WO2023137451A1 - Compositions comprenant un arn guide ciblant cd38 et leurs utilisations - Google Patents

Compositions comprenant un arn guide ciblant cd38 et leurs utilisations Download PDF

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WO2023137451A1
WO2023137451A1 PCT/US2023/060667 US2023060667W WO2023137451A1 WO 2023137451 A1 WO2023137451 A1 WO 2023137451A1 US 2023060667 W US2023060667 W US 2023060667W WO 2023137451 A1 WO2023137451 A1 WO 2023137451A1
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sequence
nucleotide
seq
nucleotides
nos
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PCT/US2023/060667
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Quinton Norman WESSELLS
Jeffrey Raymond HASWELL
Maria Fernanda ROJAS-DURAN
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Arbor Biotechnologies, Inc.
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Publication of WO2023137451A1 publication Critical patent/WO2023137451A1/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
  • the present invention provides certain advantages and advancements over the prior art.
  • the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence (e.g., a target strand sequence of a target sequence) within a cluster of differentiation 38 (CD38) 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’.
  • a target sequence e.g., a target strand sequence of a target sequence
  • CD38 cluster of differentiation 38
  • PAM protospacer adjacent motif
  • the present disclosure is based, at least in part, on the development of a system for genetic editing of a cluster of differentiation 38 (CD38) gene.
  • the system may comprise a Casl2i2 CRISPR nuclease polypeptide and an RNA guide mediating cleavage at a genetic site within the CD38 gene by the CRISPR nuclease polypeptide.
  • the gene editing system disclosed herein has achieved successful editing of CD38 gene with high editing efficiency.
  • the present disclosure features system for genetic editing of a CD38 gene, comprising (i) a Casl2i2 polypeptide or a first nucleic acid encoding the Casl2i2 polypeptide, and (ii) an RNA guide or a nucleic acid encoding the RNA guide.
  • the RNA guide comprises a spacer sequence specific to a target sequence within a CD38 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-NTTN-3’, which is located 5’ to the non-target strand of the target sequence.
  • PAM protospacer adjacent motif
  • the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that specifically binds a target sequence within a CD38 gene, wherein the target sequence comprises any one of SEQ ID NOS: 989, 967, 983, 955-966, 968-982, 984-988, or 990-1005; and (ii) a direct repeat sequence.
  • the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that specifically binds a target sequence within a CD38 gene, wherein the target sequence comprises any one of SEQ ID NOS: 989, 967, 983, 959, 988, 983, or 977 ; and (ii) a direct repeat sequence.
  • the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that is at least 90% identical to a sequence of any of SEQ ID NOs: 1040, 1018, 1034, 1006-1017, 1019-1033, 1035-1039, or 1041-1056; and (ii) a direct repeat sequence.
  • the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1040, 1018, 1034, 1010, 1039, or 1028; and (ii) a direct repeat sequence.
  • the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the CD38 gene.
  • the CD38 gene comprises the sequence NCBI Entrez Gene ID: 952, the reverse complement thereof, a variant thereof, or the reverse complement of a variant thereof.
  • the spacer sequence has a length of between 15-30 nucleotides or between 20-30 nucleotides. In some embodiments, the direct repeat sequence has a length of between 15-40 or 23-36 nucleotides.
  • 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: 287-562; 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: 287-562; 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: 287-562; d.
  • 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: 459, 326, 417, 292, 431, or 374; 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: 459, 326, 417, 292, 431, or 374; 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: 459, 326, 417, 292, 431, or 374; d.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 287-562; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 287-562; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 287-562; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 287-562; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 287-562; f.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 459, 326, 417, 292, 431, or 374; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 459, 326, 417, 292, 431, or 374; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 459, 326, 417, 292, 431, or 374; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 459, 326, 417, 292, 431, or 374; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 459, 326, 417, 292, 431, or 374.
  • the direct repeat sequence has a length of between 15-40 or 23-36 nucleotides.
  • 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; or aa.
  • 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.
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-286.
  • 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’.
  • 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 target sequence is immediately adjacent to the PAM sequence.
  • the PAM comprises the sequence 5’-GTTC-3’ and the target sequence comprises SEQ ID NO: 989 or 977. In certain embodiments, the PAM comprises the sequence 5’-GTTC-3’ and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1040 or 1028. In some embodiments, the PAM comprises the sequence 5 ’ -CTTC-3 and the target sequence comprises any one of SEQ ID NO: 967. In certain embodiments, the PAM comprises the sequence 5’-CTTC-3 and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1018. In some embodiments, the PAM comprises the sequence 5’-CTTG-3 and the target sequence comprises any one of SEQ ID NO: 959.
  • the PAM comprises the sequence 5’-CTTG-3 and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1010. In one embodiment, the PAM comprises the sequence 5’-ATTG-3 and the target sequence comprises any one of SEQ ID NO: 988 or 983. In certain embodiments, the PAM comprises the sequence 5’-ATTG-3 and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1039 or 1034.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the RNA guide has the sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the composition further comprises a Casl2i2 polypeptide or a polyribonucleotide encoding a Casl2i2 polypeptide.
  • the Casl2i2 polypeptide is a Casl2i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 890, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • the Casl2i2 polypeptide is a Casl2i2 polypeptide comprising a sequence of SEQ ID NO: 890, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • the Casl2i polypeptide comprises one or more mutations relative to SEQ ID NO: 890.
  • the one or more mutations in the Casl2i polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 890.
  • the one or more mutations are amino acid substitutions, wherein optionally the amino acid substitutions are chosen from D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • the Casl2i polypeptide comprises: (i) mutations at positions D581, D911, 1926, and V1030 of SEQ ID NO: 890, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) mutations at positions D581, 1926, and V1030 of SEQ ID NO: 890, which optionally are amino acid substitutions of D581R, I926R, and V1030G; (iii) mutations at positions D581, 1926, V1030, and S1046 of SEQ ID NO: 890, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 of SEQ ID NO: 890, which optionally are amino acid substitutions of D581R, G624R, F626R, I
  • the RNA guide and the Casl2i2 polypeptide form a ribonucleoprotein complex.
  • the composition comprises the polyribonucleotide encoding the Casl2i2 polypeptide, wherein optionally the polyribonucleotide is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • 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.
  • the RNA guide and the Casl2i2 polypeptide are encoded in a single vector or the RNA guide is encoded in a first vector and the Casl2i2 polypeptide is encoded in a second vector.
