Patent Publication Number: US-2022213456-A1

Title: Novel omni crispr nucleases

Description:
This application claims the benefit of U.S. Provisional Application Nos. 62/959,672 filed Jan. 10, 2020, 62/931,630 filed Nov. 6, 2019, 62/897,806 filed Sep. 9, 2019, and 62/841,046 filed Apr. 30, 2019, the contents of which are hereby incorporated by reference. 
    
    
     Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention. 
     REFERENCE TO SEQUENCE LISTING 
     This application incorporates-by-reference nucleotide sequences which are present in the file named “200430_90962-A-PCT SequenceListing_AWG.txt”, which is 485 kilobytes in size, and which was created on Apr. 29, 2020 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Apr. 30, 2020 as part of this application. 
     FIELD OF THE INVENTION 
     The present invention is directed to, inter alia, composition and methods for genome editing. 
     BACKGROUND OF THE INVENTION 
     The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR systems have become important tools for research and genome engineering. Nevertheless, many details of CRISPR systems have not been determined and the applicability of CRISPR nucleases may be limited by 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. For example, some CRISPR nucleases may be able to target particular genomic loci that other CRISPR nucleases cannot due to limitations of the PAM site. In addition, some CRISPR nucleases currently in use exhibit pre-immunity, which may limit in vivo applicability. See Charlesworth et al., Nature Medicine (2019) and Wagner et al., Nature Medicine (2019). Accordingly, discovery, engineering, and improvement of novel CRISPR nucleases is of importance. 
     SUMMARY OF THE INVENTION 
     Disclosed herein are compositions and methods that may be utilized for genomic engineering, epigenomic engineering, genome targeting, genome editing of cells, and/or in vitro diagnostics. 
     The disclosed compositions may be utilized for modifying genomic DNA sequences. As used herein, genomic DNA refers to linear and/or chromosomal DNA and/or plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, 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 a DNA sequence at the target site(s) in a genome. 
     Accordingly, in some embodiments, the compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) nucleases. In some embodiments, the CRISPR nuclease is a CRISPR-associated protein. 
     In some embodiments, the compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease having 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% identity to CRISPR nucleases derived from  Acetobacterium  sp. KB-1 , Alistipes  sp. An54,  Bartonella apis, Blastopirellula marina, Bryobacter aggregates  MPL3 , Algoriphagus marinus, Butyrivibrio  sp. AC2005 , bacterium  LF-3 , Aliiarcobacter faecis, Caviibacter abscessus, Arcobacter  sp. SM1702 , Arcobacter mytili, Arcobacter thereius, Carnobacterium funditum, Peptoniphilus obesi  ph1 , Carnobacterium iners, Lactobacillus allii, Bacteroides coagulans, Butyrivibrio  sp. NC3005,  Clostridium  sp. AF02-29 or  Algoriphagus antarcticus . Each possibility represents a separate embodiment. 
     OMNI Nucleases 
     Embodiments of the present invention provide for CRISPR nucleases designated as an “OMNI” nuclease as provided in Table 1. 
     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, 2, 4, and 149-166 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: 6, 7, 9, 10, 15, 16, and 177-186 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 comprising a CRISPR associated system comprising:
         a) one or more 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 NOs: 1, 2, 4, and 149-166 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and   wherein the one or more 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.       

     This invention also provides a non-naturally occurring composition comprising:
         a) a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, and 149-166 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and   b) one or more 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 CRISPR nuclease; and   ii) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence,   
           wherein 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.       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1C : The predicted secondary structure of a single guide RNA (sgRNA) (crRNA-tracrRNA) from  Butyrivibrio  sp. AC2005 (OMNI-39). The crRNA and tracrRNA portions of the sgRNA are noted.  FIG. 1A : The native pre-mature crRNA-tracrRNA duplex.  FIG. 1B : Examples of V1 and V2 of sgRNA design with the duplex shortening (indicated by triangles in A) compared with the native.  FIG. 1C : V3 guide modification within the lower stem duplex from V2 (indicated by triangles). See also sgRNA Table 2. 
         FIGS. 2A-2E : Bacterial PAM Depletion results for OMNI nucleases. The PAM logo is a schematic representation of the ratio of the depleted site. A condensed 4N window library of all possible PAM locations along an 8 bp sequence for each OMNI nuclease in  E. coli  is shown. Sequence motifs generated for bacterial PAM sites are based on depletion assay results. Activity was estimated based on the average of the two most depleted sequences and was calculated as: 1−Depletion score. Bacterial PAM depletion results for OMNI-39 sgRNA v1, v2, and v3 ( FIG. 2A ); OMNI-40 sgRNA v1, v2, and v3 ( FIG. 2B ); OMNI-51 sgRNA v1 and v2 ( FIG. 2C ); OMNI-52 sgRNA v1, v2, and v2 ( FIG. 2D ); and OMNI-51 sgRNA v1 and v2 ( FIG. 2E ) are depicted. 
         FIGS. 3A-3M : In-vitro PAM Depletion by TXTL results for OMNI nucleases. The PAM logo is a schematic representation of the ratio of the depleted site. A condensed 4N window library of all possible PAM locations along an 8 bp sequence for each OMNI nuclease in a cell-free in vitro TXTL system is shown. Sequence motifs generated for in vitro PAM sites are based on depletion assay results. Activity estimated based on the average of the two most depleted sequences and was calculated as: 1−Depletion score. In vitro PAM depletion results for OMNI-34 sgRNA v1, v2, and v3 ( FIG. 3A ); OMNI-35 sgRNA v1 and v2 ( FIG. 3B ); OMNI-36 sgRNA v1 and v2 ( FIG. 3C ); OMNI-39 sgRNA v2 ( FIG. 3D ); OMNI-40 sgRNA v2 ( FIG. 3E ); OMNI-42 sgRNA v2 ( FIG. 3F ); OMNI-43 sgRNA v1 and v2 ( FIG. 3G ); OMNI-44 sgRNA v2 ( FIG. 3H ); OMNI-46 sgRNA v1 and v2 ( FIG. 3I ); OMNI-47 sgRNA v1 and v2 ( FIG. 3J ); OMNI-51 sgRNA v1 ( FIG. 3K ); OMNI-52 sgRNA v1 ( FIG. 3L ); and OMNI-53 sgRNA v1 ( FIG. 3M ) are depicted. 
         FIG. 4 : expression of OMNI-39, OMNI-40, and OMNI-53 in mammalian cells: OMNI nucleases were transiently transfected in Hek293T cells. Cells were harvested and lysed at 72 h. the lysates were used to test OMNI expression in the mammalian cells by WB against the HA tag. SpCas9-HA that was transfected in the same manner served as a positive control. GAPDH was used to normalize loading quantities. 
         FIGS. 5A-5C : Nuclease activity in endogenous context in mammalian cells. OMNI nucleases were expressed in mammalian cell system by DNA transfection together with sgRNA expressing plasmid. Cell lysates were used for site specific genomic DNA amplification and NGS. The percentage of Indels was measured and analyzed to determine editing level. cells transfected with the OMNI nuclease without guide RNA served as a negative control for comparison and background determination. Editing levels in different genomic locations are shown.  FIG. 5A : OMNI-39 nuclease activity in endogenous context in mammalian cells.  FIG. 5B : OMNI-40 nuclease activity in endogenous context in mammalian cells.  FIG. 5C : OMNI-53 nuclease activity in endogenous context in mammalian cells. 
     
    
    
     DETAILED DESCRIPTION 
     According to some aspects of the invention, the disclosed compositions comprise a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease and/or a nucleic acid molecule comprising a sequence encoding the same. 
     In some embodiments, 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: 1-4 and 149-166. In an embodiment the sequence encoding the CRISPR nuclease has at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5-10, 14-16, and 167-186. 
     In some embodiments, 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-1 , Alistipes  sp. An54,  Bartonella apis, Blastopirellula marina, Bryobacter aggregatus  MPL3 , Algoriphagus marinus, Butyrivibrio  sp. AC2005 , bacterium  LF-3 , Aliiarcobacter faecis, Caviibacter abscessus, Arcobacter  sp. SM1702 , Arcobacter mytili, Arcobacter thereius, Carnobacterium funditum, Peptoniphilus obesi  ph1 , Carnobacterium iners, Lactobacillus allii, Bacteroides coagulans, Butyrivibrio  sp. NC3005,  Clostridium  sp. AF02-29 or  Algoriphagus antarcticus . Each possibility represents a separate embodiment. 
     According to some aspects of the invention, the disclosed compositions comprise DNA constructs or a vector system comprising nucleotide sequences that encode the CRISPR nuclease or variant CRISPR nuclease. In some embodiments, the nucleotide sequence that encode the CRISPR nuclease or variant CRISPR nuclease is operably linked to a promoter that is operable in the cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments the cell of interest is a mammalian cell. In some embodiments, the nucleic acid sequence encoding the engineered CRISPR nuclease is codon optimized for use in cells from a particular organism. In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for  E. Coli . In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for Eukaryotic cells. In some embodiments, the nucleic acid sequence encoding the nuclease is codon optimized for mammalian cells. 
     In some embodiments, the composition comprises a recombinant nucleic acid, comprising a heterologous promoter operably linked to a polynucleotide encoding a CRISPR enzyme having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% identity to any of SEQ ID NOs: 1-4 or 149-166. Each possibility represents a separate embodiment. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 1 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 6, and 7. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% 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 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 9, and 10. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% 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 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 16. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 149 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 150 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 167 and 177. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 151 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 168 and 178. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 152 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 169 and 179. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 153 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 154 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 155 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 156 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 170 and 180. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 157 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 171 and 181. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 158 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 172 and 182. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 159 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 160 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 173 and 183. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 161 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 174 and 184. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 162 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 163 or a sequence encoding the CRISPR nuclease. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 164 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 175 and 185. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 165 or the sequence encoding the CRISPR nuclease has at least a 75%, 80%, 85, 90%, 95%, or 97% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 176 and 186. 
     In an embodiment of the composition, the CRISPR nuclease has at least 75%, 80%, 85, 90%, 95%, or 97% identity to the amino acid sequence as set forth in SEQ ID NO: 166 or a sequence encoding the CRISPR nuclease. 
     According to some embodiments, there is provided an engineered or non-naturally occurring composition comprising a CRISPR nuclease comprising a sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, and 149-166 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease. Each possibility represents a separate embodiment. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, the DNA-targeting RNA molecule further comprises a nucleotide sequence that can form a complex with a CRISPR nuclease. 
     This invention also provides a non-naturally occurring composition comprising a CRISPR associated system comprising:
         a) one or more 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) a 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 NOs: 1, 2, 4, and 149-166 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease;
           wherein the one or more 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.   
               

     In an embodiment, 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. 
     In an embodiment, the composition further comprises a donor template for homology directed repair (HDR). 
     In an embodiment, the composition is capable of editing the target region in the genome of a cell. 
     In an embodiment of the composition:
         a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 1, and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 17-26 and 226-231;   b) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 2, and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 27-36 and 232-237;   c) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 4, and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 46-54, 329-334, GUUUGAGAA, and GGAUUAUCC;   d) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 150, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 187-200;   e) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 151, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 201-212;   f) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 152, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 213-225;   g) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 156, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 238-249, GUUUAAGAG, and CGAGUUUA;   h) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 157, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 250-262;   i) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 158, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 263-275;   j) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 160, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 276-288;   k) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 161, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 289-301 and GUUUGAGAG;   l) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 164, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 302-314 and GUUUGAGAG; or   m) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 165, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 315-328 and GUUUGAGAG.       

     According to some embodiments, there is provided a non-naturally occurring composition comprising:
         (a) a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, comprising: an RNA-binding portion; and
           an activity portion that exhibits site-directed enzymatic activity, wherein the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to any of SEQ ID NOs: 1, 2, 4, and 149-166; and   
           (b) one or more RNA molecules or a DNA polynucleotide encoding the one or more RNA molecules comprising:
           i) a DNA-targeting RNA sequence, comprising a nucleotide sequence that is complementary to a sequence in a target DNA sequence; and   ii) a protein-binding RNA sequence, capable of interacting with the RNA-binding portion of the CRISPR nuclease,   
           wherein the DNA targeting RNA sequence and the CRISPR nuclease do not naturally occur together. Each possibility represents a separate embodiment.       

     In some embodiments, there is provided a single RNA molecule comprising the DNA-targeting RNA sequence and the protein-binding RNA sequence, wherein the RNA molecule can form a complex with the CRISPR nuclease and serve as the DNA targeting module. In some embodiments, the RNA molecule 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. Each possibility represents a separate embodiment. In some embodiments, a first RNA molecule comprising the DNA-targeting RNA sequence and a second RNA molecule comprising the protein-binding RNA sequence interact by base pairing or alternatively fused together to form one or more RNA molecules that complex with the CRISPR nuclease and serve as the DNA targeting module. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 1, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 17-26 and 226-231. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 2, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 27-36 and 232-237. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 4, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 46-54, 329-334, GUUUGAGAA, and GGAUUAUCC. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 150, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 187-200. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 151, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 201-212. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 152, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 213-225. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 156, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 238-249, GUUUAAGAG, and CGAGUUUA. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 157, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 250-262. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 158, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 263-275. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 160, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 276-288. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 161, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 289-301 and GUUUGAGAG. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 164, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 302-314 and GUUUGAGAG. 
     In some embodiments, the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 165, and the RNA molecule comprises a sequence selected from SEQ ID NOs: 315-328 and GUUUGAGAG. 
     This invention also provides a non-naturally occurring composition comprising:
         a) a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs 1˜4 and 149-166 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and   b) one or more 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 CRISPR nuclease; and   ii) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence, wherein 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.   
               

     In an embodiment, 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. 
     In an embodiment, the CRISPR nuclease and at least one of the one or more RNA molecules do not naturally occur together. 
     In an embodiment:
         a) the CRISPR nuclease comprises an RNA-binding portion and an activity portion that exhibits site-directed enzymatic activity;   b) the DNA-targeting RNA nucleotide sequence comprises a nucleotide sequence that is complementary to a sequence in a target DNA sequence; and   c) the nuclease-binding RNA nucleotide sequence comprises a sequence that interacts with the RNA-binding portion of the CRISPR nuclease.       

