WO2022140340A1 - Compositions comprising an rna guide targeting dmd and uses thereof - Google Patents

Compositions comprising an rna guide targeting dmd and uses thereof Download PDF

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Publication number
WO2022140340A1
WO2022140340A1 PCT/US2021/064540 US2021064540W WO2022140340A1 WO 2022140340 A1 WO2022140340 A1 WO 2022140340A1 US 2021064540 W US2021064540 W US 2021064540W WO 2022140340 A1 WO2022140340 A1 WO 2022140340A1
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Prior art keywords
nucleotide
sequence
seq
nos
nucleotides
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PCT/US2021/064540
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French (fr)
Inventor
Quinton Norman WESSELLS
Jeffrey Raymond HASWELL
Tia Marie DITOMMASO
Noah Michael Jakimo
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Vertex Pharmaceuticals Incorporated
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Publication of WO2022140340A1 publication Critical patent/WO2022140340A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]

Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • CRISPR-Cas CRISPR-associated genes
  • compositions comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a Duchenne muscular dystrophy (DMD) gene and (ii) a direct repeat sequence (e.g., any of the sequences provided in Table 1 or a portion of any of the sequences provided in Table 1); wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN- 3’.
  • DMD Duchenne muscular dystrophy
  • PAM protospacer adjacent motif
  • the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53 of the DMD gene.
  • a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647;
  • a variant of SEQ ID NO: 1647 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds;
  • exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; a variant of SEQ ID NO: 1648 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds; or c.
  • exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649; a variant of SEQ ID NO: 1649 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; d.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide
  • nucleotide 1 through nucleotide
  • nucleotide 1 through nucleotide
  • nucleotide 1 through nucleotide
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e.
  • nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8;
  • nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8;
  • nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8;
  • h nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8;
  • nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m.
  • nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v.
  • nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa.
  • the direct repeat comprises any stretch of about 15-36, 15-35, 15-34, 15-32, 15-30, 15-28, 15-26, 15-23, 20-23, 18-25, 30-36, 30-35, 30-34, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-10, or comprises any stretch of about 15-36, 15-35, 15-34, 15-32, 15-30, 15-28, 15-26, 15-23, 20-23, 18-25, 30-36, 30-35, 30-34, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more contiguous nucleotides of a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-10.
  • the direct repeat comprises any stretch of about 30, 31, 32, 33, 34, 35, or 36 or more contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-10, or comprises any stretch of about 30, 31, 32, 33, 34, 35, or 36 or more contiguous nucleotides of a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-10.
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g.
  • nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m.
  • nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t.
  • the spacer sequence is substantially complementary (e.g., at least
  • the PAM comprises the sequence 5 ’-ATTA-3’
  • the target sequence is immediately adjacent to the PAM sequence.
  • composition further comprises a Casl2i2 polypeptide or a nucleic acid encoding a Casl2i2 polypeptide.
  • the Casl2i2 polypeptide comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1646.
  • the nucleic acid encoding a Casl2i2 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 1654, or a fragment of such a nucleic acid that encodes a functional Casl2i2 polypeptide.
  • the Casl2i2 polypeptide comprises a sequence of SEQ ID NO: 1650, 1651, 1652, or 1653.
  • RNA guide and the Casl2i2 polypeptide form a ribonucleoprotein complex.
  • the ribonucleoprotein complex binds a target nucleic acid.
  • composition is present within a cell.
  • RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a DMD gene and (ii) a direct repeat sequence is provided.
  • the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53.
  • a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO:
  • exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; d.
  • the spacer sequence comprises or consists of: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e.
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b.
  • nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f.
  • nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j.
  • nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n.
  • nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r.
  • nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w.
  • nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; or bb. a portion thereof.
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h.
  • nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n.
  • nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u.
  • nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197-1420.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’.
  • PAM protospacer adjacent motif
  • the PAM comprises the sequence 5 ’-ATTA-3’,
  • the target sequence is immediately adjacent to the
  • the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • the Casl2i2 polypeptide comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1646.
  • the RNA guide and the Casl2i2 polypeptide form a ribonucleoprotein complex.
  • the ribonucleoprotein complex binds a target nucleic acid.
  • the composition is present within a cell.
  • the RNA guide and the Casl2i2 polypeptide are encoded in a vector, e.g., expression vector.
  • RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a DMD gene and (ii) a direct repeat sequence.
  • target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53.
  • a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647;
  • exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c.
  • exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; b.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e.
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1- 8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d.
  • nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h.
  • nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1.
  • nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p.
  • nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u.
  • nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z.
  • the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d.
  • nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j.
  • nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q.
  • nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y.
  • nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197- 1420.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’, wherein N is any nucleotide.
  • PAM protospacer adjacent motif
  • the PAM comprises the sequence 5’-ATTA-3’, 5’-
  • ATTT-3’ 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-GITAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’.
  • the target sequence is immediately adjacent to the
  • the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • Casl2i2 polypeptide is provided.
  • RNA guide comprising such an RNA guide, a direct repeat, and/or a Casl2i2 polypeptide is provided.
  • a cell comprising a composition, an RNA guide, a nucleic acid, or a vector is provided.
  • the cell is a eukaryotic cell (e.g., an animal cell, a mammalian cell, or a human cell). In one aspect of the cell, the cell is a muscle cell. In one aspect of the cell, the cell is a primary cell, a stem cell, a T cell, or a cell line.
  • a eukaryotic cell e.g., an animal cell, a mammalian cell, or a human cell.
  • the cell is a muscle cell.
  • the cell is a primary cell, a stem cell, a T cell, or a cell line.
  • kits comprising a composition, an RNA guide, a nucleic acid, or a vector is provided.
  • a method of editing a DMD sequence such as a DMD exon
  • the method comprising contacting the DMD sequence with a composition or an RNA guide is provided.
  • the DMD sequence such as a DMD exon (e.g., exon 45, exon 51, or exon 53) is in a cell in vitro.
  • the DMD sequence such as a DMD exon (e.g., exon 45, exon 51, or exon 53) is in a cell in vivo.
  • the composition or the RNA guide induces a deletion in the DMD sequence, such as a portion of a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a DMD exon e.g., exon 45, exon 51, or exon 53.
  • the deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide.
  • the deletion is downstream of the 5’-NTTN-3’ sequence.
  • the deletion is up to about 40 nucleotides in length.
  • the deletion is from about 4 nucleotides to 40 nucleotides in length.
  • the deletion is from about 4 nucleotides to 25 nucleotides in length.
  • the deletion is from about 10 nucleotides to 25 nucleotides in length.
  • the deletion is from about 10 nucleotides to 15 nucleotides in length.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence. [0066] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence. [0081] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
  • the 5’-NTTN-3’ sequence is 5’-CTTT-3’, 5’-CTTC-3’, 5’- GTTT-3’, 5’-GTTC-3’, 5’-TTTC-3’, 5’-GTTA-3’, or 5’-GTTG-3’.
  • the deletion overlaps with a mutation in the gene.
  • the deletion overlaps with an insertion in the gene.
  • the deletion removes a repeat expansion of the gene or a portion thereof.
  • the deletion disrupts one or both alleles of the gene.
  • activity refers to a biological activity.
  • activity includes enzymatic activity, e.g., catalytic ability of an effector.
  • activity can include nuclease activity.
  • DMD refers to “Duchenne muscular dystrophy.”
  • the DMD gene encodes a protein called dystrophin, which is expressed primarily in skeletal and cardiac muscles. Absence or abnormal expression of DMD gene expression, leading to dystrophin deficiency, is a cause of myopathies including Duchenne and Becker muscular dystrophies. Exon skipping approaches to altering DMD expression can be directed to DMD exons 45, 51, and 53.
  • SEQ ID NOs: 1647, 1648, and 1649 as set forth herein provide examples of DMD exon 45, exon 51, and exon 53 sequences, respectively. It is to be understood that spacer sequences described herein can target SEQ ID NOs: 1647, 1648, or 1649, or reverse complements thereof, depending upon whether they are indicated as “+” or as set forth in Tables 2-4.
  • the term “complex” refers to a grouping of two or more molecules.
  • the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another.
  • the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Casl2i2 polypeptide).
  • the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence.
  • the term “complex” can refer to a grouping of a DMD-targeting RNA guide and a Casl2i2 polypeptide.
  • PAM protospacer adjacent motif
  • a target sequence e.g., a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)
  • a complex comprising an RNA guide (e.g., a DMD-targeting RNA guide) and a Casl2i2 polypeptide binds.
  • the RNA guide binds to a first strand of the target (e.g., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand).
  • a first strand of the target e.g., the target strand or the spacer-complementary strand
  • a PAM sequence as described herein is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand).
  • the term “adjacent” includes instances in which the RNA guide of a complex comprising an RNA guide and a Casl2i2 polypeptide specifically binds, interacts, or associates with a target sequence that is immediately adjacent to a PAM. In such instances, there are no nucleotides between the target sequence and the PAM.
  • adjacent also includes instances in which there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the target sequence, to which the RNA guide binds, and the PAM.
  • the PAM sequence as described herein is present in the non-target strand (e.g., the non-spacer-complementary strand).
  • adjacent includes a PAM sequence as described herein as being immediately adjacent to (or within a small number, e.g., 1, 2, 3, 4, or 5 nucleotides of) a sequence in the non-target strand.
  • RNA guide refers to any RNA molecule that facilitates the targeting of a polypeptide (e.g., a Casl2i2 polypeptide) to a target sequence (e.g., a sequence of a DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)).
  • a target sequence e.g., a sequence of a DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)
  • An RNA guide may comprise sequences that are complementary to a specific nucleic acid sequence (e.g., a DMD nucleic acid sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)).
  • An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence.
  • crRNA is also used herein to refer to an
  • the term “substantially complementary” refers to a polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a target sequence.
