizes to the anti-repeat of a tracrRNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the stem loop formed by the hybridization of the crRNA repeat and the anti-repeat.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the anti-repeat of a tracrRNA. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first stem of the anti-repeat of a tracrRNA.
  • the first stem of an anti-repeat includes nucleotides within an anti-repeat forming the first stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the first stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the first stem of an anti -repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • At least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the second stem of the anti-repeat of a tracrRNA.
  • the second stem of an anti-repeat includes nucleotides within an anti-repeat forming the second stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the second stem of an antirepeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of an anti-repeat.
  • BNA e.g., LNA and/or cEt
  • At least one BNA (e.g., LNA and/or cEt) modification is within the first and second stems of an anti-repeat of a tracrRNA.
  • the terminal 5' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 5' nucleotides and the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • BNA e.g., LNA and/or cEt
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least one BNA (e.g., LNA and/or cEt) modification is within the first stem and no chemical modifications are within the second stem of an anti-repeat of a tracrRNA.
  • At least one BNA (e.g., LNA and/or cEt) modification is within the third stem of the anti-repeat of a tracrRNA.
  • the third stem of an anti-repeat includes nucleotides within an anti-repeat forming the third stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein.
  • the terminal 5' nucleotide of the third stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of the third stem of an anti -repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all nucleotides of the third stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications.
  • all stems of the anti-repeat of a tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications.
  • the terminal 5' nucleotide of each stem of an antirepeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 3' nucleotide of each stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification.
  • the terminal 5' nucleotide and the terminal 3' nucleotide of each stem of an antirepeat comprises BNA (e.g., LNA and/or cEt) modifications.
  • At least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, only the first stem of the anti-repeat of a tracrRNA comprises BNA (e.g., LNA and/or cEt) modifications.
  • nucleotides of an anti-repeat within a loop, bulge, or bubble in a stem loop formed by hybridization of the anti-repeat and the crRNA repeat do not comprise BNA (e.g., LNA and/or cEt) modifications.
  • an anti-repeat of a tracrRNA does not comprise BNA (e.g., LNA and/or cEt) modifications.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide position from the 5' end, a chemical modification at the fifth nucleotide position from the 5' end, or a combination thereof.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end and a modification at the second nucleotide position from the 5' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, and a chemical modification at the third nucleotide position from the 5' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, and a chemical modification at the fourth nucleotide from the 5' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide from the 5' end, and a chemical modification at the fifth nucleotide from the 5' end.
  • a “5' region” of an RNA molecule disclosed herein includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides of the 5' end of the RNA molecule.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide position from the 3' end, a chemical modification at the fifth nucleotide position from the 3' end, or a combination thereof.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end and a chemical modification at the second nucleotide position from the 3' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3’ end, a chemical modification at the second nucleotide position from the 3' end, and a chemical modification at the third nucleotide position from the 3' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, and a chemical modification at the fourth nucleotide from the 3' end.
  • an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide from the 3' end, and a chemical modification at the fifth nucleotide from the 3' end.
  • a “3' region” of an RNA molecule disclosed herein includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides of the 3' end of the RNA molecule.
  • a 3' region of a crRNA in the context of a single guide RNA includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides from the tracrRNA or the linker that joins the crRNA and the tracrRNA of the single guide RNA.
  • the three terminal nucleotides at both the 5' region and the 3' region of a tracrRNA of the disclosure comprise MS modifications and the remaining nucleotides in the first stem of the anti -repeat of the tracrRNA comprise 2'-0-Me modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at both the 5' region and the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the anti-repeat of the tracrRNA comprise 2'-0-Me modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • the three terminal nucleotides at the 5' region of a crRNA comprise MS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise MS modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications.
  • the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA+PS modifications and the crRNA does not comprise any further chemical modifications.
  • the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt +PS modifications and the crRNA does not comprise any further chemical modifications.
  • the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise MS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications and the 3' region of the crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications and the 3' region of the crRNA comprise LNA modifications.
  • the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise BNA+PS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise LNA+PS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications and the 3' region of the crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt+PS modifications and the 3' region of the crRNA comprise LNA+PS modifications.
  • the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt +PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA are chemically modified, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA are chemically modified.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5’ region and the 3’ region of the sgRNA are chemically modified, and wherein all the nucleotides in the first stem of the crRNA repeat and all the nucleotides in the first stem of the anti-repeat of the tracrRNA are chemically modified.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3 ' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3 ' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.