     In an embodiment, 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. 
     In an embodiment, the nuclease-binding RNA nucleotide sequence is on a first RNA molecule and the DNA-targeting RNA nucleotide sequence is on a single guide RNA molecule, and wherein the first and second RNA sequence interact by base-pairing or are fused together to form one or more RNA molecules or sgRNA that complex with the CRISPR nuclease and serve as the targeting module. 
     In an embodiment, 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. 
     In an embodiment, the composition further comprises a donor template for homology directed repair (HDR). 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 1, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 5, 6, or 7, and the PAM sequence is selected from: NNGYAD, NNGYAA, and NNGHAD. Non-limiting examples of suitable PAM sequences include: TGGCAA and CAGCAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 17-26 and 226-231. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 2, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 8, 9, or 10, and the PAM sequence is selected from: NYGRV, NYGAV, and VTGAAG. Non-limiting examples of suitable PAM sequences include CTGAG, CTGAC, ACGAC, GTGAC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 27-36 and 232-237. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 4, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 14, 15, or 16, and the PAM is selected from: NRTA, NRHR, and NAWA. Non-limiting examples of suitable PAM sequences include: TGTA, AATA, TGTA, and GGTA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 46-54, 329-334, GUUUGAGAA, and GGAUUAUCC 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 150, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 167 or 177 and the PAM is NRNNNNAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 187-200. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 151, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 168 or 178 and the PAM is NRR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 201-212. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 152, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 169 or 179 and the PAM is NNYCCC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 213-225. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 156, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 170 or 180 and the PAM is selected from NNGMM and NTGCC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 238-249, GUUUAAGAG, and CGAGUUUA. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 157, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 171 or 181 and the PAM is YAAAR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 250-262. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 158, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 172 or 182 and the PAM is NRHAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 263-275. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 160, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 173 or 183 and the PAM is YAAAR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 276-288. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 161, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 174 or 184 and the PAM is selected from NVYR and NRTA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 289-301 and GUUUGAGAG. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 164, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 175 or 185 and the PAM is NRRAAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 302-314 and GUUUGAGAG. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 165, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 176 or 186 and the PAM is NRRADT. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 315-328 and GUUUGAGAG. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, the CRISPR nuclease has altered PAM specificity compared to the wild-type of the CRISPR nuclease. 
     In an embodiment, the CRISPR nuclease is non-naturally occurring. 
     In an embodiment, the CRISPR nuclease is engineered and comprises unnatural or synthetic amino acids. 
     In an embodiment, the CRISPR nuclease is engineered and comprises one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags. 
     In an embodiment, 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. 
     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. 
     In an embodiment, the cell is a eukaryotic cell. 
     In another embodiment, the cell is a prokaryotic cell. 
     In some embodiments, the one or more RNA molecules further comprises an RNA sequence 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. 
     In an embodiment, 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. In an embodiment 1-4 NLSs are fused with the CRISPR nuclease. In an embodiment, an NLS is located within the open-reading frame (ORF) of the CRISPR nuclease. 
     Methods of fusing an NLS 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. As an example, to fuse an NLS to the amino-terminus of a CRISPR nuclease, 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. Conversely, to fuse an NLS to the carboxy-terminus of a 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. 
     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. 
     The 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. 
     In an embodiment, the one or more NLSs are in tandem repeats. 
     In an embodiment, 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. 
     As discussed, the CRISPR nuclease may be engineered to comprise one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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. 
     In an embodiment, 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 use of any of the 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. 
     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, 2, 4, and 149-166 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: 6, 7, 9, 10, 15, 16 and 177-186 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. 
     In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo. In some embodiments, some steps of the method are performed ex vivo and some steps are performed in vivo. In some embodiments the mammalian cell is a human cell. 
     In an embodiment, 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. 
     In an embodiment, the DNA targeting RNA molecule is a crRNA molecule suitable to form an active complex with the CRISPR nuclease. 
     In an embodiment, the RNA molecule comprising a nuclease-binding RNA sequence is a tracrRNA molecule suitable to form an active complex with the CRISPR nuclease. 
     In an embodiment, 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. 
     In an embodiment, the method further comprises introducing into the cell: (iv) an RNA molecule comprising a sequence complementary to a protospacer sequence. 
     In an embodiment, 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). 
     In an embodiment, the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 5′ of a Protospacer Adjacent Motif (PAM). 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 1, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 5, 6, or 7, and the PAM sequence is selected from: NNGYAD, NNGYAA, and NNGHAD. Non-limiting examples of suitable PAM sequences include: TGGCAA and CAGCAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 17-26 and 226-231. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 2, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 8, 9, or 10, and the PAM sequence is selected from: NYGRV, NYGAV, and VTGAAG. Non-limiting examples of suitable PAM sequences include CTGAG, CTGAC, ACGAC, GTGAC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 27-36 and 232-237. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 4, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 14, 15, or 16, and the PAM is selected from: NRTA, NRHR, and NAWA. Non-limiting examples of suitable PAM sequences include: TGTA, AATA, TGTA, and GGTA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 46-54, 329-334, GUUUGAGAA, and GGAUUAUCC 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 150, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 167 or 177 and the PAM is NRNNNNAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 187-200. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 151, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 168 or 178 and the PAM is NRR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 201-212. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 152, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 169 or 179 and the PAM is NNYCCC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 213-225. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 156, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 170 or 180 and the PAM is selected from NNGMM and NTGCC. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 238-249, GUUUAAGAG, and CGAGUUUA. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 157, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 171 or 181 and the PAM is YAAAR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 250-262. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 158, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 172 or 182 and the PAM is NRHAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 263-275. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 160, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 173 or 183 and the PAM is YAAAR. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 276-288. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 161, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 174 or 184 and the PAM is selected from NVYR and NRTA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 289-301 and GUUUGAGAG. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 164, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 175 or 185 and the PAM is NRRAAA. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 302-314 and GUUUGAGAG. 
     In some embodiments, (a) the CRISPR nuclease has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity to SEQ ID NO: 165, or (b) the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 176 or 186 and the PAM is NRRADT. In this embodiment, the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA-targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 315-328 and GUUUGAGAG. 
     In an embodiment of any of the methods described herein, 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. 
     In an embodiment, 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. 
     DNA-Targeting RNA Molecules 
     In embodiments of the present invention, the DNA-targeting RNA sequence comprises a guide sequence portion. The “guide sequence portion” of an RNA molecule refers to a nucleotide sequence that is capable of hybridizing to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence which is fully complementary to the DNA sequence being targeted along the length of the guide sequence portion. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length, or approximately 17-24, 18-22, 19-22, 18-20, 17-20, or 21-22 nucleotides in length. The entire length of the guide sequence portion is fully complementary to the DNA sequence being targeted along the length of the guide sequence portion. The guide sequence portion may be part of an RNA molecule that can form a complex with a CRISPR nuclease with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex. When the RNA molecule having the guide sequence portion is present contemporaneously with the CRISPR molecule, the RNA molecule is capable of targeting the CRISPR nuclease to the specific target DNA sequence. Each possibility represents a separate embodiment. An RNA molecule can be custom designed to target any desired sequence. 
     In embodiments of the present invention, the CRISPR nuclease has greater cleavage activity when used with an RNA molecule comprising a guide sequence portion having 21-23 nucleotides, compared to its cleavage activity when used with an RNA molecule comprising a guide sequence portion having 20 or fewer nucleotides, and/or 24 or more nucleotides. In embodiments of the present invention, the CRISPR nuclease has greater cleavage activity when used with an RNA molecule comprising a guide sequence portion having 21-22 nucleotides, compared to its cleavage activity when used with an RNA molecule comprising a guide sequence portion having 20 or fewer nucleotides, and/or 23 or more nucleotides. In an embodiment, the CRISPR nuclease has its greatest cleavage activity when used with an RNA molecule comprising a guide sequence portion having 22 nucleotides. 
     According to some aspects of the invention, the disclosed methods comprise 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 the composition of any one of the embodiments described herein. 
     In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell. 
     According to some aspects of the invention, the disclosed methods comprise a use of any one of the compositions described herein 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. 
     According to some aspects of the invention, the disclosed methods comprise a method of treating subject having a mutation disorder comprising targeting any one of the compositions described herein to an allele associated with the mutation disorder. 
     In some embodiments, the mutation disorder is related to a disease or disorder selected from any of a neoplasia, age-related macular degeneration, schizophrenia, neurological, neurodegenerative, or movement disorder, Fragile X Syndrome, secretase-related disorders, prion-related disorders, ALS, addiction, autism, Alzheimer&#39;s Disease, neutropenia, inflammation-related disorders, Parkinson&#39;s Disease, blood and coagulation diseases and disorders, cell dysregulation and oncology diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, dermatological diseases and disorders, neurological and neuronal diseases and disorders, and ocular diseases and disorders. 
     In some embodiments, the mutation disorder is beta thalassemia or sickle cell anemia. 
     In some embodiments, the allele associated with the disease is BCL11A. 
     Diseases and Therapies 
     Certain embodiments of the invention target a nuclease to a specific genetic locus associated with a disease or disorder as a form of gene editing, method of treatment, or therapy. For example, to induce editing or knockout of a gene, a novel nucleases disclosed herein may be specifically targeted to a pathogenic mutant allele of the gene using a custom designed guide RNA molecule. The guide RNA molecule is preferably designed by first considering the PAM requirement of the nuclease, which as shown herein is also dependent on the system in which the gene editing is being performed. For example, a guide RNA molecule designed to target an OMNI-40 nuclease to a target site is designed to contain a spacer region complementary to a region neighboring the OMNI-40 PAM sequence “NYGRV.” The guide RNA molecule is further preferably designed to contain a spacer region (i.e. the region of the guide RNA molecule having complementarity to the target allele) of sufficient and preferably optimal length in order to increase specific activity of the nuclease and reduce off-target effects. 
     As a non-limiting example, the guide RNA molecule may be designed to target the nuclease to a specific region of a mutant allele, e.g. near the start codon, such that upon DNA damage caused by the nuclease a non-homologous end joining (NHEJ) pathway is induced and leads to silencing of the mutant allele by introduction of frameshift mutations. This approach to guide RNA molecule design is particularly useful for altering the effects of dominant negative mutations and thereby treating a subject. As a separate non-limiting example, the guide RNA molecule may be designed to target a specific pathogenic mutation of a mutated allele, such that upon DNA damage caused by the nuclease a homology directed repair (HDR) pathway is induced and leads to template mediated correction of the mutant allele. This approach to guide RNA molecule design is particularly useful for altering haploinsufficiency effects of a mutated allele and thereby treating a subject. 
     Non-limiting examples of specific genes which may be targeted for alteration to treat a disease or disorder are presented herein below. Specific disease-associated genes and mutations that induce a mutation disorder are described in the literature. Such mutations can be used to design a DNA-targeting RNA molecule to target a CRISPR composition to an allele of the disease associated gene, where the CRISPR composition causes DNA damage and induces a DNA repair pathway to alter the allele and thereby treat the mutation disorder. 
     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-1) infection. Accordingly, without limitation, embodiments of the invention that target CXCR4 may be used in methods of treating subjects afflicted with HIV-1 or conferring resistance to HIV-1 infection in a subject. 
     Programmed cell death protein 1 (PD-1) disruption enhances CAR-T cell mediated killing of tumor cells and PD-1 may be a target in other cancer therapies. Accordingly, without limitation, embodiments of the invention that target PD-1 may be used in methods of treating subjects afflicted with cancer. In an embodiment, the treatment is CAR-T cell therapy with T cells that have been modified according to the invention to be PD-1 deficient. 
     In addition, BCL11A is a gene that plays a role in the suppression of hemoglobin production. Globin production may be increased to treat diseases such as thalassemia or sickle cell anemia by inhibiting BCL11A. See for example, PCT International Publication No. WO 2017/077394A2; U.S. Publication No. US2011/0182867A1; Humbert et al. Sci. Transl. Med. (2019); and Canver et al. Nature (2015). Accordingly, without limitation, embodiments of the invention that target an enhancer of BCL11A may be used in methods of treating subjects afflicted with beta thalassemia or sickle cell anemia. 
     Embodiments of the invention may also be used for targeting any disease-associated gene, for studying, altering, or treating any of the diseases or disorders listed in Table A or Table B below. Indeed, any disease-associated with a genetic locus may be studied, altered, or treated by using the nucleases disclosed herein to target the appropriate disease-associated gene, for example, those listed in U.S. Publication No. 2018/0282762A1 and European Patent No. EP3079726B1. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Diseases, Disorders and their associated genes 
               
            
           
           
               
               
            
               
                 DISEASE/DISORDERS 
                 GENE(S) 
               
               
                   
               
               
                 Neoplasia 
                 PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1;  
               
               
                   
                 Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF; HIF1a;  
               
               
                   
                 HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1  
               
               
                   
                 (Wilms Tumor); FGF Receptor Family members (5 members: 1,  
               
               
                   
                 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL;  
               
               
                   
                 BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF  
               
               
                   
                 Receptor; Igf1 (4 variants); gf2 (3 variants); Igf 1 Receptor; Igf 2  
               
               
                   
                 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6,  
               
               
                   
                 7, 8, 9, 12); Kras; Apc  
               
               
                 Age-related Macular 
                 Aber; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD; Vldlr;  
               
               
                 Degeneration  
                 Ccr2  
               
               
                 Schizophrenia 
                 Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);  
               
               
                   
                 Complexin1 (Cp1x1); Tph1 Tryptophan hydroxylase; Tph2  
               
               
                   
                 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b  
               
               
                 Neurological, Neuro 
                 5-HTT (S1c6a4); COMT; DRD (Drd1a); SLC6A3; DAOA;  
               
               
                 degenerative, and  
                 DTNBP1; Dao (Dao 1)  
               
               
                 Movement Disorders 
                   
               
               
                 Trinucleotide Repeat 
                 HTT (Huntington’s Dx); SBMA/SMAX1/AR (Kennedy’s Dx);  
               
               
                 Disorders  
                 FXN/X25 (Friedrich’s Ataxia); ATX3 (Machado-Joseph’s Dx);  
               
               
                   
                 ATXN1 and ATXN2 (spinocerebellar ataxias); DMPK  
               
               
                   
                 (myotonic dystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP  
               
               
                   
                 (Creb-BP-global instability); VLDLR (Alzheimer’s); Atxn7;  
               
               
                   
                 Atxn10  
               
               
                 Fragile X Syndrome 
                 FMR2; FXR1; FXR2; mGLUR5  
               
               
                 Secretase Related  
                 APH-1 (alpha and beta); Presenilin (Psen1); nicastrin (Ncstn);  
               
               
                 Disorders  
                 PEN-2  
               
               
                 Others 
                 Nos1; Parp1; Nat1; Nat2  
               
               
                 Prion related disorders  
                 Prp  
               
               
                 ALS  
                 SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-  
               
               
                   
                 b; VEGF-c) 
               
               
                 Addiction 
                 Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5;  
               
               
                   
                 Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol)  
               
               
                 Autism  
                 Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X  
               
               
                   
                 (FMR2 (AFF2); FXR1; FXR2; Mglur5)  
               
               
                 Alzheimer’s Disease 
                 E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1;  
               
               
                   
                 SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin  
               
               
                   
                 1); Uchl1; Uchl3; APP  
               
               
                 Inflammation  
                 IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL-  
               
               
                   
                 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa;  
               
               
                   
                 NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;  
               
               
                   
                 Cx3cl1  
               
               
                 Parkinson’s Disease  
                 x-Synuclein; DJ-1; LRRK2; Parkin; PINK1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 Diseases, Disorders and their associated genes 
               
            
           
           
               
               
            
               
                 DISEASE CATEGORY  
                 DISEASE AND ASSOCIATED GENES 
               
               
                   
               
               
                 Blood and coagulation Anemia 
                 (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,  
               
               
                 diseases and disorders 
                 UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,  
               
               
                   
                 ANH1, ASB, ABCB7, ABC7, ASAT); Bare lymphocyte  
               
               
                   
                 syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11,  
               
               
                   
                 MHC2TA, C2TA, RFX5, RFXAP, RFX5), Bleeding disorders  
               
               
                   
                 (TBXA2R, P2RX1, P2X1); Factor H and factor H-like 1 (HF1,  
               
               
                   
                 CFH, HUS); Factor V and factor VIII (MCFD2); Factor VII  
               
               
                   
                 deficiency (F7); Factor X deficiency (F10); Factor XI deficiency  
               
               
                   
                 (F11); Factor XII deficiency (F12, HAF); Factor XIIIA  
               
               
                   
                 deficiency (F13A1, F13A); Factor XIIIB deficiency (F13B);  
               
               
                   
