WO2020069029A1 - Nouvelles nucléases crispr - Google Patents

Nouvelles nucléases crispr Download PDF

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WO2020069029A1
WO2020069029A1 PCT/US2019/053018 US2019053018W WO2020069029A1 WO 2020069029 A1 WO2020069029 A1 WO 2020069029A1 US 2019053018 W US2019053018 W US 2019053018W WO 2020069029 A1 WO2020069029 A1 WO 2020069029A1
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seq
sequence
crispr nuclease
nucleotide sequence
composition
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PCT/US2019/053018
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David Baram
Lior IZHAR
Asael Herman
Liat ROCKAH
Nurit MERON
Joseph GEORGESON
Nadav MARBACH-BAR
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Emendobio Inc.
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Publication of WO2020069029A1 publication Critical patent/WO2020069029A1/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
    • 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
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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

  • This application incorporates-by-reference nucleotide sequences which are present in the file named“l90925_906l7-A-PCT_Sequence_Listing_DH.txf’, which is 636 kilobytes in size, and which was created on September 24, 2019 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed September 25, 2019 as part of this application.
  • the present invention is directed to, inter alia , composition and methods for genome editing.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPRs systems have become important tools for research and genome engineering. Nevertheless, many details of CRISPR systems remain to be learned and the applicability of CRISPR nucleases may be limited by the sequence specificity requirements, expression, or delivery challenges. Different CRISPR nucleases have diverse characteristics such as: size, PAM site, on target activity, specificity, cleavage pattern (e.g. blunt, staggered ends), and prominent pattern of indel formation following cleavage. Different sets of characteristics may be useful for different applications.
  • CRISPR nucleases may be able to target particular genomic loci that other CRISPR nucleases cannot due to limitations of the PAM site.
  • CRISPR nucleases currently in use exhibit pre-immunity, which may limit in vivo applicability. See Charlesworth, C. T., et al. (2019). Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature medicine, 25(2), 249, and Wagner, D. L., et al. (2019). High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nature medicine, 25(2), 242.
  • compositions and methods that may be utilized for genomic engineering, epigenomic engineering, genome targeting, genome editing of cells, and/or in vitro diagnostics.
  • genomic DNA refers to linear and/or chromosomal DNA and/or to plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest.
  • the cell of interest is a eukaryotic cell.
  • the cell of interest is a prokaryotic cell.
  • the methods produce double-stranded breaks (DSBs) at pre- determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the target site(s) in a genome.
  • compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) nucleases.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR nuclease is CRISPR-associated protein.
  • compositions comprise a Clustered Regularly Interspaced
  • CRISPRs Short Palindromic Repeats
  • Clostridium cocleatum e.g., Clostridium cocleatum-loc2
  • Acetitomaculum ruminis Alloscardovia macacae
  • Enterococcus devriesei Enterococcus thailandicus
  • Fructobacillus ficulneus Aquimarina sp. (e.g., Aquimarina sp.
  • Leuconostoc lactis e.g., Leuconostoc lactis CCK940
  • Dolosicoccus paucivorans e.g., Dolosicoccus paucivorans UMB0860
  • Lactobacillus kefiri Lactobacillus kefiri OG2
  • Embodiments of the present invention provide for CRISPR nucleases designated
  • OMNI OMNI nucleases as provided in Table 1 hereinbelow. Column 1 of Table 1 indicates each OMNI designation; column 2 indicates the SEQ ID NO for OMNI; column 3 indicates the DNA sequence encoding each OMNI; column 4 indicates the DNA sequence encoding each OMNI codon optimized for E. coli or bacterial cultures; column 5 indicates the DNA sequence encoding each OMNI codon optimized for mammalian cells.
  • This invention provides a method of modifying a nucleotide sequence at a target site in the genome of a mammalian cell comprising introducing into the cell (i) a composition comprising a CRISPR nuclease having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9 or a nucleic acid molecule comprising a sequence encoding a CRISPR nuclease which sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:23-27 and (ii) a DNA-targeting RNA molecule, or a DNA polynucleotide encoding a DNA-targeting RNA molecule, comprising a nucleotide sequence that is complementary to a sequence in the target DNA.
  • This invention also provides a non-naturally occurring composition
  • a non-naturally occurring composition comprising a
  • CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease.
  • composition comprising a CRISPR associated system comprising:
  • RNA molecules comprising a guide sequence portion linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more RNA molecules; and b) an CRISPR nuclease comprising an amino acid sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO: l, SEQ ID NO: l, SEQ ID
  • SEQ ID NO:2 SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease;
  • RNA molecules hybridize to the target sequence, wherein the target sequence is 3' of a Protospacer Adjacent Motif (PAM), and the one or more RNA molecules form a complex with the RNA-guided nuclease.
  • PAM Protospacer Adjacent Motif
  • This invention also provides a non-naturally occurring composition comprising:
  • a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease;
  • RNA molecules or one or more DNA polynucleotide encoding the one or more RNA molecules, comprising at least one of:
  • nuclease-binding RNA nucleotide sequence capable of interacting with/binding to the CRISPR nuclease; and ii) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence,
  • the CRISPR nuclease is capable of complexing with the one or more RNA molecules to form a complex capable of hybridizing with the target DNA sequence.
  • Fig. 1A-B Fig. 1A, An example of the predicted secondary structures of the full duplex RNA elements (crRNA:tracrRNA chimera) used for identification of possible“Nexus” and “hairpins” in the design of sgRNAs for each nuclease;
  • Fig. IB an example of variations in the sequence and predicted structure between regions of two different sgRNAs, V 1 and V2, designed for use with a single nuclease.
  • the crRNA and tracrRNA were connected with tetra-loop‘gaaa’, generating different sgRNA scaffolds.
  • Figs. 2A-B Fig.2A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 4 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 2B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 4 sgRNA v2.
  • Figs. 3A-B Fig.3A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 6 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 3B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 6 sgRNA v2.
  • Figs. 4A-B Fig. 4A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 8 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 4B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 8 sgRNA v2.
  • Figs. 5A-B Fig. 5A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 10 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 5B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 10 sgRNA v2.
  • Figs. 6A-B Fig. 6A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 11 sgRNA vl in E. coli and sequence motifs generated.
  • Fig. 6B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 11 sgRNA v2.
  • Figs. 7A-B Fig. 7A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 13 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 7B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 13 sgRNA v2.
  • Figs. 8A-B Fig. 8A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 17 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 8B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 17 sgRNA v2.
  • Figs. 9A-B Fig. 9A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 18 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 9B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 18 sgRNA v2.
  • Figs. 10A-B Fig. 10A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 19 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 10B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 19 sgRNA v2.
  • Figs. 11A-B Fig. 11 A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 20 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 11B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 20 sgRNA v2.
  • Figs. 12A-B Fig. 12A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 23 sgRNA vl in E. coli and sequence motifs generated.
  • Fig. 12B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 23 sgRNA v2.
  • Figs. 13A-B Fig. 13A, a condensed 4N window library of all possible PAM locations along an 8bp sequence for OMNI 24 sgRNA vl in E. coli and sequence motifs generated. Activity estimated based on the average of the two most depleted sequences and was calculated as 1 - Depletion score; Fig. 13B discloses the sequence motifs generated for all possible PAM locations along an 8bp sequence for the OMNI 24 sgRNA v2.
  • Figure 14 Depletions of PAM sites along positions 1-4 of an 8bp sequence for spCas9 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 15 Depletions of PAM sites along positions 3-6 of an 8bp sequence for OMNI 4 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 16 Depletions of PAM sites along positions 1-4 of an 8bp sequence for OMNI 6 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 17 Depletions of PAM sites along positions 1-4 of an 8bp sequence for OMNI 8 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 18 Depletions of PAM sites along positions 1-4 (top panel) and positions 5-8 (middle panel) of an 8bp sequence for OMNI 10 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 19 Depletions of PAM sites along positions 3-7 of an 8bp sequence for OMNI 13 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 20 Depletions of PAM sites along positions 3-6 of an 8bp sequence for OMNI 17 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 21 Depletions of PAM sites along positions 1-4 of an 8bp sequence for OMNI 18 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Figure 22 Depletions of PAM sites along positions 1-4 of an 8bp sequence for OMNI
  • Figure 23 Depletions of PAM sites along positions 1-6 of an 8bp sequence for OMNI
  • Figure 24 Depletions of PAM sites along positions 2-6 of an 8bp sequence for OMNI
  • Figure 25 Depletions of PAM sites along positions 2-5 of an 8bp sequence for OMNI
  • Fig. 26A-B Fig. 26A, depletions of PAM sites along positions 1-6 of an 8bp sequence for OMNI 16 using sgRNA vl in a cell-free in vitro TXTL system. Sequence motifs generated for
  • FIG. 26B depletions of PAM sites along positions 1- 6 of an 8bp sequence for OMNI 16 using sgRNA v2 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Fig. 27A-B Fig. 27A, depletions of PAM sites along positions 2-5 of an 8bp sequence for OMNI 21 using sgRNA vl in a cell-free in vitro TXTL system. Sequence motifs generated for
  • Fig. 27B depletions of PAM sites along positions 2- 5 of an 8bp sequence for OMNI 21 using sgRNA v2 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Fig. 28A-B Fig. 28A, depletions of PAM sites along positions 4-7 of an 8bp sequence for OMNI 27 using sgRNA vl in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results; Fig.
  • Fig. 29A-B Fig. 29A, depletions of PAM sites along positions 3-6 of an 8bp sequence for OMNI 30 using sgRNA vl in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results; Fig. 29B, depletions of PAM sites along positions 3- 6 of an 8bp sequence for OMNI 30 using sgRNA v2 in a cell-free in vitro TXTL system. Sequence motifs generated for PAM sites based on depletion assay results.
  • Fig. 30A-C growth selective plates with Arabinose for nuclease complexes for OMNI 4, OMNI 6, OMNI 8, and OMNI 10 along identified PAM sites. OMNI complexes that cleave the positive plasmid targeted survive in the presence of Arabinose, whereas OMNI complexes that do not cleave the positive plasmid cannot grow on selective plates; Fig. 30B, growth selective plates for nucleases complexes for OMNI 11, OMNI 13, OMNI 17, and OMNI 18; Fig. 30C, growth selective plates for nucleases complexes for OMNI 19, OMNI 20, OMNI 23, and OMNI 24.
  • Fig. 31A-C Fig. 31A, SpCas9 as a control and OMNI 4, OMNI 6, OMNI 8, OMNI 22, OMNI 20 nucleases tested for in vitro cleavage of different PAM sites in. E. coli strain BW25141 (l ⁇ E3) co-expressing the respective OMNI nucleases and sgRNA were lysed using BugBuster lysis solution. The lysate was reacted in the recommended cleavage buffer with linear DNA substrates containing the PAM sequences flanked by a unique protospacer targeted by the sgRNA (Tl) or a non-targeted protospacer (T2) as a control with cleavage results shown; Fig.