  • a multiple nucleotide e.g., a four nucleotide
  • a chemically modified nucleotide and/or a chemical modification “within” a region of a RNA molecule of the disclosure includes all nucleotides and phosphate backbone in that region, including the first and last nucleotide positions that are considered part of that region.
  • RNA, crRNA repeat, spacer, tracrRNA, antirepeat, and/or guide RNA that have been engineered to be shorter.
  • a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, and/or a guide RNA of the present disclosure are truncated or shortened.
  • a truncated crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, and/or guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification maintains or enhances gene editing efficiency as compared to the truncated crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, and/or guide RNA lacking the at least one BNA (e.g., LNA and/or cEt) modification.
  • BNA e.g., LNA and/or cEt
  • Truncation and “deletion” in the context of engineering a spacer, crRNA repeat, crRNA, anti-repeat, tracrRNA, backbone, or guide RNA are used interchangeably herein and refer to removal of at least one nucleotide from a spacer, crRNA repeat, crRNA, anti-repeat, tracrRNA, backbone, or guide RNA.
  • an engineered spacer comprises a truncation of 1 nucleotide (nt), 2 nt,
  • an engineered spacer comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt, as compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 14, 15, 89, 90, 111, and 112.
  • an engineered crRNA repeat comprises a truncation of 1 nt, 2 nt, 3 nt,
  • an engineered crRNA repeat comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt, as compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 2, 70, 94, 241, 253, and 538.
  • an engineered crRNA repeat, absent chemical modifications has the nucleotide sequence set forth as SEQ ID NO: 38 or that differs from SEQ ID NO: 38 by 1 or 2 nucleotides. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 38 by 2 nucleotides. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 38 by 1 nucleotide. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 38.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide, wherein, with reference to SEQ ID NO: 39. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39.
  • an engineered crRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, or 14 nt, as compared to the same crRNA prior to the engineering.
  • an engineered crRNA comprises a truncation of 1 nt, 2 nt, 3 nt,
  • an engineered crRNA absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 40 or 41. In some embodiments, an engineered crRNA, absent chemical modifications, comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 40 or 41.
  • an engineered crRNA, absent chemical modifications comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 40 or 41. In some embodiments, an engineered crRNA, absent chemical modifications, comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 40 or 41.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has the sequence set forth as SEQ ID NO: 42.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified, truncated crRNA has the sequence set forth as SEQ ID NO: 43.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has the sequence set forth as SEQ ID NO: 44.
  • an engineered tracrRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt,
  • an engineered tracrRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt,
  • an engineered tracrRNA comprises a truncation of 2 nucleotides, 4 nucleotides, 6 nucleotides, 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, or 18 nucleotides, as compared to the same tracrRNA prior to the engineering.
  • an engineered chemically modified tracrRNA comprises a deletion of 1 to 12 nucleotides within the first stem of the anti -repeat, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, or 12 nt within the first stem of the anti-repeat, as compared to the same chemically modified tracrRNA prior to the deletion.
  • an engineered chemically modified tracrRNA comprises a deletion of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt within the first stem of the anti-repeat, as compared to the same chemically modified tracrRNA prior to the deletion.
  • an engineered chemically modified tracrRNA comprises a deletion of nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 6 nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion.
  • an engineered chemically modified tracrRNA comprises a deletion in a stem loop most proximal to the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 4 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion.
  • an engineered chemically modified tracrRNA comprises a deletion of 1 to 3 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nucleotide pair, 2 nucleotide pairs, or 3 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion.
  • an engineered tracrRNA, absent chemical modifications comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 45-47.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 51.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 52.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53s. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 53.
  • a gRNA of the disclosure is a sgRNA that comprises a backbone, wherein the backbone of the sgRNA comprises a crRNA repeat and a tracrRNA linked by a nucleotide linker.
  • the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG.
  • the linker has the nucleotide sequence set forth as AAAG.
  • a chemically modified sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.
  • a gRNA of the disclosure is a dgRNA that comprises a backbone, wherein the backbone of the dgRNA comprises a crRNA repeat and a tracrRNA.
  • the backbone of an engineered chemically modified sgRNA or dgRNA is 2 to 30 nucleotides shorter, as compared to the same backbone prior to the engineering.
  • the backbone of an engineered chemically modified sgRNA or dgRNA is 2 to 18 nucleotides shorter, as compared to the same backbone prior to the engineering.
  • the backbone of an engineered chemically modified sgRNA or dgRNA is 2 nucleotides, 4 nucleotides, 6 nucleotides, 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, or 18 nucleotides shorter, as compared to the same backbone prior to the engineering.