                 Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95,  
               
               
                   
                 FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2,  
               
               
                   
                 FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE,  
               
               
                   
                 FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9,  
               
               
                   
                 FANCL, FANCM, KIAA1596); Hemophagocytic  
               
               
                   
                 lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D,  
               
               
                   
                 MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C,  
               
               
                   
                 HEMA); Hemophilia B (F9, HEMB), Hemorrhagic disorders  
               
               
                   
                 (PI, ATT, F5); Leukocyde deficiencies and disorders (ITGB2,  
               
               
                   
                 CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,  
               
               
                   
                 EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia  
               
               
                   
                 (HBB); Thalassemia (HBA2, HBB, HBD, LCRB, HBA1)  
               
               
                 Cell dysregulation and  
                 B-cell non-Hodgkin lymphoma (BCL7A, BCL7); Leukemia  
               
               
                 oncology diseases and  
                 (TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1,  
               
               
                 disorders  
                 IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT,  
               
               
                   
                 KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG,  
               
               
                   
                 KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL,  
               
               
                   
                 FLT3, KIT, PBT, LPP, NPM1, NUP214, D9546E, CAN, CAN,  
               
               
                   
                 RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q,  
               
               
                   
                 NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10,  
               
               
                   
                 CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML,  
               
               
                   
                 PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11,  
               
               
                   
                 PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,  
               
               
                   
                 GATA1, GF1, ERYF1, NFE1, ABL1, NQ01, DIA4, NMOR1,  
               
               
                   
                 NUP214, D9S46E, CAN, CAIN)  
               
               
                 Inflammation and immune  
                 AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG,  
               
               
                 related diseases and  
                 CXCL12, SDF1); Autoimmune lymphoproliferative syndrome  
               
               
                 disorders  
                 (TNFRSF6, APT1, FAS, CD95, ALPS1A); Combined  
               
               
                   
                 immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1  
               
               
                   
                 (CCL5, SCYA5, D175136E, TCP228), HIV susceptibility or  
               
               
                   
                 infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5  
               
               
                   
                 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID,  
               
               
                   
                 HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5,  
               
               
                   
                 CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX,  
               
               
                   
                 TNFRSF14B, TACI); Inflammation (IL-10, IL-1 (IL-1a, IL-1b),  
               
               
                   
                 IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL- 17d, IL-  
               
               
                   
                 17f), 11-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD,  
               
               
                   
                 IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3c11); Severe  
               
               
                   
                 combined immunodeficiencies (SCIDs)(JAK3, JAKL,  
               
               
                   
                 DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC,  
               
               
                   
                 CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX,  
               
               
                   
                 IMD4)  
               
               
                 Metabolic, liver, kidney  
                 Amyloid neuropathy (TTR, PALB); Amyloidosis (APOA1,  
               
               
                 and protein diseases and  
                 APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB);  
               
               
                 disorders  
                 Cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292,  
               
               
                   
                 KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);  
               
               
                   
                 Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT,  
               
               
                   
                 G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2,  
               
               
                   
                 PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A,  
               
               
                   
                 MODY3), Hepatic failure, early onset, and neurologic disorder  
               
               
                   
                 (SCOD1, SC01), Hepatic lipase deficiency (LIPC),  
               
               
                   
                 Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL,  
               
               
                   
                 PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1,  
               
               
                   
                 IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidney  
               
               
                   
                 disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2);  
               
               
                   
                 Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystic  
               
               
                   
                 kidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1,  
               
               
                   
                 PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63)  
               
               
                 Muscular/Skeletal  
                 Becker muscular dystrophy (DMD, BMD, MYF6), Duchenne  
               
               
                 diseases and disorders  
                 Muscular Dystrophy (DMD, BMD); Emery-Dreifuss muscular  
               
               
                   
                 dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS,  
               
               
                   
                 LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A);  
               
               
                   
                 Facioscapulohumeral muscular dystrophy (FSHMD1A,  
               
               
                   
                 FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I,  
               
               
                   
                 LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD,  
               
               
                   
                 TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG,  
               
               
                   
                 LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D,  
               
               
                   
                 DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L,  
               
               
                   
                 TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,  
               
               
                   
                 FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J,  
               
               
                   
                 POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1,  
               
               
                   
                 PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7, LR3,  
               
               
                   
                 OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL,  
               
               
                   
                 TCIRG1, TIRC7, OC116, OPTB1); Muscular atrophy (VAPB,  
               
               
                   
                 VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2,  
               
               
                   
                 SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2,  
               
               
                   
                 SMUBP2, CATF1, SMARD1)  
               
               
                 Dermatological diseases  
                 Albinisim (TYR, OCA2, TYRP1, SLC45A2, LYST),  
               
               
                 and disorders  
                 Ectodermal dysplasias (EDAR, EDARADD, WNT10A), Ehlers-  
               
               
                   
                 Danlos syndrome (COL5A1, COL5A2, COL1A1, COL1A2,  
               
               
                   
                 COL3A1, TNXB, ADAMTS2, PLOD1, FKBP14), Ichthyosis-  
               
               
                   
                 associated disorders (FLG, STS, TGM1, ALOXE3/ALOX12B,  
               
               
                   
                 KRT1, KRT10, ABCA12, KRT2, GJB2, TGM1, ABCA12,  
               
               
                   
                 CYP4F22, ALOXE3, CERS3, NSHDL, EBP, MBTPS2, GJB2,  
               
               
                   
                 SPINK5, AGHD5, PHYH, PEX7, ALDH3A2, ERCC2, ERCC3,  
               
               
                   
                 GFT2H5, GBA), Incontinentia pigmenti (IKBKG, NEMO),  
               
               
                   
                 Tuberous sclerosis (TSC1, TSC2), Premature aging syndromes  
               
               
                   
                 (POLR3A, PYCR1, LMA, POLD1, WRN, DMPK)  
               
               
                 Neurological and Neuronal  
                 ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a,  
               
               
                 diseases and disorders  
                 VEGF-b, VEGF-c); Alzheimer disease (APP, AAA, CVAP,  
               
               
                   
                 AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1,  
               
               
                   
                 NO53, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1,  
               
               
                   
                 PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism  
               
               
                   
                 (Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1,  
               
               
                   
                 MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,  
               
               
                   
                 KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1,  
               
               
                   
                 FXR2, mGLUR5); Huntington’s disease and disease like  
               
               
                   
                 disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP,  
               
               
                   
                 SCA17); Parkinson disease (NR4A2, NURR1, NOT, TINUR,  
               
               
                   
                 SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1,  
               
               
                   
                 PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARKS,  
               
               
                   
                 SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH,  
               
               
                   
                 NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16,  
               
               
                   
                 MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,  
               
               
                   
                 MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1  
               
               
                   
                 (Nrg1), Erb4 (receptor for Neuregulin), Complexin1 (Cp1x1),  
               
               
                   
                 Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2,  
               
               
                   
                 Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT,  
               
               
                   
                 DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1));  
               
               
                   
                 Secretase Related Disorders (APH-1 (alpha and beta), Presenilin  
               
               
                   
                 (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Natl, Nat2);  
               
               
                   
                 Trinucleotide Repeat Disorders (HTT (Huntington’s Dx),  
               
               
                   
                 SBMA/SMAX1/AR (Kennedy’s Dx), FXN/X25 (Friedrich’s  
               
               
                   
                 Ataxia), ATX3 (Machado-Joseph’s Dx), ATXN1 and ATXN2  
               
               
                   
                 (spinocerebellar ataxias), DMPK (myotonic dystrophy),  
               
               
                   
                 Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP-global  
               
               
                   
                 instability), VLDLR (Alzheimer’s), Atxn7, Atxn10)  
               
               
                 Ocular diseases and  
                 Age-related macular degeneration (Abcr, Ccl2, Cc2, cp  
               
               
                 disorders  
                 (ceruloplasmin), Timp3, cathepsinD, Vldlr, Ccr2); Cataract  
               
               
                   
                 (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49,  
               
               
                   
                 CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1,  
               
               
                   
                 CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,  
               
               
                   
                 CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP,  
               
               
                   
                 AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4,  
               
               
                   
                 CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1,  
               
               
                   
                 GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM,  
               
               
                   
                 KRIT1); Corneal clouding and dystrophy (APOA1, TGFBI,  
               
               
                   
                 CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2,  
               
               
                   
                 M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,  
               
               
                   
                 PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA,  
               
               
                   
                 CNA2); Glaucoma (MYOC, TIGR, GLC1A, JOAG, GPOA,  
               
               
                   
                 OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1,  
               
               
                   
                 NTG, NPG, CYP1B1, GLC3A); Leber congenital  
               
               
                   
                 amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1,  
               
               
                   
                 LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D,  
               
               
                   
                 GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy  
               
               
                   
                 (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2,  
               
               
                   
                 PRPH, AVMD, AOFMD, VMD2) 
               
               
                   
               
            
           
         
       
     
     Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. 
     In the discussion unless otherwise stated, 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. Unless otherwise indicated, 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. 
     It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application. 
     For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 
     It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated. 
     In the description and claims of the present application, 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. 
     The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer 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. The following are non-limiting examples of 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 (shRNA), 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. If present, 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. 
     The term “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. Each of the RNA sequences described herein may comprise one or more nucleotide analogs. 
     As used herein, the following nucleotide identifiers are used to represent a referenced nucleotide base(s): 
     
       
         
           
               
               
               
               
               
            
               
                   
               
               
                 Nucleotide 
                   
                   
                   
                   
               
            
           
           
               
               
            
               
                 reference 
                 Base(s) represented 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 A 
                 A 
                   
                   
                   
               
               
                 C 
                   
                 C 
                   
                   
               
               
                 G 
                   
                   
                 G 
                   
               
               
                 T 
                   
                   
                 T 
                   
               
               
                 W 
                 A 
                   
                   
                 T 
               
               
                 S 
                   
                 C 
                 G 
                   
               
               
                 M 
                 A 
                 C 
                   
                   
               
               
                 K 
                   
                   
                 G 
                 T 
               
               
                 R 
                 A 
                   
                 G 
                   
               
               
                 Y 
                   
                 C  
                   
                 T 
               
               
                 B 
                   
                 C 
                 G 
                 T 
               
               
                 D 
                 A 
                   
                 G 
                 T 
               
               
                 H 
                 A 
                 C 
                   
                 T 
               
               
                 V 
                 A 
                 C 
                 G 
                   
               
               
                 N 
                 A 
                 C 
                 G 
                 T 
               
               
                   
               
            
           
         
       
     