  • the invention further provides a vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas 12i2 polypeptide.
  • the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Casl2i2 polypeptide.
  • the vectors may be expression vectors.
  • the present disclosure provides a system comprising: (i) an RNA guide described herein, or a nucleic acid encoding the RNA guide, and (ii) a Casl2i2 polypeptide, or a nucleic acid encoding the Casl2i2 polypeptide.
  • the present disclosure provides a system comprising a pharmaceutical composition comprising a composition or system described herein.
  • the invention further provides 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 (e.g., a target strand sequence of a target sequence) within a CD38 gene and (ii) a direct repeat sequence.
  • a target sequence e.g., a target strand sequence of a target sequence
  • a direct repeat sequence e.g., a target strand sequence of a target sequence
  • the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the CD38 gene.
  • the CD38 gene comprises the sequence of NCBI Entrez Gene ID: 952, the reverse complement thereof, a variant thereof, or the reverse complement of a variant thereof.
  • 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: 287-562; 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: 287-562; 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: 287-562; d.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 287-562; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 287-562; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 287-562; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 287-562; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 287-562; f.
  • 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; or aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or 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 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; or aa. SEQ ID NO: 10 or a portion thereof.
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-286.
  • 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’, 5’-ATTT-3’, 5’- ATTG-3’, 5’-ATTC-3’, 5 ’-TITA-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 target sequence is immediately adj acent to the PAM sequence .
  • the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • the Casl2i2 polypeptide is a Casl2i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 890, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • the Casl2i2 polypeptide is a Casl2i2 polypeptide comprising a sequence of SEQ ID NO: 890, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • 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.
  • the RNA guide and the Casl2i2 polypeptide are encoded in a single vector or the RNA guide is encoded in a first vector and the Casl2i2 polypeptide is encoded in a second vector.
  • the invention further provides a vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas 12i2 polypeptide.
  • the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Casl2i2 polypeptide.
  • the vectors may be expression vectors.
  • RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence (e.g., a target strand sequence of a target sequence) within a CD38 gene and (ii) a direct repeat sequence.
  • the target sequence is within exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the CD38 gene.
  • the CD38 gene comprises the sequence of NCBI Entrez Gene ID: 952, the reverse complement thereof, a variant thereof, or the reverse complement of a variant thereof.
  • 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: 287-562; 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: 287-562; 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: 287-562; d.
  • the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 287-562; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 287-562; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 287-562; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 287-562; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 287-562; f.
  • 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; or aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or 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 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; or aa. SEQ ID NO: 10 or a portion thereof.
  • the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-286.
  • 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’-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 target sequence is immediately adjacent to the PAM sequence.
  • the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the RNA guide has the sequence of any one of SEQ ID NOs: 726- 888 or 904-954.
  • the invention yet further provides a nucleic acid encoding an RNA guide as described herein.
  • the invention yet further provides a vector comprising such an RNA guide as described herein.
  • the invention yet further provides a cell comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.
  • the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • a cell comprising a disrupted CD38 gene, which can be produced by contacting a host cell with the system disclosed herein to genetically edit the CD38 gene in the host cell.
  • a population of cells wherein a plurality of host cells in the population comprise a disrupted CD38 gene, which was produced by contacting the population of cells with the system disclosed herein to genetically edit the CD38 gene a plurality of cells in the population.
  • the population of cells may also comprise cells without an edit in the CD38 gene.
  • the invention yet further provides a kit comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.
  • the invention yet further provides a method of editing a CD38 sequence, the method comprising contacting a CD38 sequence with a composition or an RNA guide as described herein.
  • the method is carried out in vitro.
  • the method is carried out ex vivo.
  • the method is carried out in vivo.
  • the method is carried out ex vivo.
  • the invention yet further provides a method of binding a Casl2i polypeptide and an RNA guide to a target sequence, the method comprising contacting the target sequence with a composition, system, vector, or vector system described herein.
  • the composition comprises the polyribonucleotide encoding the Casl2i polypeptide, and the contacting results in production of the Casl2i polypeptide in the cell.
  • the CD38 sequence is in a cell, which optionally is a T cell.
  • the cell is cultured in vitro.
  • the contacting step is performed by administering the system to a subject comprising the host cell.
  • the method comprises contacting the cell with the composition, system, or RNA guide as described herein.
  • the host cell is cultured in vitro.
  • the contacting step is performed by administering the system for editing the CD38 gene to a subject comprising the host cell.
  • the composition or the RNA guide induces a deletion in the CD38 sequence.
  • 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 50 nucleotides in length.
  • 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.
  • 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. In another aspect of the method, 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.
  • 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. In another 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.
  • 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 CD38 sequence.
  • the deletion overlaps with an insertion in the CD38 sequence.
  • the deletion removes a repeat expansion of the CD38 sequence or a portion thereof.
  • the deletion disrupts one or both alleles of the CD38 sequence.
  • RNA guide nucleic acid, vector, cell, kit, or method described herein
  • the RNA guide comprises the sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the invention yet further provides a method of treating a disease or condition in a subject, the method comprising administering a composition, an RNA guide, or a cell described herein to the subject.
  • the RNA guide, the cell, the kit, or the method described herein, the RNA guide and/or the polyribonucleotide encoding the Casl2i2 polypeptide are comprised within a lipid nanoparticle.
  • the RNA guide and the polyribonucleotide encoding the Casl2i2 polypeptide are comprised within the same lipid nanoparticle.
  • the RNA guide and the polyribonucleotide encoding the Casl2i2 polypeptide are comprised within separate lipid nanoparticles.
  • RNA guide comprising (i) a spacer sequence that is complementary to a target sequence within an CD38 gene and (ii) a direct repeat sequence, wherein the target sequence is a sequence of any one of SEQ ID NOs: 563-725 or 955-1005 or the reverse complement thereof.
  • the direct repeat sequence 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 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; or aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or 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 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; or aa. SEQ ID NO: 10 or a portion thereof.
  • the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 904-954.
  • the RNA guide has the sequence of any one of SEQ ID NOs: 904- 954.
  • each of the first three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification.
  • each of the last three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification.
  • each of the last four nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification.
  • each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification, and the last nucleotide of the RNA guide is unmodified.
  • the invention yet further provides a nucleic acid encoding an RNA guide described herein.
  • the invention yet further provides a vector comprising a nucleic acid described herein.