  • Fig. 32A-C Fig. 32A, OMNI 4 and OMNI 6 open-reading frames were cloned into bacterial expression plasmids and expressed in G10 cells. Purity of OMNI proteins expressed was measured by SDS-PAGE analysis; Fig. 32B, Synthetic sgRNA of OMNI 4 and OMNI 6 ribonucleoproteins (RNPs) were formed. The RNPs were reacted with a cleavage buffer with lOOng of linear DNA substrates containing the protospacer targeted by the sgRNA flanking each OMNI PAM sequence; Fig.
  • Fig. 33A-H Activity of OMNI nucleases in Mammalian cells was assayed using a GFP Fluorescent gain-based reporter system in HEK 293T cells. Negative control cells were transfected with the reporter vector with only the OMNI nuclease or only the guide; Fig. 33A, OMNI 4 GFP signal for the nuclease + guide and guide only at PAM 4D2 with sgRNA vl; Fig. 33B, OMNI 13 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 4D1 with sgRNA v2; Fig.
  • OMNI 17 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 17D2 with sgRNA v2
  • Fig. 33D OMNI 18 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 1 with sgRNA v4
  • Fig. 33E OMNI 19 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 4D2 with sgRNA v2
  • Fig. 33F OMNI 20 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 20D2 with sgRNA v3
  • Fig. 20 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 20D2 with sgRNA v3
  • Fig. 20 GFP signal for the nuclease + guide, guide only, and nuclease
  • OMNI 23 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 6D2 with sgRNA v2
  • Fig. 33H OMNI 24 GFP signal for the nuclease + guide, guide only, and nuclease only at PAM 8D2 with sgRNA v2.
  • Fig. 34A-C Activity of OMNI nucleases with optimized expression vectors for E. coli and Human cells were assayed using a GFP Fluorescent gain-based reporter system. Negative control cells were transfected with the reporter vector with no guide.
  • Fig. 34A OMNI 6 activity with optimized expression vectors for E. coli and Human cells and sgRNAs sgRNA v2, sgRNA v2, sgRNA v4 and sgRNA v5;
  • Fig. 34B Activity of OMNI 18 encoded by a human optimized expression vector was compared to spCas9 activity.
  • OMNI 18 was assayed with sgRNA v2, sgRNA v3, and sgRNA v4. Negative controls for OMNI 18 and spCas9 were run with either no guide or no nuclease; Fig. 34C, Activity of OMNI 20 encoded by a human optimized expression vector was compared to spCas9 activity. OMNI 20 was assayed utilizing sgRNA-PAM pairings V2-0, V2-20D2,V3-0, and V3-20D2. SpCas9 activity was assayed at Pam site 1. Negative controls for OMNI 30 and spCas9 were run with no nuclease.
  • Fig. 35A-D In order to overcome potential transcriptional and structural constrains and to assess the plasticity of the sgRNA scaffold in the human cellular environmental context, several versions of sgRNA were tested. In each case the modifications represent small variations in the nucleotide sequence within the predicted duplex and/or hairpins that were introduced to several synthetic sgRNA; Fig. 35A, the predicted secondary structure of the full duplex RNA elements of OMNI 6 sgRNA v2 (SEQ ID NO:88); Fig.35B, the predicted secondary structure of the full duplex RNA elements of OMNI 6 sgRNA v3 (SEQ ID NO:89); Fig.
  • Fig. 36A-B The intrinsic fidelity of OMNI6 was measured by conducting an activity assay as described; Fig. 36A, an intrinsic fidelity assay of OMNI 6 at site ELANE g35 in HeLa cells as compared to SpCas9.
  • the on target (SEQ ID NO:383) and off target (SEQ ID NO:384) editing efficiency ratio obtained by OMNI 6 was 2.43: 1 while spCas9 on/off ratio was 1 : 1.
  • Fig. 36B an intrinsic fidelity assay of OMNI 6 at site ELANE g58 in HEK293 FRT cells as compared to SpCas9.
  • the on target (SEQ ID NO: 101) and off target (SEQ ID NO:385) editing efficiency ratio obtained by OMNI 6 was 17.64: 1 while spCas9 on/off ratio was 1.58: 1.
  • Figure 37 synthetic sgRNAs for OMNI 4 and OMNI 6 were synthesized and expressed in U20S cells. RNPs were formed. Cells were lysed and their genomic DNA content was used in PCR reaction, amplifying the corresponding putative genomic targets. Amplicons were subjected to NGS and the resulting sequences were then used calculate the percentage of editing events.
  • Figure 37, left panel , OMNI 4 % editing at genomic site EMX1 on target (SEQ ID NO:386) was 50.4% as compared to 0.1% for the negative control with no sgRNA.
  • Figure 37, right panel , OMNI 6 % editing at genomic site EMX1 on target (SEQ ID NO: 101) was 48.1% as compared to 1.6% for the negative control with no sgRNA.
  • compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease and/or a nucleotide sequence encoding the same.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR nuclease comprises an amino acid sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, or 82% amino acid sequence identity to a CRISPR nuclease as set forth in any of SEQ ID NOs: 1-22.
  • the CRISPR nuclease comprises an amino acid sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75% amino acid sequence identity to a CRISPR nucleases derived from Acetobacterium sp. KB-l, Alistipes sp.An54, Bartonella apis, Blastopirellula marina, Bryobacter aggregatus MPL3, Algoriphagus marinus, Butyrivibrio sp.
  • the CRISPR nuclease of the invention exhibits increased specificity to a target site compared to a spCas9 nuclease when complexed with the one or more RNA molecules.
  • the complex of the CRISPR nuclease of the invention and one or more RNA molecules exhibits at least maintained on-target editing activity of the target site and reduced off-target activity compared to spCas9 nuclease.
  • the CRISPR nuclease is engineered or non-naturally occurring.
  • the CRISPR nuclease may also be recombinant.
  • Such CRISPR nucleases are produced using laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.
  • This invention provides a non-naturally occurring composition
  • a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease.
  • the CRISPR nuclease further comprises an RNA-binding portion capable of interacting with a DNA-targeting RNA molecule (gRNA) and an activity portion that exhibits site-directed enzymatic activity.
  • gRNA DNA-targeting RNA molecule
  • the composition further comprises a DNA-targeting RNA molecule or a DNA polynucleotide encoding a DNA-targeting RNA molecule, wherein the DNA-targeting RNA molecule comprises a nucleotide sequence that is complementary to a sequence in a target region, wherein the DNA-targeting RNA molecule and the CRISPR nuclease do not naturally occur together.
  • the DNA-targeting RNA molecule further comprises a nucleotide sequence that can form a complex with a CRISPR nuclease.
  • the composition further comprises an RNA molecule comprising a nucleotide sequence that can form a complex with a CRISPR nuclease (tracrRNA) or a DNA polynucleotide comprising a sequence encoding an RNA molecule that can form a complex with the CRISPR nuclease.
  • tracrRNA CRISPR nuclease
  • DNA polynucleotide comprising a sequence encoding an RNA molecule that can form a complex with the CRISPR nuclease.
  • the composition further comprises a donor template for homology directed repair (HDR).
  • HDR homology directed repair
  • the composition is capable of editing the target region in the genome of a cell.
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO: 73;
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO: 105.
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO: 127.
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 10 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO:229;
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 11 the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO:238;
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 12 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO:248; or
  • the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 13 the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule is SEQ ID NO:258.
  • composition comprising a CRISPR associated system comprising:
  • RNA molecules comprising a guide sequence portion linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more RNA molecules; and b) an CRISPR nuclease comprising an amino acid sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and
  • RNA molecules hybridize to the target sequence, wherein the target sequence is 3' of a Protospacer Adjacent Motif (PAM), and the one or more RNA molecules form a complex with the RNA-guided nuclease.
  • PAM Protospacer Adjacent Motif
  • the composition further comprises an RNA molecule comprising a nucleotide molecule that can form a complex with the RNA nuclease (tracrRNA) or a DNA polynucleotide encoding an RNA molecule comprising a nucleotide sequence that can form a complex with the CRISPR nuclease.
  • RNA nuclease RNA nuclease
  • the composition further comprises a donor template for homology directed repair (HDR).
  • HDR homology directed repair
  • This invention also provides a non-naturally occurring composition comprising:
  • a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NOs:4-22 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease;
  • RNA molecules or one or more DNA polynucleotide encoding the one or more RNA molecules, comprising at least one of: i) a nuclease-binding RNA nucleotide sequence capable of interacting with/binding to the
  • RNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence
  • the CRISPR nuclease is capable of complexing with the one or more RNA molecules to form a complex capable of hybridizing with the target DNA sequence.
  • the CRISPR nuclease and the one or more RNA molecules form a CRISPR complex that is capable of binding to the target DNA sequence to effect cleavage of the target DNA sequence.
  • the CRISPR nuclease and at least one of the one or more RNA molecules do not naturally occur together.
  • the CRISPR nuclease comprises an RNA-binding portion and an activity portion that exhibits site-directed enzymatic activity
  • the DNA-targeting RNA nucleotide sequence comprises a nucleotide sequence that is complementary to a sequence in a target DNA sequence
  • the nuclease-binding RNA nucleotide sequence comprises a sequence that interacts with the RNA-binding portion of the CRISPR nuclease.
  • the nuclease-binding RNA nucleotide sequence and the DNA- targeting RNA nucleotide sequence are on a single guide RNA molecule (sgRNA), wherein the sgRNA molecule can form a complex with the CRISPR nuclease and serve as the DNA targeting module.
  • sgRNA single guide RNA molecule
  • the sgRNA has a length of up to 1000 bases, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases.
  • the nuclease-binding RNA nucleotide sequence is on a first RNA molecule and the DNA-targeting RNA nucleotide sequence is on a single guiide RNA molecule, and wherein the first and second RNA sequence interact by base-paring or are fused together to fonn one or more RNA molecules or sgRNA that complex with the CRISPR nuclease and serve as the targeting module.
  • the composition further comprises a donor template for homology directed repair (HDR).
  • HDR homology directed repair
  • the CRISPR nuclease comprises 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, or 140-150 amino acid substitutions, deletions, and/or insertions compared to the amino acid sequence of the wild-type of the CRISPR nuclease.
  • the CRISPR nuclease exhibits at least 2%, 5%, 7% 10%, 15%, 20%, 25%, 30, or 35% increased specificity compared the wild-type of the CRISPR nuclease.