  • the backbone of an engineered sgRNA, absent chemical modifications has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 32-34.
  • an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has the nucleotide sequence set forth as SEQ ID NO: 35.
  • an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 36.
  • an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 37.
  • an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 296.
  • an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 297.
  • a gRNA of the disclosure is a sgRNA that comprises a spacer and a backbone, wherein the backbone of the sgRNA comprises a crRNA repeat and a tracrRNA linked by a nucleotide linker.
  • an engineered chemically modified sgRNA comprises a truncation in the spacer and/or a truncation in the backbone, as compared to the same chemically modified sgRNA prior to the engineering.
  • an engineered chemically modified sgRNA comprises a truncation in the spacer.
  • an engineered chemically modified sgRNA comprises a truncation in the backbone.
  • an engineered chemically modified sgRNA comprises a truncation in the spacer and a truncation in the backbone. In some embodiments, an engineered chemically modified sgRNA comprises a deletion of 1 to 25 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering. In some embodiments, an engineered chemically modified sgRNA comprises a deletion of 13 to 23 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering.
  • an engineered chemically modified sgRNA comprises a deletion of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering.
  • an engineered chemically modified sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 60-68, 298, and 299.
  • a “substitution” or “replacement” as used herein in the context of engineering a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA refers to either substituting or replacing a given number of nucleotides with a same number of nucleotides (e.g., replacing 3 nucleotides with 3 nucleotides) or substituting or replacing a given number of nucleotides with a different number of nucleotides.
  • the different number of nucleotides can be greater or less than the given number of nucleotides (e.g., replacing 8 nucleotides with 3 nucleotides, or replacing 3 nucleotides with 8 nucleotides).
  • an “addition” as used herein in the context of engineering a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA refers to adding at least one nucleotide to a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA at any location(s) of a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA.
  • the engineering described herein, with or without chemical modifications allows use of dgRNA in prime editing. In some embodiments, this engineering, with or without chemical modifications, allows use of dgRNA in prime editing with delivery of an RNA-guided nuclease as an mRNA.
  • the first stem of an engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence from a native precursor CRISPR RNA (pre- crRNA).
  • pre- crRNA a native precursor CRISPR RNA
  • a native pre-crRNA includes a series of alternating crRNA repeats and spacers and is transcribed from a CRISPR array in an organism.
  • the native pre-crRNA is further processed by enzymes to a mature crRNA form that is used for genome modification in the organism.
  • the native pre-crRNA contains sequences that are not present in an intermediate processed or mature crRNA.
  • nucleotide sequences that are used to lengthen the first stem of an APG05586 crRNA repeat can be from an Enterococcus sp. APG05586 pre-crRNA.
  • the first stem of an engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence that is GC-rich. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the content of G or C in the 3' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the 3' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of an engineered crRNA repeat further comprises at least one chemical modification. In some embodiments, the first stem of an engineered crRNA repeat further comprises MS, BNA, or BNA+PS modifications at the 3' region.
  • the first stem of an engineered anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA). In some embodiments, the first stem of an engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence. In some embodiments, the first stem of an engineered anti -repeat comprises at the 5 ' region a GC-rich nucleotide sequence, wherein the content of G or C in the 5' region is at least 60%, at least 80%, or 100%.
  • the first stem of an engineered anti-repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.
  • the first stem of an engineered anti-repeat further comprises at least one chemical modification.
  • the first stem of an engineered anti-repeat further comprises BNA (e.g., LNA and/or cEt) modifications on all nucleotides.
  • GC-rich nucleotide sequence refers to a nucleotide sequence that has at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% GC content.
  • a GC-rich nucleotide sequence refers to a nucleotide sequence that comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs when the nucleotide sequence comprises at most 5 nucleotides.
  • an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 380.
  • an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 381.
  • an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 382.
  • an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 383.
  • Chemical modification (e.g., LNA modification of all nucleotides at the first stem of the antirepeat) of a dgRNA does not improve gene editing for some RGN systems when an RNA-guided nuclease is delivered as an mRNA (see FIGs. 10B and 10C).
  • gene editing efficiency is improved or gene editing is rescued by adding nucleotides to the first stem of the anti- repeat, adding nucleotides to the first stem of the crRNA repeat, and chemically modifying the lengthened first stems of the crRNA repeat and anti-repeat.
  • an engineered crRNA repeat comprises an addition of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering.
  • an engineered crRNA repeat comprises an addition of 2 to 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering.
  • an engineered crRNA repeat comprises an addition of 2 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 4 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering.