  • the level of complementarity is such that the polynucleotide can hybridize to the target sequence with sufficient affinity to permit an effector polypeptide (e.g., Casl2i2) that is complexed with the polynucleotide to act (e.g., cleave) on the target sequence.
  • target and target sequence refer to a nucleic acid sequence to which an RNA guide specifically binds.
  • target and target sequence refer to a nucleic acid to which an RNA guide directs a nuclease, e.g., a Casl2i2.
  • the DNA targeting sequence (e.g., spacer) of an RNA guide binds to a target sequence.
  • the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand).
  • the target strand i.e., the spacer-complementary strand
  • the target sequence is a sequence within a DMD exon (e.g., exon 45, exon 51, or exon 53), including, but not limited to, the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or a reverse complement thereof.
  • the present disclosure relates to an RNA guide capable of binding to a DMD gene sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), and methods of use thereof.
  • a composition comprising an RNA guide having one or more characteristics is described herein.
  • a method of producing the RNA guide is described.
  • a method of delivering a composition comprising the RNA guide is described.
  • compositions comprising an RNA guide targeting a DMD gene or a portion of the DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • the RNA guide is comprised of a direct repeat component and a spacer component.
  • the RNA guide binds a Casl2i2 polypeptide.
  • the spacer component is substantially complementary to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), wherein the DMD target sequence is adjacent to a 5’-NTTN-3’ PAM sequence as described herein.
  • the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand).
  • the disclosure provides for compositions comprising a complex, wherein the complex comprises an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • the disclosure provides for a complex comprising an RNA guide and a Casl2i2 polypeptide.
  • the RNA guide and the Casl2i2 polypeptide bind to each other in a molar ratio of about 1:1.
  • a complex comprising an RNA guide and a Casl2i2 polypeptide binds to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a complex comprising an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), and a Casl2i2 polypeptide binds to the DMD target sequence at a molar ratio of about 1 : 1.
  • the complex comprises enzymatic activity, such as nuclease activity, that can cleave the DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • the RNA guide, the Casl2i2 polypeptide, and the DMD target sequence (such as a DMD exon (e.g., exon 45, exon 51, or exon 53)), either alone or together, do not naturally occur.
  • the composition described herein comprises an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a DMD exon e.g., exon 45, exon 51, or exon 53.
  • the composition described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • the RNA guide may direct the Casl2i2 polypeptide as described herein to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a DMD target sequence such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • Two or more RNA guides may target two or more separate Casl2i2 polypeptides (e.g., Casl2i2 polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) DMD target sequences, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a DMD exon e.g., exon 45, exon 51, or exon 53.
  • an RNA guide is DMD target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more DMD target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).
  • the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5’ to 3’ direction.
  • the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5 ’ to 3 ’ direction.
  • the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.
  • the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule.
  • the spacer and direct repeat sequences are linked directly to one another.
  • a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length.
  • the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.
  • RNA guides Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.
  • the RNA guide comprises a direct repeat sequence.
  • the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14- 50, or 15-40 nucleotides (e.g., 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, or 40 nucleotides).
  • the direct repeat sequence is or comprises a sequence of Table 1 or a portion of a sequence of Table 1.
  • the direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ
  • the direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NO:
  • the direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can comprise nucleotide 12 through nucleotide 34 ofSEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.
  • the direct repeat sequence has or comprises a sequence comprising at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9.
  • the direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.
  • the direct repeat sequence is or comprises a sequence that is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is or comprises the reverse complement of any one of SEQ ID NOs: 1-10.
  • the RNA guide comprises a DNA targeting or spacer sequence.
  • the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14- 50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary a specific target sequence.
  • the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.
  • the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence.
  • the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence.
  • the percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
  • the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence.
  • the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
  • the spacer sequence is or comprises a sequence of Table 2 or a portion of a sequence of Table 2. It should be understood that an indication of SEQ ID NOs: 289-566, 882-1196, or 1421-1644 should be considered as equivalent to a listing of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644, with each of the intervening numbers present in the listing, i.e., 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,
  • the spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
  • the spacer sequence has or comprises a sequence having at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
  • the RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this disclosure.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof.
  • the internucleoside linkage e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone
  • the RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.
  • the modification may include a chemical or cellular induced modification.
  • intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
  • nucleotide modifications may exist at various positions in the sequence.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased.
  • the sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%).
  • any intervening percentage e.g.
  • sugar modifications e.g., at the 2’ position or 4’ position
  • replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages.
  • Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • a sequence will include ribonucleotides with a phosphorus atom in its intemucleoside backbone.
  • Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3 ’-amino phosphoramidate and aminoalky Iphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 ’-5’ linkages, 2 ’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’- 2’.
  • the sequence may be negatively or positively charged.
  • the modified nucleotides which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone).
  • the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • a modified nucleoside includes an alpha-thio-nucleoside (e . g. , 5 ’ - O-(l-thiophosphate)-adenosine, 5 ’-O-(l -thiophosphate) -cytidine (a-thio-cytidine), 5’-O-(l- thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l-thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e . g. , 5 ’ - O-(l-thiophosphate)-adenosine, 5 ’-O-(l -thiophosphate) -cytidine (a-thio-cytidine), 5’-O-(l- thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-ur
  • the sequence may include one or more cytotoxic nucleosides.
  • cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification.
  • Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5 -azacytidine, 4’-thio- aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C- cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5 -fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l- (tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)
  • Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-l-beta-D- arabinofuranosy Icy tosine, N4-palmitoy 1- 1 -(2-C-cy ano-2-deoxy-beta-D-arabino-pentofuranosy 1) cytosine, and P-4055 (cytarabine 5 ’-elaidic acid ester).
  • the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc).
  • the one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).
  • the RNA Modification Database 1999 update.
  • the first isolated nucleic acid comprises messenger RNA (mRNA).
  • the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5 -aza-uridine, 2 -thiouridine, 4-thio-pseudouridine, 2 -thio -pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propyny 1-uridine, 1 -propyny 1-pseudouridine, 5 -taurinomethy luridine, 1 -taurinomethy 1-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5 -methy
  • the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methy 1-pseudoisocytidine, pyrrolo -cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocy tidine, 1 -methyl- 1 -deaza-pseudoisocy tidine, zebularine, 5 -aza-
  • the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6 -diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl
  • mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8- aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methy lino sine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
  • the sequence may or may not be uniformly modified along the entire length of the molecule.
  • nucleotide e.g., naturally -occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the sequence includes a pseudouridine.
  • the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by AD ARI marks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
  • the composition of the present disclosure includes a Casl2i2 polypeptide as described in PCT/US2019/022375 (WO2019178427A1), which is incorporated herein in its entirety, including the sequences. Nucleic acid encoding Casl2i2 polypeptides are also provided. [00135] In some embodiments, the composition of the present disclosure includes a Casl2i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1646 and/or encoded by SEQ ID NO: 1645).
  • a nucleic acid sequence encoding the Casl2i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1645.
  • the Casl2i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1645.
  • the percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
  • One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions (e.g., within a range of medium to high stringency).
  • the Casl2i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1645.
  • the Casl2i2 polypeptide of the present disclosure comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1646.
  • the present disclosure describes a Casl2i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1646.
  • Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • Casl2i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1646 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • the Casl2i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653.
  • the Casl2i2 polypeptide of the present disclosure comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653.
  • the present disclosure describes a Casl2i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
  • a Casl2i2 polypeptide (e.g., a Casl2il2 polypeptide of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO: 155) comprises a RuvC domain.
  • Casl2i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
  • enzymatic activity e.g., nuclease or endonuclease activity
  • the Casl2i2 polypeptide has the same enzymatic activity (e.g., nuclease or endonuclease activity) as, or within 20%, 15%, 10%, 5%, or 2% of the same enzymatic activity as, a polypeptide having the amino acid sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653 in an enzymatic activity (e.g., nuclease or endonuclease activity) assay (e.g., target cleavage assay, or a TIDE assay).
  • an enzymatic activity e.g., nuclease or endonuclease activity
  • changes to the Casl2i2 polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions.
  • the Casl2i2 polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a poly histidine tag (His-tag), Myc, and FLAG.
  • the Casl2i2 polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • the Casl2i2 polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.
  • the nucleotide sequence encoding the Casl2i2 polypeptide described herein can be codon-optimized for use in a particular host cell or organism.
  • the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA).
  • the target sequence is within a DMD gene or a locus of a DMD gene.
  • the DMD gene is a mammalian gene.
  • the DMD gene is a human gene.
  • the target sequence is within a DMD exon (e.g., exon 45, exon 51, or exon 53) as set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complements thereof.
  • target sequences within DMD exons are set forth in Tables 2, 3, and 4, respectively.
  • the target sequence is within a variant (e.g., a polymorphic variant) of a DMD exon (e.g., exon 45, exon 51, or exon 53) set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complements thereof.
  • the DMD exon (e.g., exon 45, exon 51, or exon 53) sequence is a homolog of a sequence set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complement thereof.
  • the DMD exon (e.g., exon 45, exon 51, or exon 53) sequence is a non-human DMD exon (corresponding to, e.g., human exon 45, exon 51, or exon 53) sequence.
  • the target sequence is adjacent to a 5’-NTTN-3’ PAM sequence, wherein N is any nucleotide.
  • the 5’-NTTN-3’ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence.
  • the 5’-NTTN-3’ sequence is 5’-NTTY-3’, 5’-NTTC-3’, 5’-NTTT-3’, 5’-NTTA-3’, 5’-NTTB-3’, 5’-NTTG-3’, 5’-CTTY-3’, 5’-DTTR’3’, 5’-CTTR-3’, 5’-DTTT-3’, 5’- ATTN-3’, or 5’-GTTN-3’, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G.
  • the 5’-NTTN-3’ sequence is 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-G TAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’.
  • the target sequence is single-stranded (e.g., single-stranded DNA).