  • the invention yet further provides a vector system comprising one or more vectors encoding (i) an RNA guide as described herein and (ii) a Casl2i polypeptide, optionally wherein the vector system comprises a first vector encoding the RNA guide and a second vector encoding the Casl2i polypeptide.
  • the invention yet further provides a cell comprising an RNA guide described herein, a nucleic acid described herein, a vector described herein, or a vector system described herein.
  • the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.
  • the invention yet further provides a kit comprising an RNA guide described herein, a nucleic acid described herein, a vector described herein, or a vector system described herein.
  • the invention yet further provides a method of editing an CD38 sequence, the method comprising contacting a CD38 sequence with an RNA guide described herein.
  • the CD38 sequence is in a cell.
  • the RNA guide induces an indel in the CD38 sequence.
  • the invention yet further provides a method of treating a disease in a subject in need thereof, the method comprising administering the RNA guide described herein or a cell described herein to the subject.
  • activity refers to a biological activity.
  • activity includes enzymatic activity, e.g., catalytic ability of a nuclease, such as a Casl2i polypeptide.
  • activity can include nuclease activity.
  • CD38 refers to cluster of differentiation 38 and is also known in the field as cyclic ADP ribose hydrolase. Proteins in this family commonly function in cell adhesion, signal transduction, and calcium signaling.
  • An example of a CD38 gene sequence is provided at www.ncbi.nlm.nih.gov/gene/952, which is incorporated herein by reference as of the date of this filing.
  • NCBI Entrez Gene ID 912
  • NCBI Reference Sequence NC_000004.12 with reference to the following chromosomal location: Homo sapiens chromosome 4, nucleotides 15778275-15853232 (GRCh38.pl3 Primary Assembly). It is understood that spacer and guide sequences described herein can target this sequence or the reverse complement thereof, depending upon whether they are indicated as “+” or as set forth in Table 2A or as “TS” or “BS” as set forth in Table 2B.
  • the target sequences listed in Table 2A, Table 2B, and Table 3 are on the non-target strand of the CD38 gene.
  • Casl2i2 polypeptide refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Casl2i2 polypeptide.
  • the Casl2i2 polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 99% or 100% sequence identity with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Patent No. 10,808,245, which is incorporated by reference herein in its entirety.
  • a Casl2i2 polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 99% or 100% sequence identity with SEQ ID NO: 5 (Casl2i2) of U.S. Patent No. 10,808,245, corresponding to SEQ ID NO: 890 of the present application.
  • a Casl2i2 polypeptide of the disclosure is a Casl2i2 polypeptide as described in PCT/US2021/025257.
  • the Casl2i2 polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).
  • 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 Cas 12i2 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 CD38-targeting RNA guide and a Casl2i2 polypeptide.
  • the term “protospacer adjacent motif’ or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., a CD38 target sequence) to which a complex comprising an RNA guide (e.g., a CD38-targeting RNA guide) and a Casl2i2 polypeptide binds.
  • a target sequence e.g., a CD38 target sequence
  • a complex comprising an RNA guide (e.g., a CD38-targeting RNA guide) and a Casl2i2 polypeptide binds.
  • the PAM may be adjacent to the non-target strand of the double stranded target sequence.
  • 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 RNA guide may associate with the target strand of the double stranded target sequence.
  • the PAM sequence as described herein is present in the non-target strand (e.g., the non-spacer-complementary strand).
  • 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.
  • RNA guide refers to any RNA molecule that facilitates the targeting of a polypeptide (e.g., a Casl2i2 polypeptide) described herein to a target sequence (e.g., a sequence of a CD38 gene).
  • a target sequence e.g., a sequence of a CD38 gene.
  • An RNA guide may be designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., a CD38 nucleic acid sequence).
  • 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 RNA guide.
  • a spacer sequence is complementary to a target sequence.
  • the spacer sequence may be complementary to the target strand of the double stranded target sequence.
  • the term “complementary” refers to the ability of nucleobases of a first nucleic acid molecule, such as an RNA guide, to base pair with nucleobases of a second nucleic acid molecule, such as a target sequence. Two complementary nucleic acid molecules are able to non-covalently bind under appropriate temperature and solution ionic strength conditions.
  • a first nucleic acid molecule (e.g., a spacer sequence of an RNA guide) comprises 100% complementarity to a second nucleic acid (e.g., a target sequence).
  • a first nucleic acid molecule (e.g., a spacer sequence of an RNA guide) is complementary to a second nucleic acid molecule (e.g., a target sequence) if the first nucleic acid molecule comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second nucleic acid.
  • 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 a nuclease (e.g., Casl2i2) that is complexed with the polynucleotide to act (e.g., cleave) on the target sequence.
  • a spacer sequence that is substantially complementary to a target sequence has less than 100% complementarity to the target sequence.
  • a spacer sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence.
  • an RNA guide with a spacer sequence that is substantially complementary to a target sequence has 100% complementarity to the target sequence.
  • the terms “target” and “target sequence” refer to a nucleic acid sequence to which an RNA guide specifically binds.
  • the DNA targeting sequence (e.g., spacer) of an RNA guide binds to a target sequence.
  • the spacer may bind the target strand of the double stranded 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) comprises a 5’- NAAN-3’ sequence.
  • the target sequence is a sequence within a CD38 gene sequence, including, but not limited, to the sequence set forth in NCBI Entrez Gene ID: 952 or the reverse complement thereof. The target sequences listed in Table 2A, Table 2B, and Table 3 are on the non-target strand of the CD38 gene.
  • upstream and downstream refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5’ to 3’ direction, respectively, in which RNA transcription occurs.
  • a first sequence is upstream of a second sequence when the 3 ’ end of the first sequence occurs before the 5 ’ end of the second sequence.
  • a first sequence is downstream of a second sequence when the 5’ end of the first sequence occurs after the 3’ end of the second sequence.
  • the 5’-NTTN-3’ sequence is upstream of an indel described herein, and a Casl2i2-induced indel is downstream of the 5’-NTTN-3’ sequence.
  • FIG. 1 shows indel activity by variant Casl2i2 RNP targeting CD38 in primary T cells.
  • FIG. 2 shows cell viability for all RNP conditions. Error bars represent variability within three technical replicates.
  • FIG. 3 shows a significant decrease in CD38 expression via flow cytometry for two top performing RNA guides.