  • the first stem of an engineered crRNA repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, nucleotides in the 3' region of the first stem of a crRNA repeat further comprise MS modifications, BNA modifications, or BNA+PS modifications.
  • three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise MS modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA+PS modifications. In some embodiments, the BNA modifications comprise 2', 4' BNA modifications.
  • the 2', 4' BNA modifications are 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.
  • the 2', 4' BNA modifications are LNA modifications.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512.
  • an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 301.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 302.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 303.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 305.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 306.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 307.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 309.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 310.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 311.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 313.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 314.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 315.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 321.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 322.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 323.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 345.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 346.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 347.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 349.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 350.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 351.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 353.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 354.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 355.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 357.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 358.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 359.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 361.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 362.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 363.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 325.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 326.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 327.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 329.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 330.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 331.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 333.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 334.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 335.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 337.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 338.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 339.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 466.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 467.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 468.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 470.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 471.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 472.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 474.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 475.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 476.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 478.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 479.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 480.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 482.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 483.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 484.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 509.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 510.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 511.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 513.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 514.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 515.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 517.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 518.
  • an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 519.
  • an engineered tracrRNA comprises an addition of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering.
  • an engineered tracrRNA comprises an addition of 2 to 6 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering.
  • an engineered tracrRNA comprises an addition of 2 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA is lengthened by addition of 4 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA comprises an addition of 6 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering.
  • the first stem of an engineered anti -repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of the engineered anti -repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.
  • all nucleotides of the first stem of the engineered anti-repeat further comprise BNA (e.g., LNA and/or cEt) modifications.
  • BNA e.g., LNA and/or cEt
  • the BNA modifications comprise 2', 4' BNA modifications.
  • the 2', 4' BNA modifications are 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.
  • the 2', 4' BNA modifications are LNA modifications.
  • the 2', 4' BNA modifications are cEt modifications.
  • the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise MS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA+PS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA+PS modifications.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 364.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 365.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 366.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 367.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 369.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 375.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 376.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 377.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 378.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 379.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 371.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 372.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 373.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 710.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 711.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 499.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 500.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 501.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 504.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 505.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 534.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 535.
  • an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 537.
  • RNA-guided nuclease systems comprising the presently disclosed guide RNAs, particularly guide RNAs modified with BNA modifications (e.g., guide RNAs modified with LNA modifications).
  • the RNA-guided nucleases bind the presently disclosed gRNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification.
  • BNA bridged nucleic acid
  • the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA.
  • the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti -repeat of the tracrRNA.
  • BNA e.g., LNA and/or cEt
  • RNA-guided nuclease refers to a polypeptide that binds to a particular target sequence (e.g., target DNA sequence) in a sequence -specific manner and is directed to the target sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target strand of the target sequence (e.g., target DNA sequence).
  • RGNs Active fragments or variants thereof of naturally-occurring RGNs maintain binding to a target sequence in an RNA-guided sequence -specific manner.
  • an RGN can be capable of cleaving the target strand and/or nontarget strand of a target sequence upon binding, the term RGN also encompasses nuclease-dead RGNs that are capable of binding to, but not cleaving, a target sequence. Cleavage of a target strand and/or non-target strand of a target sequence by an RGN can result in a single- or double -stranded break.
  • RGNs only capable of cleaving a single strand of a double -stranded target nucleic acid molecule are referred to herein as nickases.
  • CRISPR-Cas systems are RGN systems classified into Class 1 or Class 2.
  • the Class 1 and 2 systems are subdivided into types (Types I, II, III, IV, V, VI), with some types further divided into subtypes (e.g., Type II-A, Type II-B, Type II-C, Type V-A, Type V-B).
  • Class 2 systems comprise a single effector nuclease and include Types II, V, and VI.
  • the RGN is a naturally-occurring Type II CRISPR-Cas protein or an active variant or fragment thereof.
  • Type II CRISPR-Cas protein refers to an RGN that requires a trans-activating RNA (tracrRNA) and comprises two nuclease domains (i.e., RuvC and HNH), each of which is responsible for cleaving a single strand of a double -stranded DNA molecule.
  • a representative type II RGN includes a Cas9 protein, such as Streptococcus pyogenes Cas9 (SpCas9 or SpyCas9) or a SpCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 279 and 280, respectively, and are described in U.S. Pat. Nos. 10,000,772 and 8,697,359, each of which is herein incorporated by reference in its entirety.
  • a Cas9 protein such as Streptococcus pyogenes Cas9 (SpCas9 or SpyCas9) or a SpCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 279 and 280, respectively, and are described in U.S. Pat. Nos. 10,000,772 and 8,697,359, each of which is herein incorporated by reference in its entirety.