  • the target sequence is double-stranded (e.g., double-stranded DNA). In some embodiments, the target sequence comprises both single-stranded and double-stranded regions. In some embodiments, the target sequence is linear. In some embodiments, the target sequence is circular. In some embodiments, the target sequence comprises one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotides analogs. In some embodiments, the target sequence is not modified.
  • the RNA guide binds to a first strand of a doublestranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5’-NTTN- 3’ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non- spacer-complementary strand).
  • the RNA guide binds adjacent to a 5 ’-NAAN-3’ sequence on the target strand (e.g., the spacer-complementary strand).
  • the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5' or 3' untranslated region, etc. In some embodiments, the target sequence is a plasmid.
  • the target sequence is present in a readily accessible region of the target sequence.
  • the target sequence is in an exon of a target gene.
  • the target sequence is across an exon-intron junction of a target gene.
  • the target sequence is present in a non-coding region, such as a regulatory region of a gene.
  • the target sequence comprises a sequence that is not found in the genome of the cell.
  • the target sequence is exogenous to a cell. In some embodiments, the target sequence is a horizontally transferred plasmid. In some embodiments, the target sequence is integrated in the genome of the cell. In some embodiments, the target sequence is not integrated in the genome of the cell. In some embodiments, the target sequence is a plasmid in the cell. In some embodiments, the target sequence is present in an extrachromosomal array.
  • the target sequence is an isolated nucleic acid, such as an isolated DNA or an isolated RNA. In some embodiments, the target sequence is present in a cell-free environment. In some embodiments, the target sequence is an isolated vector, such as a plasmid. In some embodiments, the target sequence is an ultrapure plasmid.
  • the target sequence is a locus of the DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53) sequence, that hybridizes to the RNA guide.
  • a cell has only one copy of the target sequence.
  • a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.
  • the present disclosure includes methods for production of the RNA guide, methods for production of the Casl2i2 polypeptide, and methods for complexing the RNA guide and Casl2i2 polypeptide.
  • the RNA guide is made by in vitro transcription of a DNA template.
  • the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence).
  • the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide.
  • the RNA guide is made using chemical synthetic methods.
  • the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide.
  • the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Casl2i2 polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Casl2i2 polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.
  • the Casl2i2 polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Casl2i2 polypeptide of the present disclosure, isolating the Casl2i2 polypeptide, optionally, purifying the Casl2i2 polypeptide, and complexing the Casl2i2 polypeptide with an RNA guide.
  • the Casl2i2 polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Casl2i2 polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell.
  • the Casl2i2 polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.
  • a host cell is used to express the Casl2i2 polypeptide.
  • the host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells).
  • the method for transferring the expression vector described above into host cells i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.
  • the host cells may be cultured, cultivated or bred, for production of the Casl2i2 polypeptide.
  • the host cells can be collected and Casl2i2 polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).
  • the methods for Casl2i2 polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Casl2i2 polypeptide.
  • the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Casl2i2 polypeptide.
  • a variety of methods can be used to determine the level of production of a Casl2i2 polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Casl2i2 polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).
  • the present disclosure provides methods of in vivo expression of the Casl2i2 polypeptide in a cell, comprising providing a polyribonucleotide encoding the Casl2i2 polypeptide to a host cell wherein the polyribonucleotide encodes the Casl2i2 polypeptide, expressing the Casl2i2 polypeptide in the cell, and obtaining the Casl2i2 polypeptide from the cell.
  • an RNA guide targeting DMD such as a DMD exon (e.g., exon 45, exon 51, or exon 53), is complexed with a Casl2i2 polypeptide to form a ribonucleoprotein.
  • complexation of the RNA guide and Casl2i2 polypeptide occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C.
  • the RNA guide does not dissociate from the Casl2i2 polypeptide at about 37°C over an incubation period of at least about any one of lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Jackpot, 2hr, 3hr, 4hr, or more hours.
  • the RNA guide and Casl2i2 polypeptide are complexed in a complexation buffer.
  • the Casl2i2 polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide.
  • the Casl2i2 polypeptide is stored in a complexation buffer.
  • the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.
  • the Casl2i2 polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein.
  • mRNA or DNA encoding the Casl2i2 polypeptide is introduced into a cell so that the Casl2i2 polypeptide is expressed in the cell.
  • the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.
  • compositions or complexes described herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.).
  • a carrier such as a carrier and/or a polymeric carrier, e.g., a liposome
  • transfection e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers
  • electroporation or other methods of membrane disruption e.g., nucleofection
  • viral delivery e.g., lentivirus, retrovirus, adenovirus, AAV
  • microinjection microprojectile bombardment (“gene gun”)
  • fugene direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.
  • the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Casl2i2 polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Casl2i2 polypeptide complex to a cell, where a ternary complex is formed.
  • nucleic acids e.g., nucleic acids encoding the Casl2i2 polypeptide, RNA guide, donor DNA, etc.
  • Exemplary intracellular delivery methods include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particlebased methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection.
  • the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
  • compositions or complexes described herein can be delivered to a variety of cells.
  • the cell is an isolated cell.
  • the cell is in cell culture or a co-culture of two or more cell types.
  • the cell is ex vivo.
  • the cell is obtained from a living organism and maintained in a cell culture.
  • the cell is a single-cellular organism.
  • the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.
  • the cell is a eukaryotic cell.
  • the cell is a plant cell or derived from a plant cell.
  • the cell is a fungal cell or derived from a fungal cell.
  • the cell is an animal cell or derived from an animal cell.
  • the cell is an invertebrate cell or derived from an invertebrate cell.
  • the cell is a vertebrate cell or derived from a vertebrate cell.
  • the cell is a mammalian cell or derived from a mammalian cell.
  • the cell is a human cell.
  • the cell is a zebra fish cell.
  • the cell is a rodent cell.
  • the cell is synthetically made, sometimes termed an artificial cell.
  • the cell is derived from a cell line.
  • a wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).
  • the cell is an immortal or immortalized cell.
  • the cell is a primary cell.
  • the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell.
  • the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC.
  • iPSC induced pluripotent stem cell
  • the cell is a differentiated cell.
  • the differentiated cell is a muscle cell (e.g., a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell, or a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a liver cell (e.g., a hepatocyte), a fibroblast, or a sex cell.
  • a muscle cell e.g., a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell, or
  • the cell is a terminally differentiated cell.
  • the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell.
  • the cell is an immune cell.
  • the immune cell is a T cell.
  • the immune cell is a B cell.
  • the immune cell is a Natural Killer (NK) cell.
  • the immune cell is a Tumor Infiltrating Lymphocyte (TIL).
  • the cell is a mammalian cell, e.g., a human cell or a murine cell.
  • the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease -specific mouse model.
  • the cell is a cell within a living tissue, organ, or organism.
  • the disclosure also provides methods of modifying a target sequence within the DMD gene.
  • the methods comprise introducing a DMD-targeting RNA guide and a Casl2i2 polypeptide or nucleic acid encoding a Casl2i2 polypeptide into a cell.
  • the DMD-targeting RNA guide and Casl2i2 polypeptide can be introduced as a ribonucleoprotein complex into a cell.
  • the DMD-targeting RNA guide and Casl2i2 polypeptide can be introduced on a nucleic acid vector.
  • the Casl2i2 polypeptide can be introduced as an mRNA.
  • the RNA guide can be introduced directly into the cell.
  • the target sequence is in a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • the DMD exon e.g., exon 45, exon 51, or exon 53
  • the DMD exon has a sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complement thereof.
  • the sequence of the DMD exon (e.g., exon 45, exon 51, or exon 53) is a variant of the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 51) (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53) (or the reverse complement thereof).
  • the target sequence is a polymorphic variant of a DMD exon (e.g., exon 45, exon 51, or exon 53) sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53) (or the reverse complement thereof) or a non-human form of a DMD exon (e.g., an exon corresponding to human exon 45, exon 51, or exon 53).
  • a DMD exon e.g., exon 45, exon 51, or exon 53
  • a non-human form of a DMD exon e.g., an exon corresponding to human exon 45, exon 51, or exon 53.
  • an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5’-NTTN-3’ PAM sequence.
  • the 5’-NTTN-3’ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence.
  • the 5’-NTTN-3’ sequence is 5’- NTTY-3’, 5’-NTTC-3’, 5’-NTTT-3’, 5’-NTTA-3’, 5’-NTTB-3’, 5’-NTTG-3’, 5’-CTTY-3’, 5’- DTTR’3’, 5’-CTTR-3’, 5’-DTTT-3’, 5’-ATTN-3’, or 5’-GTTN-3’, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G.
  • the 5’-NTTN-3’ sequence is 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT- 3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’- CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’.
  • the RNA guide is designed to bind to a first strand of a double-stranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5’-NTTN-3’ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand).
  • the RNA guide binds adjacent to a 5’-NAAN-3’ sequence on the target strand (e.g., the spacer-complementary strand).
  • the Casl2i2 polypeptide has enzymatic activity (e.g., nuclease activity).
  • the Casl2i2 polypeptide induces one or more DNA double -stranded breaks in the cell. In some embodiments, the Casl2i2 polypeptide induces one or more DNA singlestranded breaks in the cell. In some embodiments, the Casl2i2 polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).
  • an RNA guide disclosed herein forms a complex with the Casl2i2 polypeptide and directs the Casl2i2 polypeptide to a target sequence adjacent to a 5’-NTTN-3’ sequence.
  • the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5’-NTTN-3’ sequence.
  • the complex induces a deletion adjacent to a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • the complex induces a deletion adjacent to a T/C-rich sequence.
  • the deletion is downstream of a 5’-NTTN-3’ sequence. In some embodiments, the deletion is downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.