  • FIG. 4 shows correlation of indels and cells expressing CD38+ for CD38 target screening with variant Casl2i2.
  • FIG. 5 shows indel activity seven days following transfection of Casl2i2 RNPs targeting CD 38 in primary NK cells.
  • FIG. 6 shows cell viability (via dead cell staining and flow) seven days following Casl2i2 RNP in primary NK cells. Each circle represents the average of 3 technical replicates for one target, and each bar represents the average across targets.
  • FIG. 7 shows CD56+ CD38+ expressing cells upon Casl2i2-mediated RNP targeting CD38.
  • the present disclosure relates to an RNA guide capable of binding to CD38 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.
  • the invention described herein comprises compositions comprising an RNA guide targeting CD38.
  • 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 CD38 target sequence, wherein the CD38 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 nontarget strand or the non-spacer-complementary strand).
  • the invention described herein comprises compositions comprising a complex, wherein the complex comprises an RNA guide targeting CD38.
  • the invention comprises 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 CD38 target sequence.
  • a complex comprising an RNA guide targeting CD38 and a Casl2i2 polypeptide binds to a CD38 target sequence at a molar ratio of about 1 : 1.
  • the complex comprises enzymatic activity, such as nuclease activity, that can cleave the CD38 target sequence.
  • the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the RNA guide has a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the invention described herein comprises compositions comprising an RNA guide as described herein and/or an RNA encoding a Casl2i2 polypeptide as described herein.
  • the RNA guide and the RNA encoding a Casl2i2 polypeptide are comprised together within the same composition.
  • the RNA guide and the RNA encoding a Casl2i2 polypeptide are comprised within separate compositions.
  • the RNA guide comprises a direct repeat and/or a spacer sequence described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 726-888 or 904-954. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • Casl2i2 polypeptides are smaller than other nucleases.
  • Casl2i2 is 1,054 amino acids in length
  • .S'. pyogenes Cas9 (SpCas9) is 1,368 amino acids in length
  • .S'. thermophilus Cas9 (StCas9) is 1,128 amino acids in length
  • FnCpfl is 1,300 amino acids in length
  • AsCpfl is 1,307 amino acids in length
  • LbCpfl is 1,246 amino acids in length.
  • Casl2i2 RNA guides which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides.
  • the smaller Casl2i2 polypeptide and RNA guide sizes are beneficial for delivery.
  • Compositions comprising a Casl2i2 polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See PCT/US2021/025257, which is incorporated by reference in its entirety.
  • indels induced by compositions comprising a Casl2i2 polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide.
  • SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length.
  • Casl2i2 polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of a gene such as CD38.
  • the composition described herein comprises an RNA guide targeting a CD38 gene or a portion of CD38 gene. 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 CD38.
  • the RNA guide may direct the Casl2i2 polypeptide as described herein to a CD38 target sequence.
  • 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) CD38 target sequences.
  • Casl2i2 polypeptides e.g., Casl2i2 polypeptides having the same or different sequence
  • CD38 target sequences e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more
  • an RNA guide binds specifically to one or more CD38 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 direct repeat sequence followed by a spacer sequence, referring to the sequences in the 5’ to 3’ direction. 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.
  • 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 at least 23 nt in length. In some embodiments, the direct repeat sequence is about 23 nt in length
  • 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 of SEQ 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 of SEQ ID NO: 9.
  • the direct repeat sequence is set forth in SEQ ID NO: 10.
  • 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. In some embodiments, compositions comprising a Casl2i2 polypeptide and an RNA guide comprising the direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides are capable of introducing indels into a CD38 target sequence. See, e.g., Example 2, where indels were measured in CD38 target sequences following delivery of Casl2i2 RNP to primary T cells by transfection.
  • 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.
  • a direct repeat sequence described herein comprises a uracil (U). In some embodiments, a direct repeat sequence described herein comprises a thymine (T). In some embodiments, a direct repeat sequence according to Table 1 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 1.
  • 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 2A, or a portion of a sequence of Table 2A.
  • the target sequences listed in Table 2A, Table 2B, and Table 3 are on the non-target strand of the CD38 sequence. It should be understood that an indication of SEQ ID NOs: 287-562 should be considered as equivalent to a listing of all of the SEQ ID NOs in the ranges: SEQ ID NOs: 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,
  • the spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 287- 562.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 287- 562.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 287- 562.
  • the spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 287-
  • the spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 287-
  • 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 2A or a portion of a sequence of Table 2A.
  • 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: 287- 562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • 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: 287-562.
  • the invention includes RNA guides that comprise any and all combinations of the direct repeats and spacers described herein.
  • the invention includes RNA guides that comprise any and all combinations of the direct repeats and spacers listed above (e.g., in Tables 2A) or comprise the guide RNA sequences listed above (e.g., in Tables 2B), consistent with the disclosure herein.
  • the sequence of an RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • an RNA guide has a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • a spacer sequence (e.g., Table 2A), a guide sequence (e.g., Table 2B), or any combination of a spacer sequence and a direct repeat sequence (e.g., a guide sequence) described herein comprises a uracil (U).
  • a spacer sequence (e.g., Table 2A), a guide sequence (e.g., Table 2B), or any combination of a spacer sequence and a direct repeat sequence (e.g., a guide sequence) described herein comprises a thymine (T).
  • a spacer sequence according to Table 2A or a guide sequence according to Table 2B comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 2A or Table 2B.
  • 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 invention.
  • Exemplary modifications can include any modification to the sugar, the nucleobase, the intemucleoside 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.
  • 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
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the modification may include a chemical or cellular induced modification.
  • 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 l% to 90%, from l% 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
  • 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 intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Sequences having modified backbones include, among others, those that do not have a phosphoms atom in the backbone.
  • modified RNAs that do not have a phosphoms atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • a sequence will include ribonucleotides with a phosphoms 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 aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs ofthese, 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.
  • the sequence may be negatively or positively charged
  • the modified nucleotides which may be incorporated into the sequence, can be modified on the intemucleoside 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 intemucleoside 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 ’ -()-( 1 -thiophosphate)-cytidine (a-thio-cytidine), 5 ’ -O-( 1 -thiophosphate)- guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l-thiophosphate)-pseudouridine).