     As used herein, 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 a targeting RNA molecule that can form a complex with a CRISPR nuclease with the targeting sequence serving as the targeting portion of the CRISPR complex. 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. Each possibility represents a separate embodiment. A targeting RNA molecule can be custom designed to target any desired sequence. 
     The term “targets” as used herein, refers to preferential hybridization of a targeting sequence or a targeting molecule 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. 
     In the context of targeting a DNA sequence that is present in a plurality of cells, it is understood that 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. Accordingly, it is understood that 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. As used herein, the term “modified cells” refers to cells in which a double strand break is affected 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. 
     As used herein the term “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. In embodiments of the present invention, 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 the CRISPR nucleases indicated in Table 1. 
     The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate human manipulation. The terms, when referring to 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. 
     As used herein the term “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. 
     As used herein, “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. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, 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. 
     The term “nuclease” as used herein 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. 
     The term “PAM” as used herein 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. 
     The term “mutation disorder” or “mutation disease” as used herein refers to any disorder or disease that is related to dysfunction of a gene caused by a mutation. A dysfunctional gene manifesting as a mutation disorder contains a mutation in at least one of its alleles and is referred to as a “disease-associated gene.” The mutation may be in any portion of the disease-associated gene, for example, in a regulatory, coding, or non-coding portion. The mutation may be any class of mutation, such as a substitution, insertion, or deletion. The mutation of the disease-associated gene may manifest as a disorder or disease according to the mechanism of any type of mutation, such as a recessive, dominant negative, gain-of-function, loss-of-function, or a mutation leading to haploinsufficiency of a gene product. 
     A skilled artisan will appreciate that embodiments of the present invention disclose 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). The nuclease then mediates cleavage of target DNA to create a double-stranded break within the protospacer. 
     In embodiments of the present invention, 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. A CRISPR nuclease may form a CRISPR complex comprising the CRISPR nuclease and RNA molecule without a further, separate tracrRNA molecule. Alternatively, CRISPR nucleases may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule. 
     The term “protein binding sequence” or “nuclease binding sequence” refers to a sequence capable of binding with a CRISPR nuclease to form a CRISPR complex. A skilled artisan will understand that 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. 
     An RNA molecule may comprise 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 U.S. Pat. No. 8,906,616). In embodiments of the present invention, the RNA molecule may further comprise a portion having a tracr mate sequence. 
     In embodiments of the present invention, the targeting molecule may further comprise the sequence of a tracrRNA molecule. Such embodiments 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). (See Jinek et al., Science (2012)). 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. In such embodiments the tracrRNA molecule may hybridize with the RNA molecule via base pairing and may be advantageous in certain applications of the invention described herein. 
     In embodiments of the present invention an RNA molecule may comprise a “ nexus ” region and/or “hairpin” regions which may further define the structure of the RNA molecule. (See Briner et al., Molecular Cell (2014)). 
     As used herein, the term “direct repeat sequence” refers to two or more repeats of a specific amino acid sequence of nucleotide sequence. 
     As used herein, 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. 
     As used herein, the term “operably linked” refers to a relationship (i.e. fusion, hybridization) between two sequences or molecules permitting them to function in their intended manner. In embodiments of the present invention, 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. 
     As used herein, the term “heterologous promoter” refers to a promoter that does not naturally occur together with the molecule or pathway being promoted. 
     As used herein, 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. For example, 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 Sequences 
     The terms “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. In addition, a polypeptide having an NLS coupled by its N- or C-terminus to amino acid side chains located randomly along the amino acid sequence of the polypeptide will be translocated. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. Non-limiting examples of NLSs include an NLS sequence derived from: the SV40 virus large T-antigen, nucleoplasmin, c-myc, the hRNPA1 M9 NLS, the IBB domain from importin-alpha, myoma T protein, human p53, mouse c-abl IV, influenza vims NS1, Hepatitis virus delta antigen, mouse Mx1 protein, human poly(ADP-ribose) polymerase, and the steroid hormone receptors (human) glucocorticoid. Such NLS sequences are listed as SEQ ID NOs: 69-84. 
     Delivery 
     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. In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2′-0-methyl (M), 2′-0-methyl, 3′phosphorothioate (MS) or 2′-0-methyl, 3′thioPACE (MSP), pseudouridine, and 1-methyl pseudo-uridine. Each possibility represents a separate embodiment of the present invention. 
     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. 
     In some embodiments, 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 a 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 a donor template for gene editing via, for example, homology directed repair. In some embodiments, the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA. In some embodiments, the composition to be delivered includes mRNA of the nuclease, DNA-targeting RNA and the tracrRNA and a 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 a donor template for gene editing via, for example, 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. In certain embodiments, 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. For a review of gene therapy procedures, see Anderson, Science (1992); Nabel and Felgner, TIBTECH (1993); Mitani and Caskey, TIBTECH (1993); Dillon, TIBTECH (1993); Miller, Nature (1992); Van Brunt, Biotechnology (1988); Vigne et al., Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin (1995); Haddada et al., Current Topics in Microbiology and Immunology (1995); and Yu et al., Gene Therapy 1:13-26 (1994). 
     Methods of non-viral delivery of nucleic acids and/or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic 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 al. Trends Plant Sci. (2006). 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 al., Nat. Biotechnol. (2015), Coelho et al., N. Engl. J. Med. (2013); Judge et al., Mol. Ther. (2006); and Basha et al., Mol. Ther. (2011). 
     Additional exemplary 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. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in PCT International Publication Nos. WO/1991/017424 and WO/1991/016024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). 
     The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science (1995); Blaese et al., Cancer Gene Ther. (1995); Behr et al., Bioconjugate Chem. (1994); Remy et al., Bioconjugate Chem. (1994); Gao and Huang, Gene Therapy (1995); Ahmad and Allen, Cancer Res., (1992); U.S. Pat. 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 EnGenelC delivery vehicles (EDVs). 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., Nature Biotechnology (2009)). 
     The use of 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, recombinant retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. However, an RNA virus is preferred for delivery of the RNA compositions described herein. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. Nucleic acid of the invention may be delivered by non-integrating lentivirus. Optionally, RNA delivery with Lentivirus is utilized. Optionally the lentivirus includes mRNA of the nuclease, RNA of the guide. Optionally the lentivirus includes mRNA of the nuclease, RNA of the guide and a donor template. Optionally, the lentivirus includes the nuclease protein, guide RNA. Optionally, the lentivirus includes the nuclease protein, guide RNA and/or a donor template for gene editing via, for example, homology directed repair. Optionally the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA. Optionally the lentivirus includes mRNA of the nuclease, DNA-targeting RNA, and the tracrRNA, and a donor template. Optionally, the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA. Optionally, the lentivirus includes the nuclease protein, DNA-targeting RNA, and the tracrRNA, and a donor template for gene editing via, for example, homology directed repair. 
     As mentioned above, the compositions described herein may be delivered to a target cell using a non-integrating lentiviral particle method, e.g. a LentiFlash® system. Such a method may be used to deliver mRNA or other types of RNAs into the target cell, such that delivery of the RNAs to the target cell results in assembly of the compositions described herein inside of the target cell. See also PCT International Publication Nos. WO2013/014537, WO2014/016690, WO2016185125, WO2017194902, and WO2017194903. 
     The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. 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 Panganiban, J. Virol. (1992); Johann et al., J. Virol. (1992); Sommerfelt et al., Virol. (1990); Wilson et al., J. Virol. (1989); Miller et al., J. Virol. (1991); PCT International Publication No. WO/1994/026877A1). 
     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 al., Blood (1995); Kohn et al., Nat. Med. (1995); Malech et al., PNAS (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. (1997); Dranoff et al., Hum. Gene Ther. (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. For example, 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. 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. Pat. No. 7,479,554). 
     In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, 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 al., Proc. Natl. Acad. Sci. USA (1995), reported that 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. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells. 
     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. Alternatively, 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. In some embodiments, 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 (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with an RNA composition, and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, “Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010)) and the references cited therein for a discussion of how to isolate and culture cells from patients). 
     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-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, 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 . In certain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, 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. 
     In one embodiment, 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 al., J. Exp. Med. (1992)). 
     Stem cells are isolated for transduction and differentiation using known methods. For example, 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-1 (granulocytes), and Iad (differentiated antigen presenting cells) (as a non-limiting example see Inaba et al., J. Exp. Med. (1992)). Stem cells that have been modified may also be used in some embodiments. 
     Notably, any one of the CRISPR nucleases 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.) containing therapeutic RNA compositions can also be administered directly to an organism for transduction of cells in vivo. Alternatively, 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 (e.g., T-cells) include non-integrating lentivirus vectors. See, for example, U.S. Patent Publication No. 2009/0117617. 
     Pharmaceutically acceptable carriers 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&#39;s Pharmaceutical Sciences, 17th ed., 1989). 
     DNA Repair by Homologous Recombination 
     The term “homology-directed repair” or “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. In some embodiments, 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. 
     The terms “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. In some embodiment, 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. In some embodiment, 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. In some embodiment, the nucleic acid template comprises modified ribonucleotides. 
     Insertion of an exogenous sequence (also called a “donor sequence,” donor template” or “donor”), for example, for correction of a mutant gene or for increased expression of a wild-type gene can also be carried out. It will be readily apparent that the 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. Additionally, 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. 2010/0047805; 2011/0281361; 2011/0207221; and 2019/0330620. 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 dideoxynucleotide 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 and Wilson, Proc. Natl. Acad. Sci. USA (1987); Nehls et al., Science (1996). 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 internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. 
     Accordingly, embodiments of the present invention using a donor template for repair may use a DNA or RNA, single-stranded and/or double-stranded donor template that can be introduced into a cell in linear or circular form. In embodiments of the present invention a gene-editing composition comprises: (1) an RNA molecule comprising a guide sequence to affect a double strand break in a gene prior to repair and (2) a donor RNA template for repair, the RNA molecule comprising the guide sequence is a first RNA molecule and the donor RNA template is a second RNA molecule. In some embodiments, the guide RNA molecule and template RNA molecule are connected as part of a single 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. Moreover, 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 recombinant viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLY)). 
     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. However, it will be apparent that 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. For example, 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. In other embodiments, the transgene (e.g., with or without additional coding sequences such as for the endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus, for example a CCR5 gene, a CXCR4 gene, a PPP1R12c (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Pat. Nos. 7,951,925 and 8,110,379; U.S. Publication Nos. 2008/0159996; 20100/0218264; 2010/0291048; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983 and 2013/0177960 and U.S. Provisional Application No. 61/823,689). 
     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. 
     Furthermore, although not required for expression, 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. 
     In certain embodiments, 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. 
     For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment. For example, it is understood that any of the RNA molecules or compositions of the present invention may be utilized in any of the methods of the present invention. 
     As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. 
     Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. 
     It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. 
     Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, Sambrook et al., “Molecular Cloning: A laboratory Manual” (1989); Ausubel, R. M. (Ed.), “Current Protocols in Molecular Biology” Volumes I-III (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley &amp; Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (Eds.), “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); Methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; Cellis, J. E. (Ed.), “Cell Biology: A Laboratory Handbook”, Volumes I-III (1994); Freshney, “Culture of Animal Cells—A Manual of Basic Technique” Third Edition, Wiley-Liss, N.Y. (1994); Coligan J. E. (Ed.), “Current Protocols in Immunology” Volumes I-III (1994); Stites et al. (Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton &amp; Lange, Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); Clokie and Kropinski (Eds.), “Bacteriophage Methods and Protocols”, Volume 1: Isolation, Characterization, and Interactions (2009), all of which are incorporated by reference. Other general references are provided throughout this document. 
     Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only. 
     Experimental Details 
     Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only. 
     CRISPR repeat (crRNA), transactivating crRNA (tracrRNA), nuclease polypeptide, and 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 were predicted is provided in Table 1. 
     Construction of OMNI Nuclease Polypeptides 
     For construction of OMNI nuclease polypeptides, the open reading frame of several identified OMNI nucleases (OMNIs) were codon optimized for human cell line expression. The ORF was cloned into the bacterial plasmid pb-NNC and into the mammalian plasmid pmOMNI (Table 4). 
     Prediction and Construction of sgRNA 
     For each OMNI the sgRNA was predicted by detection of the CRISPR repeat array sequence (crRNA) and a trans-activating crRNA (tracrRNA) in the respective 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 tool. 
     The predicted secondary structures of the full duplex RNA elements (crRNA-tracrRNA chimera) was used for identification of possible tracr sequences for the design of a sgRNA having various versions for each OMNI nuclease. By shortening the duplex at the upper stem at different locations, the crRNA and tracrRNA were connected with tetra-loop ‘gaaa’, thereby generating possible sgRNA scaffolds (sgRNA designs of all OMNIs are listed in Table 2). At least two versions of possible designed scaffolds for each OMNI were synthesized and connected downstream to a 22 nt universal unique spacer sequence (T2, SEQ ID NO: 56) and cloned into a bacterial expressing plasmid under a constitutive promoter and into a mammalian expression plasmid under a U6 promoter (pbGuide and pmGuide, respectively, Table 4). 
     In order to overcome potential transcriptional and structural constraints and to assess the plasticity of the sgRNA scaffold in the human cellular environmental context, several versions of the sgRNA were tested. In each case the modifications represent small variations in the nucleotide sequence of the possible sgRNA ( FIG. 1C , Table 2). 
     
       
         
           
               
               
            
               
                   
                 T1- 
               
               
                   
                 (SEQ ID NO: 55) 
               
               
                   
                 GGTGCGGTTCACCAGGGTGTCG 
               
               
                   
                   
               
               
                   
                 T2- 
               
               
                   
                 (SEQ ID NO: 56) 
               
               
                   
                 GGAAGAGCAGAGCCTTGGTCTC 
               
            
           
         
       
     
     Bacterial PAM Depletion Assay 
     To confirm that each of the identified nucleases are functional CRISPR-OMNI nuclease systems and to identify their PAM sequences,  E. coli  strain BW25141 (1DE3) were co-transformed with: (1) a library plasmid pool containing randomized PAM sequences of 8 N&#39;s flanking a unique protospacer (pbPOS T2 library, Table 4); (2) plasmids encoding  E. coli  codon-optimized OMNI nucleases, pbNNC2 (Table 4); and (3) a plasmid encoding a designed sgRNA targeting the protospacer of the library, or a non-targeting gRNA as control (pbGuide, T2 and T1, respectively, Table 4). Next, cells were selected for all three plasmids by recovering them on media containing the appropriate antibiotics. In this this assay, 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. The surviving 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 samples. By comparing the frequency of a sequence in the library after selection of the targeting guide (T2) relative to the non-targeting (T1), individual PAM sequences were be identified ( FIG. 2A-2E ). The presented data reflect a condensed 4N window library with all possible locations along the 8 bp sequence. Sequence motifs were generated using the Weblogo tool. Activity of the OMNI nuclease was estimated based on the average of the two most depleted sequences and was calculated as: 
       1−Depletion score (Depletion score−Average of the ratios from the two most depleted sites)
 
     OMNI nucleases with scores that are higher than 0.6 were considered to be active. Following deep sequencing we detected depletion in the tested OMNI systems, indicating functional DNA interference in a heterologous host ( FIGS. 2A-2E , Table 3). 
     In-Vitro Depletion Assay by TXTL 
     Depletion of PAM sequences in-vitro was followed by Maxwell et al, Methods. 2018. Briefly, linear DNA expressing the OMNI nucleases and an sgRNA under T7 promoter were added to a TXTL mix (Arbor Bioscience) together with a linear construct expressing T7 polymerase. 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 T2 library, Table 4) flanked by an 8N randomized set of potential PAM sequences. Depletion of PAM sequences from the library was measured by high-throughput sequencing upon using PCR to add the necessary adapters and indices to both the cleaved library and to a control library expressing a non-targeting gRNA (T1). Following deep sequencing, the in-vitro activity was confirmed by the fraction of the depleted sequences having the same PAM sequence relative to their occurrence in the control by the OMNI nuclease indicating functional DNA cleavage by an in-vitro system ( FIGS. 3A-3M , Table 3). 
     PAM Library in Mammalian System 
     While a PAM sequence preference is considered as an inherent property of the nuclease, it may be affected, to some extent, by the cellular environment, genomic composition and genome size. Since the human cellular environment is significantly different from the bacterial environment with respect to those properties, a “fine tuning” step has been introduced to address potential differences in PAM preferences in the human cellular context. To this end, a PAM library was constructed in a human cell line. The PAM library was introduced to the cells using a viral vector (see Table 4), as a constant target sequence followed by a stretch of 6N. Upon introduction of an OMNI and an sgRNA targeting the library constant target site, NGS analysis was used to identify the edited sequences and the PAM associated with them. The enriched edited sequences were then used to define the PAM consensus. We apply this methodology to determine the optimized PAM requirements of OMNI nuclease in mammalian cells (Table 3, “mammalian refinements”). The OMNI-53 PAM is a reduced version of the PAM identified by TXTL. On the other hand, OMNI-40 shows a stricter PAM compared with TXTL results. The OMNI-39 PAM could not be determined using the mammalian system due to a low number of editing events. 
     Expression of OMNI Nucleases Coded by an Optimized DNA Sequence in Mammalian Cells 
     First, expression of each of the optimized DNA sequences coding for OMNI-39, OMNI-40, and OMNI-53 in mammalian cells was validated. To this end, an expression vector coding for an HA-tagged OMNI nuclease or  Streptococcus Pyogenes  Cas9 (SpCas9) linked to mCherry by a P2A peptide (pmOMNI, Table 4) was introduced into Hek293T cells using the Jet-optimus™ transfection reagent (polyplus-transfection). The P2A peptide is a self-cleaving peptide which can induce the cleaving of the recombinant protein in a cell such that the OMNI nuclease and the mCherry are separated upon expression. The mCherry serves as indicator for transcription efficiency of the OMNI from expression vector. Expression of all OMNI proteins was confirmed by a western blot assay using anti-HA antibody ( FIG. 4 ). 
     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. To this end, for each OMNI a corresponding OMNI-P2A-mCherry expression vector (pmOMNI, Table 4) was transfected into HeLa cells together with an sgRNA designed to target a specific location in the human genome (pmGuide, Table 4). At 72 h, cells were harvested. Half of the cells were used for quantification of transfection efficiency by FACS using mCherry fluorescence as a marker. The other half of the cells were lysed, and their genomic DNA content was used to PCR amplify the corresponding putative genomic targets. Amplicons were subjected to NGS and the resulting sequences were then used to 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 percent editing was deduced from the fraction of indel-containing sequences within each amplicon. All editing values were normalized to the transfection and translation efficacy obtained for each experiment 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 eleven unique sgRNAs each designed to target a different genomic location. The results of these experiments are summarized in Table 5. As can be seen in the table (column 6, “% editing”), all of the OMNIs exhibit high and significant editing levels compared to the negative control (column 9, “% editing in neg control”) in all or most target sites tested. OMNI-39 exhibited high and significant editing levels in two out of four sites tested. OMNI-40 and OMN-53 exhibited high and significant editing levels in three of four sites tested. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 OMNI nuclease sequences 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ ID 
                   
                 SEQ ID NO 
                 SEQ ID NO of DNA 
               
               
                   
                 NO of 
                   
                 of DNA 
                 sequence codon 
               
               
                   
                 Amino 
                   
                 sequence 
                 optimized for 
               
               
                 “OMNI” 
                 Acid 
                   
                 encoding 
                 encoding OMNI in 
               
               
                 Name 
                 Sequence 
                 Source Organism 
                 OMNI 
                 human cells 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 OMNI-32 
                 149 
                   Acetobacterium  sp. 
                 335 
                 343 
               
               
                   
                   
                 KB-1 
                   
                   
               
               
                 OMNI-34 
                 150 
                   Alistipes  sp. An54 
                 167 
                 177 
               
               
                 OMNI-35 
                 151 
                   Bartonella  apis 
                 168 
                 178 
               
               
                 OMNI-36 
                 152 
                   Blastopirellula marina  169 
                 179 
                   
               
               
                 OMNI-37 
                 153 
                 Bryobacter aggregatus 
                 336 
                 344 
               
               
                   
                   
                 MPL3 
                   
                   
               
               
                 OMNI-38 
                 154 
                   Algoriphagus  marinus 
                 337 
                 345 
               
               
                 OMNI-39 
                 1 
                   Butyrivibrio  sp. 
                 5 
                 6, 7 
               