  • the deletion alters expression of the DMD gene. In some embodiments, the deletion alters function of the DMD gene. In some embodiments, the deletion inactivates the DMD gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5’-ATTA-3’, 5
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7,
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3,
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’- TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g.,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA- 3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’- CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence. [00189] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12,
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-G TAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence. [00191] In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’- NTTN-3’ sequence.
  • the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’- TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG- 3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.
  • the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23 , 24, 25 , 26, 27, or 28 nucleotides) of a 5 ’ -ATTA-3 ’ , 5 ’ -ATTT-3 ’ , 5 ’ - ATTG-3 ’ , 5 ’ -ATTC-3 ’ , 5 ’ - TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, 5’
  • the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25
  • the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’- TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (
  • the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 25 to
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C- rich sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA- 3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • ends
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C- rich sequence.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTAS’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’,
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA- 3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5 ’-ATTA-3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’, 5 ’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’- ATTA-3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21 , 22, 23 , 24, 25 , 26, 27, or 28 nucleotides) of a 5 ’ -ATTA-3 ’ , 5 ’ -ATTT-3 ’ , 5 ’ - ATTG-3 ’ , 5 ’- ATTC-3 ’ , 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA- 3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’, 5 ’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’- ATTC-3’, 5
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA- 3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’- ATTC-3’, 5’-
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’- NTTN-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’- TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG- 3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’, 5
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’,
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
  • nucleotides e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides
  • the deletion is up to about 50 nucleotides in length (e.g., about 1, 2, 3, 4, 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).
  • the deletion is up to about 40 nucleotides in length (e.g., about 1, 2, 3, 4, 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 3, 4, 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, or 45 nucleotides).
  • the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).
  • the methods described herein are used to engineer a cell comprising a deletion as described herein in a DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
  • a DMD exon e.g., exon 45, exon 51, or exon 53.
  • kits or systems that can be used, for example, to carry out a method described herein.
  • the kits or systems include an RNA guide and a Casl2i2 polypeptide.
  • the kits or systems include a polynucleotide that encodes such a Casl2i2 polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein.
  • the Casl2i2 polypeptide and the RNA guide can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use.
  • the kits or systems can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Casl2i2 polypeptide.
  • This Example describes transfection followed by indel assessment on multiple DMD target sequences, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), using a Casl2i2 polypeptide in mammalian cells.
  • DMD exon e.g., exon 45, exon 51, or exon 53
  • Casl2i2 polypeptide in mammalian cells.
  • a Casl2i2 polypeptide-encoding sequence is cloned into a pcda3.1 backbone (Invitrogen). The plasmid is then maxi-prepped and diluted to 1 pg/pL.
  • a dsDNA fragment encoding an RNA guide described herein is derived by ultramers containing the target sequence scaffold, and the U6 promoter. Ultramers are resuspended in 10 mM Tris «HCl at a pH of 7.5 to a final stock concentration of 100 pM. Working stocks are subsequently diluted to 10 pM. again using 10 mM Tris*HCl to serve as the template for the PCR reaction.
  • the amplification of the RNA guide is done in 50 pL reactions with the following components: 0.02 pl of aforementioned template, 2.5 pl forward primer, 2.5 pl reverse primer, 25 pL NEB HiFi Polymerase, and 20 pl water. Cycling conditions are: 1 x (30s at 98°C), 30 x (10s at 98°C, 15s at 67°C), 1 x (2min at 72°C). PCR products are cleaned up with a 1.8X SPRI treatment and normalized to 25 ng/pL.
  • the crRNA is not included in Solution 2.
  • the solution 1 and solution 2 mixtures are mixed by pipetting up and down and then incubated at room temperature for 25 minutes. Following incubation, 20 pL of the Solution 1 and Solution 2 mixture are added dropwise to each well of a 96 well plate containing the cells. 72 hours post transfection, cells are trypsinized by adding 10 pL of TrypLE to the center of each well and incubated for approximately 5 minutes. 100 pL of D10 media is then added to each well and mixed to resuspend cells. The cells are then spun down at 500g for 10 minutes, and the supernatant is discarded. QuickExtract buffer is added to 1/5 the amount of the original cell suspension volume. Cells are incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.
  • NGS Next Generation Sequencing
  • the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence.
  • a kmer-scanning algorithm is used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence.
  • the first 30nt of each read is required to match the reference and reads where over half of the mapping nucleotides are mismatches are filtered out as well. Up to 50,000 reads passing those filters are used for analysis, and reads are counted as an indel read if they contained an insertion or deletion.
  • the indel % is calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters is 10,000. [00224] Percentages of indels in a DMD exon (e.g., exon 45, exon 51, or exon 53) following transfection are calculated. Indel percentages over background are indicative of Casl2i2 activity in mammalian cells.

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Abstract

The present disclosure relates to compositions comprising RNA guides useful with a Cas12i2 polypeptide or nucleic acid encoding a Cas12i2 polypeptide for targeting DMD, processes for characterizing the compositions, cells comprising the compositions, and methods of using the compositions.

Description

COMPOSITIONS COMPRISING AN RNA GUIDE TARGETING DMD AND USES THEREOF
[0001] This application claims the benefit of priority to United States Provisional Application No. 63/129,076, filed December 22, 2020, and United States Provisional Application No. 63/142,665, filed January 28, 2021, which are incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on January 27, 2021, is named 01245-0032-00PCT_Sequence_Listing_l_27_21_ST25 and is 379,292 bytes in size.
BACKGROUND
[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements. There remains a need for new CRISPR-Cas systems that comprise novel RNA guides and/or novel combinations of Cas enzymes and RNA guides. Adding the novel DNA-targeting systems described herein to the toolbox of techniques for genome and epigenome manipulation enables broad applications for specific, programmed perturbations.
SUMMARY OF THE DISCLOSURE
[0004] It is against the above background that the present disclosure provides certain advantages and advancements over the prior art. Although this disclosure provided herein is not limited to specific advantages or functionalities, the disclosure provides compositions comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a Duchenne muscular dystrophy (DMD) gene and (ii) a direct repeat sequence (e.g., any of the sequences provided in Table 1 or a portion of any of the sequences provided in Table 1); wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN- 3’.
[0005] In one aspect, the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53 of the DMD gene.
[0006] In one aspect, a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647; a variant of SEQ ID NO: 1647 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds; b. exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; a variant of SEQ ID NO: 1648 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649; a variant of SEQ ID NO: 1649 includes, for example, any stretch of about 16 or more contiguous nucleotides to which a spacer sequence described herein binds.
[0007] In another aspect, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421- 1644; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
[0008] In another aspect, the spacer sequence comprises: a. nucleotide 1 through nucleotide
16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide
17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide
18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide
Figure imgf000003_0001
Figure imgf000004_0001
21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide
22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide
23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
[0009] In another aspect, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; bb. or a portion thereof. In some embodiments, the direct repeat comprises any stretch of about 15-36, 15-35, 15-34, 15-32, 15-30, 15-28, 15-26, 15-23, 20-23, 18-25, 30-36, 30-35, 30-34, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-10, or comprises any stretch of about 15-36, 15-35, 15-34, 15-32, 15-30, 15-28, 15-26, 15-23, 20-23, 18-25, 30-36, 30-35, 30-34, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more contiguous nucleotides of a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-10. In some embodiments, the direct repeat comprises any stretch of about 30, 31, 32, 33, 34, 35, or 36 or more contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-10, or comprises any stretch of about 30, 31, 32, 33, 34, 35, or 36 or more contiguous nucleotides of a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1-10.
[0010] In another aspect, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof. [0011] In another aspect, the spacer sequence is substantially complementary (e.g., at least
90%, 95%, 96%, 97%, 98%, 99% or 100% complementary) to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197-1420.
[0012] In another aspect of the composition, the PAM comprises the sequence 5 ’-ATTA-3’,
5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’.
[0013] In another aspect, the target sequence is immediately adjacent to the PAM sequence.
[0014] In another aspect, the composition further comprises a Casl2i2 polypeptide or a nucleic acid encoding a Casl2i2 polypeptide.
[0015] In another aspect, the Casl2i2 polypeptide comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1646. In some embodiments, the nucleic acid encoding a Casl2i2 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 1654, or a fragment of such a nucleic acid that encodes a functional Casl2i2 polypeptide.
[0016] In another aspect of the composition, the Casl2i2 polypeptide comprises a sequence of SEQ ID NO: 1650, 1651, 1652, or 1653.
[0017] In another aspect, the RNA guide and the Casl2i2 polypeptide form a ribonucleoprotein complex.
[0018] In another aspect, the ribonucleoprotein complex binds a target nucleic acid.
[0019] In another aspect, the composition is present within a cell.
[0020] In some embodiments, a composition comprising an RNA guide and a Casl2i2 polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a DMD gene and (ii) a direct repeat sequence is provided.
[0021] In one aspect, the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53.
[0022] In another aspect, a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO:
1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647; b. exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649.
[0023] In another aspect, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421- 1644; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
[0024] In another aspect, the spacer sequence comprises or consists of: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. [0025] In another aspect, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; or bb. a portion thereof.
[0026] In another aspect, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide
Figure imgf000008_0001
1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
[0027] In another aspect, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197-1420.
[0028] In another aspect, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’.
[0029] In another aspect of the composition, the PAM comprises the sequence 5 ’-ATTA-3’,
5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’.
[0030] In another aspect of the composition, the target sequence is immediately adjacent to the
PAM sequence.
[0031] In another aspect of the composition, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
[0032] In another aspect of the composition, the Casl2i2 polypeptide comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1646.
[0033] In another aspect of the composition, the RNA guide and the Casl2i2 polypeptide form a ribonucleoprotein complex.
[0034] In another aspect of the composition, the ribonucleoprotein complex binds a target nucleic acid.
[0035] In another aspect of the composition, the composition is present within a cell.