  • the CRISPR nuclease exhibits at least 2%, 5%, 7% 10%, 15%, 20%, 25%, 30, or 35% increased activity compared the wild-type of the CRISPR nuclease.
  • the CRISPR nuclease has altered PAM specificity compared to the wild-type of the CRISPR nuclease.
  • the CRISPR nuclease is non-naturally occurring.
  • the CRISPR nuclease is engineered and comprises unnatural or synthetic amino acids.
  • the CRISPR nuclease is engineered and comprises one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags.
  • NLS nuclear localization sequences
  • cell penetrating peptide sequences cell penetrating peptide sequences
  • affinity tags affinity tags
  • the CRISPR nuclease comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of a CRISPR complex comprising the CRISPR nuclease in a detectable amount in the nucleus of a eukaryotic cell.
  • the CRISPR nuclease comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near carboxy- terminus, or a combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino- terminus and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near carboxy-terminus.
  • 1-4 NLSs are fused with the CRISPR nuclease.
  • an NLS is located within the open-reading frame (ORF) of the CRISPR nuclease.
  • NLSs Methods of fusing NLSs at or near the amino-terminus, at or near carboxy-terminus, or within the ORF of an expressed protein are well known in the art.
  • the nucleic acid sequence of the NLS is placed immediately after the start codon of the CRISPR nuclease on the nucleic acid encoding the NLS- fused CRISPR nuclease.
  • the nucleic acid sequence of the NLS is placed after the codon encoding the last amino acid of the CRISPR nuclease and before the stop codon.
  • NLSs any combination of NLSs, cell penetrating peptide sequences, and/or affinity tags at any position along the ORF of the CRISPR nuclease is contemplated in this invention.
  • amino acid sequences and nucleic acid sequences of the CRISPR nucleases provided herein may include NLS and/or TAGs inserted so as to interrupt the contiguous amino acid or nucleic acid sequences of the CRISPR nucleases.
  • the one or more NLSs are in tandem repeats.
  • the one or more NLSs are considered in proximity to the N- or C- terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • CRISPR nuclease may be engineered to comprise one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags.
  • NLS nuclear localization sequences
  • cell penetrating peptide sequences cell penetrating peptide sequences
  • affinity tags affinity tags
  • the CRISPR nuclease exhibits increased specificity to a target site compared to the wild-type of the CRISPR nuclease when complexed with the one or more RNA molecules.
  • the complex of the CRISPR nuclease and one or more RNA molecules exhibits at least maintained on-target editing activity of the target site and reduced off-target activity compared to the wild-type of the CRISPR nuclease.
  • the composition further comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to the nucleotide acid molecule comprising the sequence encoding the CRISPR nuclease.
  • the CRISPR nuclease or nucleic acid molecule comprising a sequence encoding the CRISPR nuclease is non-naturally occurring or engineered.
  • This invention also provides a non-naturally occurring or engineered composition comprising a vector system comprising the nucleic acid molecule comprising a sequence encoding any of the CRISPR nucleases of the invention.
  • This invention also provides a method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell any of the compositions of the invention.
  • the cell is a eukaryotic cell.
  • compositions of the invention for the treatment of a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subject.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: l or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:23.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: l or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO: 51.
  • the composition comprises an RNA molecule comprising a nuclease-binding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs:73-79 and is suitable to form an active complex with the CRISPR nuclease.
  • the composition comprises an RNA molecule comprising a nuclease-binding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs: 88-91 and is suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant selected from the group consisting of AGGGNNNN, CGGGNNNN, TGGGNNNN, GGGTNNNN, NGGNNNNN, CGGTCGAA, TGGTCCGC, and AGGACCTC.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 80 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:8l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 82 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 83 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 87-91 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO:88.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of AGGGNNNN, CGGGNNNN, TGGGNNNN, GGGTNNNN, NGGNNNNN, CGGTCGAA, TGGTCCGC, AGGACCTC, NGGNN, NGGNM, and NGG.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:2 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 24. [00115] In an embodiment of the composition, the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:2 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO: 52.
  • the composition comprises an RNA molecule comprising a nuclease-binding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs: 105-107 and is suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant selected from the group consisting of NGCACNNN, NATACNNN, NGTACNNN, CGTANNNN, NRTAHNNN, TGTACTAA, TATACGAA, TGCACTAA.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 108 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 109 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 110 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 111 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 115-116 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 115.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NGCACNNN, NATACNNN, NGTACNNN, CGTANNNN, NRTAHNNN, TGTACTAA, TATACGAA, TGCACTAA, NVYAH, and YGTAM.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 3 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO:31 or SEQ ID NO:25.
  • composition wherein the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 3 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to SEQ ID NO:53.
  • the composition comprises an RNA molecule comprising a nuclease-binding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs: 127-128 and is suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant selected from the group consisting of NNAAACNN, NCAAANNN, CGGANNNN, NNGAAGNN, NRRARNNN, TGGAAGCT, and AAAAAGCT.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 129 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 130 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 131 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 132 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 133-134 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-137 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 136.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NNAAACNN, NCAAANNN, CGGANNNN, NNGAAGNN, NRRARNNN, TGGAAGCT, AAAAAGCT, NVVRR, NRRRR, NVVRV, NRRAV, CGGGAGAG, TAAGGTCC, TGGTCCGC and GGATGAT.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:4 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 26, and SEQ ID NO: 54.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 143 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 144 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 145 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 146 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 147, GGCUUCGCC, and SEQ ID NO: 148 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 149-150 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 150.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of TGCACTAA, AGGACCTC, NVNVMY, NRNACY, NVDNMY, NRKACY.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:5 or the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease has at least a 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 55.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 157 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 158 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 159 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 160 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 161, AUUAUUAU, and SEQ ID NO: 162 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 163-164 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 164.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NNYVVH, NNYAAH, GGTAATAG, and GGCAAAAG.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 6 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 56.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 170 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 171 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 172 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 173 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 174, AGCUUAUGC, and SEQ ID NO: 175 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 176-179 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 177.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NGGNN, NGGNM, TGGAAGCT, TGGTCCGC, TGGTTGAT, and CGGTCGAA .
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 7 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:27, and SEQ ID NO:57.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 186 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 187 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 188 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 189 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 193-194 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 194.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NRTAN, TGCACTAA, TATACGAA.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 8 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO:58.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 199 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:200 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:20l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:202 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 203-205 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 206-207 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO:207.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NGGNR, NGGNG, AGGACCTC, TGGCGTTG.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 9 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO:59.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 214 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 215 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 216 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 217 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 218, 219, and 220 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 221-222 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO:222 .
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NVRNH, NRRNC, NVRNC, AGGACCTC, TGGCGTTG, and TAGGCTCT.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 10 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:38 and SEQ ID NO:60.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:229 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:230 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:23 l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:232 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:233 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:234, AGUUUACU, and SEQ ID NO: 235, and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:236-237 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 11 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:28, and SEQ ID NO:6l.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:238 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:239 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:240 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:24l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:242 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:243, 244, and 245 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:246-247 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence SEQ ID NO:247 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NGGNN, NGGYK, NGGN, NGGY, and TGGTTGAT.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 12 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:40 and SEQ ID NO:62.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:248 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:249 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:250 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:25l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:252 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:253, 254, and 255 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 256-257 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence SEQ ID NO:257 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NVVHHY, NRRTTT, CAGTTTAA, and CAATTTAA.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 13 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:4l and SEQ ID NO:63.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:258 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:259 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:260 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:26l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:262 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:263, 264, and 265 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:266-267 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence SEQ ID NO:267 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NNGWWB, NHGWWY, GTGTACTC, and GTGTTCTC.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 14 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:64.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:268 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:269 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:270 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:27l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 272, 273 and 274 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 275-276 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 15 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:43 and SEQ ID NO:65.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:277 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:278 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:279 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:280 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:28l, 282, and 283 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:284-285 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence SEQ ID NO:285 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NRAVR and NRHAAC.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 16 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:44 and SEQ ID NO:66.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:286 to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:287 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:288 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:289 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 290, 291, and 292 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 293-294 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence SEQ ID NO:293 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NRAVR and NAARG.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 17 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:45 and SEQ ID NO:67.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:295 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:296 to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:297 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:298 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence SEQ ID NO:299 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID N0s:300-30l suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 18 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:46 and SEQ ID NO:68.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:302 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:303 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:304 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:305 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 306, GCUUAAAGC, and SEQ ID NO:307, and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID N0s:308-309 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 19 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:69.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:3 lO suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:3 l l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:3 l2 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:3 l3 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 314, 315 and 316 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:3 l7-3 l8 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NNDVYY and NNDAYT.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:20 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:70 and SEQ ID NO: 319.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:320 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:32l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:322 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:323 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:324, 325, and 326 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 327-328 suitable to form an active complex with the CRISPR nuclease.
  • OMNI 30 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 327-328 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:2l or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:49 and SEQ ID NO:7l.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:329 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:330 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:33 l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:332 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:333, 334, and 335 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:336-337 suitable to form an active complex with the CRISPR nuclease.
  • the CRISPR nuclease may use a PAM site or variant as selected from the group consisting of NNNVYT and NNNACT.
  • the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:22 or the sequence encoding the CRISPR nuclease has at least a 95% sequence identity to nucleotide sequence selected from the group consisting of SEQ ID NO:50 and SEQ ID NO:72.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 338 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 339 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:340 suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:34l suitable to form an active complex with the CRISPR nuclease.
  • the composition further comprises a single guide
  • RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 342, 343, and 344 and is suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:345-346 suitable to form an active complex with the CRISPR nuclease.
  • This invention provides a method of modifying a nucleotide sequence at a target site in the genome of a mammalian cell comprising introducing into the cell (i) a composition comprising a CRISPR nuclease having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9 or a nucleic acid molecule comprising a sequence encoding a CRISPR nuclease which sequence has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:23-27 and (ii) a DNA-targeting RNA molecule, or a DNA polynucleotide encoding a DNA-targeting RNA molecule, comprising a nucleotide sequence that is complementary to a sequence in the target DNA.
  • the method further comprises introducing into the cell: (iii) an RNA molecule comprising a nuclease-binding RNA sequence or a DNA polynucleotide encoding an RNA molecule comprising a nuclease-binding RNA that interacts with the CRISPR nuclease.
  • the DNA targeting RNA molecule is a crRNA molecule suitable to form an active complex with the CRISPR nuclease.
  • the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule suitable to form an active complex with the CRISPR nuclease.
  • the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule.
  • the method further comprises introducing into the cell: (iv) an RNA molecule comprising a sequence complementary to a protospacer sequence.
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a Protospacer Adjacent Motif (PAM).