               
                   
                   
                 AC2005 
                   
                   
               
               
                 OMNI-40 
                 2 
                 bacterium LF-3 
                 8 
                  9, 10 
               
               
                 OMNI-41 
                 155 
                 Aliiarcobacter faecis 
                 338 
                 346 
               
               
                 OMNI-42 
                 156 
                 Caviibacter abscessus 
                 170 
                 180 
               
               
                 OMNI-43 
                 157 
                   Arcobacter  sp. 
                 171 
                 181 
               
               
                   
                   
                 SM1702 
                   
                   
               
               
                 OMNI-44 
                 158 
                 
                   Arcobacter 
                   mytili 
                 
                 172 
                 182 
               
               
                 OMNI-45 
                 159 
                 
                   Arcobacter thereius 
                 
                 339 
                 347 
               
               
                 OMNI-46 
                 160 
                 
                   Carnobacterium 
                 
                 173 
                 183 
               
               
                   
                   
                 
                   funditum 
                 
                   
                   
               
               
                 OMNI-47 
                 161 
                   Peptoniphilus   obesiph 1 
                 174 
                 184 
               
               
                 OMNI-48 
                 162 
                 Carnobacterium iners 
                 340 
                 348 
               
               
                 OMNI-49 
                 163 
                 
                   Lactobacillus allii 
                 
                 341 
                 349 
               
               
                 OMNI-51 
                 164 
                 
                   Bacteroides coagulans 
                 
                 175 
                 185 
               
               
                 OMNI-52 
                 165 
                   Butyrivibrio  sp. 
                 176 
                 186 
               
               
                   
                   
                 NC3005 
                   
                   
               
               
                 OMNI-53 
                 4 
                   Clostridium  sp. AF02- 
                 14 
                 15, 16 
               
               
                   
                   
                 29 
                   
                   
               
               
                 OMNI-54 
                 166 
                 
                   Algoriphagus 
                 
                 342 
                 350 
               
               
                   
                   
                 
                   antarcticus 
                 
                   
                   
               
               
                   
               
               
                 Table 1. OMNI nuclease sequences: Table 1 lists the organism from which the OMNI nuclease was identified, its protein sequence, its DNA sequence, and its human optimized DNA sequence(s). 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 OMNI Guide Sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 OMNI-34 
                 OMNI-35 
                 OMNI-36 
                 OMNI-39 
               
               
                   
               
               
                 Minimal 
                 crRNA 
                 GUUGUGGU 
                 GUUGCGGCUU 
                 GCUGUGGCUU 
                 GUUUUAGUA 
               
               
                 crRNA: 
                 (Repeat) 
                 UUG 
                 G 
                 GGAGGGA 
                 CC 
               
               
                 tracrRNA 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
               
               
                 duplex 
                   
                 187) 
                 201) 
                 213) 
                 226) 
               
               
                   
                 tracrRNA 
                 CUUACCAC 
                 CUGGCUGUUA 
                 UGCUUCGCAA 
                 GACCUACUAA 
               
               
                   
                 (Antirepeat) 
                 AAU 
                 AC 
                 GUCAUAGU 
                 AAU 
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
               
               
                   
                   
                 188) 
                 202) 
                 214) 
                 227) 
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUGUGGU 
                 GUUGCGGCUU 
                 GCUGUGGCUU 
                 GUUUUAGUA 
               
               
                 tracrRNA 
                 (Repeat) 
                 UUGAUGUA 
                 GACCGC 
                 GGAGGGAAU 
                 CCUAGAG 
               
               
                 duplex V1 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 CGU 
                 (SEQ ID NO: 
               
               
                   
                   
                 189) 
                 203) 
                 (SEQ ID NO: 
                 17) 
               
               
                   
                   
                   
                   
                 215) 
                   
               
               
                   
                 tracrRNA 
                 UACAUCUU 
                 GCGGUCUGGC 
                 ACGAUUGCUU 
                 CUUUAGACCU 
               
               
                   
                 (Antirepeat) 
                 ACCACAAU 
                 UGUUAAC 
                 CGCAAGUCAU 
                 ACUAAAAU 
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 AGU 
                 (SEQ ID NO: 
               
               
                   
                   
                 190) 
                 204) 
                 (SEQ ID NO: 
                 18) 
               
               
                   
                   
                   
                   
                 216) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUGUGGU 
                 GUUGCGGCUU 
                 GCUGUGGCUU 
                 GUUUUAGUA 
               
               
                 tracrRNA 
                 (Repeat) 
                 UUGAUGUA 
                 GACCGCAUU 
                 GGAGGGAAU 
                 CCUAGAGAAA 
               
               
                 duplex V2 
                   
                 GAA 
                 (SEQ ID NO: 
                 CGUCGC 
                 (SEQ ID NO: 
               
               
                   
                   
                 (SEQ ID NO: 
                 205) 
                 (SEQ ID NO: 
                 19) 
               
               
                   
                   
                 191) 
                   
                 217) 
                   
               
               
                   
                 tracrRNA 
                 UUCUACAU 
                 AAUGCGGUCU 
                 GCGACGAUUG 
                 UUUCUUUAG 
               
               
                   
                 (Antirepeat) 
                 CUUACCAC 
                 GGCUGUUAAC 
                 CUUCGCAAGU 
                 ACCUACUAAA 
               
               
                   
                   
                 AAU 
                 (SEQ ID NO: 
                 CAUAGU 
                 AU  
               
               
                   
                   
                 (SEQ ID NO: 
                 206) 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
               
               
                   
                   
                 192) 
                   
                 218) 
                 20) 
               
               
                   
               
               
                 TracrRNA 
                 TracrRNA 
                 AAGGCUAU 
                 AAGCUAGAU 
                 AAAGCAAUA 
                 AAGGCUUUA 
               
               
                 sequences 
                 Portion 1 
                 AUGCC 
                 AUGC 
                 GUCAGCG 
                 UGCC 
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
               
               
                   
                   
                 193) 
                 207) 
                 219) 
                 228) 
               
               
                   
                 TracrRNA 
                 GAAGGUUU 
                 ACCAAAUAAG 
                 AAAGGUUUG 
                 GAGAUUAAA 
               
               
                   
                 Portion 2 
                 UCAACCU 
                 ACAGCUCCUC 
                 CUCACGGAGC 
                 GGAUGCCGAC 
               
               
                   
                   
                 (SEQ ID NO:  
                 CGGGGGCUGU 
                 AUUCCGUCGA 
                 GGGCAUCCUU 
               
               
                   
                   
                 194) 
                 UUUUU 
                 GUACCCUUU 
                 UUUU 
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
               
               
                   
                   
                   
                 208) 
                 220) 
                 229) 
               
               
                   
                 TracrRNA 
                 ACCGUCUCC 
                 Not listed 
                 GACGCCUCCC 
                 Not listed 
               
               
                   
                 Portion 3 
                 GCGUAUUC 
                   
                 AGCGGGGCGU 
                   
               
               
                   
                   
                 CGUGGAGA 
                   
                 CUUUUUUU 
                   
               
               
                   
                   
                 CUUUUUU 
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 221) 
                   
               
               
                   
                   
                 195) 
                   
                   
                   
               
               
                   
                 TracrRNA 
                 Not listed 
                 Not listed 
                 Not listed 
                 Not listed 
               
               
                   
                 Portion 4 
                   
                   
                   
                   
               
               
                   
                 Full 
                 UACAUCUU 
                 GCGGUCUGGC 
                 ACGAUUGCUU 
                 CUUUAGACCU 
               
               
                   
                 tracrRNA 
                 ACCACAAU 
                 UGUUAACAA 
                 CGCAAGUCAU 
                 ACUAAAAUA 
               
               
                   
                 V1 
                 AAGGCUAU 
                 GCUAGAUAU 
                 AGUAAAGCA 
                 AGGCUUUAU 
               
               
                   
                   
                 AUGCCGAA 
                 GCACCAAAUA 
                 AUAGUCAGCG 
                 GCCGAGAUUA 
               
               
                   
                   
                 GGUUUUCA 
                 AGACAGCUCC 
                 AAAGGUUUG 
                 AAGGAUGCCG 
               
               
                   
                   
                 ACCUACCG 
                 UCCGGGGGCU 
                 CUCACGGAGC 
                 ACGGGCAUCC 
               
               
                   
                   
                 UCUCCGCG 
                 GUUUUUU 
                 AUUCCGUCGA 
                 UUUUUU 
               
               
                   
                   
                 UAUUCCGU 
                 (SEQ ID NO: 
                 GUACCCUUUG 
                 (SEQ ID NO: 
               
               
                   
                   
                 GGAGACUU 
                 209) 
                 ACGCCUCCCA 
                 230) 
               
               
                   
                   
                 UUUU 
                   
                 GCGGGGCGUC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 UUUUUUU 
                   
               
               
                   
                   
                 196) 
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 222) 
                   
               
               
                   
                 Full 
                 UUCUACAU 
                 AAUGCGGUCU 
                 GCGACGAUUG 
                 UUUCUUUAG 
               
               
                   
                 tracrRNA 
                 CUUACCAC 
                 GGCUGUUAAC 
                 CUUCGCAAGU 
                 ACCUACUAAA 
               
               
                   
                 V2 
                 AAUAAGGC 
                 AAGCUAGAU 
                 CAUAGUAAA 
                 AUAAGGCUU 
               
               
                   
                   
                 UAUAUGCC 
                 AUGCACCAAA 
                 GCAAUAGUCA 
                 UAUGCCGAGA 
               
               
                   
                   
                 GAAGGUUU 
                 UAAGACAGCU 
                 GCGAAAGGU 
                 UUAAAGGAU 
               
               
                   
                   
                 UCAACCUA 
                 CCUCCGGGGG 
                 UUGCUCACGG 
                 GCCGACGGGC 
               
               
                   
                   
                 CCGUCUCCG 
                 CUGUUUUUU 
                 AGCAUUCCGU 
                 AUCCUUUUUU 
               
               
                   
                   
                 CGUAUUCC 
                 (SEQ ID NO: 
                 CGAGUACCCU 
                 (SEQ ID NO: 
               
               
                   
                   
                 GUGGAGAC 
                 210) 
                 UUGACGCCUC 
                 231) 
               
               
                   
                   
                 UUUUUU 
                   
                 CCAGCGGGGC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 GUCUUUUUU 
                   
               
               
                   
                   
                 197) 
                   
                 U 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 223) 
                   
               
               
                   
               
               
                 sgRNA 
                 sgRNA V1 
                 GUUGUGGU 
                 GUUGCGGCUU 
                 GCUGUGGCUU 
                 GUUUUAGUA 
               
               
                 Versions 
                   
                 UUGAUGUA 
                 GACCGCgaaaG 
                 GGAGGGAAU 
                 CCUAGAGgaaa 
               
               
                   
                   
                 gaaaUACAUC  
                 CGGUCUGGCU 
                 CGUgaaaACGA 
                 CUUUAGACCU 
               
               
                   
                   
                 UUACCACA 
                 GUUAACAAGC 
                 UUGCUUCGCA 
                 ACUAAAAUA 
               
               
                   
                   
                 AUAAGGCU 
                 UAGAUAUGC 
                 AGUCAUAGU 
                 AGGCUUUAU 
               
               
                   
                   
                 AUAUGCCG 
                 ACCAAAUAAG 
                 AAAGCAAUA 
                 GCCGAGAUUA 
               
               
                   
                   
                 AAGGUUUU 
                 ACAGCUCCUC 
                 GUCAGCGAAA 
                 AAGGAUGCCG 
               
               
                   
                   
                 CAACCUACC 
                 CGGGGGCUGU 
                 GGUUUGCUCA 
                 ACGGGCAUCC 
               
               
                   
                   
                 GUCUCCGC 
                 UUUUU 
                 CGGAGCAUUC 
                 UUUUUU  
               
               
                   
                   
                 GUAUUCCG 
                 (SEQ ID NO: 
                 CGUCGAGUAC 
                 (SEQ ID NO: 
               
               
                   
                   
                 UGGAGACU 
                 211) 
                 CCUUUGACGC 
                 24) 
               
               
                   
                   
                 UUUUU 
                   
                 CUCCCAGCGG 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 GGCGUCUUUU 
                   
               
               
                   
                   
                 198) 
                   
                 UUU 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 224) 
                   
               
               
                   
                 sgRNA V2 
                 GUUGUGGU 
                 GUUGCGGCUU 
                 GCUGUGGCUU 
                 GUUUUAGUA 
               
               
                   
                   
                 UUGAUGUA 
                 GACCGCAUUg 
                 GGAGGGAAU 
                 CCUAGAGAAA 
               
               
                   
                   
                 GAAgaaaUUC 
                 aaaAAUGCGGU 
                 CGUCGCgaaaG 
                 gaaaUUUCUUU 
               
               
                   
                   
                 UACAUCUU 
                 CUGGCUGUUA 
                 CGACGAUUGC 
                 AGACCUACUA 
               
               
                   
                   
                 ACCACAAU 
                 ACAAGCUAGA 
                 UUCGCAAGUC 
                 AAAUAAGGC 
               
               
                   
                   
                 AAGGCUAU 
                 UAUGCACCAA 
                 AUAGUAAAG 
                 UUUAUGCCGA 
               
               
                   
                   
                 AUGCCGAA 
                 AUAAGACAGC 
                 CAAUAGUCAG 
                 GAUUAAAGG 
               
               
                   
                   
                 GGUUUUCA 
                 UCCUCCGGGG 
                 CGAAAGGUU 
                 AUGCCGACGG 
               
               
                   
                   
                 ACCUACCG 
                 GCUGUUUUU 
                 UGCUCACGGA 
                 GCAUCCUUUU 
               
               
                   
                   
                 UCUCCGCG 
                 U 
                 GCAUUCCGUC 
                 UU 
               
               
                   
                   
                 UAUUCCGU 
                 (SEQ ID NO: 
                 GAGUACCCUU 
                 (SEQ ID NO: 
               
               
                   
                   
                 GGAGACUU 
                 212) 
                 UGACGCCUCC 
                 25) 
               
               
                   
                   
                 UUUU 
                   
                 CAGCGGGGCG 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 UCUUUUUUU 
                   
               
               
                   
                   
                 199) 
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 225) 
                   
               
               
                   
               
               
                 Other 
                 sgRNA V3 
                 GUUGUGGU 
                 Not listed 
                 Not listed 
                 GUUUAAGUA 
               
               
                 sgRNA 
                   
                 UUGAUGUA 
                   
                   
                 CCUAGAGAAA 
               
               
                 Optimi- 
                   
                 GAAgaaaUUC 
                   
                   
                 gaaaUUUCUUU 
               
               
                 zations 
                   
                 UACAUCUU 
                   
                   
                 AGACCUACUU 
               
               
                   
                   
                 ACCACAAU 
                   
                   
                 AAAUAAGGC 
               
               
                   
                   
                 AAGGCUAU 
                   
                   
                 UUUAUGCCGA 
               
               
                   
                   
                 AUGCCGAA 
                   
                   
                 GAUUAAAGG 
               
               
                   
                   
                 GGUUAUCA 
                   
                   
                 AUGCCGACGG 
               
               
                   
                   
                 ACCUACCG 
                   
                   
                 GCAUCCUUUU 
               
               
                   
                   
                 UCUCCGCG 
                   
                   
                 UU 
               
               
                   
                   
                 UAUUCCGU 
                   
                   
                 (SEQ ID NO: 
               