[0036] In another aspect of the composition, the RNA guide and the Casl2i2 polypeptide are encoded in a vector, e.g., expression vector.
[0037] The disclosure yet further provides an RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a DMD gene and (ii) a direct repeat sequence. [0038] In one aspect of the RNA guide, the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53.
[0039] In another aspect of the RNA guide, a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647; b. exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649. [0040] In another aspect of the RNA guide, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421- 1644; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289- 566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
[0041] In another aspect of the RNA guide, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
[0042] In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1- 8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; or bb. a portion thereof. [0043] In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
[0044] In another aspect of the RNA guide, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197- 1420.
[0045] In another aspect of the RNA guide, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’, wherein N is any nucleotide.
[0046] In another aspect of the RNA guide, the PAM comprises the sequence 5’-ATTA-3’, 5’-
ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-GITAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’.
[0047] In another aspect of the RNA guide, the target sequence is immediately adjacent to the
PAM sequence. [0048] In another aspect of the RNA guide, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
[0049] In some embodiments, a nucleic acid encoding an RNA guide, a direct repeat, and/or a
Casl2i2 polypeptide is provided.
[0050] In some embodiments, a vector comprising such an RNA guide, a direct repeat, and/or a Casl2i2 polypeptide is provided.
[0051] In some embodiments, a cell comprising a composition, an RNA guide, a nucleic acid, or a vector is provided.
[0052] In one aspect of the cell, the cell is a eukaryotic cell (e.g., an animal cell, a mammalian cell, or a human cell). In one aspect of the cell, the cell is a muscle cell. In one aspect of the cell, the cell is a primary cell, a stem cell, a T cell, or a cell line.
[0053] In some embodiments, a kit comprising a composition, an RNA guide, a nucleic acid, or a vector is provided.
[0054] In some embodiments, a method of editing a DMD sequence, such as a DMD exon
(e.g., exon 45, exon 51, or exon 53), the method comprising contacting the DMD sequence with a composition or an RNA guide is provided.
[0055] In one aspect of the method, the DMD sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), is in a cell in vitro. In one aspect of the method, the DMD sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53) is in a cell in vivo.
[0056] In one aspect of the method, the composition or the RNA guide induces a deletion in the DMD sequence, such as a portion of a DMD exon (e.g., exon 45, exon 51, or exon 53).
[0057] In one aspect of the method, the deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide.
[0058] In one aspect of the method, the deletion is downstream of the 5’-NTTN-3’ sequence.
[0059] In one aspect of the method, the deletion is up to about 40 nucleotides in length.
[0060] In one aspect of the method, the deletion is from about 4 nucleotides to 40 nucleotides in length.
[0061] In one aspect of the method, the deletion is from about 4 nucleotides to 25 nucleotides in length.
[0062] In one aspect of the method, the deletion is from about 10 nucleotides to 25 nucleotides in length.
[0063] In one aspect of the method, the deletion is from about 10 nucleotides to 15 nucleotides in length.
[0064] In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
[0065] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence. [0066] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
[0067] In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0068] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0069] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0070] In one aspect of the method, the deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
[0071] In one aspect of the method, the deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence.
[0072] In one aspect of the method, the deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
[0073] In one aspect of the method, the deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0074] In one aspect of the method, the deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0075] In one aspect of the method, the deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0076] In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0077] In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0078] In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0079] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0080] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence. [0081] In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0082] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0083] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0084] In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
[0085] In one aspect of the method, the 5’-NTTN-3’ sequence is 5’-CTTT-3’, 5’-CTTC-3’, 5’- GTTT-3’, 5’-GTTC-3’, 5’-TTTC-3’, 5’-GTTA-3’, or 5’-GTTG-3’.
[0086] In one aspect of the method, the deletion overlaps with a mutation in the gene.
[0087] In one aspect of the method, the deletion overlaps with an insertion in the gene.
[0088] In one aspect of the method, the deletion removes a repeat expansion of the gene or a portion thereof.
[0089] In one aspect of the method, the deletion disrupts one or both alleles of the gene.
Definitions
[0090] The disclosure provides particular embodiments, but the disclosure is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.
[0091] As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, activity can include nuclease activity.
[0092] As used herein the term “DMD” refers to “Duchenne muscular dystrophy.” The DMD gene encodes a protein called dystrophin, which is expressed primarily in skeletal and cardiac muscles. Absence or abnormal expression of DMD gene expression, leading to dystrophin deficiency, is a cause of myopathies including Duchenne and Becker muscular dystrophies. Exon skipping approaches to altering DMD expression can be directed to DMD exons 45, 51, and 53. SEQ ID NOs: 1647, 1648, and 1649 as set forth herein provide examples of DMD exon 45, exon 51, and exon 53 sequences, respectively. It is to be understood that spacer sequences described herein can target SEQ ID NOs: 1647, 1648, or 1649, or reverse complements thereof, depending upon whether they are indicated as “+” or as set forth in Tables 2-4.
[0093] As used herein, the term “complex” refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. As used herein, the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Casl2i2 polypeptide). As used herein, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence. As used herein, the term “complex” can refer to a grouping of a DMD-targeting RNA guide and a Casl2i2 polypeptide.
[0094] As used herein, the term “protospacer adjacent motif’ or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)) to which a complex comprising an RNA guide (e.g., a DMD-targeting RNA guide) and a Casl2i2 polypeptide binds. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (e.g., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand). As used herein, the term “adjacent” includes instances in which the RNA guide of a complex comprising an RNA guide and a Casl2i2 polypeptide specifically binds, interacts, or associates with a target sequence that is immediately adjacent to a PAM. In such instances, there are no nucleotides between the target sequence and the PAM. The term “adjacent” also includes instances in which there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the target sequence, to which the RNA guide binds, and the PAM. In some embodiments, the PAM sequence as described herein is present in the non-target strand (e.g., the non-spacer-complementary strand). In such a case, the term “adjacent” includes a PAM sequence as described herein as being immediately adjacent to (or within a small number, e.g., 1, 2, 3, 4, or 5 nucleotides of) a sequence in the non-target strand.
[0095] As used herein, the term “RNA guide” refers to any RNA molecule that facilitates the targeting of a polypeptide (e.g., a Casl2i2 polypeptide) to a target sequence (e.g., a sequence of a DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)). An RNA guide may be designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., a DMD nucleic acid sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53)). An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence. The term “crRNA” is also used herein to refer to an RNA guide.
[0096] As used herein, the term “substantially complementary” refers to a polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a target sequence. In some embodiments, the level of complementarity is such that the polynucleotide can hybridize to the target sequence with sufficient affinity to permit an effector polypeptide (e.g., Casl2i2) that is complexed with the polynucleotide to act (e.g., cleave) on the target sequence.
[0097] As used herein, the terms “target” and “target sequence” refer to a nucleic acid sequence to which an RNA guide specifically binds. In some embodiments, the terms “target” and “target sequence” refer to a nucleic acid to which an RNA guide directs a nuclease, e.g., a Casl2i2. In some embodiments, the DNA targeting sequence (e.g., spacer) of an RNA guide binds to a target sequence. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand). In some embodiments, the target strand (i.e., the spacer-complementary strand) comprises a 5 ’-NAAN-3’ sequence. In some embodiments, the target sequence is a sequence within a DMD exon (e.g., exon 45, exon 51, or exon 53), including, but not limited to, the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or a reverse complement thereof.
DETAILED DESCRIPTION
[0098] The present disclosure relates to an RNA guide capable of binding to a DMD gene sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), and methods of use thereof. In some aspects, a composition comprising an RNA guide having one or more characteristics is described herein. In some aspects, a method of producing the RNA guide is described. In some aspects, a method of delivering a composition comprising the RNA guide is described.
COMPOSITION
[0099] In some aspects, the disclosure provides for compositions comprising an RNA guide targeting a DMD gene or a portion of the DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). In some embodiments, the RNA guide is comprised of a direct repeat component and a spacer component. In some embodiments, the RNA guide binds a Casl2i2 polypeptide. In some embodiments, the spacer component is substantially complementary to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), wherein the DMD target sequence is adjacent to a 5’-NTTN-3’ PAM sequence as described herein. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand).
[00100] In some embodiments, the disclosure provides for compositions comprising a complex, wherein the complex comprises an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). In some embodiments, the disclosure provides for a complex comprising an RNA guide and a Casl2i2 polypeptide. In some embodiments, the RNA guide and the Casl2i2 polypeptide bind to each other in a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide and a Casl2i2 polypeptide binds to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). In some embodiments, a complex comprising an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), and a Casl2i2 polypeptide binds to the DMD target sequence at a molar ratio of about 1 : 1. In some embodiments, the complex comprises enzymatic activity, such as nuclease activity, that can cleave the DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). The RNA guide, the Casl2i2 polypeptide, and the DMD target sequence (such as a DMD exon (e.g., exon 45, exon 51, or exon 53)), either alone or together, do not naturally occur.
RNA Guide
[00101] In some embodiments, the composition described herein comprises an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). In some embodiments, the composition described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
[00102] The RNA guide may direct the Casl2i2 polypeptide as described herein to a DMD target sequence, such as a DMD exon (e.g., exon 45, exon 51, or exon 53). Two or more RNA guides may target two or more separate Casl2i2 polypeptides (e.g., Casl2i2 polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) DMD target sequences, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
[00103] Those skilled in the art reading the below examples of particular kinds of RNA guides will understand that, in some embodiments, an RNA guide is DMD target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more DMD target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).
[00104] In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5’ to 3’ direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5 ’ to 3 ’ direction. In some embodiments, the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.
[00105] In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. In some embodiments, the spacer and direct repeat sequences are linked directly to one another. In some embodiments, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.
[00106] Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.
[00107] Direct Repeat
[00108] In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14- 50, or 15-40 nucleotides (e.g., 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, or 40 nucleotides). [00109] In some embodiments, the direct repeat sequence is or comprises a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ
ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 12 through nucleotide 34 ofSEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.