  • a representative type II RGN includes a Streptococcus thermophilus Cas9 (StCas9) or a StCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 281 and 282, respectively, and are disclosed in U.S. Pat. No. 10,113,167, which is herein incorporated by reference in its entirety.
  • a representative type II RGN includes a Streptococcus aureus Cas9 (SaCas9) or a SaCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 283 and 284, respectively, and are disclosed in U.S. Pat. No. 9,752,132, which is herein incorporated by reference in its entirety.
  • the CRISPR-Cas protein is a naturally-occurring Type V CRISPR-Cas protein or an active variant or fragment thereof.
  • Type V CRISPR-Cas protein refers to an RGN that cleaves dsDNA and comprises a single RuvC nuclease domain or a split-RuvC nuclease domain and lacks an HNH domain (Zetsche et al 2015, Cell doi: 10.1016/j .cell.2015.09.038; Shmakov et al 2017, Nat Rev Microbiol doi: 10.1038/nrmicro.2016.184; Yan et al 2018, Science doi: 10.1126/science.aav7271; Harrington et al 2018, Science doi: 10.1126/science.aav4294).
  • a presently disclosed RGN protein comprises a Casl2 (e.g., Casl2a).
  • Casl2a is also referred to as Cpfl, and does not require a tracrRNA, although other Type V CRISPR-Cas proteins, such as Casl2b, do require a tracrRNA.
  • Most Type V effectors can also target ssDNA (single-stranded DNA), often without a PAM requirement (Zetsche et al 2015; Yan et al 2018; Harrington et al 2018).
  • Type V CRISPR-Cas protein and “Type V RGN” encompasses the unique RGNs comprising split RuvC nuclease domains, such as those disclosed in U.S. Provisional Application Nos. 62/955,014 filed December 30, 2019 and 63/058,169 filed July 29, 2020, and PCT International Application No. PCT/US2020/067138 filed December 28, 2020, the contents of each of which are incorporated herein by reference in its entirety.
  • the present invention provides a RGN protein comprising Francisella novicida Casl2a (FnCasl2a), the sequence of which is set forth as SEQ ID NO: 408 and is disclosed in U.S. Pat. No. 9,790,490, which is herein incorporated by reference in its entirety, or any of the nuclease -inactivating mutants of FnCasl2a disclosed within U.S. Pat. No. 9,790,490.
  • FnCasl2a Francisella novicid
  • the presently disclosed RGN systems comprise an RGN that binds to a target sequence, e.g., such as those that are disclosed herein.
  • the RGN recognizes a PAM having a consensus nucleotide sequence including a NNNNCC 3' of the target sequence on its non-target strand (where N is A, C, T, or G; R is G or A), and active fragments or variants thereof.
  • the RGN recognizes a PAM having a consensus nucleotide sequence including NNGRR at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G; R is G or A), and active fragments or variants thereof.
  • the RGN recognizes a PAM having a consensus nucleotide sequence including NNRYA at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G; R is G or A; Y is C or T/U), and active fragments or variants thereof.
  • the RGN recognizes a PAM having a consensus nucleotide sequence including NGG at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G), and active fragments or variants thereof.
  • the active fragment or variant of an RGN recognizing such PAM sequences is capable of binding and in some embodiments, cleaving or nicking a target sequence.
  • an RGN capable of binding a target sequence adjacent to a PAM consensus sequence (i.e., capable of recognizing the PAM consensus sequence) set forth as NNNNCC is used in the presently disclosed compositions and methods.
  • the PAM sequence is at 3' of the target sequence on its non-target strand.
  • the RGN binds to a guide RNA comprising a crRNA repeat set forth in SEQ ID NO: 2 or an active variant or fragment thereof, and a tracrRNA set forth in SEQ ID NO: 3 or an active variant or fragment thereof.
  • Non-limiting examples of RGN systems useful in the presently disclosed compositions and methods along with corresponding crRNA sequences and tracrRNA sequences (if needed), are presented in Table 1 below and described further in Examples 1-18, and FIGs. 2-34 of the present specification.
  • RNA-guided nucleases and corresponding crRNA repeat sequences, tracrRNA sequences, and PAM sequences.
  • an active variant of an RGN disclosed herein requires a tracrRNA for activity.
  • an active variant of an RGN disclosed herein includes a type II RGN.
  • an active variant of an RGN disclosed herein comprises an amino acid 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 amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, or 252.
  • an active fragment of the APG07433.1 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,
  • an active fragment of the APG01604 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 69.