               
                   
                   
                 GGAGACUU 
                   
                   
                 26) 
               
               
                   
                   
                 UUUU 
                   
                   
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                   
                   
               
               
                   
                   
                 200) 
               
               
                   
               
               
                   
                   
                 OMNI-40 
                 OMNI-42 
                 OMNI-43 
               
               
                   
               
               
                 Minimal 
                 crRNA 
                 GUUUUGUUA 
                 GUUUAAGAG 
                 GUUUUAAUA 
                   
               
               
                 crRNA: 
                 (Repeat) 
                 CC 
                   
                 CCCCUACA 
                   
               
               
                 tracrRNA 
                   
                 (SEQ ID NO: 
                   
                 (SEQ ID NO: 
                   
               
               
                 duplex 
                   
                 232) 
                   
                 250) 
                   
               
               
                   
                 tracrRNA 
                 GACCUAACAA 
                 CGAGUUUA 
                 UAAUAGGGG 
                   
               
               
                   
                 (Antirepeat) 
                 AAC 
                   
                 UAUUAAAC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 233) 
                   
                 251) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUUGUUA 
                 GUUUAAGAG 
                 GUUUUAAUA 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 CCAUAUG 
                 UUAUG 
                 CCCCUACAAA 
                   
               
               
                 duplex V1 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 CUG 
                   
               
               
                   
                   
                 27) 
                 238) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 252) 
                   
               
               
                   
                 tracrRNA 
                 UAUAUGACCU 
                 CAUAACGAGU 
                 CAGUUUAAU 
                   
               
               
                   
                 (Antirepeat) 
                 AACAAAAC 
                 UUA 
                 AGGGGUAUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 AAAC 
                   
               
               
                   
                   
                 28) 
                 239) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 253) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUUGUUA 
                 GUUUAAGAG 
                 GUUUUAAUA 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 CCAUAUGAUU 
                 UUAUGUAA 
                 CCCCUACAAA 
                   
               
               
                 duplex V2 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 CUGCUA 
                   
               
               
                   
                   
                 29) 
                 240) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 254) 
                   
               
               
                   
                 tracrRNA 
                 AUUUAUAUG 
                 UUACAUAACG 
                 UAACAGUUU 
                   
               
               
                   
                 (Antirepeat) 
                 ACCUAACAAA 
                 AGUUUA 
                 AAUAGGGGU 
                   
               
               
                   
                   
                 AC 
                 (SEQ ID NO: 
                 AUUAAAC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 241) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 30) 
                   
                 255) 
                   
               
               
                   
               
               
                 TracrRNA 
                 TracrRNA 
                 AAGGGUUUA 
                 AAUAAAAAU 
                 UAAGGUUGC 
                   
               
               
                 sequences 
                 Portion 1 
                 UCCC 
                 UUAUUGAAA 
                 UAUUUUAGC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UC 
                 AACU 
                   
               
               
                   
                   
                 234) 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 242) 
                 256) 
                   
               
               
                   
                 TracrRNA 
                 GGACUCGGCU 
                 GUCAAAUUA 
                 GACUUUAGGC 
                   
               
               
                   
                 Portion 2 
                 CUUCGGAGCC 
                 UUUUUGAC 
                 AGUGGUUUC 
                   
               
               
                   
                   
                 UUUUU 
                 (SEQ ID NO: 
                 GACCACUUGC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 243) 
                 CCUUUUUU 
                   
               
               
                   
                   
                 235) 
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 257) 
                   
               
               
                   
                 TracrRNA 
                 Not listed 
                 UAGCCUCUUU 
                 Not listed 
                   
               
               
                   
                 Portion 3 
                   
                 UUGAAGAGG 
                   
                   
               
               
                   
                   
                   
                 UUUUUUU 
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                   
                 244) 
                   
                   
               
               
                   
                 TracrRNA 
                 Not listed 
                 Not listed 
                 Not listed 
                   
               
               
                   
                 Portion 4 
                   
                   
                   
                   
               
               
                   
                 Full 
                 UAUAUGACCU 
                 CAUAACGAGU 
                 CAGUUUAAU 
                   
               
               
                   
                 tracrRNA 
                 AACAAAACAA 
                 UUAAAUAAA 
                 AGGGGUAUU 
                   
               
               
                   
                 V1 
                 GGGUUUAUCC 
                 AAUUUAUUG 
                 AAACUAAGG 
                   
               
               
                   
                   
                 CGGACUCGGC 
                 AAAUCGUCAA 
                 UUGCUAUUU 
                   
               
               
                   
                   
                 UCUUCGGAGC 
                 AUUAUUUUU 
                 UAGCAACUGA 
                   
               
               
                   
                   
                 CUUUUU 
                 GACUAGCCUC 
                 CUUUAGGCAG 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UUUUUGAAG 
                 UGGUUUCGAC 
                   
               
               
                   
                   
                 236) 
                 AGGUUUUUU 
                 CACUUGCCCU 
                   
               
               
                   
                   
                   
                 U 
                 UUUUU 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 245) 
                 258) 
                   
               
               
                   
                 Full 
                 AUUUAUAUG 
                 UUACAUAACG 
                 UAACAGUUU 
                   
               
               
                   
                 tracrRNA 
                 ACCUAACAAA 
                 AGUUUAAAU 
                 AAUAGGGGU 
                   
               
               
                   
                 V2 
                 ACAAGGGUU 
                 AAAAAUUUA 
                 AUUAAACUA 
                   
               
               
                   
                   
                 UAUCCCGGAC 
                 UUGAAAUCG 
                 AGGUUGCUA 
                   
               
               
                   
                   
                 UCGGCUCUUC 
                 UCAAAUUAU 
                 UUUUAGCAAC 
                   
               
               
                   
                   
                 GGAGCCUUUU 
                 UUUUGACUA 
                 UGACUUUAG 
                   
               
               
                   
                   
                 U 
                 GCCUCUUUUU 
                 GCAGUGGUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 GAAGAGGUU 
                 UCGACCACUU 
                   
               
               
                   
                   
                 237) 
                 UUUUU 
                 GCCCUUUUUU 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 246) 
                 259) 
                   
               
               
                   
               
               
                 sgRNA 
                 sgRNA V1 
                 GUUUUGUUA 
                 GUUUAAGAG 
                 GUUUUAAUA 
                   
               
               
                 Versions 
                   
                 CCAUAUGgaaa 
                 UUAUGgaaaCA 
                 CCCCUACAAA 
                   
               
               
                   
                   
                 UAUAUGACCU 
                 UAACGAGUU 
                 CUGgaaaCAGU 
                   
               
               
                   
                   
                 AACAAAACAA 
                 UAAAUAAAA 
                 UUAAUAGGG 
                   
               
               
                   
                   
                 GGGUUUAUCC 
                 AUUUAUUGA 
                 GUAUUAAAC 
                   
               
               
                   
                   
                 CGGACUCGGC 
                 AAUCGUCAAA 
                 UAAGGUUGC 
                   
               
               
                   
                   
                 UCUUCGGAGC 
                 UUAUUUUUG 
                 UAUUUUAGC 
                   
               
               
                   
                   
                 CUUUUU 
                 ACUAGCCUCU 
                 AACUGACUUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UUUUGAAGA 
                 AGGCAGUGG 
                   
               
               
                   
                   
                 34) 
                 GGUUUUUUU 
                 UUUCGACCAC 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 UUGCCCUUUU 
                   
               
               
                   
                   
                   
                 247) 
                 UU 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 260) 
                   
               
               
                   
                 sgRNA V2 
                 GUUUUGUUA 
                 GUUUAAGAG 
                 GUUUUAAUA 
                   
               
               
                   
                   
                 CCAUAUGAUU 
                 UUAUGUAAga 
                 CCCCUACAAA 
                   
               
               
                   
                   
                 gaaaAUUUAUA 
                 aaUUACAUAA 
                 CUGCUAgaaaU 
                   
               
               
                   
                   
                 UGACCUAACA 
                 CGAGUUUAA 
                 AACAGUUUA 
                   
               
               
                   
                   
                 AAACAAGGG 
                 AUAAAAAUU 
                 AUAGGGGUA 
                   
               
               
                   
                   
                 UUUAUCCCGG 
                 UAUUGAAAU 
                 UUAAACUAA 
                   
               
               
                   
                   
                 ACUCGGCUCU 
                 CGUCAAAUUA 
                 GGUUGCUAU 
                   
               
               
                   
                   
                 UCGGAGCCUU 
                 UUUUUGACU 
                 UUUAGCAACU 
                   
               
               
                   
                   
                 UUU 
                 AGCCUCUUUU 
                 GACUUUAGGC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UGAAGAGGU 
                 AGUGGUUUC 
                   
               
               
                   
                   
                 35) 
                 UUUUUU 
                 GACCACUUGC 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 CCUUUUUU 
                   
               
               
                   
                   
                   
                 248) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 261) 
                   
               
               
                   
               
               
                 Other 
                 sgRNA V3 
                 GUUUAGUUA 
                 GUUUAAGAG 
                 GUUUAAAUA 
                   
               
               
                 sgRNA 
                   
                 CCAUAUGAUU 
                 UUAUGUAAga 
                 CCCCUACAAA 
                   
               
               
                 Optimi- 
                   
                 gaaaAUUUAUA 
                 aaUUACAUAA 
                 CUGCUAgaaaU 
                   
               
               
                 zations 
                   
                 UGACCUAACU 
                 CGAGUUUAA 
                 AACAGUUUA 
                   
               
               
                   
                   
                 AAACAAGGG 
                 AUAAAAAUU 
                 AUAGGGGUA 
                   
               
               
                   
                   
                 UUUAUCCCGG 
                 UAUUGAAAU 
                 UUUAAACUA 
                   
               
               
                   
                   
                 ACUCGGCUCU 
                 CGUCAAAUUA 
                 AGGUUGCUA 
                   
               
               
                   
                   
                 UCGGAGCCUU 
                 UcUUUGACUA 
                 UCUUAGCAAC 
                   
               
               
                   
                   
                 UUU 
                 GCCUCUUAUU 
                 UGACUUUAG 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 GAAGAGGUU 
                 GCAGUGGUU 
                   
               
               
                   
                   
                 36) 
                 UUUUU 
                 UCGACCACUU 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                 GCCCUUUUUU 
                   
               
               
                   
                   
                   
                 249) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 262) 
               
               
                   
               
               
                   
                   
                 OMNI-44 
                 OMNI-46 
                 OMNI-47 
               
               
                   
               
               
                 Minimal 
                 crRNA 
                 GUUUUAAUA 
                 GCUAUACGUU 
                 GUUUGAGAG 
                   
               
               
                 crRNA: 
                 (Repeat) 
                 CCCCUAUA 
                 CCUUAC 
                   
                   
               
               
                 tracrRNA 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
                   
               
               
                 duplex 
                   
                 263) 
                 276) 
                   
                   
               
               
                   
                 tracrRNA 
                 UAAUAGGGG 
                 GCAAGGAACG 
                 UGAGUUCAA 
                   
               
               
                   
                 (Antirepeat) 
                 UAUUAAAC 
                 UAUAGU 
                 AU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 264) 
                 277) 
                 289) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUUAAUA 
                 GCUAUACGUU 
                 GUUUGAGAG 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 CCCCUAUAAA 
                 CCUUACAAAA 
                 UUAUG 
                   
               
               
                 duplex V1 
                   
                 CUA 
                 U 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 290) 
                   
               
               
                   
                   
                 265) 
                 278) 
                   
                   
               
               
                   
                 tracrRNA 
                 UAGUUUAAU 
                 ACUUUGCAAG 
                 CAUGAUGAG 
                   
               
               
                   
                 (Antirepeat) 
                 AGGGGUAUU 
                 GAACGUAUA 
                 UUCAAAU 
                   
               
               
                   
                   
                 AAAC 
                 GU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 291) 
                   
               
               
                   
                   
                 266) 
                 279) 
                   
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUUAAUA 
                 GCUAUACGUU 
                 GUUUGAGAG 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 CCCCUAUAAA 
                 CCUUACAAAA 
                 UUAUGUAA 
                   
               
               
                 duplex V2 
                   
                 CUACUA 
                 UCGG 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 292) 
                   
               
               
                   
                   
                 267) 
                 280) 
                   
                   
               
               
                   
                 tracrRNA 
                 UAGUAGUUU 
                 CCGACUUUGC 
                 UUACAUGAU 
                   
               
               
                   
                 (Antirepeat) 
                 AAUAGGGGU 
                 AAGGAACGU 
                 GAGUUCAAA 
                   
               
               
                   
                   
                 AUUAAAC 
                 AUAGU 
                 U 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 268) 
                 281) 
                 293) 
                   
               
               
                   
               
               
                 TracrRNA 
                 TracrRNA 
                 UAAGACUACU 
                 AAAGGGAGU 
                 AAAAAUUUA 
                   
               
               
                 sequences 
                 Portion 1 
                 UUAAUAGUA 
                 GCUCUGCACU 
                 UUCAAAUC 
                   
               
               
                   
                   
                 GUU 
                 CUCCU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 294) 
                   
               
               
                   
                   
                 269) 
                 282) 
                   
                   
               
               
                   
                 TracrRNA 
                 GAUUUUAGG 
                 GUAAAGCACU 
                 GCCCAUUAUG 
                   
               
               
                   
                 Portion 2 
                 AGAUAGUUU 
                 AACCCCAUUU 
                 GGC 
                   
               
               
                   
                   
                 UUCUAUCUCC 
                 UCUUCGGAGA 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 CUUUUU 
                 AUGGGGUUA 
                 295) 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UCUUUUU 
                   
                   
               
               
                   
                   
                 270) 
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                   
                 283) 
                   
                   
               
               
                   
                 TracrRNA 
                 Not listed 
                 Not listed 
                 CGCAGAUGUU 
                   
               
               
                   
                 Portion 3 
                   
                   
                 CUGC 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 296) 
                   
               
               
                   
                 TracrRNA 
                 Not listed 
                 Not listed 
                 AUUAUAUGC 
                   
               
               
                   
                 Portion 4 
                   
                   
                 UUGCAAGUU 
                   
               
               
                   
                   
                   
                   
                 GCAAGCUUUU 
                   
               
               
                   
                   
                   
                   
                 UUU 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 297) 
                   
               
               
                   
                 Full 
                 UAGUUUAAU 
                 ACUUUGCAAG 
                 CAUGAUGAG 
                   
               
               
                   
                 tracrRNA 
                 AGGGGUAUU 
                 GAACGUAUA 
                 UUCAAAUAA 
                   
               
               
                   
                 V1 
                 AAACUAAGAC 
                 GUAAAGGGA 
                 AAAUUUAUU 
                   
               
               
                   
                   
                 UACUUUAAU 
                 GUGCUCUGCA 
                 CAAAUCGCCC 
                   
               
               
                   
                   
                 AGUAGUUGA 
                 CUCUCCUGUA 
                 AUUAUGGGCC 
                   
               
               
                   
                   
                 UUUUAGGAG 
                 AAGCACUAAC 
                 GCAGAUGUUC 
                   
               
               
                   
                   
                 AUAGUUUUU 
                 CCCAUUUUCU 
                 UGCAUUAUA 
                   
               
               
                   
                   
                 CUAUCUCCCU 
                 UCGGAGAAU 
                 UGCUUGCAAG 
                   
               
               
                   
                   
                 UUUU 
                 GGGGUUAUC 
                 UUGCAAGCUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UUUUU 
                 UUUUU 
                   