[00110] In some embodiments, the direct repeat sequence has or comprises a sequence comprising at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.
[00111] In some embodiments, the direct repeat sequence is or comprises a sequence that is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is or comprises the reverse complement of any one of SEQ ID NOs: 1-10.
[00112] Table 1. Direct repeat sequences
Figure imgf000021_0001
Spacer
[00113] In some embodiments, the RNA guide comprises a DNA targeting or spacer sequence. In some embodiments, the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14- 50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary a specific target sequence. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.
[00114] In some embodiments, the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence. In some embodiments, the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
[00115] In some embodiments, the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence. In some embodiments, the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.
[00116] In some embodiments, the spacer sequence is or comprises a sequence of Table 2 or a portion of a sequence of Table 2. It should be understood that an indication of SEQ ID NOs: 289-566, 882-1196, or 1421-1644 should be considered as equivalent to a listing of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644, with each of the intervening numbers present in the listing, i.e., 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148. [00117] The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882- 1196, or 1421-1644.
[00118] In some embodiments, the spacer sequence has or comprises a sequence having at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
Table 2. Target and Spacer Sequences - Exon 45
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
[00119] The disclosure provides for all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein.
Modifications
[00120] The RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this disclosure.
[00121] Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.
[00122] The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein. [00123] In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
[00124] Different sugar modifications, nucleotide modifications, and/or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
[00125] In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its intemucleoside backbone.
[00126] Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3 ’-amino phosphoramidate and aminoalky Iphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 ’-5’ linkages, 2 ’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’- 2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged. [00127] The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
[00128] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
[00129] In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e . g. , 5 ’ - O-(l-thiophosphate)-adenosine, 5 ’-O-(l -thiophosphate) -cytidine (a-thio-cytidine), 5’-O-(l- thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l-thiophosphate)-pseudouridine). [00130] Other intemucleoside linkages that may be employed according to the present disclosure, including intemucleoside linkages which do not contain a phosphorous atom, are described herein.
[00131] In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5 -azacytidine, 4’-thio- aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C- cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5 -fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l- (tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’- methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-l-beta-D- arabinofuranosy Icy tosine, N4-palmitoy 1- 1 -(2-C-cy ano-2-deoxy-beta-D-arabino-pentofuranosy 1) cytosine, and P-4055 (cytarabine 5 ’-elaidic acid ester).
[00132] In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5 -aza-uridine, 2 -thiouridine, 4-thio-pseudouridine, 2 -thio -pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propyny 1-uridine, 1 -propyny 1-pseudouridine, 5 -taurinomethy luridine, 1 -taurinomethy 1-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5 -methy 1-uridine, 1-methyl- pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l -methy 1-pseudouridine, 1 -methy 1-1 -deazapseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methy 1-pseudoisocytidine, pyrrolo -cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocy tidine, 1 -methyl- 1 -deaza-pseudoisocy tidine, zebularine, 5 -aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy- 1 -methy 1- pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6 -diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy -adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8- aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methy lino sine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
[00133] The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., naturally -occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by AD ARI marks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.
Casl2i2 Polypeptide
[00134] In some embodiments, the composition of the present disclosure includes a Casl2i2 polypeptide as described in PCT/US2019/022375 (WO2019178427A1), which is incorporated herein in its entirety, including the sequences. Nucleic acid encoding Casl2i2 polypeptides are also provided. [00135] In some embodiments, the composition of the present disclosure includes a Casl2i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1646 and/or encoded by SEQ ID NO: 1645).
[00136] A nucleic acid sequence encoding the Casl2i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1645. In some embodiments, the Casl2i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 1645. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions (e.g., within a range of medium to high stringency).
[00137] In some embodiments, the Casl2i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 1645.
[00138] In some embodiments, the Casl2i2 polypeptide of the present disclosure comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1646.
[00139] In some embodiments, the present disclosure describes a Casl2i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1646. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
[00140] Also provided is a Casl2i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1646 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.
[00141] In some embodiments, the Casl2i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653.
[00142] In some embodiments, the Casl2i2 polypeptide of the present disclosure comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653.
[00143] In some embodiments, the present disclosure describes a Casl2i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.
[00144] In some embodiments, a Casl2i2 polypeptide (e.g., a Casl2il2 polypeptide of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO: 155) comprises a RuvC domain.
[00145] Also provided is a Casl2i2 polypeptide of the present disclosure having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods. In some embodiments, the Casl2i2 polypeptide has the same enzymatic activity (e.g., nuclease or endonuclease activity) as, or within 20%, 15%, 10%, 5%, or 2% of the same enzymatic activity as, a polypeptide having the amino acid sequence of SEQ ID NO: 1650, SEQ ID NO: 1651, SEQ ID NO: 1652, or SEQ ID NO: 1653 in an enzymatic activity (e.g., nuclease or endonuclease activity) assay (e.g., target cleavage assay, or a TIDE assay).
[00146] Although the changes described herein may be one or more amino acid changes, changes to the Casl2i2 polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions. For example, the Casl2i2 polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a poly histidine tag (His-tag), Myc, and FLAG. In some embodiments, the Casl2i2 polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).
[00147] In some embodiments, the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Casl2i2 polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
[00148] In some embodiments, the Casl2i2 polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.
[00149] In some embodiments, the nucleotide sequence encoding the Casl2i2 polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA).
Target Sequence
[00150] In some embodiments, the target sequence is within a DMD gene or a locus of a DMD gene. In some embodiments, the DMD gene is a mammalian gene. In some embodiments, the DMD gene is a human gene. In some embodiments, the target sequence is within a DMD exon (e.g., exon 45, exon 51, or exon 53) as set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complements thereof. Examples of target sequences within DMD exons (e.g., exon 45, exon 51, or exon 53) of SEQ ID NO: 1647 (exon 45), 1648 (exon 51), and 1649 (exon 53) (and the reverse complements thereof) are set forth in Tables 2, 3, and 4, respectively. In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of a DMD exon (e.g., exon 45, exon 51, or exon 53) set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complements thereof. In some embodiments, the DMD exon (e.g., exon 45, exon 51, or exon 53) sequence is a homolog of a sequence set forth in SEQ ID NOs: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complement thereof. For examples, in some embodiments, the DMD exon (e.g., exon 45, exon 51, or exon 53) sequence is a non-human DMD exon (corresponding to, e.g., human exon 45, exon 51, or exon 53) sequence.
[00151] In some embodiments, the target sequence is adjacent to a 5’-NTTN-3’ PAM sequence, wherein N is any nucleotide. The 5’-NTTN-3’ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5’-NTTN-3’ sequence is 5’-NTTY-3’, 5’-NTTC-3’, 5’-NTTT-3’, 5’-NTTA-3’, 5’-NTTB-3’, 5’-NTTG-3’, 5’-CTTY-3’, 5’-DTTR’3’, 5’-CTTR-3’, 5’-DTTT-3’, 5’- ATTN-3’, or 5’-GTTN-3’, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5’-NTTN-3’ sequence is 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-G TAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’. [00152] In some embodiments, the target sequence is single-stranded (e.g., single-stranded DNA). In some embodiments, the target sequence is double-stranded (e.g., double-stranded DNA). In some embodiments, the target sequence comprises both single-stranded and double-stranded regions. In some embodiments, the target sequence is linear. In some embodiments, the target sequence is circular. In some embodiments, the target sequence comprises one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotides analogs. In some embodiments, the target sequence is not modified. In some embodiments, the RNA guide binds to a first strand of a doublestranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5’-NTTN- 3’ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non- spacer-complementary strand). In some embodiments, the RNA guide binds adjacent to a 5 ’-NAAN-3’ sequence on the target strand (e.g., the spacer-complementary strand).
[00153] In some embodiments, the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5' or 3' untranslated region, etc. In some embodiments, the target sequence is a plasmid.
[00154] In some embodiments, the target sequence is present in a readily accessible region of the target sequence. In some embodiments, the target sequence is in an exon of a target gene. In some embodiments, the target sequence is across an exon-intron junction of a target gene. In some embodiments, the target sequence is present in a non-coding region, such as a regulatory region of a gene. In some embodiments, wherein the target sequence is exogenous to a cell, the target sequence comprises a sequence that is not found in the genome of the cell.
[00155] In some embodiments, the target sequence is exogenous to a cell. In some embodiments, the target sequence is a horizontally transferred plasmid. In some embodiments, the target sequence is integrated in the genome of the cell. In some embodiments, the target sequence is not integrated in the genome of the cell. In some embodiments, the target sequence is a plasmid in the cell. In some embodiments, the target sequence is present in an extrachromosomal array.
[00156] In some embodiments, the target sequence is an isolated nucleic acid, such as an isolated DNA or an isolated RNA. In some embodiments, the target sequence is present in a cell-free environment. In some embodiments, the target sequence is an isolated vector, such as a plasmid. In some embodiments, the target sequence is an ultrapure plasmid.
[00157] The target sequence is a locus of the DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53) sequence, that hybridizes to the RNA guide. In some embodiments, a cell has only one copy of the target sequence. In some embodiments, a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.
PRODUCTION
[00158] The present disclosure includes methods for production of the RNA guide, methods for production of the Casl2i2 polypeptide, and methods for complexing the RNA guide and Casl2i2 polypeptide.
RNA Guide
[00159] In some embodiments, the RNA guide is made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence). In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Casl2i2 polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Casl2i2 polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.
Casl2i2 Polypeptide
[00160] In some embodiments, the Casl2i2 polypeptide of the present disclosure can be prepared by (a) culturing bacteria which produce the Casl2i2 polypeptide of the present disclosure, isolating the Casl2i2 polypeptide, optionally, purifying the Casl2i2 polypeptide, and complexing the Casl2i2 polypeptide with an RNA guide. The Casl2i2 polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Casl2i2 polypeptide of the present disclosure from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell. Alternatively, the Casl2i2 polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.