  • alpha-thio-nucleoside e.g., 5’-O-(l- thiophosphate)-adenosine, 5 ’ -()-( 1 -thiophosphate)-cytidine (a-thio-cytidine), 5 ’ -O-( 1 -thiophosphate)- guanosine, 5’-O-(l-thiophosphate)-uridine,
  • intemucleoside linkages that may be employed according to the present invention, including intemucleoside linkages which do not contain a phosphorous atom, are described herein.
  • 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)- dione),
  • Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4- octadecyl- 1 -beta-D-arabinofuranosylcytosine, N4-palmitoyl- 1 -(2-C-cyano-2-deoxy-beta-D-arabino- pentofuranosyl) 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. Nucl Acids Res 27: 196-197)
  • the first isolated nucleic acid comprises messenger RNA (mRNA).
  • the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-
  • 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 -methyl -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-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebul
  • 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 carbamoyladeno
  • 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-methylinosine, 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.
  • nucleoside
  • the sequence may or may not be uniformly modified along the entire length of the molecule.
  • nucleotides 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.
  • an RNA guide comprises a) one or more modified nucleotides at or near its 5 ’ end and/or b) one or more modified nucleotides at or near its 3’ end. In some embodiments, an RNA guide comprises at least one modified nucleotide within five nucleotides from its 5 ’ end. In some embodiments, an RNA guide comprises at least one modified nucleotide within five nucleotides from its 3’ end. In some embodiments, an RNA guide comprises at least two modified nucleotides within five nucleotides from its 5 ’ end. In some embodiments, an RNA guide comprises at least two modified nucleotides within five nucleotides from its 3’ end.
  • an RNA guide comprises at least three modified nucleotides within five nucleotides from its 5’ end. In some embodiments, an RNA guide comprises at least three modified nucleotides within five nucleotides from its 3’ end. In some embodiments, an RNA guide comprises at least four modified nucleotides within five nucleotides from its 5’ end. In some embodiments, an RNA guide comprises at least four modified nucleotides within five nucleotides from its 3’ end. In some embodiments, an RNA guide comprises five modified nucleotides at its 5’ end. In some embodiments, an RNA guide comprises five modified nucleotides at its 3’ end.
  • the first three nucleotides of an RNA guide are modified nucleotides.
  • the last three nucleotides of an RNA guide are modified nucleotides.
  • the last four nucleotides of an RNA guide are modified nucleotides.
  • the first to last, second to last, and third to last nucleotides of an RNA guide (at the 3’ end) are modified nucleotides, and the last (3’) nucleotide is unmodified.
  • the modified nucleotide has a modification to a phosphodiester linkage, a sugar, or both.
  • the modified nucleotide is a 2’-O-methyl phosphorothioate modification.
  • composition of the present invention includes a Casl2i2 polypeptide as described in PCT/US2019/022375, the contents of which are incorporated herein by reference in its entirety.
  • the composition of the present invention includes a Casl2i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 890 and/or encoded by SEQ ID NO: 889).
  • the Casl2i2 polypeptide comprises at least one RuvC domain.
  • 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: 889.
  • 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: 889.
  • 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 of temperature and ionic strength (e.g., within a range of medium to high stringency). See, e.g., Tijssen, “Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation” (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).
  • the Casl2i2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 890 and comprises one or more mutations relative to SEQ ID NO: 890.
  • the one or more mutations in the Casl2i2 polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 890.
  • the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.
  • the Casl2i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G). In some embodiments, the Casl2i2 polypeptide comprises mutations at positions D581, 1926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In some embodiments, the Casl2i2 polypeptide comprises mutations at positions D581, 1926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G).
  • the Casl2i2 polypeptide comprises mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).
  • 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: 889.
  • the Casl2i2 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 890.
  • the present invention 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: 890.
  • 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 invention 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: 890 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: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • the Casl2i2 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • the Casl2i2 polypeptide of the present invention comprises a polypeptide sequence having at least 95% identity to SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • a Casl2i2 polypeptide having at least 95% identity to SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate the polypeptide from its respective parent/reference sequence.
  • the present invention 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: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903.
  • 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 of the present invention 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: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, or SEQ ID NO: 903 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.
  • changes to the Cas 12i2 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 polyhistidine 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.oqp/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 double stranded
  • the spacer sequence can bind to the target sequence by virtue of the spacer sequence being substantially complementary to and hybridizing with a first strand ofthe target sequence (i.e., the target strand orthe spacer-complementary strand).
  • the target sequence can be conveniently described by providing the sequence of the target strand or the non-target strand.
  • a nucleic acid can bind to a double-standed target sequence by hybridizing with either strand of the double-standed target sequence.
  • the target sequence is within a CD38 gene or a locus of a CD38 gene.
  • the CD38 gene is a mammalian gene.
  • the CD38 gene is a human gene.
  • the target sequence is within the sequence of NCBI Entrez Gene ID: 952 or the reverse complement thereof.
  • the target sequence is within an exon of the CD38 gene of NCBI Entrez Gene ID: 952 (or the reverse complement thereof), e.g., within a sequence of SEQ ID NO: 891, 892, 893, 894, 895, 896, 897, or 898 (or a reverse complement thereof).
  • Target sequences within an exon of the CD38 gene of NCBI Entrez Gene ID: 952 (and the reverse complement thereof) are set forth in Table 2A and Table 2B.
  • the target sequence is within a variant (e.g., a polymorphic variant) of the CD38 gene sequence of NCBI Entrez Gene ID: 952 or the reverse complement thereof.
  • the CD38 gene sequence is a homolog of the sequence of NCBI Entrez Gene ID: 952 or the reverse complement thereof.
  • the CD38 gene sequence is a non-human CD38 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 ’-TITA-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 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.
  • the RNA guide binds 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).
  • 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. 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.
  • 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 CD38 gene 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 cell has exactly two copies of the target sequence.
  • a CD38 target sequence is selected to be edited by a Casl2i2 polypeptide and an RNA guide using one or more of the following criteria.
  • a target sequence near the 5’ end of the CD38 coding sequence is selected.
  • an RNA guide is designed to target a sequence in exon 1 (SEQ ID NO: 891) or exon 2 (SEQ ID NO: 892).
  • a target sequence adjacent to a 5’-CTTY-3’ PAM sequence is selected.
  • an RNA guide is designed to target a sequence adjacent to a 5’-CTTT-3’ or 5’-CTTC-3’ sequence.
  • a target sequence having low sequence similarity to other genomic sequences is selected.