               
               
                   
                   
                 271) 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 284) 
                 298) 
                   
               
               
                   
                 Full 
                 UAGUAGUUU 
                 CCGACUUUGC 
                 UUACAUGAU 
                   
               
               
                   
                 tracrRNA 
                 AAUAGGGGU 
                 AAGGAACGU 
                 GAGUUCAAA 
                   
               
               
                   
                 V2 
                 AUUAAACUA 
                 AUAGUAAAG 
                 UAAAAAUUU 
                   
               
               
                   
                   
                 AGACUACUUU 
                 GGAGUGCUCU 
                 AUUCAAAUCG 
                   
               
               
                   
                   
                 AAUAGUAGU 
                 GCACUCUCCU 
                 CCCAUUAUGG 
                   
               
               
                   
                   
                 UGAUUUUAG 
                 GUAAAGCACU 
                 GCCGCAGAUG 
                   
               
               
                   
                   
                 GAGAUAGUU 
                 AACCCCAUUU 
                 UUCUGCAUUA 
                   
               
               
                   
                   
                 UUUCUAUCUC 
                 UCUUCGGAGA 
                 UAUGCUUGCA 
                   
               
               
                   
                   
                 CCUUUUU 
                 AUGGGGUUA 
                 AGUUGCAAGC 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UCUUUUU 
                 UUUUUUU 
                   
               
               
                   
                   
                 272) 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 285) 
                 299) 
                   
               
               
                   
               
               
                 sgRNA 
                 sgRNA V1 
                 GUUUUAAUA 
                 GCUAUACGUU 
                 GUUUGAGAG 
                   
               
               
                 Versions 
                   
                 CCCCUAUAAA 
                 CCUUACAAAA 
                 UUAUGgaaaCA 
                   
               
               
                   
                   
                 CUAgaaaUAGU 
                 UgaaaACUUUG 
                 UGAUGAGUU 
                   
               
               
                   
                   
                 UUAAUAGGG 
                 CAAGGAACGU 
                 CAAAUAAAA 
                   
               
               
                   
                   
                 GUAUUAAAC 
                 AUAGUAAAG 
                 AUUUAUUCA 
                   
               
               
                   
                   
                 UAAGACUACU 
                 GGAGUGCUCU 
                 AAUCGCCCAU 
                   
               
               
                   
                   
                 UUAAUAGUA 
                 GCACUCUCCU 
                 UAUGGGCCGC 
                   
               
               
                   
                   
                 GUUGAUUUU 
                 GUAAAGCACU 
                 AGAUGUUCU 
                   
               
               
                   
                   
                 AGGAGAUAG 
                 AACCCCAUUU 
                 GCAUUAUAU 
                   
               
               
                   
                   
                 UUUUUCUAUC 
                 UCUUCGGAGA 
                 GCUUGCAAGU 
                   
               
               
                   
                   
                 UCCCUUUUU 
                 AUGGGGUUA 
                 UGCAAGCUUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UCUUUUU 
                 UUUU 
                   
               
               
                   
                   
                 273) 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                 286) 
                 300) 
                   
               
               
                   
                 sgRNA V2 
                 GUUUUAAUA 
                 GCUAUACGUU 
                 GUUUGAGAG 
                   
               
               
                   
                   
                 CCCCUAUAAA 
                 CCUUACAAAA 
                 UUAUGUAAga 
                   
               
               
                   
                   
                 CUACUAgaaaU 
                 UCGGgaaaCCG 
                 aaUUACAUGA 
                   
               
               
                   
                   
                 AGUAGUUUA 
                 ACUUUGCAAG 
                 UGAGUUCAA 
                   
               
               
                   
                   
                 AUAGGGGUA 
                 GAACGUAUA 
                 AUAAAAAUU 
                   
               
               
                   
                   
                 UUAAACUAA 
                 GUAAAGGGA 
                 UAUUCAAAUC 
                   
               
               
                   
                   
                 GACUACUUUA 
                 GUGCUCUGCA 
                 GCCCAUUAUG 
                   
               
               
                   
                   
                 AUAGUAGUU 
                 CUCUCCUGUA 
                 GGCCGCAGAU 
                   
               
               
                   
                   
                 GAUUUUAGG 
                 AAGCACUAAC 
                 GUUCUGCAUU 
                   
               
               
                   
                   
                 AGAUAGUUU 
                 CCCAUUUUCU 
                 AUAUGCUUGC 
                   
               
               
                   
                   
                 UUCUAUCUCC 
                 UCGGAGAAU 
                 AAGUUGCAA 
                   
               
               
                   
                   
                 CUUUUU 
                 GGGGUUAUC 
                 GCUUUUUUU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UUUUU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 274) 
                 (SEQ ID NO: 
                 301) 
                   
               
               
                   
                   
                   
                 287) 
                   
                   
               
               
                   
               
               
                 Other 
                 sgRNA V3 
                 GUUUAAAUA 
                 GCUAUACGUU 
                 Not listed 
                   
               
               
                 sgRNA 
                   
                 CCCCUAUAAA 
                 CCUUACAAAA 
                   
                   
               
               
                 Optimi- 
                   
                 CUACUAgaaaU 
                 UCGGgaaaCCG 
                   
                   
               
               
                 zations 
                   
                 AGUAGUUUA 
                 ACUUUGCAAG 
                   
                   
               
               
                   
                   
                 AUAGGGGUA 
                 GAACGUAUA 
                   
                   
               
               
                   
                   
                 UUUAAACUA 
                 GUAAAGGGA 
                   
                   
               
               
                   
                   
                 AGACUACUUU 
                 GUGCUCUGCA 
                   
                   
               
               
                   
                   
                 AAUAGUAGU 
                 CUCUCCUGUA 
                   
                   
               
               
                   
                   
                 UGAUAUUAG 
                 AAGCACUAAC 
                   
                   
               
               
                   
                   
                 GAGAUAGUU 
                 CCCAUUCUCU 
                   
                   
               
               
                   
                   
                 AUUCUAUCUC 
                 UCGGAGAAU 
                   
                   
               
               
                   
                   
                 CCUUUUU 
                 GGGGUUAUC 
                   
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 UUUU 
                   
                   
               
               
                   
                   
                 275) 
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                   
                 288) 
               
               
                   
               
               
                   
                   
                 OMNI-51 
                 OMNI-52 
                 OMNI-53 
               
               
                   
               
               
                 Minimal 
                 crRNA 
                 GUUUGAGAG 
                 GUUUGAGAG 
                 GUUUGAGAA 
                   
               
               
                 crRNA: 
                 (Repeat) 
                   
                   
                   
                   
               
               
                 tracrRNA 
                 tracrRNA 
                 CAGAGUUCAA 
                 CGAGUGCAAA 
                 UGAGUGCAA 
                   
               
               
                 duplex 
                 (Antirepeat) 
                 AU 
                 U 
                 AU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 302) 
                 315) 
                 329) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUGAGAG 
                 GUUUGAGAG 
                 GUUUGAGAA 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 UUAUG 
                 CUUUG 
                 CCAUG 
                   
               
               
                 duplex V1 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO:  
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 303) 
                 316) 
                 46) 
                   
               
               
                   
                 tracrRNA 
                 CAUGACAGAG 
                 CAAAGCGAGU 
                 CAUGGUGAG 
                   
               
               
                   
                 (Antirepeat) 
                 UUCAAAU 
                 GCAAAU 
                 UGCAAAU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 304) 
                 317) 
                 47) 
                   
               
               
                   
               
               
                 crRNA: 
                 crRNA 
                 GUUUGAGAG 
                 GUUUGAGAG 
                 GUUUGAGAA 
                   
               
               
                 tracrRNA 
                 (Repeat) 
                 UUAUGUAA 
                 CUUUGUUA 
                 CCAUGUAA 
                   
               
               
                 duplex V2 
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 305) 
                 318) 
                 48) 
                   
               
               
                   
                 tracrRNA 
                 UUACAUGACA 
                 UAACAAAGCG 
                 UUACAUGGU 
                   
               
               
                   
                 (Antirepeat) 
                 GAGUUCAAA 
                 AGUGCAAAU 
                 GAGUGCAAA 
                   
               
               
                   
                   
                 U 
                 (SEQ ID NO: 
                 U 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 319) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 306) 
                   
                 49) 
                   
               
               
                   
               
               
                 TracrRNA 
                 TracrRNA 
                 AAAAAUUUA 
                 AAGGUUUUA 
                 AAGGAUUAU 
                   
               
               
                 sequences 
                 Portion 1 
                 UUCAAACC 
                 CCGGAAUC 
                 CCGAAAU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 307) 
                 320) 
                 330) 
                   
               
               
                   
                 TracrRNA 
                 GCCUAUUUAA 
                 GUCUUUAUU 
                 UGUAUGCCCG 
                   
               
               
                   
                 Portion 2 
                 UUAUAGGC 
                 AAGA 
                 CAUUGUGCGG 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 CAAUA 
                   
               
               
                   
                   
                 308) 
                 321) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 331) 
                   
               
               
                   
                 TracrRNA 
                 CGCAGAUGUU 
                 ACCGCAUGGU 
                 AAAAGGCUCG 
                   
               
               
                   
                 Portion 3 
                 CUGC 
                 GCGG 
                 AAAGAGUCU 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 UUUU 
                   
               
               
                   
                   
                 309) 
                 322) 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                   
                   
                 332) 
                   
               
               
                   
                 TracrRNA 
                 ACUAUGCUUG 
                 AUUAUUUAG 
                 Not listed 
                   
               
               
                   
                 Portion 4 
                 CAAGGUUGCA 
                 AAGCCAUUUA 
                   
                   
               
               
                   
                   
                 AGCUUUUUU 
                 GAUGGCUUCU 
                   
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 AUUUU 
                   
                   
               
               
                   
                   
                 310) 
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                   
                 323) 
                   
                   
               
               
                   
                 Full 
                 CAUGACAGAG 
                 CAAAGCGAGU 
                 CAUGGUGAG 
                   
               
               
                   
                 tracrRNA 
                 UUCAAAUAA 
                 GCAAAUAAG 
                 UGCAAAUAA 
                   
               
               
                   
                 V1 
                 AAAUUUAUU 
                 GUUUUACCGG 
                 GGAUUAUCCG 
                   
               
               
                   
                   
                 CAAACCGCCU 
                 AAUCGUCUUU 
                 AAAUUGUAU 
                   
               
               
                   
                   
                 AUUUAAUUA 
                 AUUAAGAACC 
                 GCCCGCAUUG 
                   
               
               
                   
                   
                 UAGGCCGCAG 
                 GCAUGGUGCG 
                 UGCGGCAAUA 
                   
               
               
                   
                   
                 AUGUUCUGCA 
                 GAUUAUUUA 
                 AAAAGGCUCG 
                   
               
               
                   
                   
                 CUAUGCUUGC 
                 GAAGCCAUUU 
                 AAAGAGUCU 
                   
               
               
                   
                   
                 AAGGUUGCA 
                 AGAUGGCUUC 
                 UUUU 
                   
               
               
                   
                   
                 AGCUUUUUU 
                 UAUUUU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 333) 
                   
               
               
                   
                   
                 311) 
                 324) 
                   
                   
               
               
                   
                 Full 
                 UUACAUGACA 
                 UAACAAAGCG 
                 UUACAUGGU 
                   
               
               
                   
                 tracrRNA 
                 GAGUUCAAA 
                 AGUGCAAAU 
                 GAGUGCAAA 
                   
               
               
                   
                 V2 
                 UAAAAAUUU 
                 AAGGUUUUA 
                 UAAGGAUUA 
                   
               
               
                   
                   
                 AUUCAAACCG 
                 CCGGAAUCGU 
                 UCCGAAAUUG 
                   
               
               
                   
                   
                 CCUAUUUAAU 
                 CUUUAUUAA 
                 UAUGCCCGCA 
                   
               
               
                   
                   
                 UAUAGGCCGC 
                 GAACCGCAUG 
                 UUGUGCGGCA 
                   
               
               
                   
                   
                 AGAUGUUCU 
                 GUGCGGAUU 
                 AUAAAAAGG 
                   
               
               
                   
                   
                 GCACUAUGCU 
                 AUUUAGAAG 
                 CUCGAAAGAG 
                   
               
               
                   
                   
                 UGCAAGGUU 
                 CCAUUUAGAU 
                 UCUUUUU 
                   
               
               
                   
                   
                 GCAAGCUUUU 
                 GGCUUCUAUU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 UU 
                 UU 
                 334) 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                 312) 
                 325) 
                   
                   
               
               
                   
               
               
                 sgRNA 
                 sgRNA V1 
                 GUUUGAGAG 
                 GUUUGAGAG 
                 GUUUGAGAA 
                   
               
               
                 Versions 
                   
                 UUAUGgaaaCA 
                 CUUUGgaaaCA 
                 CCAUGgaaaCA 
                   
               
               
                   
                   
                 UGACAGAGU 
                 AAGCGAGUGC 
                 UGGUGAGUG 
                   
               
               
                   
                   
                 UCAAAUAAA 
                 AAAUAAGGU 
                 CAAAUAAGG 
                   
               
               
                   
                   
                 AAUUUAUUC 
                 UUUACCGGAA 
                 AUUAUCCGAA 
                   
               
               
                   
                   
                 AAACCGCCUA 
                 UCGUCUUUAU 
                 AUUGUAUGCC 
                   
               
               
                   
                   
                 UUUAAUUAU 
                 UAAGAACCGC 
                 CGCAUUGUGC 
                   
               
               
                   
                   
                 AGGCCGCAGA 
                 AUGGUGCGG 
                 GGCAAUAAA 
                   
               
               
                   
                   
                 UGUUCUGCAC 
                 AUUAUUUAG 
                 AAGGCUCGAA 
                   
               
               
                   
                   
                 UAUGCUUGCA 
                 AAGCCAUUUA 
                 AGAGUCUUU 
                   
               
               
                   
                   
                 AGGUUGCAA 
                 GAUGGCUUCU 
                 UU 
                   
               
               
                   
                   
                 GCUUUUUU 
                 AUUUU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                 53) 
                   
               
               
                   
                   
                 313) 
                 326) 
                   
                   
               
               
                   
                 sgRNA V2 
                 GUUUGAGAG 
                 GUUUGAGAG 
                 GUUUGAGAA 
                   
               
               
                   
                   
                 UUAUGUAAga 
                 CUUUGUUAgaa 
                 CCAUGUAAgaa 
                   
               
               
                   
                   
                 aaUUACAUGA 
                 aUAACAAAGC 
                 aUUACAUGGU 
                   
               
               
                   
                   
                 CAGAGUUCAA 
                 GAGUGCAAA 
                 GAGUGCAAA 
                   
               
               
                   
                   
                 AUAAAAAUU 
                 UAAGGUUUU 
                 UAAGGAUUA 
                   
               
               
                   
                   
                 UAUUCAAACC 
                 ACCGGAAUCG 
                 UCCGAAAUUG 
                   
               
               
                   
                   
                 GCCUAUUUAA 
                 UCUUUAUUA 
                 UAUGCCCGCA 
                   
               
               
                   
                   
                 UUAUAGGCCG 
                 AGAACCGCAU 
                 UUGUGCGGCA 
                   
               
               
                   
                   
                 CAGAUGUUCU 
                 GGUGCGGAU 
                 AUAAAAAGG 
                   
               
               
                   
                   