  • PAM Protospacer Adjacent Motif
  • the method is for treating a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subject.
  • the method comprises first selecting a subject afflicted with a disease associated with a genomic mutation, and obtaining the cell from the subject.
  • This invention also provides a modified cell or cells obtained by any of the methods described herein. In an embodiment these modified cell or cells are capable of giving rise to progeny cells. In an embodiment these modified cell or cells are capable of giving rise to progeny cells after engraftment.
  • This invention also provides a composition comprising these modified cells and a pharmaceutically acceptable carrier. Also provided is an in vitro or ex vivo method of preparing this, comprising mixing the cells with the pharmaceutically acceptable carrier.
  • the CRISPR nuclease has at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 1 or the sequence encoding the CRISPR nuclease has at least 95% identity to the nucleic acid sequence as set forth in SEQ ID NO: 23.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 80 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 81 suitable to form an active complex with the CRISPR nuclease;
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 82 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 83 suitable to form an active complex with the CRISPR nuclease; or
  • the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86 and is suitable to form an active complex with the CRISPR nuclease; optionally the single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 87-91 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO:88; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of AGGGNNNN, CGGGNNNN, TGGGNNNN, GGGTNNNN, NGGNNNNN, CGGTCGAA, TGGTCCGC, and AGGACCTC, to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is ELANE g58;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence AGG or AGGACCCA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO:92;
  • the target site in the genome is ELANE g35;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence GGGGAGCA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO:93;
  • the target site in the genome is ELANE g39;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence GGGGACGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO:94;
  • the target site in the genome is ELANE g62;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence GGGACAGA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 95;
  • the target site in the genome is CXCR4 sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CGGAGGAG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 96;
  • the target site in the genome is CXCR4 s2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence AGGATGGC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 97;
  • the target site in the genome is PD 1 sl ;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence TGGCCAGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 98; or h)
  • the target site in the genome is PD1 s2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence TGGGCGGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 99.
  • the CRISPR nuclease has at least 95% identity to an amino acid sequence as set forth in SEQ ID NO:2 or the sequence encoding the CRISPR nuclease has at least 95% identity to the nucleic acid sequence as set forth in SEQ ID NO: 24.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 108 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 109 suitable to form an active complex with the CRISPR nuclease; b) the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 110 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 111 suitable to form an active complex with the CRISPR nuclease
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 115; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NGCACNNN, NATACNNN, NGTACNNN, CGTANNNN, NRTAHNNN, TGTACTAA, TATACGAA, TGCACTAA to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is 9q3 l.2 sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CATACTTG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 117;
  • the target site in the genome is 9q3 l.2 s3;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CCTACAAA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 118;
  • the target site in the genome is HBB;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence GATACCAA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 119;
  • the target site in the genome is 20ql 1.1;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence ACTACAGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 120;
  • the target site in the genome is FANCF sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence ACTACCTA; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 121; or
  • the target site in the genome is VISTA Enhancer hs267 s2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence TTTACAGG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 122.
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO:3 or the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease has least 95% identity to the nucleic acid sequence as set forth in SEQ ID NO:25.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 129 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 130 suitable to form an active complex with the CRISPR nuclease;
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 131 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 132 suitable to form an active complex with the CRISPR nuclease; or
  • the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule comprising the nucleotide sequence as set forth in SEQ ID NOs: 133 or l34and is suitable to form an active complex with the CRISPR nuclease; optionally the single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-137 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 136; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NNAAACNN, NCAAANNN, CGGANNNN, NNGAAGNN, NRRARNNN, TGGAAGCT, AAAAAGCT, NVVRR, NRRRR, NVVRV, NRRAV, CGGGAGAG, TAAGGTCC, TGGTCCGC and GGATGAT to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR s9;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in AAGAGACC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 140;
  • the target site in the genome is CXCR sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in CGGAGGAG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 141; or c) (i) the target site in the genome is ELANE
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO:4 or the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease has least 95% identity to the nucleic acid sequence as set forth in SEQ ID NO: 26.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth SEQ ID NO: 143 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 144 suitable to form an active complex with the CRISPR nuclease;
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 145 suitable to form an active complex with the
  • the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 146 suitable to form an active complex with the CRISPR nuclease; or c) the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 147, GGCUUCGCC, or SEQ ID NO: 148 and is suitable to form an active complex with the CRISPR nuclease; optionally the single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 149-150 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 150; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of TGCACTAA, AGGACCTC, NVNVMY, NRNACY, NVDNMY, NRKACY to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR4 s3;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in GACACTCC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 151;
  • the target site in the genome is CXCR4 s4;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in CCCACTAC;
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 152;
  • the target site in the genome is PD1 s3;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break
  • the target site in the genome is PD1 s4;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in GCCACTCC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 156.
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 5.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth SEQ ID NO : 157 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 158 suitable to form an active complex with the CRISPR nuclease; b) the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 159 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 160 suitable to form an active complex with the CRISPR nuclease
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 164; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NNYVVH, NNYAAH, GGTAATAG, and GGCAAAAG to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR4 s6;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CCCAATAT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 167;
  • the target site in the genome is PD1 s5;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence GACAATGG;
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 168; or c) (i) the target site in the genome is PD1 s6; (i
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO:6.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 170 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 171 suitable to form an active complex with the CRISPR nuclease; b) the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 172 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 173 suitable to form an active complex with the CRISPR nucleas
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 177; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NGGNN, NGGNM, TGGAAGCT, TGGTCCGC, TGGTTGAT, and CGGTCGAA to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR4 sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CGGAGGAG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 182;
  • the target site in the genome is CXCR4 s2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence AGGATGGC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 183;
  • the target site in the genome is PD 1 sl ;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence TGGCCAGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 184; or
  • the target site in the genome is PD1 s2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence TGGGCGGT; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 185.
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 7 or the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease has least 95% identity to the nucleic acid sequence as set forth in SEQ ID NO:27. [00335] In an embodiment of the method:
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 186 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 187 suitable to form an active complex with the CRISPR nuclease;
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 188 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 189 suitable to form an active complex with the CRISPR nuclease; or
  • the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule comprising the nucleotide sequence as set forth in SEQ ID NOs: l90, 191, or 192 and is suitable to form an active complex with the CRISPR nuclease; optionally the single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 193-194 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 194; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NRTAN, TGCACTAA, TATACGAA to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR s7;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in AATATACC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 197; or
  • the target site in the genome is CXCR s8;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in GATAAACA;
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 198.
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 8.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 199 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:200 suitable to form an active complex with the CRISPR nuclease; b) the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:20l suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO:202 suitable to form an active complex with the CRISPR nuclease;
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO:207; optionally the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NGGNR, NGGNG, AGGACCTC, TGGCGTTG to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR4 sl;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence CGGAGGAG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO:2lO;
  • the target site in the genome is CXCR4 slO;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence AGGAGCGC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO:2l 1;
  • the target site in the genome is PD 1 sl
  • the CRISPR nuclease has least 95% identity to an amino acid sequence as set forth in SEQ ID NO:9.
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 214 suitable to form an active complex with the CRISPR nuclease and/or the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 215 suitable to form an active complex with the CRISPR nuclease;
  • the DNA targeting RNA molecule is a crRNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 216 suitable to form an active complex with the CRISPR nuclease; and/or the RNA molecule comprising a nuclease-binding RNA sequence is a
  • the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-biding RNA sequence are fused in the form of a single guide RNA molecule comprising the nucleotide sequence as set forth in SEQ ID NOs: 218, 219, or 220 and is suitable to form an active complex with the CRISPR nuclease; optionally the single guide RNA molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 221-222 suitable to form an active complex with the CRISPR nuclease.
  • the single guide RNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO:222
  • the active complex of the CRISPR nuclease may use a PAM site or variant comprising a sequence selected from the group consisting of NVRNH, NRRNC, NVRNC, AGGACCTC, TGGCGTTG, and TAGGCTCT to modify the nucleotide sequence at the target site in the cell.
  • the target site in the genome is CXCR4 sl2;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in CAAACGCG; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 225;
  • the target site in the genome is CXCR4 sl3;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in TAAACACG;
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 226;
  • the target site in the genome is PD1 sl3;
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 226;
  • the target site in the genome is PD 1 sl4;
  • the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a PAM site comprising the nucleotide sequence as set forth in CAGACGAC; and
  • the protospacer region comprises a nucleotide sequence as set forth in SEQ ID NO: 228.
  • Certain embodiments of the invention target specific genetic loci associated with disease or disease therapies.
  • Mutations in the ELANE gene are associated with neutropenia. Accordingly, without limitation, embodiments of the invention that target ELANE may be used in methods of treating subjects afflicted with neutropenia.
  • CXCR4 is a co-receptor for the human immunodeficiency virus type 1 (HIV-l) infection. Accordingly, without limitation, embodiments of the invention that target CXCR4 may be used in methods of treating subjects afflicted with HIV-l or conferring resistance to HIV-l infection in a subject.
  • HIV-l human immunodeficiency virus type 1
  • PD-l Programmed cell death protein 1
  • PD-l may be a target in other cancer therapies. Accordingly, without limitation, embodiments of the invention that target PD-l may be used in methods of treating subjects afflicted with cancer.
  • the treatment is CAR T cell therapy with T cells that have been modified according to the invention to be PD-l deficient.
  • 20ql l.l and 9q3 l are genetic loci associated with cancer. Warren, H., et al. (2012). 9q3 l. 2-rs865686 as a susceptibility locus for estrogen receptor-positive breast cancer: evidence from the Breast Cancer Association Consortium. Cancer Epidemiology and Prevention Biomarkers , 27(10), 1783-1791. Accordingly, without limitation, embodiments of the invention that target 9q31 or 20ql 1.1 may be used in methods of treating subjects afflicted with cancer.
  • HBB is a globin protein. Mutations in the gene produce several variants of the proteins which are implicated with genetic disorders such as sickle-cell disease and beta thalassemia, as well as beneficial traits such as genetic resistance to malaria. Accordingly, without limitation, embodiments of the invention that target HBB may be used in methods of treating subj ects afflicted with sickle-cell disease and beta thalassemia or to confer genetic resistance to malaria.
  • Embodiments of the invention may also be used for studying any of the above diseases or any disease associated with the genetic locus that is targeted. Definitions
  • adjectives such as“substantially” and“about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended.
  • the word“or” in the specification and claims is considered to be the inclusive“or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.
  • each of the verbs,“comprise,” “include” and“have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • Other terms as used herein are meant to be defined by their well-known meanings in the art.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonueleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, in Irons, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shR A), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers,
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • nucleotide analog or “modified nucleotide” refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions), in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)), in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate.
  • RNA sequences described herein may comprise one or more nucleotide analogs.