  • potential non-target sites can be identified by searching for other genomic sequences adjacent to a PAM sequence and calculating the Levenshtein distance between the target sequence and the PAM-adjacent sequences.
  • the Levenshtein distance corresponds to the minimum number of edits (e.g., insertions, deletions, or substitutions) required to change one sequence into another (e.g., to change the sequence of a potential non-target locus into the sequence of the on-target locus).
  • RNA guides are designed for target sequences that do not have potential off-target sequences with a Levenshtein distance of 0 or 1.
  • the present invention includes methods for production of the RNA guide, methods for production of the 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.
  • RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Casl2i2 polypeptide.
  • the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Casl2i2 polypeptide.
  • the RNA guide is purchased from a commercial vendor.
  • the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.
  • the Casl2i2 polypeptide of the present invention can be prepared by (a) culturing bacteria which produce the Casl2i2 polypeptide of the present invention, 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 invention 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 transcriptiontranslation 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 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.).
  • 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.
  • the present disclosure further provides methods of in vivo expression of a Casl2i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Casl2i polypeptide to a host cell wherein the polyribonucleotide encodes the Casl2i polypeptide and expressing the Casl2i polypeptide in the cell.
  • the polyribonucleotide encoding the Casl2i polypeptide is delivered to the cell with an RNA guide and, once expressed in the cell, the Casl2i polypeptide and the RNA guide form a complex.
  • the polyribonucleotide encoding the Casl2i polypeptide and the RNA guide are delivered to the cell within a single composition. In some embodiments, the polyribonucleotide encoding the Casl2i polypeptide and the RNA guide are comprised within separate compositions. In some embodiments, the host cell is present in a subject, e.g., a human patient.
  • an RNA guide targeting CD38 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 Cas 12i2 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, adeno- associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno- associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno- associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno- associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus, adeno- associated virus (AAV)
  • microinjection e.g., lentivirus, retrovirus, adenovirus,
  • a nucleic acid encoding the RNA guide may be located in a viral vector.
  • a nucleic acid encoding the Casl2i2 polypeptide may be located in a viral vector.
  • the viral vector comprises the both the nucleic acid encoding the Casl2i2 polypeptide and the nucleic acid encoding the RNA guide.
  • any of the systems described herein may comprise a nucleic acid encoding the Casl2i2 polypeptide, which is located in a first vector, and a nucleic acid encoding the RNA guide, which is located on a second vector.
  • the first and/or second vector is a viral vector.
  • the first and second vectors are the same type of vector. In other examples, the first and second vectors are different types of vectors.
  • 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 preformed 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.
  • a preformed RNA guide/Casl2i2 polypeptide complex to a cell, where a ternary complex is formed.
  • an RNA guide and an RNA encoding a Casl2i2 polypeptide are delivered together in a single composition.
  • an RNA guide and an RNA encoding a Casl2i2 polypeptide are delivered in separate compositions.
  • an RNA guide and an RNA encoding a Casl2i2 polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Casl2i2 polypeptide delivered in separate compositions are delivered using different delivery technologies.
  • Exemplary intracellular delivery methods include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, 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; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection.
  • viruses such as AAV, or virus-like agents
  • chemical-based transfection methods such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethyleni
  • a lipid nanoparticle comprises an mRNA encoding a Casl2i2 polypeptide, an RNA guide, or an mRNA encoding a Casl2i2 polypeptide and an RNA guide.
  • the mRNA encoding the Casl2i2 polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 889 or a variant thereof.
  • the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
  • the Casl2i2 component and the RNA guide component are delivered together.
  • the Casl2i2 component and the RNA guide component are packaged together in a single AAV particle.
  • the Casl2i2 component and the RNA guide component are delivered together via lipid nanoparticles (LNPs).
  • the Casl2i2 component and the RNA guide component are delivered separately.
  • the Casl2i2 component and the RNA guide are packaged into separate AAV particles.
  • the Casl2i2 component is delivered by a first delivery mechanism and the RNA guide is delivered by a second delivery mechanism.
  • any of the systems described herein may comprise one or more LNPs, wherein the LNP comprises the Casl2i2 polypeptide or the nucleic acid encoding the Casl2i2 polypeptide, the RNA guide or the nucleic acid encoding the RNA guide, or both.
  • the system described herein may comprise an LNP, wherein the LNP comprises the Casl2i2 polypeptide or the nucleic acid encoding the Casl2i2 polypeptide, and a viral vector comprising the nucleic acid encoding the RNA guide.
  • the viral vector is an AAV vector.
  • the system described herein may comprise an LNP, which comprises the RNA guide or the nucleic acid encoding the RNA guide, and a viral vector comprising the nucleic acid encoding the Casl2i2 polypeptide.
  • the viral vector is an AAV vector.
  • the gene editing system disclosed herein may comprise a Casl2i2 polypeptide as disclosed herein.
  • the gene editing system may comprise a nucleic acid encoding the Cas 12i2 polypeptide.
  • the gene editing system may comprise a vector (e.g. , a viral vector such as an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11 and AAV 12) encoding the Casl2i2 polypeptide.
  • the gene editing system may comprise a mRNA molecule encoding the Casl2i2 polypeptide. In some instances, the mRNA molecule may be codon- optimized.
  • 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 in vivo.
  • 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. 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.
  • 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.
  • the cell is derived from a cell line.
  • a wide variety of cell lines fortissue 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.)).
  • ATCC American Type Culture Collection
  • the cell is an immortal or immortalized cell.
  • the cell is an immune cell.
  • the immune cell is a lymphoid progenitor 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 Infdtrating Lymphocyte (TIL).
  • TIL Tumor Infdtrating Lymphocyte
  • the immune cell is a memory cell.
  • the immune cell is a plasma cell.
  • the immune cell is a myeloid progenitor cell.
  • the immune cell is a neutrophil.
  • the immune cell is an eosinophil.
  • the immune cell is a monocyte. In some embodiments, the immune cell is a mast cell. In some embodiments, the immune cell is a basophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the cell is a differentiated cell.
  • the differentiated cell is a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet).
  • the differentiated cell is a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a nerve cell (e.g., a neuron), an epithelial cell, a liver cell (e.g., a hepatocyte), a fibroblast, or a sex cell.
  • 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 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.
  • 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 CD38 gene.
  • the methods comprise introducing a CD38-targeting RNA guide and a Casl2i2 polypeptide into a cell.