  • an active fragment of the APG05586 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 93.
  • an active fragment of the APG07991 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 252.
  • compositions and methods can utilize an RGN comprising at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and/or RNA binding domain to interact with guide RNAs.
  • nuclease domain e.g., DNase, RNase domain
  • RNA recognition and/or RNA binding domain to interact with guide RNAs.
  • Further domains that can be found in RGNs useful in the presently disclosed compositions and methods include, but are not limited to: DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains.
  • the RGNs useful in the presently disclosed compositions and methods comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of a DNA binding domain, helicase domain, protein-protein interaction domain, and dimerization domain.
  • the presently disclosed target sequences are bound by an RGN.
  • the target strand of the target sequence hybridizes with the guide RNA associated with the RGN.
  • the target strand and/or the non-target strand of the target sequence e.g., target DNA sequence
  • cleave or “cleavage” refer to the hydrolysis of at least one phosphodiester bond within the backbone of one or both strands of a double-stranded target sequence (e.g., target DNA sequence) that can result in either single-stranded or double-stranded breaks within the target sequence.
  • the cleavage of a presently disclosed target sequence can result in staggered breaks or blunt ends.
  • RGNs useful in the presently disclosed compositions and methods can be wild-type sequences derived from bacterial or archaeal species.
  • the RGNs can be variants or fragments of wild-type polypeptides.
  • the wild-type RGN can be modified to alter nuclease activity or alter PAM specificity, for example.
  • the RGN is not naturally-occurring.
  • an RGN useful in the presently disclosed compositions and methods can function with its own cognate guide RNA or a guide RNA that is heterologous to the RGN.
  • the APG07991 RGN (SEQ ID NO: 252) can bind to engineered and chemically modified APG07991 crRNAs and tracrRNAs disclosed herein to effect cleavage or modification of a target nucleic acid molecule (e.g., a APG07991 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, and 482-484; and/or a APG07991 tracrRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, and 505; see FIGs.
  • the APG07991 RGN also can bind to engineered and chemically modified SpyCas9 crRNAs and tracrRNAs disclosed herein to effect cleavage or modification of a target nucleic acid molecule (e.g., a SpyCas9 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 509-511, 513- 515, and 517-519; and/or a SpyCas9 tracrRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 534, 535, and 537; see FIG. 29).
  • a target nucleic acid molecule e.g., a SpyCas9 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 509-511, 513- 515, and 517-519; and/or a SpyCas9 tracrRNA having a nucleotide sequence set forth as any one
  • an RGN useful in the presently disclosed compositions and methods functions as a nickase, only cleaving a single strand of a double-stranded target sequence (e.g., target DNA sequence).
  • a double-stranded target sequence e.g., target DNA sequence
  • Such RGNs have a single functioning nuclease domain.
  • the nickase is capable of cleaving the target strand or the non-target strand of the double -stranded target sequence (e.g., target sequence).
  • additional nuclease domains have been mutated such that the nuclease activity is reduced or eliminated.
  • nickase in order to effect a double-stranded cleavage of a double -stranded target sequence (e.g., target DNA sequence), two nickases are needed, each of which nicks a single strand within the doublestranded target sequence.
  • a double -stranded target sequence e.g., target DNA sequence
  • the RGN lacks nuclease activity altogether and is referred to herein as nuclease-dead or nuclease inactive.
  • Any method known in the art for introducing mutations into an amino acid sequence such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used for generating nickases or nuclease-dead RGNs. See, e.g., U.S. Publ. No. 2014/0068797 and U.S. Pat. No. 9,790,490; each of which is incorporated by reference in its entirety.
  • RGNs that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location.
  • the RGN polypeptide or guide RNA can be fused to a detectable label to allow for detection of a particular sequence.
  • the detectable label or purification tag can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease, either directly or indirectly via a linker peptide.
  • the RGN component of the fusion protein is a nuclease-dead RGN.
  • the RGN component of the fusion protein is an RGN with nickase activity.
  • a detectable label is a molecule that can be visualized or otherwise observed.
  • the detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the RGN polypeptide that can be detected visually or by other means.
  • Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody.
  • Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl).
  • Non-limiting examples of small molecule detectable labels include radioactive labels, such as 3 H and 35 S.
  • RGN polypeptides can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium).
  • purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tag.
  • nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired gene (i.e., target gene).
  • the binding of a nuclease-dead RGN to a target sequence results in the reduction in expression of the target gene by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region.
  • the RGN e.g., a nuclease-dead RGN
  • its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence within a target gene, serves to either repress or activate the expression of the target gene.