  • nucleotide identifiers are used to represent a referenced nucleotide base(s):
  • the term“targeting sequence” or“targeting molecule” refers a nucleotide sequence or molecule comprising a nucleotide sequence that is capable of hybridizing to a specific target sequence, e.g., the -targeting sequence has a nucleotide sequence which is at least partially complementary to the sequence being targeted along the length of the targeting sequence .
  • the targeting sequence or -targeting molecule may be part of an RNA molecule that can form a complex with a CRISPR nuclease with the targeting sequence serving as the targeting portion of the CRISPR complex.
  • RNA molecule When the molecule having the targeting sequence is present contemporaneously with the CRISPR molecule the RNA molecule is capable of targeting the CRISPR nuclease to the specific target sequence.
  • RNA molecule can be custom designed to target any desired sequence.
  • targets refers to a targeting sequence or targeting molecule’s preferential hybridization to a nucleic acid having a targeted nucleotide sequence. It is understood that the term “targets” encompasses variable hybridization efficiencies, such that there is preferential targeting of the nucleic acid having the targeted nucleotide sequence, but unintentional off-target hybridization in addition to on-target hybridization might also occur. It is understood that where an RNA molecule targets a sequence, a complex of the RNA molecule and a CRISPR nuclease molecule targets the sequence for nuclease activity.
  • the targeting encompasses hybridization of the guide sequence portion of the RNA molecule with the sequence in one or more of the cells, and also encompasses hybridization of the RNA molecule with the target sequence in fewer than all of the cells in the plurality of cells. Accordingly, it is understood that where an RNA molecule targets a sequence in a plurality of cells, a complex of the RNA molecule and a CRISPR nuclease is understood to hybridize with the target sequence in one or more of the cells, and also may hybridize with the target sequence in fewer than all of the cells.
  • the complex of the RNA molecule and the CRISPR nuclease introduces a double strand break in relation to hybridization with the target sequence in one or more cells and may also introduce a double strand break in relation to hybridization with the target sequence in fewer than all of the cells.
  • modified cells refers to cells in which a double strand break is effected by a complex of an RNA molecule and the CRISPR nuclease as a result of hybridization with the target sequence, i.e. on- target hybridization.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. Accordingly, as used herein, where a sequence of amino acids or nucleotides refers to a wild type sequence, a variant refers to variant of that sequence, e.g., comprising substitutions, deletions, insertions.
  • an engineered CRISPR nuclease is a variant CRISPR nuclease comprising at least one amino acid modification (e.g., substitution, deletion and/or insertion) compared to the CRISPR nuclease of any of SEQ ID NOs: l-22.
  • nucleic acid molecules or polypeptides may mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or I, optical isomers, and amino acid analogs and peptidomimetics.
  • genomic DNA refers to linear and/or chromosomal DNA and/or to plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest.
  • the cell of interest is a eukaryotic cell.
  • the cell of interest is a prokaryotic cell.
  • the methods produce double-stranded breaks (DSBs) at pre-determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the target site(s) in a genome.
  • Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
  • nuclease refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acid.
  • a nuclease may be isolated or derived from a natural source. The natural source may be any living organism. Alternatively, a nuclease may be a modified or a synthetic protein which retains the phosphodiester bond cleaving activity.#
  • PAM refers to a nucleotide sequence of a target DNA located in proximity to the targeted DNA sequence and recognized by the CRISPR nuclease.
  • the PAM sequence may differ depending on the nuclease identity.
  • RNA molecules capable of complexing with a nuclease, e.g. a CRISPR nuclease, such as to associate with a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • a CRISPR nuclease and a targeting molecule form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence.
  • CRISPR nucleases may form a CRISPR complex comprising a CRISPR nuclease and RNA molecule without a further tracrRNA molecule.
  • CRISPR nucleases may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule.
  • protein binding sequence or“nuclease binding sequence” refers to a sequence capable of binding with a CRISPR nuclease to form a CRISPR complex.
  • a tracrRNA capable of binding with a CRISPR nuclease to form a CRISPR complex comprises a protein or nuclease binding sequence.
  • An“RNA binding portion” of a CRISPR nuclease refers to a portion of the CRISPR nuclease which may bind to an RNA molecule to form a CRISPR complex, e.g. the nuclease binding sequence of a tracrRNA molecule.
  • An“activity portion” or“active portion” of a CRISPR nuclease refers to a portion of the CRISPR nuclease which effects a double strand break in a DNA molecule, for example when in complex with a DNA-targeting RNA molecule.
  • RNA molecule refers to a sequence sufficiently complementary to a tracrRNA molecule so as to hybridize to the tracrRNA via basepairing and promote the formation of a CRISPR complex. ⁇ See US Patent No. 8906616).
  • the RNA molecule may further comprise a portion having a tracr mate sequence.
  • the targeting molecule may further comprise the sequence of a tracrRNA molecule.
  • a tracrRNA molecule may be designed as a synthetic fusion of the guide portion of the RNA molecule (gRNA or crRNA) and the trans-activating crRNA (tracrRNA), together forming a single guide RNA (sgRNA).
  • gRNA or crRNA the guide portion of the RNA molecule
  • tracrRNA trans-activating crRNA
  • sgRNA single guide RNA
  • Embodiments of the present invention may also form CRISPR complexes utilizing a separate tracrRNA molecule and a separate RNA molecule comprising a guide sequence portion.
  • the tracrRNA molecule may hybridize with the RNA molecule via base pairing and may be advantageous in certain applications of the invention described herein.
  • an RNA molecule may comprise a“nexus” region and/or“hairpin” regions which may further define the structure of the RNA molecule.
  • direct repeat sequence refers to two or more repeats of a specific amino acid sequence of nucleotide sequence.
  • an RNA sequence or molecule capable of“interacting with” or“binding” with a CRISPR nuclease refers to the RNA sequence or molecules ability to form a CRISPR complex with the CRISPR nuclease.
  • operably linked refers to a relationship (i.e. fusion, hybridization) between two sequences or molecules permitting them to function in their intended manner.
  • a promoter when an RNA molecule is operably linked to a promoter, both the RNA molecule and the promotor are permitted to function in their intended manner.
  • heterologous promoter refers to a promoter that does not naturally occur together with the molecule or pathway being promoted.
  • a sequence or molecule has an X%“sequence identity” to another sequence or molecule if X% of bases or amino acids between the sequences of molecules are the same and in the same relative position.
  • a first nucleotide sequence having at least a 95% sequence identity with a second nucleotide sequence will have at least 95% of bases, in the same relative position, identical with the other sequence.
  • nuclear localization sequence and "NLS” are used interchangeably to indicate an amino acid sequence/peptide that directs the transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier.
  • the term “NLS” is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier.
  • NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide.
  • NLS nucleoplasmin
  • the nucleoplasm bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:348); the c-myc NLS having the amino acid sequence PAA RV LD (SEQ ID NO:349) or RQRR.NELKRSP (SEQ ID NO:350); the hRNPAl M9 NLS having the sequence NQSSNFGPM GGNF GGRS S GP Y GGGGQ YF AKPRNQGGY (SEQ ID NO:35 l); the sequence R RIZFK KGKDTALLRRRRVE 7S 7ELRKAKKDEQILKRRNV (SEQ ID NO:352) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:353) and PPKKARED (SEQ ID NO:354) of the myoma T protein; the sequence PQP KKPL (SEQ ID NO:355) of human p53; the sequence SAII KKKKM AP (SEQ ID NO:35
  • the CRISPR nuclease or CRISPR compositions described herein may be delivered as a protein, DNA molecules, RNA molecules, Ribonucleoproteins (RNP), nucleic acid vectors, or any combination thereof.
  • the RNA molecule comprises a chemical modification.
  • suitable chemical modifications include 2'-0-methyl (M), 2'-0 ⁇ methyl, 3'phosphorothioate (MS) or 2'-0-m ethyl, 3 'thioPACE (MSP), pseudouridine, and 1- methyl pseudo-uridine.
  • M 2'-0-methyl
  • MS 3'phosphorothioate
  • MSP 3 'thioPACE
  • pseudouridine pseudouridine
  • 1- methyl pseudo-uridine 1- methyl pseudo-uridine.
  • the CRISPR nucleases and/or polynucleotides encoding same described herein, and optionally additional proteins (e.g., ZFPs, TALENs, transcription factors, restriction enzymes) and/or nucleotide molecules such as guide RNA may be delivered to a target cell by any suitable means.
  • the target cell may be any type of cell e.g., eukaryotic or prokaryotic, in any environment e.g., isolated or not, maintained in culture, in vitro, ex vivo, in vivo or in planta.
  • the composition to be delivered includes mRNA of the nuclease and RNA of the guide. In some embodiments, the composition to be delivered includes mRNA of the nuclease, RNA of the guide and DNA donor template. In some embodiments, the composition to be delivered includes the CRISPR nuclease and guide RNA. In some embodiments, the composition to be delivered includes the CRISPR nuclease, guide RNA and DNA donor template for homology directed repair. In some embodiments, the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA.
  • the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA and DNA donor template. In some embodiments, the composition to be delivered includes the CRISPR nuclease DNA-targeting RNA and the tracrRNA. In some embodiments, the composition to be delivered includes the CRISPR nuclease, DNA-targeting RNA and the tracrRNA and DNA donor template for homology directed repair.
  • Any suitable viral vector system may be used to deliver RNA compositions.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids and/or CRISPR nuclease in cells (e.g., mammalian cells, plant cells, etc.) and target tissues. Such methods can also be used to administer nucleic acids encoding and/or CRISPR nuclease protein to cells in vitro.
  • nucleic acids and/or CRISPR nuclease are administered for in vivo or ex vivo gene therapy uses.
  • Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Methods of non-viral delivery of nucleic acids and/or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, artificial virions, and agent- enhanced uptake of nucleic acids or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus. See, e.g., Chung et ak (2006) Trends Plant Sci.
  • bacteria or viruses e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus. See,
  • Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
  • Cationic-lipid mediated delivery of proteins and/or nucleic acids is also contemplated as an in vivo or in vitro delivery method. See Zuris et ak (2015) Nat. Biotechnol. 33(l):73-80. See also Coelho et ak (2013) N. Engl. J. Med. 369, 819-829; Judge et ak (2006) Mol. Ther. 13, 494-505; and Basha et ak (2011) Mol. Ther. 19, 2186-2200.
  • nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc., (see for example U.S. Patent No. 6,008,336).
  • Lipofection is described in e.g., U.S. Patent No. 5,049,386, U.S. PatentNo. 4,946,787; and U.S. Patent No. 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM., Lipofectin.TM. and Lipofectamine.TM. RNAiMAX).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes
  • Boese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4, 186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs).