  • the CD38 -targeting RNA guide and Casl2i2 polypeptide can be introduced as a ribonucleoprotein complex into a cell.
  • the CD38-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 sequence of the CD38 gene is ofNCBI Entrez Gene ID: 952 (or the reverse complement thereof).
  • the target sequence is in an exon of a CD38 gene, such as an exon having a sequence set forth in any one of SEQ ID NOs: 891, 892, 893, 894, 895, 896, 897, or 898 (or the reverse complement thereof).
  • the sequence of the CD38 gene is a variant of the sequence of NCBI Entrez Gene ID: 952 (or the reverse complement thereof) or a homolog of the sequence ofNCBI Entrez Gene ID: 952 (or the reverse complement thereof).
  • the target sequence is polymorphic variant of the CD38 sequence ofNCBI Entrez Gene ID: 952 (or the reverse complement thereof) or a non-human form of the CD38 gene.
  • 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). 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 single-stranded 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).
  • 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’-AHT-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 CD38 gene. In some embodiments, the deletion alters function of the CD38 gene. In some embodiments, the deletion inactivates the CD38 gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a nonframeshifting 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’-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
  • 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 ’-TITA-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 nucle
  • 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 (
  • 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 ofthe 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 ’ , 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) ofthe 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 ’-TITA-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.
  • the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10,
  • 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. 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’-
  • 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 ’-TITA-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 embodiment
  • 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 ofthe 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 ’-TITA-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 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 (
  • 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.
  • 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
  • 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.
  • 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. 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.
  • the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10,
  • nucleotides 11, 12, 13, 14, 15, 16, or 17 nucleotides and ends within about 20 to about 30 nucleotides (e.g., about 17, 18,
  • 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, 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 ’-TITA-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 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 a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19,
  • 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,
  • 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 ’-TITA-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,
  • 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.
  • 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,
  • 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 ’-TITA-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
  • 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 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 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 ’-TITA-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
  • 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.
  • 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 ’-TITA-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.
  • 5 -ATTA-3
  • 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 ’-TITA-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
  • the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6,
  • 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 ’-TITA-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’-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 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 ’-TITA-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’-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 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.
  • 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 ’-TITA-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 ’-TITA-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 ’-TITA-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 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).
  • 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).
  • 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 CD38 gene.
  • the methods are carried out using a complex comprising a Casl2i2 enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein.
  • the sequence of the RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • an RNA guide has a sequence of any one of SEQ ID NOs: 726-888 or 904-954.
  • the RNA guide targeting CD38 is encoded in a plasmid. In some embodiments, the RNA guide targeting CD38 is synthetic or purified RNA. In some embodiments, the Casl2i2 polypeptide is encoded in a plasmid. In some embodiments, the Casl2i2 polypeptide is encoded by an RNA that is synthetic or purified.
  • compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy.
  • the compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used as a therapy for treating or alleviating a cancer, such as multiple myeloma or lymphoma.
  • Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject (e.g., multiple myeloma or lymphoma). Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein.
  • Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing a CD38 sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.
  • modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of treatment.
  • the modified cells may comprise a substitution, insertion, and/or deletion described herein.
  • the modified cells may be a heterogeneous population comprising cells with different types of gene edits.
  • the modified cells may comprise a substantially homogenous cell population, e.g., at least 80% of the cells in the whole population comprising one particular gene edit in the CD38 gene.
  • the cells can be suspended in a suitable media.
  • compositions comprising the gene editing system or components thereof.
  • a composition can be a pharmaceutical composition.
  • a pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • a pharmaceutical composition comprising the gene editing system or components thereof as described herein may be administered to a subject in need thereof.
  • the gene editing system or components thereof may be delivered to specific cells or tissue, where the gene editing system could function to genetically modify the CD38 gene in such cells.
  • a formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative.
  • Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations.
  • Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may comprise, in addition to the system or cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or saline.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • 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 kits or systems include a polynucleotide that encodes an RNA guide disclosed 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.
  • the kit may be useful for research purposes.
  • the kit may be useful to study gene function.
  • This Example describes indel assessment on CD38 target sequences using a variant Casl2i2 polypeptide and RNA guides introduced into mammalian cells (e.g., HEK293T cells) by transient transfection.
  • a Casl2i2 polypeptide is cloned into a PCDNATM3. 1 backbone (Invitrogen). The plasmid is then maxiprepped and diluted.
  • a dsDNA fragment encoding a crRNA is derived by ultramers containing the target sequence scaffold, and the U6 promoter. Ultramers are resuspended in Tris*HCl at a pH of 7.5. Working stocks are subsequently diluted using Tris*HCl to serve as the template for the PCR reaction.
  • the amplification of the crRNA is done with the following components: the aforementioned template, forward primer, reverse primer, NEB HiFi Polymerase, and 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.
  • 25,000 HEK293T 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.
  • LIPOFECTAMINE® 2000 transfection reagent; ThermoFisher
  • OPTI- MEM® reduced-serum medium; ThermoFisher
  • the LIPOFECTAMINE® transfection reagent; ThermoFisher
  • OPTI- MEM® reduced-serum medium; ThermoFisher
  • 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, the Solution 1 and Solution 2 mixture are added dropwise to each well of a 96 well plate containing the cells.
  • TRYPLETM recombinant cell-dissociation enzymes; ThermoFisher
  • D10 media is then added to each well and mixed to resuspend cells.
  • the cells are then spun down for 10 minutes, and the supernatant is discarded.
  • QUICKEXTRACTTM DNA extraction buffer; Lucigen
  • PCR1 PCR1 products are purified by column purification.
  • Round 2 PCR PCR2 is done to add Illumina adapters and indexes. Reactions are then pooled, loaded onto a 2% E-GELTM EX for 10 minutes and gel extracted. Sequencing runs are done with a 150 cycle NEXTSEQTM v2.5 mid or high output kit (Illumina).
  • Presence of indels in the CD38 gene indicates that an RNA guide and Casl2i2 polypeptide are successfully delivered by transfection and active in the cells.
  • This example describes RNP transfection followed by FACS staining and indel assessment on CD38 using a variant Casl2i2 (SEQ ID NO: 900) in primary T cells.