[00161] In some embodiments, a host cell is used to express the Casl2i2 polypeptide. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.
[00162] After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Casl2i2 polypeptide. After expression of the Casl2i2 polypeptide, the host cells can be collected and Casl2i2 polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).
[00163] In some embodiments, the methods for Casl2i2 polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Casl2i2 polypeptide. In some embodiments, the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Casl2i2 polypeptide.
[00164] A variety of methods can be used to determine the level of production of a Casl2i2 polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Casl2i2 polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).
[00165] The present disclosure provides methods of in vivo expression of the Casl2i2 polypeptide in a cell, comprising providing a polyribonucleotide encoding the Casl2i2 polypeptide to a host cell wherein the polyribonucleotide encodes the Casl2i2 polypeptide, expressing the Casl2i2 polypeptide in the cell, and obtaining the Casl2i2 polypeptide from the cell.
Complexing
[00166] In some embodiments, an RNA guide targeting DMD, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), is complexed with a Casl2i2 polypeptide to form a ribonucleoprotein. In some embodiments, complexation of the RNA guide and Casl2i2 polypeptide occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C. In some embodiments, the RNA guide does not dissociate from the Casl2i2 polypeptide at about 37°C over an incubation period of at least about any one of lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Ihr, 2hr, 3hr, 4hr, or more hours.
[00167] In some embodiments, the RNA guide and Casl2i2 polypeptide are complexed in a complexation buffer. In some embodiments, the Casl2i2 polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the Casl2i2 polypeptide is stored in a complexation buffer.
[00168] In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.
[00169] In some embodiments, the Casl2i2 polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In some embodiments, mRNA or DNA encoding the Casl2i2 polypeptide is introduced into a cell so that the Casl2i2 polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.
DELIVERY
[00170] Compositions or complexes described herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.
[00171] In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Casl2i2 polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Casl2i2 polypeptide complex to a cell, where a ternary complex is formed. Exemplary intracellular delivery methods, include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particlebased methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
Cells
[00172] Compositions or complexes described herein can be delivered to a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.
[00173] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.
[00174] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.
[00175] In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.
[00176] In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a muscle cell (e.g., a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell, or a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a liver cell (e.g., a hepatocyte), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease -specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.
METHODS
[00177] The disclosure also provides methods of modifying a target sequence within the DMD gene. In some embodiments, the methods comprise introducing a DMD-targeting RNA guide and a Casl2i2 polypeptide or nucleic acid encoding a Casl2i2 polypeptide into a cell. The DMD-targeting RNA guide and Casl2i2 polypeptide can be introduced as a ribonucleoprotein complex into a cell. The DMD-targeting RNA guide and Casl2i2 polypeptide can be introduced on a nucleic acid vector. The Casl2i2 polypeptide can be introduced as an mRNA. The RNA guide can be introduced directly into the cell.
[00178] In some embodiments, the target sequence is in a DMD exon (e.g., exon 45, exon 51, or exon 53). In some embodiments, the DMD exon (e.g., exon 45, exon 51, or exon 53) has a sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53), or the reverse complement thereof. In some embodiments, the sequence of the DMD exon (e.g., exon 45, exon 51, or exon 53) is a variant of the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 51) (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53) (or the reverse complement thereof). For example, in some embodiments, the target sequence is a polymorphic variant of a DMD exon (e.g., exon 45, exon 51, or exon 53) sequence set forth in SEQ ID NO: 1647 (exon 45), 1648 (exon 51), or 1649 (exon 53) (or the reverse complement thereof) or a non-human form of a DMD exon (e.g., an exon corresponding to human exon 45, exon 51, or exon 53).
[00179] In some embodiments, an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5’-NTTN-3’ PAM sequence. The 5’-NTTN-3’ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5’-NTTN-3’ sequence is 5’- NTTY-3’, 5’-NTTC-3’, 5’-NTTT-3’, 5’-NTTA-3’, 5’-NTTB-3’, 5’-NTTG-3’, 5’-CTTY-3’, 5’- DTTR’3’, 5’-CTTR-3’, 5’-DTTT-3’, 5’-ATTN-3’, or 5’-GTTN-3’, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5’-NTTN-3’ sequence is 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT- 3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’- CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’. In some embodiments, the RNA guide is designed to bind to a first strand of a double-stranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5’-NTTN-3’ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand). In some embodiments, the RNA guide binds adjacent to a 5’-NAAN-3’ sequence on the target strand (e.g., the spacer-complementary strand). [00180] In some embodiments, the Casl2i2 polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Casl2i2 polypeptide induces one or more DNA double -stranded breaks in the cell. In some embodiments, the Casl2i2 polypeptide induces one or more DNA singlestranded breaks in the cell. In some embodiments, the Casl2i2 polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).
[00181] In some embodiments, an RNA guide disclosed herein forms a complex with the Casl2i2 polypeptide and directs the Casl2i2 polypeptide to a target sequence adjacent to a 5’-NTTN-3’ sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5’-NTTN-3’ sequence. In some embodiments, the complex induces a deletion adjacent to a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the complex induces a deletion adjacent to a T/C-rich sequence.
[00182] In some embodiments, the deletion is downstream of a 5’-NTTN-3’ sequence. In some embodiments, the deletion is downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.
[00183] In some embodiments, the deletion alters expression of the DMD gene. In some embodiments, the deletion alters function of the DMD gene. In some embodiments, the deletion inactivates the DMD gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).
[00184] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.
[00185] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’- ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT- 3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.
[00186] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
[00187] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’- TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence.
[00188] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA- 3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’- CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence. [00189] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11,
12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’- ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5' -OTTOS'. 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12,
13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.
[00190] In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5 ’-G TAS’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence. [00191] In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’- NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’- TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG- 3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.
[00192] In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23 , 24, 25 , 26, 27, or 28 nucleotides) of a 5 ’ -ATTA-3 ’ , 5 ’ -ATTT-3 ’ , 5 ’ - ATTG-3 ’ , 5 ’ -ATTC-3 ’ , 5 ’ - TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
[00193] In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a T/C-rich sequence.
[00194] In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’- TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC- 3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
[00195] In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.
[00196] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
[00197] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00198] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C- rich sequence.
[00199] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA- 3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
[00200] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C- rich sequence.
[00201] In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTAS’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00202] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’- ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA- 3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence. [00203] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5 ’-ATTA-3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’, 5 ’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’- ATTA-3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’, 5 ’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC- 3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00204] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21 , 22, 23 , 24, 25 , 26, 27, or 28 nucleotides) of a 5 ’ -ATTA-3 ’ , 5 ’ -ATTT-3 ’ , 5 ’ - ATTG-3 ’ , 5 ’ - ATTC-3 ’ , 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’- GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.
[00205] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA- 3’, 5 ’-ATTT-3’, 5 ’-ATTG-3’, 5 ’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’- ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5' -OTTOS'. 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
[00206] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
[00207] In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5’-ATTA- 3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’- GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’- ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5' -OTTOS'. 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00208] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’- NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’- TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG- 3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
[00209] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’- TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA- 3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’- CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00210] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.
[00211] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’- GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.
[00212] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG- 3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’- GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.
[00213] In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5’-NTTN-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-NTTN-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5 ’-ATTA-3’, 5’-ATTT-3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’- TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT- 3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5’- GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.
[00214] In some embodiments, the deletion is up to about 50 nucleotides in length (e.g., about 1, 2, 3, 4, 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 some embodiments, the deletion is up to about 40 nucleotides in length (e.g., about 1, 2, 3, 4, 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 3, 4, 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, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).
[00215] In some embodiments, the methods described herein are used to engineer a cell comprising a deletion as described herein in a DMD gene, such as a DMD exon (e.g., exon 45, exon 51, or exon 53).
KITS
[00216] The present disclosure also provides kits or systems that can be used, for example, to carry out a method described herein. In some embodiments, the kits or systems include an RNA guide and a Casl2i2 polypeptide. In some embodiments, the kits or systems include a polynucleotide that encodes such a Casl2i2 polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. The Casl2i2 polypeptide and the RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits or systems can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Casl2i2 polypeptide. [00217] All references and publications cited herein are hereby incorporated by reference.
EXAMPLES
[00218] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
Example 1 - Editing of DMD in a Mammalian Cell
[00219] This Example describes transfection followed by indel assessment on multiple DMD target sequences, such as a DMD exon (e.g., exon 45, exon 51, or exon 53), using a Casl2i2 polypeptide in mammalian cells.
[00220] A Casl2i2 polypeptide-encoding sequence is cloned into a pcda3.1 backbone (Invitrogen). The plasmid is then maxi-prepped and diluted to 1 pg/pL. For RNA guide preparation, a dsDNA fragment encoding an RNA guide described herein is derived by ultramers containing the target sequence scaffold, and the U6 promoter. Ultramers are resuspended in 10 mM Tris«HCl at a pH of 7.5 to a final stock concentration of 100 pM. Working stocks are subsequently diluted to 10 pM. again using 10 mM Tris*HCl to serve as the template for the PCR reaction. The amplification of the RNA guide is done in 50 pL reactions with the following components: 0.02 pl of aforementioned template, 2.5 pl forward primer, 2.5 pl reverse primer, 25 pL NEB HiFi Polymerase, and 20 pl water. Cycling conditions are: 1 x (30s at 98°C), 30 x (10s at 98°C, 15s at 67°C), 1 x (2min at 72°C). PCR products are cleaned up with a 1.8X SPRI treatment and normalized to 25 ng/pL.