  • the expression modulator of the fusion protein comprises a transcriptional repressor domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene.
  • Transcriptional repressor domains are known in the art and include, but are not limited to, Spl-like repressors, IKB, and Kriippel associated box (KRAB) domains.
  • the expression modulator of the fusion protein comprises a transcriptional activation domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene.
  • Transcriptional activation domains are known in the art and include, but are not limited to, a herpes simplex virus VP 16 activation domain and an NFAT activation domain.
  • the expression modulator modulates the expression of the target sequence or regulated gene through epigenetic mechanisms.
  • an epigenetic modulator covalently modifies DNA or histone proteins to alter histone structure and/or chromosomal structure without altering the DNA sequence, leading to changes in gene expression (e.g., upregulation or downregulation).
  • epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA.
  • epigenetic modulators include histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases, DNA methyltransferases, and DNA demethylases.
  • the nuclease-dead RGNs or an RGN with nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, for example a deaminase polypeptide or active variant or fragment thereof, that directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleobase to another.
  • the base-editing polypeptide can be fused to the RGN at its amino-terminal (N-terminal) or carboxy-terminal (C-terminal) end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker.
  • Base editors are fusion proteins comprising a DNA-targeting polypeptide, such as an RGN, and a base-editing polypeptide, such as a deaminase.
  • Non-limiting examples of deaminases or base editors that are useful for such compositions and methods includes a cytosine deaminase, an adenine deaminase, or a base editor (such as the adenine deaminase or base editor described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No.
  • WO 2018/027078 or any of the deaminases or base editors disclosed in International Publ. Nos. WO 2020/139783, WO 2022/056254, WO 2022/204093, U.S. provisional application no. 63/382,344 (fded November 4, 2022), and U.S. provisional application no. 63/485,642 (fded February 17, 2023), each of which is herein incorporated by reference in its entirety).
  • the deaminase that is useful for such presently disclosed compositions and methods is a deaminase disclosed in Table 17 of International Publ. No. WO 2020/139783, which is incorporated herein by reference in its entirety.
  • a deaminase useful for such presently disclosed compositions and methods includes a deaminase set forth as any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707.
  • a deaminase useful for such presently disclosed compositions and methods includes a deaminase having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707.
  • a base editor useful for such presently disclosed compositions and methods includes a base editor set forth as any one of the polypeptides of SEQ ID NOs: 550-591.
  • a base editor useful for such presently disclosed compositions and methods includes a base editor having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 550-591.
  • certain base editor comprising an RGN and a base-editing polypeptide e.g., cytosine deaminase
  • Non-limiting examples of uracil stabilizing polypeptides include those disclosed in PCT Publication No. WO 2021/217002 and PCT Publication No. WO 2022/015969, each of which is herein incorporated by reference in its entirety.
  • the disclosed uracil stabilizing polypeptides include USP2, and a uracil glycosylase inhibitor (UGI) domain, which may increase base editing efficiency. Therefore, a base editor may comprise an RGN described herein or variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2.
  • the RGN that is fused to the base-editing polypeptide is a nickase that cleaves the DNA strand that is not acted upon by the base-editing polypeptide (e.g., deaminase).
  • a gRNA modified with BNA modifications can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN.
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti- repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification.
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification.
  • BNA e.g., LNA and/or cEt
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification.
  • BNA e.g., LNA and/or cEt
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications.
  • BNA e.g., LNA and/or cEt
  • a gRNA modified with BNA modifications can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN.
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5 ' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS BNA
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • BNA
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the crRNA repeat and the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • BNA
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the crRNA repeat and the antirepeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • a gRNA modified with BNA modifications can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN.
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti -repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS)
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti- repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.
  • MS MS
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti -repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered crRNA repear and the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered crRNA repear and the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered antirepeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or c
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/
  • a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered antirepeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or c
  • an RGN may be fused to a prime editing polypeptide (e.g., a reverse transcriptase).
  • Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein working in association with a polymerase (described in, e.g., US 11,447,77OB1; WO2021072328; WO2021226558; WO2020156575; W02021042047; US11193123; each incorporated by reference in its entirety herein).
  • the prime editing system uses an RGN that is a nickase, and the system is programmed with a prime editing (PE) guide RNA (“PEgRNA”).
  • PEgRNA prime editing guide RNA
  • the PEgRNA is a guide RNA that both specifies the target sequence and provides the template for polymerization of the replacement strand containing the edit by way of an extension engineered onto the guide RNA (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA).