  • EDVs EnGeneIC delivery vehicles
  • These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV.
  • the antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiamid et al (2009) Nature Biotechnology 27(7) p. 643).
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer.
  • an RNA virus is preferred for delivery of the RNA compositions described herein.
  • Nucleic acid of the invention may be delivered by non integrating lentivirus.
  • RNA delivery with Lentivirus is utilized.
  • the lentivirus includes mRNA of the nuclease, RNA of the guide.
  • the lentivirus includes mRNA of the nuclease, RNA of the guide and DNA donor template.
  • the lentivirus includes the nuclease protein, guide RNA.
  • the lentivirus includes the nuclease protein, guide RNA and/or DNA donor template for homology directed repair.
  • the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA.
  • the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA, and DNA donor template.
  • the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA.
  • the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA, and DNA donor template for homology directed repair.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g. Buchscher et ah, J. Virol. 66:2731-2739 (1992); Johann et ak, J. Virol. 66: 1635-1640 (1992); Sommerfelt et ah, Virol. 176:58-59 (1990); Wilson et ah, J. Virol. 63 :2374-2378 (1989); Miller et ah, J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immunodeficiency virus
  • HAV human immunodeficiency virus
  • At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
  • pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et ah, Blood 85:3048-305 (1995); Kohn et ah, Nat. Med. 1 : 1017-102 (1995); Malech et ah, PNAS 94:22 12133-12138 (1997)).
  • PA3 l7/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1): 10-20 (1997); Dranoff et al., Hum. Gene Ther. 1 : 111-2 (1997).
  • Packaging cells are used to form virus particles that are capable of infecting a host cell.
  • Such cells include 293 cells, which package adenovirus, AAV, and psi.2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed.
  • the missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
  • ITR inverted terminal repeat
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced at clinical scale using baculovirus systems (see U.S. Patent No. 7,479,554).
  • a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et ak, Proc. Natl. Acad. Sci.
  • Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • delivery of mRNA in-vivo and ex-vivo, and RNPs delivery may be utilized.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with an RNA composition, and re-infused back into the subject organism (e.g., patient).
  • RNA composition e.g., RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA-derived RNA composition
  • RNA composition e.g., RNA composition
  • RNA composition e.g., RNA composition
  • RNA composition e.g., RNA composition
  • RNA composition e.g., RNA composition
  • RNA composition e.g., RNA composition suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et ah, Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of
  • Suitable cells include but not limited to eukaryotic and prokaryotic cells and/or cell lines.
  • Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB 11, CHO-DUKX, CHOK1 SV), VERO, MDCK, WI38, V79, B14AF28-G3, BEK, HaK, NSO, SP2/0-Agl4, HeLa, HEK293 (e.g, HEK293-F, HEK293-H, HEK293-T), and perC6 cells, any plant cell (differentiated or undifferentiated) as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces.
  • COS e.g., CHO-S, CHO-K1, CHO-DG44, CHO
  • the cell line is a CHO- Kl, MDCK or HEK293 cell line.
  • primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with the nucleases (e.g. ZFNs or TALENs) or nuclease systems (e.g. CRISPR).
  • Suitable primary cells include peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in-vitro or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-gamma. and TNF-alpha are known (as a non-limiting example see, Inaba et ak, J. Exp. Med. 176: 1693-1702 (1992)).
  • cytokines such as GM-CSF, IFN-gamma. and TNF-alpha are known (as a non-limiting example see, Inaba et ak, J. Exp. Med. 176: 1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods.
  • stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-l (granulocytes), and lad (differentiated antigen presenting cells) (as a non-limiting example see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).
  • T cells CD4+ and CD8+
  • CD45+(panB cells) CD45+(panB cells)
  • GR-l granulocytes
  • lad differentiated antigen presenting cells
  • any one of the CRISPR nuclease described herein may be suitable for genome editing in post-mitotic cells or any cell which is not actively dividing, e.g., arrested cells.
  • Examples of post-mitotic cells which may be edited using a CRISPR nuclease of the present invention include, but are not limited to, myocyte, a cardiomyocyte, a hepatocyte, an osteocyte and a neuron.
  • Vectors e.g., retroviruses, liposomes, etc.
  • therapeutic RNA compositions can also be administered directly to an organism for transduction of cells in vivo.
  • naked RNA or mRNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Vectors suitable for introduction of transgenes into immune cells include non-integrating lentivirus vectors. See, for example, ET.S. Patent Publication No. 2009/0117617.
  • compositions are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, l7th ed., 1989). DNA Repair by Homologous Recombination
  • HDR refers to a mechanism for repairing DNA damage in cells, for example, during repair of double-stranded and single-stranded breaks in DNA.
  • HDR requires nucleotide sequence homology and uses a "nucleic acid template” (nucleic acid template or donor template used interchangeably herein) to repair the sequence where the double- stranded or single break occurred (e.g., DNA target sequence). This results in the transfer of genetic information from, for example, the nucleic acid template to the DNA target sequence.
  • HDR may result in alteration of the DNA target sequence (e.g., insertion, deletion, mutation) if the nucleic acid template sequence differs from the DNA target sequence and part or all of the nucleic acid template polynucleotide or oligonucleotide is incorporated into the DNA target sequence.
  • an entire nucleic acid template polynucleotide, a portion of the nucleic acid template polynucleotide, or a copy of the nucleic acid template is integrated at the site of the DNA target sequence.
  • nucleic acid template and“donor”, refer to a nucleotide sequence that is inserted or copied into a genome.
  • the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that will be added to or will template a change in the target nucleic acid or may be used to modify the target sequence.
  • a nucleic acid template sequence may be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), preferably between about 100 and 1,000 nucleotides in length (or any integer there between), more preferably between about 200 and 500 nucleotides in length.
  • a nucleic acid template may be a single stranded nucleic acid, a double stranded nucleic acid.
  • the nucleic acid template comprises a nucleotide sequence, e.g., of one or more nucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.
  • the nucleic acid template comprises a ribonucleotide sequence, e.g., of one or more ribonucleotides, that corresponds to wild type sequence of the target nucleic acid, e.g., of the target position.
  • the nucleic acid template comprises modified ribonucleotides.
  • donor sequence also called a "donor sequence,” donor template” or “donor”
  • donor sequence is typically not identical to the genomic sequence where it is placed.
  • a donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • the donor polynucleotide can be DNA or RNA, single-stranded and/or double- stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 20100047805; 20110281361; and 20110207221. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxy nucleotide residues are added to the 3' terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • a donor DNA template for HDR may use a DNA or RNA, single-stranded and/or double- stranded and can be introduced into a cell in linear or circular form.
  • a donor RNA template for HDR the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule.
  • a donor sequence may also be an oligonucleotide and be used for gene correction or targeted alteration of an endogenous sequence.
  • the oligonucleotide may be introduced to the cell on a vector, may be electroporated into the cell, or may be introduced via other methods known in the art.
  • the oligonucleotide can be used to correct ' a mutated sequence in an endogenous gene (e.g., the sickle mutation in beta globin), or may be used to insert sequences with a desired purpose into an endogenous locus.
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
  • viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)
  • the donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted.
  • the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
  • the donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
  • a transgene as described herein may be inserted into an endogenous locus such that some (N-terminal and/or C-terminal to the transgene) or none of the endogenous sequences are expressed, for example as a fusion with the transgene.
  • the transgene (e.g., with or without additional coding sequences such as forthe endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus, for example a CCR5 gene, a CXCR4 gene, a PPPlRl2c (also known as AAVS1) gene, an albumin gene or a Rosa gene.
  • a safe-harbor locus for example a CCR5 gene, a CXCR4 gene, a PPPlRl2c (also known as AAVS1) gene, an albumin gene or a Rosa gene.
  • the endogenous sequences When endogenous sequences (endogenous or part of the transgene) are expressed with the transgene, the endogenous sequences may be full-length sequences (wild-type or mutant) or partial sequences. Preferably the endogenous sequences are functional. Non- limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier.
  • the transgene e.g., therapeutic gene
  • exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the donor molecule comprises a sequence selected from the group consisting of a gene encoding a protein (e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein), a regulatory sequence and/or a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
  • each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment.
  • any of the RNA molecules or compositions of the present invention may be utilized in any of the methods of the present invention.
  • Nt-HA tag-OMNI nuclease open reading frame (Table 2) was cloned into pbNNC plasmid under the T7 promoter (Table 3). The sgRNA was cloned into bacterial plasmid under constitutive promoter. E. coli strain BW25141 (l ⁇ E3) was co-transformed with OMNI 6 and the corresponding guide and were co-expressed over-night by addition of 0.1 mM IPTG during mid exponential growth at 18 degrees Celsius. The cells were then pelleted and lysed, and the expression of OMNI 6 was detected by Western blot analysis using anti-HA antibodies.
  • Example IB PAM Depletion Assay of OMNI nucleases in bacterial
  • E. coli strain BW25141 (l ⁇ E3) were co-transformed with: (1) a library of plasmids containing a randomized PAM sequences of 8 N’s flanked by a unique protospacer, (2) plasmids encoding the OMNI 6 nuclease, and (3) a plasmid encoding gRNA targeting the protospacer of the library or a non-targeting gRNA as control. Next, cells were selected for all three plasmids by recovering them on media containing appropriate antibiotics.
  • Plasmids containing a PAM are cleaved and the cells that contain them could not grow, while cells containing plasmids with non- PAMs were able to propagate.
  • individual PAM sequences could be identified.
  • the most depleted PAM sites of OMNI 6 nuclease are presented in column 1 of Table 4. Based on these results the suggested consensus PAM sequence for OMNI 6 is NGGNNNNN.
  • E. coli strain BW25141 (l ⁇ E3) co-expressing the OMNI 6 nuclease and sgRNA were lysed using BugBusterTM lysis solution. The lysate was reacted in the recommended cleavage buffer with linear DNA substrates containing the PAM sequences flanked by a unique protospacer targeted by the sgRNA or a non-targeted protospacer as a control. Tested PAMs are summarized in the 3 rd column of Table 5, and editing activity in comparison to SpCas9 was demonstrated.
  • Example IE OMNI expression in mammalian cells
  • OMNI 6 The transcription levels of OMNI 6 was about 30% and approached 40% for 6f2, compared to less than 10% for Native and E+F (pCDNA3. l sp), as determined by the percentage of mCherry expressing cells out of the transfected population and after normalization to BFP levels to account for variation in transfection efficiency.
  • HEK 293T cells were transfected with pm-OMNI 6, sgRNA expressing vector pm- pGuide encoding for either 6a, 6al, 6a2 or 6f2, and the reporting vector.