[00221] Approximately 16 hours prior to transfection, 100 pl of 25,000 mammalian cells in DMEM/10%FBS+Pen/Strep are plated into each well of a 96-well plate. On the day of transfection, the cells are 70-90% confluent. For each well to be transfected, a mixture of 0.5 pl of Lipofectamine 2000 and 9.5 pl of Opti-MEM is prepared and then incubated at room temperature for 5-20 minutes (Solution 1). After incubation, the lipofectamine :OptiMEM mixture is added to a separate mixture containing 182 ng of effector plasmid and 14 ng of crRNA and water up to 10 pL (Solution 2). In the case of negative controls, the crRNA is not included in Solution 2. The solution 1 and solution 2 mixtures are mixed by pipetting up and down and then incubated at room temperature for 25 minutes. Following incubation, 20 pL of the Solution 1 and Solution 2 mixture are added dropwise to each well of a 96 well plate containing the cells. 72 hours post transfection, cells are trypsinized by adding 10 pL of TrypLE to the center of each well and incubated for approximately 5 minutes. 100 pL of D10 media is then added to each well and mixed to resuspend cells. The cells are then spun down at 500g for 10 minutes, and the supernatant is discarded. QuickExtract buffer is added to 1/5 the amount of the original cell suspension volume. Cells are incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.
[00222] Samples for Next Generation Sequencing (NGS) are prepared by rounds of PCR. The first round (PCR I) is used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) is used to add NGS indexes. Reactions are then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing runs are done using a 150 cycle NGS instrument (NextSeq v2.5) mid or high output kit and run on an NGS instrument (NextSeq 550).
[00223] For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm is used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30nt of each read is required to match the reference and reads where over half of the mapping nucleotides are mismatches are filtered out as well. Up to 50,000 reads passing those filters are used for analysis, and reads are counted as an indel read if they contained an insertion or deletion. The indel % is calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters is 10,000. [00224] Percentages of indels in a DMD exon (e.g., exon 45, exon 51, or exon 53) following transfection are calculated. Indel percentages over background are indicative of Casl2i2 activity in mammalian cells.
Table 5 - Casl2i2 and DMD Exon 45, 51, and 53 Sequences
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a Duchenne muscular dystrophy (DMD) gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’.
2. The composition of claim 1, wherein the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53 of the DMD gene.
3. The composition of claim 2, wherein: a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647; b. exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649.
4. The composition of any one of claims 1 to 3, wherein the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; l. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
5. The composition of any one of claims 1 to 4, wherein the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; l. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
6. The composition of any one of claims 1 to 5, wherein the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; bb. or a portion thereof.
7. The composition of any one of claims 1 to 6, wherein the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 84 b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
8. The composition of any one of claims 1 to 7, wherein the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197-1420.
9. The composition of claim 1, wherein the PAM comprises the sequence 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA- 3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’.
10. The composition of claim 1 or 9, wherein the target sequence is immediately adjacent to the PAM sequence.
11. The composition of any one of claims 1 to 10, wherein the composition further comprises a Cas12i2 polypeptide.
12. The composition of claim 11, wherein the Cas12i2 polypeptide comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1646.
13. The composition of claim 11 or 12, wherein the Cas12i2 polypeptide comprises a sequence of SEQ ID NO: 1650, 1651, 1652, or 1653.
14. The composition of any one of claims 11 to 13, wherein the RNA guide and the Cas12i2 polypeptide form a ribonucleoprotein complex.
15. The composition of claim 14, wherein the ribonucleoprotein complex binds a target nucleic acid.
16. The composition of claim 14 or 15, wherein the composition is present within a cell.
17. The composition of any one of claims 11 to 16, wherein the RNA guide and the Cas12i2 polypeptide are encoded in a vector, e.g., expression vector.
18. An RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a DMD gene and (ii) a direct repeat sequence.
19. The RNA guide of claim 18, wherein the target sequence is within an exon of the DMD gene, which optionally is exon 45, exon 51, or exon 53 of the DMD gene.
20. The RNA guide of claim 18 or 19, wherein: a. exon 45 of the DMD gene comprises the sequence of SEQ ID NO: 1647, the reverse complement of SEQ ID NO: 1647, a variant of SEQ ID NO: 1647, or the reverse complement of a variant of SEQ ID NO: 1647; b. exon 51 of the DMD gene comprises the sequence of SEQ ID NO: 1648, the reverse complement of SEQ ID NO: 1648, a variant of SEQ ID NO: 1648, or the reverse complement of a variant of SEQ ID NO: 1648; or c. exon 53 of the DMD gene comprises the sequence of SEQ ID NO: 1649, the reverse complement of SEQ ID NO: 1649, a variant of SEQ ID NO: 1649, or the reverse complement of a variant of SEQ ID NO: 1649.
21. The RNA guide of any one of claims 18 to 20, wherein the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; l. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
22. The RNA guide of any one of claims 18 to 21, wherein the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; l. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 289-566, 882-1196, or 1421-1644.
23. The RNA guide of any one of claims 18 to 22, wherein the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 88 c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10; or bb. a portion thereof.
24. The RNA guide of any one of claims 18 to 23, wherein the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; 90 v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; aa. SEQ ID NO: 10; or bb. a portion thereof.
25. The RNA guide of any one of claims 18 to 24, wherein the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-288, 567-881, or 1197-1420.
26. The RNA guide of any one of claims 18 to 25, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’, wherein N is any nucleotide.
27. The RNA guide of claim 26, wherein the PAM comprises the sequence 5’-ATTA-3’, 5’-ATTT- 3’, 5’-ATTG-3’, 5’-ATTC-3’, 5’-TTTA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA- 3’, 5’-GTTT-3’, 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’- CTTC-3’.
28. The RNA guide of claim 26 or 27, wherein the target sequence is immediately adjacent to the PAM sequence.
29. A nucleic acid encoding an RNA guide of any one of claims 18 to 28.
30. The nucleic acid of claim 29, wherein the nucleic acid further comprises a nucleotide sequence encoding an endonuclease.
31. The nucleic acid of claim 30, wherein the endonuclease is a Cas12i2 polypeptide.
32. The nucleic acid of claim 31, wherein the Cas12i2 polypeptide comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 1646, 1650, 1651, 1652, or 1653.
33. The nucleic acid of claim 32, wherein the Cas12i2 polypeptide comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1646.
34. A vector comprising the nucleic acid of any one of claims 29 to 33.
35. A cell comprising the composition of any one of claims 1 to 17, the RNA guide of any one of claims 18 to 28, a nucleic acid of any one of claims 29 to 33, or a vector of claim 34.
36. The cell of claim 35, wherein the cell is a eukaryotic cell (e.g., an animal cell, a mammalian cell, or a human cell), a muscle cell, a primary cell, a stem cell, a T cell, or a cell line.
37. A kit comprising the composition of any one of claims 1 to 17, the RNA guide of any one of claims 18 to 28, a nucleic acid of any one of claims 29 to 33, or a vector of claim 34.
38. A method of editing a DMD sequence, the method comprising contacting a DMD sequence with a composition of any one of claims 1 to 17 or an RNA guide of any one of claims 18 to 28.
39. The method of claim 38, wherein the DMD sequence is in a cell.
40. The method of claim 38 or 39, wherein the composition or the RNA guide induces a deletion in the DMD sequence.
41. The method of any one of claims 38 to 40, wherein the deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide.
42. The method of claim 40 or 41, wherein the deletion is downstream of the 5’-NTTN-3’ sequence.
43. The method of any one of claims 40 to 42, wherein the deletion is up to about 50 nucleotides in length.
44. The method of any one of claims 40 to 43, wherein the deletion is up to about 40 nucleotides in length.
45. The method of any one of claims 40 to 44, wherein the deletion is from about 4 nucleotides to 40 nucleotides in length. 92
46. The method of any one of claims 40 to 45, wherein the deletion is from about 4 nucleotides to 25 nucleotides in length.
47. The method of any one of claims 40 to 46, wherein the deletion is from about 10 nucleotides to 25 nucleotides in length.
48. The method of any one of claims 40 to 47, wherein the deletion is from about 10 nucleotides to 15 nucleotides in length.
49. The method of any one of claims 40 to 48, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
50. The method of any one of claims 40 to 49, wherein the deletion starts within about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence.
51. The method of any one of claims 40 to 50, wherein the deletion starts within about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence.
52. The method of any one of claims 40 to 51, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
53. The method of any one of claims 40 to 52, wherein the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence.
54. The method of any one of claims 40 to 53, wherein the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence.
55. The method of any one of claims 40 to 54, wherein the deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
56. The method of any one of claims 40 to 55, wherein the deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence.
57. The method of any one of claims 40 to 56, wherein the deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.
58. The method of any one of claims 40 to 57, wherein the deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
59. The method of any one of claims 40 to 58, wherein the deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
60. The method of any one of claims 40 to 59, wherein the deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
61. The method of any one of claims 40 to 60, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
62. The method of any one of claims 40 to 61, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
63. The method of any one of claims 40 to 62, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
64. The method of any one of claims 40 to 63, wherein the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
65. The method of any one of claims 40 to 64, wherein the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
66. The method of any one of claims 40 to 65, wherein the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
67. The method of any one of claims 40 to 66, wherein the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
68. The method of any one of claims 40 to 67, wherein the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence.
69. The method of any one of claims 40 to 68, wherein the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.
70. The method of any one of claims 40 to 69, wherein the 5’-NTTN-3’ sequence is 5’-CTTT-3’, 5’-CTTC-3’, 5’-GTTT-3’, 5’-GTTC-3’, 5’-TTTC-3’, 5’-GTTA-3’, or 5’-GTTG-3’.
71. The method of any one of claims 40 to 70, wherein the deletion overlaps with a mutation in the gene.
72. The method of any one of claims 40 to 71, wherein the deletion overlaps with an insertion in the gene.
73. The method of any one of claims 40 to 72, wherein the deletion removes a repeat expansion of the gene or a portion thereof.
74. The method of any one of claims 40 to 73, wherein the deletion disrupts one or both alleles of the gene.
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