  • PBMCs Peripheral Blood Mononuclear Cells
  • T cells were isolated from PBMCs using the EASYSEPTM Human T Cell Isolation Kit (StemCell Technologies; #17951). Following isolation, a sample was collected and stained for CD3s for flow cytometry analysis of surface expression to determine T cell purity of the isolated cells. Cell density was adjusted to le6 cells/mL, and cells were stimulated for 3 days with a cocktail of anti-CD3:CD28 antibodies.
  • IMMUNOCULTTM-XF Cell Expansion Medium (StemCell Technologies; #10981) with IL-2 and L-Glutamine and supplemented with IMMUNOCULTTM Human CD3/CD28 T Cell Activator (StemCell Technologies; #10971).
  • Casl2i2 RNP complexation reactions were made by mixing purified variant Casl2i2 with individual crRNAs targeting CD38 (in NaCl; see sequences in Table 1) at a 1: 1 (Casl2i2:crRNA) volume ratio (2.5: 1 crRNA:Casl2i2 molar ratio).
  • variant Casl2i2 was mixed with NaCl at the same volume ratio as the crRNA.
  • Complexations were incubated on ice for 30-60 min. Following incubation, Casl2i2 RNPs were added to activated T cells resuspended in P3 buffer (Lonza # V4SP-3096).
  • Activated T cell suspensions were collected and counted using an automated cell counter. A sample of cells was collected and stained for CD25 for flow cytometry analysis to determine activation efficiency. Cell density was adjusted to l.le7 cells/mL in P3 buffer (Lonza #V4SP-3096) and was dispensed at 2e5 cells/reaction into each well in a 96-well NUCLEOCUVETTE® plate (Lonza Bioscience). The final concentration of Casl2i2 RNPs was 20 pM. The plates were electroporated using an electroporation device (program EO-115, Lonza 4D-NUCLEOFECTOR®).
  • IMMUNOCULTTM-XF pre-warmed IMMUNOCULTTM-XF, IL-2, and L-Glutamine were added to cells and mixed gently by pipetting.
  • approximately 50,000 diluted nucleofected cells were plated into the pre-warmed 96- well plate with wells containing IMMUNOCULTTM-XF, IL-2, and L-Glutamine. Plates were incubated for 7 days at 37°C with media replacement at day 4.
  • 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 was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence.
  • the first 3 Ont of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well.
  • Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion.
  • the indel % was 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 was 10,000.
  • Indel activity by variant Casl2i2 RNP targeting CD 38 in primary T cells is shown in Figure 1. Indels were observed in over 60% of NGS reads for E6T1. E2T7 resulted in indels in over 40% of NGS reads. Indel activity was over 20% for E8T2, E1T5, E5T1, E4T2, E6T3, E1T1, and E3T6. Cell viability was high for all RNP conditions ( Figure 2). Casl2i2-only samples showed lower viability compared to all RNP samples. The two top performing RNA guides demonstrated a significant decrease in CD38 expression via flow cytometry (Figure 3). S17015F and HIT2 anti-CD38 antibodies were used for staining. Correlation of indels and cells expressing CD38+ for CD38 target screening with variant Casl2i2 is shown in Figure 4.
  • This example describes RNP transfection followed by FACS staining and indel assessment on CD38 using an engineered Casl2i2 nuclease (SEQ ID NO: 900) in primary NK cells.
  • PBMCs Peripheral Blood Mononuclear Cells
  • stemCell Technologies StemCell Technologies; #70025
  • NK cells were isolated from PBMCs using the EasySepTM Human NK Cell Isolation Kit (StemCell Technologies; #17955). Following isolation, a sample was collected and stained for CD56 and CD3 for flow cytometry analysis of surface expression to determine NK cell purity. Cells were cultured in fresh ImmunoCultTM NK Cell Expansion Kit (StemCell Technologies Cat # 100-071).
  • Casl2i2 RNP complexation reactions were made by mixing purified variant Casl2i2 of SEQ ID NO: 900 with individual crRNAs targeting CD38 in 250 mM NaCl at a 1: 1 (Casl2i2:crRNA) volume ratio (2.5: 1 crRNA:Casl2i2 molar ratio). For “effector only” controls, variant was mixed with 250 mM NaCl at the same volume ratio as the crRNA. Complexations were incubated on ice for 30-60 min. Following incubation, Casl2i2 RNPs were added to activated T cells resuspended in P3 buffer (Lonza # V4SP-3096).
  • NK cell suspensions were collected and counted using an automated cell counter. Cell density was adjusted to 1. Ie7 cells/mL in P3 buffer (Lonza #V4SP-3096) and was dispensed at 2e5 cells/reaction into each well in a 96-well Nucleocuvette plate. Final concentration of Casl2i2 RNPs was 20 pM. The plates were electroporated using an electroporation device (program EN-138, CM-137, FA-100, EH-115 and EO-115, Lonza 4D-Niiclcofcctor '). Immediately following electroporation, pre-warmed ImmunoCultTM NK Cell Expansion media was added to cells and mixed gently by pipetting.
  • the data present the % live cells after treatment with several guides including E6T 1 , E2T7, E4T2, E3T6, and GFP (aggregate shown for NK_EN-138, NK_CM-137, and NK_FH-115), cells electroporation alone (EN-138 EC-NK, CM-137 EC_NK, and FH-115 EC_NK), and non-electroporated cells (NK-NEC).
  • E6T 1 , E2T7, E4T2, E3T6, and GFP aggregate shown for NK_EN-138, NK_CM-137, and NK_FH-115
  • NK-NEC non-electroporated cells
  • Table 3 crRNA sequences for RNP transfection In Table 3, the double stranded target sequence is described by providing the sequence of the non-target strand of the target sequence

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Abstract

La présente invention concerne des compositions comprenant des ARN guides ciblant CD38, des méthodes de caractérisation des compositions, des cellules comprenant les compositions, et des méthodes d'utilisation des compositions.
PCT/US2023/060667 2022-01-14 2023-01-13 Compositions comprenant un arn guide ciblant cd38 et leurs utilisations WO2023137451A1 (fr)

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WO2021257730A2 (fr) * 2020-06-16 2021-12-23 Arbor Biotechnologies, Inc. Cellules modifiées par un polypeptide cas12i

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WO2021226363A1 (fr) * 2020-05-08 2021-11-11 Metagenomi Ip Technologies, Llc Enzymes à domaines ruvc
WO2021257730A2 (fr) * 2020-06-16 2021-12-23 Arbor Biotechnologies, Inc. Cellules modifiées par un polypeptide cas12i

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