  • Cells were analyzed by FACS 72 hours post transfection for GFP fluorescent quantification.
  • OMNI 6 showed editing activity on transient target DNA in mammalian cells, measured by gain of expression of GFP.
  • Native and E+F pCDNA3.1 sp
  • GFP disruption assay Activity of OMNI 6 in mammalian cells was also demonstrated using disruption of genomic integrated GFP reporter (GGGCACGGGCAGCTTGCCGGTGG - SEQ ID NO:370).
  • HEK 293T cells carrying a stably integrated GFP gene were transfected with a mix of pm-OMNI, encoding for SpCas9 or OMNI 6 or pcDNA3-SpCas9 and pm-Guide, as indicated in Fig. 4A. Cells were analyzed by FACS 72 hours post transfection for GFP fluorescent quantification.
  • OMNI 6 with guides 6a and 6f showed a GFP disruption of greater than 50%, similar to the disruption of the guide + pcDNA 3.1 sp and guide + pm-OMNIO -sp, calculated as percentage loss of mean GFP intensity (compared to the initial intensity deduced from the analysis of mock transfected, GFP integrated cells).
  • rs376l005 was performed. To this end, Hela cells were co-transfected with a plasmid encoding SpCas9 nuclease or OMNI 6, a pm-Guide encoding for rs376l005 spacer sequence (GCTGCGGGAAAGGGATTCCC - SEQ ID NO: 92), and SpCas9 guide or improved OMNI 6 guide (‘6f2’). Transient expression was achieved in a 24 well plate format using Turbofect reagent (Thermo fisher scientific). For negative control, Hela cells were transfected with a plasmid encoding SpCas9 nuclease or OMNI 6 without a guide. Cells were harvested 72 hours post DNA transfection. On target activity was demonstrated by DNA capillary electrophoresis.
  • Example 2 A Expression of OMNI 4 and OMNI 20 nuclease in bacterial
  • Example 2B PAM Depletion Assay of OMNI nucleases in bacterial
  • E. coli strain BW25141 (l ⁇ E3) were co-transformed with: (1) a library of plasmids containing a randomized PAM sequences of 8 N’s flanked by a unique protospacer, (2) plasmids encoding the OMNI nuclease, and (3) a plasmid encoding gRNA targeting the protospacer of the library or a non-targeting gRNA as control. Next, cells were selected for all three plasmids by recovering them on media containing appropriate antibiotics.
  • Plasmids containing a PAM are cleaved and the cells that contain them could not grow, while cells containing plasmids with non- PAMs were able to propagate.
  • individual PAM sequences could be identified.
  • the most depleted PAM sites of OMNI nuclease are presented in column 1 of Table 6B. Based on these results the suggested consensus PAM sequence for OMNI 4 is NRTAHNNN and the suggested consensus PAM sequence for OMNI 20 is NRRARNNN.
  • E. coli strain BW25141 (l ⁇ E3) co-expressing each of the OMNI nucleases and sgRNA were lysed using BugBusterTM lysis solution. The lysate was reacted in the recommended cleavage buffer with linear DNA substrates containing the PAM sequences flanked by a unique protospacer targeted by the sgRNA or a non-targeted protospacer as a control. Tested PAMs are summarized in the 3 rd column of Table 6B, and editing activity in comparison to SpCas9 was demonstrated.
  • Example 2E OMNI expression in mammalian cells
  • An expression vector carrying HA tagged OMNI nucleases linked by P2A peptide to mCherry was introduced into 293T cells using the TurbofectTMtransfection reagent (Thermo Fisher). The Relative level of transcription for OMNI was determined using Flow cytometry. Cell were transfected with pm-OMNI OR PCDNA3.1 and a carrier plasmid expressing BFP. Transfection was done as described above and cells were subjected to Flow cytometry analysis 72 hours post transfection. The mCherry protein expressed from the P2A polycentric transcript served as an indication for OMNI nucleases transcript levels.
  • OMNI 4 and OMNI 20 were above 30% and approached 40% for 20T 1 -D2 (pmOMNI20), similar to the expression level of 0T 1 pcDNA3.1 sp, as determined by the percentage of mCherry expressing cells out of the transfected population and after normalization to BFP levels to account for variation in transfection efficiency.
  • CRISPR repeat crRNA
  • transactivating crRNA tracrRNA
  • nucleases polypeptide PAM sequences were predicted from different metagenomic databases of sequences of environmental samples.
  • the list of bacterial species/strains from which the CRISPR repeat, tracRNA sequence and nucleases polypeptide sequence where predicted is provided in Table 1 and designated an OMNI identification number.
  • Example 3A Construction of OMNI-nuclease polypeptides.
  • Example 3B Prediction and construction of sgRNA.
  • the sgRNA was predicted by detection of the CRISPR repeat array sequence (crRNA) and a trans-activating crRNA (tracrRNA) in the bacterial genome.
  • the native pre-mature crRNA and tracrRNA sequences were connected in-silico with tetra-loop‘gaaa’ and the secondary structure elements of the duplex were predicted by using an RNA Secondary Structure prediction Web Tool: available at ma.urmc.roley.edu.
  • crRNA-tracrRNA chimera The predicted secondary structures of the full duplex RNA elements (crRNA-tracrRNA chimera) was used for identification of possible“Nexus” and“hairpins” the design of sgRNA for each nuclease with various versions (See, e.g., For example, Fig. 1A).
  • the crRNA and tracrRNA were connected with tetra-loop ‘gaaa’, thus generating possible sgRNA scaffolds (For example, Fig. IB).
  • At least 2 versions (labeled VI, V2, V3, etc.) of possible designed scaffolds for each OMNI was synthesized and 5’ connected to a 22 bps universal unique spacer sequence (SEQ ID NO:373 or 374) and was cloned into a bacterial Guide expressing plasmid under the constitutive promoter and mammalian expressing plasmid under U6 promoter (pbGuide and pmGuide according to Table 7 above).
  • Table 8 crRNA and tracrRNA for VI sgRNAs for each OMNI.
  • Table 11 Additional sgRNA designs for OMNI 18.
  • Table 12 Additional sgRNA designs for OMNI 20.
  • nexus and hairpin regions for sgRNAs for OMNI nucleases were identified as indicated in Table 13 below, with each nexus region indicated in bold and each hairpin region indicated in underline. Table 13 : nexus and hairpin regions for sgRNAs for OMNI nucleases
  • Example 3C Bacterial PAM Depletion Assay.
  • E.coli strain BW25141 (l ⁇ E3) were co-transformed with: (1) a library of plasmid pool containing a randomized PAM sequences of 8 N’s flanking a unique protospacer (pbPOS Tl library, Table 7 above), (2) plasmids encoding E.coli codon-optimized OMNI nucleases, pbNNC (Table 7) and (3) a plasmid encoding designed sgRNA targeting the protospacer of the library or a non-targeting gRNA as control (pbGuide, Tl and T2 respectively, Table 7).
  • plasmids containing a PAM are cleaved and the cells that contain them cannot grow, while cells containing plasmids with non-PAMs are able to propagate.
  • Survived plasmid DNA pool was isolated, and the library was sequenced using a 75-cycle NextSeq kit (Illumina).
  • PAM representation in the library was determined using a custom script and compared between OMNI and control. By comparing the frequency of a sequence in the library after selection of the targeting guide relative to a non-targeting guide, individual PAM sequences could be identified (Fig. 2A - Fig. 13B).
  • the presented data reflects a condensed 4N window library with all possible locations along the 8bp sequence. Sequence motifs were generated using the Weblogo tool (weblogo.berkeley.edu). Activity of the OMNI nuclease was estimated based on the average of the two most depleted sequences and was calculated as: l - Depletion score (Depletion score - Average of the ratios from 2 most depleted sites). OMNI nucleases with scores that are higher than 0.6 were considered as active. Following deep sequencing we detected depletion in 12 of the 22 tested OMNI systems, indicating functional DNA interference in a heterologous host (Fig. 2A - Fig. 13B). PAM sites and depletion assay results for each OMNI nuclease are shown in Table 14 below.
  • Table 14 OMNI Bacterial PAM Depletion Assay results.
  • Example 3D In-vitro Depletion assay by TXTL.
  • RNA expression and protein translation by the TXTL mix result in the formation of the RNP complex. Since linear DNA was used, Chi6 sequences, a RecBCD inhibitor were added to protect the DNA from degradation.
  • the sgRNA spacer is designed to target a library of plasmids containing the targeting protospacer (pbPOS Tl library, Table 7) flanked by an 8N’ randomized set of potential PAM sequences.
  • Depletion of PAM sequences from the library is measured by adding the adapters and indices necessary for high-throughput sequencing using PCR to both the cleaved library and to a control library expressing a non-targeting gRNA.
  • OMNI 4 OMNI 6, OMNI 8, OMNI 10, OMNI 13, OMNI 17, OMNI 18, OMNI 19, OMNI 20, OMNI 23, and OMNI 24 showing similar PAM pattern as discovered in E.coli ( Figure 14 - Figure 25).
  • we discover activity in 4 new OMNI nucleases OMNI 16, OMNI 21, OMNI 27 and OMNI 30), indicating functional DNA interference by in-vitro system (Fig. 26A - Fig. 29B).
  • PAM sites and depletion assay results for each tested OMNI nuclease are shown in Table 15 below.
  • Example 3D Verification of PAM by bacterial survival assay and in vitro
  • OMNI nucleases were further cloned downstream to the protospacer Tl into a positive selection plasmid harboring a toxic gene CcdB under control of BAD promoter (pbPOS Tl PAM site, Table 7). Electro-competent cells were prepared from Escherichia coli strain BW25141 (l ⁇ E3) co-transformed with each OMNI nuclease and the positive selection plasmid.
  • the targeting guide Tl spacer, pbGuide Tl
  • OMNI nucleases that cleave the positive plasmid can survive in the presence of Arabinose, whereas non-targeting guide (pbGuide T2) cannot.
  • OMNI nucleases were also tested for in vitro cleavage of different PAM sites.
  • E. coli strain BW25141 (l ⁇ E3) co-expressing the OMNI nucleases and sgRNA were lysed using BugBuster lysis solution. The lysate was reacted in the recommended cleavage buffer with linear DNA substrates containing the PAM sequences flanked by a unique protospacer targeted by the sgRNA (Tl) or a non-targeted protospacer (T2) as a control (Fig. 31A-C). Results of the bacterial survival assay and in vitro activity assay are shown in Table 16 below.
  • Table 16 Bacterial survival assay and in vitro activity assay results.
  • the PAM site identified for OMNI 20 was verified as NRRRR by full in vitro cleavage of TGGAA, CAAGG, TAAGG, partial cleavage of CGGAT, GGAAT, and no cleavage of TGGTC (Fig. 31B). This activity is dependent on the expression and transcription of the OMNI nucleases and their guide.