  • the RGN nickase/prime editing polypeptide fusion is guided to the target sequence by the PEgRNA and nicks the non-target strand upstream of sequence to be edited and upstream of the PAM, creating a 3' flap on the non-target strand.
  • the PEgRNA includes a primer binding site (PBS) that is complementary to the 3' flap of the non-target strand.
  • PBS primer binding site
  • a PBS is at least about 5, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the PEgRNA comprises a PBS that is at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length.
  • the PEgRNA may comprise a PBS that is at least 8 nucleotides in length.
  • Hybridrization of the PBS and 3' flap of the non-target strand allows polymerization of the replacement strand containing the edit using the extension of the PEgRNA as template.
  • the extension of the PEgRNA can be formed from RNA or DNA.
  • the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase).
  • the polymerase of the prime editor may be a DNA-dependent DNA polymerase.
  • the replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the non-target strand of the target sequence to be edited (with the exception that it includes the desired edit).
  • the non-target strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit.
  • prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors not only search and locate the desired target sequence to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding non-target strand of the target sequence.
  • a guide RNA of the disclosure comprises an extension comprising an edit template for prime editing.
  • a prime editing polypeptide that can be fused to an RGN includes a DNA polymerase.
  • the DNA polymerase is a reverse transcriptase.
  • the RGN is a nickase.
  • the presently disclosed guide RNAs are combined with an RGN nickase comprising a mutation in the RuvC domain (e.g., a D10A mutation) which renders the RGN capable of cleaving only the target strand (the strand which does not comprise the PAM) of a nucleic acid duplex.
  • D10A nickases are not able to cleave the non-target strand of the DNA, i.e., the strand where base editing is desired.
  • the RGN nicks the target strand, while the complementary, non-target strand is modified by the deaminase.
  • Cellular DNA-repair machinery may repair the nicked, target strand using the modified non-target strand as a template, thereby introducing a mutation in the DNA.
  • the nickase comprises an inactive RuvC domain.
  • RuvC domains have an RNase H fold structure (see, e.g., Nishimasu et al. (2014) Cell 156(5):935-949, which is incorporated by reference in its entirety).
  • RuvC domains of RGNs are often split RuvC domains, comprising two or more non-adjacent regions within the linear amino acid sequence.
  • the RuvC domain of Streptococcus pyogenes Cas9 comprises amino acid residues 1-59, 718-769 and 909- 1098 of SEQ ID NO: 279.
  • nAPG07433.1 (set forth as SEQ ID NO: 543) is a nickase variant of APG07433.1, which is set forth as SEQ ID NO: 1, and is described in WO 2019/236566 (incorporated by reference in its entirety herein).
  • D10A mutant (i.e., inactive RuvC/active HNH domain) nickases include, but are not limited to those set forth as SEQ ID NOs: 546-548.
  • the nickase RGN of the fusion protein comprises a mutation (e.g., a H840A mutation, wherein amino acid numbering is based on the Streptococcus pyogenes Cas9 sequence set forth as SEQ ID NO: 279), which renders the RGN capable of cleaving only the nontarget strand (the strand which comprises the PAM) of a nucleic acid duplex.
  • the nickase comprises an inactive HNH nuclease domain.
  • the HNH nuclease domain of RGNs have a PPa-metal fold (see, e.g., Nishimasu et al. 2014).

Abstract

L'invention concerne des compositions comprenant un ARN transactivateur CRISPR (ARNtracr), un ARN guide (ARNg) et/ou un ARN CRISPR (ARNcr) modifiés génétiquement et/ou chimiquement. L'ARNtracr, l'ARNg et/ou l'ARNcr modifiés chimiquement intègrent des modifications d'acide nucléique ponté (BNA) et d'autres modifications chimiques. Les modifications de BNA comprennent des modifications d'acide nucléique à verrouillage (LNA) en 2',4'. Les modifications chimiques supplémentaires comprennent des modifications 2'-O-méthyl (2'-O-Me), 2'-O-méthyl 3'phosphorothioate (MS) et phosphorothioate (PS). L'ARNtracr, l'ARNg et/ou l'ARNcr modifiés génétiquement et/ou chimiquement de l'invention améliorent l'efficacité de l'édition de gènes (y compris l'édition de base et l'édition primaire "prime editing") d'un système de nucléase guidée par l'ARN (RGN).
PCT/IB2023/058418 2022-08-25 2023-08-25 Modification chimique d'arn guides avec un acide nucléique verrouillé pour l'édition de gènes à médiation par nucléase guidée par l'arn WO2024042489A1 (fr)

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