  • Example 3E Purification of OMNI proteins.
  • OMNI 4 and OMNI 6 open-reading frames were cloned into bacterial expression plasmids (T7-NLS-OMNI-NLS-HA-His-tag, pET9a, Table 8) and expressed in G10 cells (HI- ControlTM 10G Competent Cells, Lucigen). Cells were grown in Terrific Broth to mid-log phase and the temperature lowered to 18 °C. Expression was induced at 0.6 OD with O. lmM IPTG for 16-20 h before harvesting and freezing cells at -80 °C.
  • Cell paste was resuspended in lysis buffer (50 mM NaFbPOr, 300 mM NaCl,l0 mM imidazole pH8.0, lmM TCEP) supplemented with EDTA-free complete protease inhibitor cocktail set III (Calbiochem).
  • Cells were lysed using Constant systems TS-75 and cleared lysate was incubated with Ni-NTA resin.
  • the resin was loaded onto gravity column and washed with wash buffer (50 mM NaH2P0 4 ,300 mM NaCl, 50 mM imidazole pH8.0, lmM TCEP) and OMNI protein eluted with wash buffer supplemented with l00-500mM Imidazole.
  • OMNI protein Fractions containing OMNI protein were pooled and concentrated and loaded onto a centricone (Amicon ETltra l5ml 100K, Merck), and buffer exchanged to GF buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 10% glycerol, 0.4M Arginine).
  • the concentrated OMNI protein was further purified by SEC on HiLoad 16/600 Superdex 200 pg-SEC, ART A Pure (GE Healthcare Life Sciences) with a 50mM Tris-HCl pH 7.5, 500mM NaCl, 10% glycerol, 0.4M Arginine.
  • OMNI protein Fractions containing OMNI protein were pooled and concentrated and loaded onto a centricone (Amicon ETltra l5ml 100K, Merck) with a final storage buffer of lOmM Tris-HCl, pH 7.5, l50mM NaCl, 10% glycerol and lmM TCEP. Purified OMNI protein was concentrated to 1 - 4ug/ul stocks and flash-frozen in liquid nitrogen and stored at -80 °C. Purity of OMNI proteins was measured by SDS-PAGE analysis (Fig. 32A).
  • Example 3F In vitro activity assay by RNP.
  • Synthetic sgRNA of OMNI 4 and OMNI 6 were synthesized with three 2’-0-methyl 3’- phosphorothioate at the 3’ and 5’ ends (Agilent). RNPs were formed by incubating 1 mg/mL protein with ImM synthetic sgRNA at Room -temp for 10 min. RNPs were stored on ice until reacted with target DNA. The RNPs were reacted in the recommended cleavage buffer with lOOng of linear DNA substrates containing the protospacer targeted by the sgRNA flanking each OMNI PAM sequence (on-targets shown in Fig. 32B). The RNP of OMNI 6 was 5-fold diluted in storage buffer and reacted with both On and Off targets (single mismatch).
  • Example 3G Expression of OMNI nucleases coded by a codon optimized DNA sequence in mammalian cells.
  • P2A peptide is a self-cleaving peptide which can induce the cleaving of the recombinant protein in cell, such that the OMNI nucleases and the mCherry are separated upon expression and the mCherry can serve as indicator for transcription efficiency of the OMNI from expression vector.
  • the level of transcription for OMNI in the activity assays was determined using Flow cytometry.
  • Example 3H Activity in mammalian cells on plasmid target.
  • HEK 293T cells were transfected with the expression vector coding for an HA tagged OMNI nuclease linked by P2A to mCherry which serves as indicator for transfection efficiency of the vector.
  • An sgRNA expressing vector with a Tl spacer (pmGuide, Table 7), and the reporting vector expressing silenced GFP that is expressed upon editing and BFP which is constantly expressed and serves as indicator for transfection efficiency of the target plasmid (pMSS4c.2, Table 7).
  • pMSS4c.2, Table 7 For negative control cells were transfected with the reporter vector with only the OMNI nuclease or only the guide. Cells were analyzed by FACS 72 hours post transfection for GFP fluorescent quantification BFP and mCherry fluorescent quantification.
  • % editing presented in Figures 33 and 34 are determined by quantification of GFP signal as fraction of BFP signal and mCherry signal. As demonstrated in Figures 33 and 34 all of the OMNIs exhibited editing activity in HEK293T cells to various degrees, indicated by the normalized GFP signal which is significantly higher compared to the relevant control.
  • Table 18 Bacterial survival assay and in vitro activity assay results.
  • Example 31 Activity in human cells on endogenous genomic targets.
  • OMNIs were also assayed for their ability to promote editing on specific genomic locations in human cells.
  • the corresponding OMNI-P2A-mCherry expression vector (pmOMNI, Table 7) was transfected into HEK293 or Hela cells together with sgRNA designed to target specific location in the human genome (pmGuide, Table 7).
  • sgRNA designed to target specific location in the human genome
  • Amplicons were subjected to NGS and the resulting sequences were then used calculate the percentage of editing events in each target site. Short Insertions or deletions (InDels) around the cut site are the typical outcome of repair of DNA ends following nuclease induced DNA cleavage. The calculation of % editing was therefore deduced from the fraction of Indels containing sequences within each amplicon. With the exception of OMNI 4, all editing values were normalized to the transfection and translation eficasy obtained for each experimen and deduced from the percentage of mCherry expressing cells. The normalized values represent the effective editing levels within the population of cells that expressed the nucleases.
  • Genomic activity of each ONMI was assessed using a panel of 4 to 10 unique sgRNA each design to target a different genomic location. Each respective sgRNA target is shown in Table 20 below.
  • Table 20 sgRNA targets for mammalian assays.
  • Table 20 Genomic activity of each OMNI in mammalian cells.
  • OMNIs for example OMNI 4 and OMNI 6 exhibit high and significant editing levels compared to the negative control as seen in column 6 of Table 20“% editing in neg control”. In all or most target sites tested (3.85%-47.9%, on 8/8 sites; l.5%-33.8%, on 7/10 sites, OMNI 4 and OMNI 6 respectively).
  • OMNI 20 exhibited high and significant editing levels in 2/5 sites tested, OMNI 18 exhibit low yet significant activity across most tested sites (4/5) and OMNI 13, OMNI 23, and OMNI 24 showed significant yet low activity on one of the six sites tested.
  • Example 3J Intrinsic fidelity in human cell
  • the intrinsic fidelity of a nuclease is a measure of its cleavage specificity.
  • a high-fidelity nuclease is a nuclease that promotes cleavage on the intended target (“on-target”) with minimal or no cleavage of the unintended target (“off-target”).
  • the target is acquired based on sequence complementarity to the spacer element of the guide RNA. Off-targeting results from similarity of the unintended target to the spacer sequence.
  • the intrinsic fidelity of OMNIs at the genomic level in human cells was measured by conducting an activity assay as described in section xi, following PCR amplification NGS and InDel analysis for both the on-target region and a pre validated off target region.
  • a measurement of intrinsic fidelity for OMNI 6 is provided in Figure 36.
  • OMNI 6 fidelity was measured using two guide RNAs independently. In each case a side-by-side measurement of spCas9 is provided for reference.
  • the first site was targeted using the ELANE g35 gRNA targeting the ELANE g35 site specified in Table 19, which has a defined on-target upstream to the ELANE gene on chrl9 and an off-target on chrl5.
  • the on/off target editing efficiency ratio obtained by OMNI 6 was 17: 1 while spCas9 on/off ratio is 1.6: 1 (24.7%/l.4%; 64.8%/4l% respectively).
  • the second site was targeted using the ELANE g58 gRNA targeting the ELANE g25 site specified in Table 19.
  • This gRNA spacer sequence has a defined off-target in the genome in the form of heterozygous single nucleotide polymorphism (SNP) in the tested cell line. As a result, one allele is identical to the spacer (on-target) and the other allele contains a single mismatch and makes an extremely challenging off-target.
  • the On/Off ratio obtained by OMNI 6 was 2.43: 1 compared to the 1 : 1 ratio obtained by spCas9 (Fig. 36A).
  • the intrinsic high fidelity of OMNI- 6 in vitro was already at the RNP level (Fig. 32C). Taken together these results demonstrate that OMNI 6 has a significantly higher intrinsic fidelity in comparison to spCas9 in both in vitro and human cellular environment.
  • Example 3K Activity in human cells as RNPs.
  • Synthetic sgRNAs for OMNI 4 and OMNI 6 were synthesized with three 2’-0-methyl 3’-phosphorothioate at the 3’ and 5’ ends (Agilent). RNPs were formed by incubating 1 mg/mL protein with ImM synthetic sgRNA at room -temp for 10 min. RNPs were stored on ice until reacting with cells. U20S cells (ATCC HTB-96) were suspend, wash, and electroporate using Lonza SE Cell Line 4D-NucleofectorTM X Kit with DN100 program, according to the manufacture protocol. Negative control cells were electroporate with the OMNI nucleases only.

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Abstract

La présente invention concerne une méthode de modification d'une séquence nucléotidique, au niveau d'un site cible, dans le génome d'une cellule de mammifère, cette méthode consistant à introduire dans la cellule : (i) une composition comprenant une nucléase CRISPR possédant au moins 95 % d'identité avec une séquence d'acides aminés choisie dans le groupe constitué par les SEQ ID NO: 1-9, ou une molécule d'acides nucléiques comprenant une séquence codant pour une nucléase CRISPR, laquelle séquence possède au moins 95 % d'identité avec une séquence d'acides nucléiques choisie dans le groupe constitué par les SEQ ID NO : 23-27 ; et (ii) une molécule d'ARN ciblant l'ADN, ou un polynucléotide d'ADN codant pour une molécule d'ARN ciblant l'ADN, comprenant une séquence nucléotidique qui est complémentaire d'une séquence dans l'ADN cible.
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Publication number Priority date Publication date Assignee Title
WO2022098693A1 (fr) * 2020-11-04 2022-05-12 Emendobio Inc. Nouveaux complexes d'arn-nucléase crispr omni-50
WO2023107946A3 (fr) * 2021-12-07 2023-08-03 Emendobio Inc. Complexes nucléase crispr omni-103-arn
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites

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Cited By (3)

* Cited by examiner, † Cited by third party
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US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
WO2022098693A1 (fr) * 2020-11-04 2022-05-12 Emendobio Inc. Nouveaux complexes d'arn-nucléase crispr omni-50
WO2023107946A3 (fr) * 2021-12-07 2023-08-03 Emendobio Inc. Complexes nucléase crispr omni-103-arn

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