                 GCACUAUGCU 
                 UAUUUAGAA 
                 CUCGAAAGAG 
                   
               
               
                   
                   
                 UGCAAGGUU 
                 GCCAUUUAGA 
                 UCUUUUU 
                   
               
               
                   
                   
                 GCAAGCUUUU 
                 UGGCUUCUAU 
                 (SEQ ID NO: 
                   
               
               
                   
                   
                 UU 
                 UUU 
                 54) 
                   
               
               
                   
                   
                 (SEQ ID NO: 
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                 314) 
                 327) 
                   
                   
               
               
                   
               
               
                 Other 
                 sgRNA V3 
                 Not listed 
                 GUUUGAGAG 
                 Not listed 
                   
               
               
                 sgRNA 
                   
                   
                 CUUUGUUAgaa 
                   
                   
               
               
                 Optimi- 
                   
                   
                 aUAACAAAGC 
                   
                   
               
               
                 zations 
                   
                   
                 GAGUGCAAA 
                   
                   
               
               
                   
                   
                   
                 UAAGGAUUU 
                   
                   
               
               
                   
                   
                   
                 ACCGGAUUCG 
                   
                   
               
               
                   
                   
                   
                 UCUUUAUUA 
                   
                   
               
               
                   
                   
                   
                 AGAACCGCAU 
                   
                   
               
               
                   
                   
                   
                 GGUGCGGAU 
                   
                   
               
               
                   
                   
                   
                 UAUUUAGAA 
                   
                   
               
               
                   
                   
                   
                 GCCAUUUAGA 
                   
                   
               
               
                   
                   
                   
                 UGGCUUCUAU 
                   
                   
               
               
                   
                   
                   
                 UUU 
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 
                   
                   
               
               
                   
                   
                   
                 328) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 OMNI PAM Sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 OMNI-34 
                 OMNI-35 
                 OMNI-36 
                   
               
               
                   
               
               
                 Bacterial 
                 PAM General 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                 Depletion 
                 PAM Specific 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                   
                 Activity 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
               
               
                 TXTL 
                 PAM General 
                 NRNNNNAA 
                 NRR 
                 NNYCCC 
                   
               
               
                 Depletion 
                 PAM Specific 
                 No data shown 
                 NRR 
                 No data shown 
                   
               
               
                   
                 Activity 
                 0.82 
                 0.97 
                 0.99 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
                 sgRNA 
                 V1, V2, V3 
                 V1, V2 
                 V1, V2 
                   
               
               
                   
               
               
                 Mammalian 
                 PAM 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                 refinements 
                 Mammlian 
               
               
                   
               
               
                   
                   
                 OMNI-39 
                 OMNI-40 
                 OMNI-42 
               
               
                   
               
               
                 Bacterial 
                 PAM General 
                 NNGYAD 
                 NYGRV 
                 No data shown 
                   
               
               
                 Depletion 
                 PAM Specific 
                 NNGYAA 
                 NYGAV 
                 No data shown 
                   
               
               
                   
                 Activity 
                 0.99 
                 0.95 
                 No data shown 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
               
               
                 TXTL 
                 PAM General 
                 NNGHAD 
                 NYGRV 
                 NNGMM 
                   
               
               
                 Depletion 
                 PAM Specific 
                 NNGYAA 
                 NYGRV 
                 NTGCC 
                   
               
               
                   
                 Activity 
                 0.95 
                 0.97 
                 0.91 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
                 sgRNA 
                 V1, V2 
                 V1, V2 
                 V1 
                   
               
               
                   
               
               
                 Mammalian 
                 PAM 
                 No data shown 
                 VTGAAG 
                 No data shown 
                   
               
               
                 refinements 
                 Mammlian 
               
               
                   
               
               
                   
                   
                 OMNI-43 
                 OMNI-44 
                 OMNI-46 
               
               
                   
               
               
                 Bacterial 
                 PAM General 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                 Depletion 
                 PAM Specific 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                   
                 Activity 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
               
               
                 TXTL 
                 PAM General 
                 YAAAR 
                 NRHAA 
                 YAAAR 
                   
               
               
                 Depletion 
                 PAM Specific 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                   
                 Activity 
                 0.91 
                 0.96 
                 0.95 
                   
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
                 sgRNA 
                 V1, V2 
                 V3 
                 V2, V3 
                   
               
               
                   
               
               
                 Mammalian 
                 PAM 
                 No data shown 
                 No data shown 
                 No data shown 
                   
               
               
                 refinements 
                 Mammlian 
               
               
                   
               
               
                   
                   
                 OMNI-47 
                 OMNI-51 
                 OMNI-52 
                 OMNI-53 
               
               
                   
               
               
                 Bacterial 
                 PAM General 
                 No data 
                 No data 
                 No data 
                 NRTA 
               
               
                 Depletion 
                   
                 shown 
                 shown 
                 shown 
                   
               
               
                   
                 PAM Specific 
                 No data 
                 No data 
                 No data 
                 NRTA 
               
               
                   
                   
                 shown 
                 shown 
                 shown 
                   
               
               
                   
                 Activity 
                 No data 
                 No data 
                 No data 
                 1.00 
               
               
                   
                 (1-Depletion 
                 shown 
                 shown 
                 shown 
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
               
               
                 TXTL 
                 PAM General 
                 NVYR 
                 NRRAAA 
                 NRRADT 
                 NRHR 
               
               
                 Depletion 
                 PAM Specific 
                 NRTA 
                 No data 
                 No data 
                 NAWA 
               
               
                   
                   
                   
                 shown 
                 shown 
                   
               
               
                   
                 Activity 
                 0.98 
                 1.00 
                 1.00 
                 0.97 
               
               
                   
                 (1-Depletion 
                   
                   
                   
                   
               
               
                   
                 score)* 
                   
                   
                   
                   
               
               
                   
                 sgRNA 
                 V1, V2 
                 V1, V2 
                 V1, V2, V3 
                 V1, V2 
               
               
                   
               
               
                 Mammalian 
                 PAM 
                 No data 
                 No data 
                 No data 
                 NRTA 
               
               
                 refinements 
                 Mammlian 
                 shown 
                 shown 
                 shown 
               
               
                   
               
               
                 *Depletion score-Average of the ratios from two most depleted sites 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Plasmids and Constructs 
               
            
           
           
               
               
               
               
            
               
                 Plasmid 
                 Purpose 
                 Elements 
                 Example 
               
               
                   
               
               
                 pbNNC-2 
                 Expressing OMNI 
                 T7 promoter HA Tag- 
                 pbNNC2 OMNI39 
               
               
                   
                 polypeptide in the bacterial 
                 Linker-OMNI 
                 ORF 
               
               
                   
                 system 
                 (Human optimized)-T7 
                   
               
               
                   
                   
                 terminator 
                   
               
               
                 pbGuide 
                 Expressing OMNI sgRNA 
                 J23119 promoter-T1/T2 
                 pbGuide OMNI39 T2 
               
               
                 T1/T2 
                 in the bacterial system 
                 spacer sgRNA scaffold- 
                 sgRNA V2 
               
               
                   
                   
                 rrnB Ti terminator 
                   
               
               
                 pbPOS T2 
                 Bacterial/TXTL depletion 
                 T2 protospacer-8N PAM 
                 pbPOS T2 library 
               
               
                 library 
                 assay 
                 library-chloramphenicol 
                   
               
               
                   
                   
                 acetyltransferase 
                   
               
               
                 pET9a 
                 Expression and purification 
                 T7 promoter-SV40 NLS- 
                 pET9a OMNI39-HisTag 
               
               
                   
                 of OMNI proteins 
                 OMNI ORF (human 
                   
               
               
                   
                   
                 optimized)-HA-SV40 
                   
               
               
                   
                   
                 NLS-8 His-tag-T7 
                   
               
               
                   
                   
                 terminator 
                   
               
               
                 pmOMNI 
                 Expressing OMNI 
                 CMV promoter-Kozak- 
                 pmOMNI OMNI39 
               
               
                   
                 polypeptide in the 
                 SV40 NLS-OMNI ORF 
                   
               
               
                   
                 mammalian system 
                 (human optimized)-HA- 
                   
               
               
                   
                   
                 SV40 NLS-P2A- 
                   
               
               
                   
                   
                 mCherry-bGH poly(A) 
                   
               
               
                   
                 signal 
                   
                   
               
               
                 pmGuide 
                 Expressing OMNI sgRNA 
                 U6 promoter-Endogenic 
                 pmGuide OMNI39 
               
               
                 Endogenic 
                 in the mammalian system 
                 spacer sgRNA scaffold 
                 CXCR4 sgRNA V3 
               
               
                 site 
                   
                   
                   
               
               
                 pPMLI3.1 
                 Viral vector for PAM 
                 LTR - HIV-1    -CMV 
                 pPML13.1 
               
               
                   
                 library in mammalian cells  
                 promoter-T2-PAM 
                   
               
               
                   
                   
                 library (6N)-GFP-SV40 
                   
               
               
                   
                   
                 promoter-blastocydin S 
                   
               
               
                   
                   
                 deaminase-LTR 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Appendix-Details of construct elements 
               
            
           
           
               
               
               
            
               
                 Element 
                 Protein Sequence 
                 DNA sequence 
               
               
                   
               
               
                 HA Tag 
                 SEQ ID NO: 63 
                 SEQ ID NO: 64 
               
               
                 NLS 
                 SEQ ID NO: 65 
                 SEQ ID NO: 66 
               
               
                 P2A 
                 SEQ ID NO: 85 
                 SEQ ID NO: 86 
               
               
                 mCherry 
                 SEQ ID NO: 67 
                 SEQ ID NO: 68 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Activity of OMNIs in human cells on endogenous genomic targets 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 3′ (PAM 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 con- 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                 taining) 
                   
                   
                   
                   
                 % 
                   
               
               
                   
                   
                 Corre- 
                   
                 genomic 
                   
                   
                   
                 % 
                 trans- 
                 Norm. % 
               
               
                   
                   
                 sponding 
                   
                 sequence 
                   
                 % 
                   
                 editing 
                 fection 
                 editing 
               
               
                   
                 Genomic 
                 Spacer 
                 Spacer 
                 (PAM 
                   
                 trans- 
                 Norm. % 
                 in neg 
                 in neg 
                 in neg 
               
               
                 Nuclease 
                 site 
                 name 
                 sequence 
                 bolded) 
                 % indels 
                 fection 
                 editing 
                 control 
                 control 
                 control 
               
               
                   
               
               
                 OMNI-39 
                 CXCR4 
                 CXCR4g1_ 
                 CCAAGUGAUA 
                   TGGCAA GA 
                 49.8-73.2 
                 67.13 
                   
                 0.08 
                 76.70 
                 0.107791557 
               
               
                   
                 site 1 
                 OMNI39 
                 AACACGAGGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 89) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 EMX1 
                 EMX1g1_ 
                 GUCACCUCCA 
                   GGGCAA CC 
                 3.9-6.3 
                   
                   
                   
                   
                   
               
               
                   
                 site 1 
                 OMNI39 
                 AUGACUAGGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 U (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 90) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 EMX1 
                 EMX1g2_ 
                 GCCGCCAUUG 
                   AAGCAA TG 
                 22.8-54.7 
                   
                   
                   
                   
                   
               
               
                   
                 site 2 
                 OMNI39 
                 ACAGAGGGAC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 91) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 PDCD1 
                 PDCD1g1_ 
                 AACUGGUACC 
                   CAGCAA CC 
                  3.71 
                 73.67 
                  5.03 
                 0.07 
                 76.70 
                 0.086641622 
               
               
                   
                 site 1 
                 OMNI39 
                 GCAUGAGCCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 92) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 OMNI-40 
                 EMX1 
                 EMX1g1_ 
                 CAUCAGGCUC 
                   CTGAG TGT 
                   25-37.5 
                 50.33 
                   
                 0.12 
                 53.37 
                 0.231979262 
               
               
                   
                 site 3 
                 OMNI40 
                 UCAGCUCAGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 93) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CXCR4 
                 CXCR4g2_ 
                 AGGUGCCGUU 
                   CTGAC ACT 
                  0.20 
                 53.60 
                  0.37 
                 0.21 
                 53.37 
                 0.396940137 
               
               
                   
                 site 2 
                 OMNI40 
                 UGUUCAUUUU 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 94) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 PDCD1 
                 PDCD1g1_ 
                 CCAGUUGUAG 
                   ACGAC TGG 
                 23.59 
                 28.33 
                 83.25 
                 0.09 
                 53.37 
                 0.174121445 
               
               
                   
                 site 2 
                 OMNI40 
                 CACCGCCCAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 95) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 PDCD1 
                 PDCD1g2_ 
                 UCUCCCCAGC 
                   GTGAC CGA 
                 16.66 
                 49.00 
                 34.00 
                 0.01 
                  0.18 
                 8.107932801 
               
               
                   
                 site 3 
                 OMNI40 
                 CCUGCUCGUG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 96) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 OMNI-53 
                 EMX1 
                 EMXg1_ 
                 GCCUGGGGCC 
                   TGTA GCCT 
                 18.3-36.7 
                 49.63 
                   
                 0.15 
                 43.80 
                 0.333213614 
               
               
                   
                 site 4 
                 OMNI53 
                 CCUAACCCUA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 108) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CXCR4 
                 CXCR4g2_ 
                 AUUUUCUGAC 
                   AATA TACC 
                 14.1-12.5 
                 38.33 
                   
                 0.22 
                 43.80 
                 0.509942217 
               
               
                   
                 site 3 
                 OMNI53 
                 ACUCCCGCCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 109) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 PDCD1 
                 PDCD1g1_ 
                 AUCCUGGCCG 
                   TGTA GCAC 
                 11.5  
                 51.27 
                 22.50 
                 0.05 
                 43.80 
                 0.105935337 
               
               
                   
                 site 4 
                 OMNI53 
                 CCAGCCCAGU 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 110) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 PDCD1 
                 PDCD1g2_ 
                 GGAGAGCUUC 
                   GGTA CCGC 
                  1.93 
                 30.30 
                  6.38 
                 0.01 
                 43.80 
                 0.019429028 
               
               
                   
                 site 5 
                 OMNI53 
                 GUGCUAAACU 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 NO: 111) 
               
               
                   
               
               
                 Table 5. Nuclease activity in endogenous context in mammalian cells: OMNI nucleases were expressed in mammalian cell system (HeLa) by DNA transfection together with an sgRNA expressing plasmid. Cell lysates were used for site specific genomic DNA amplification and NGS. The percentage of indels was measured and analyzed to determine the editing level. Each sgRNA is composed of the tracrRNA (see Table 2) and the spacer detailed here. The spacer 3′ genomic sequence contains the expected PAM relevant for each OMNI nuclease. Transfection efficiency (% transfection) was measured by flow cytometry of the mCherry signal, as described above. The transfection efficiency was used to normalize the editing level (% indels norm). All tests were performed in triplicates. OMNI nuclease only (no guide) transfected cells served as a negative control. 
               
            
           
         
       
     
     REFERENCES 
     
         
         1. Ahmad and Allen (1992) “Antibody-mediated Specific Binging and Cytotoxicity of Lipsome-entrapped Doxorubicin to Lung Cancer Cells in Vitro”, Cancer Research 52:4817-20. 
         2. Anderson (1992) “Human gene therapy”, Science 256:808-13. 
         3. Basha et al. (2011) “Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells”, Mol. Ther. 19(12):2186-200. 
         4. Behr (1994) “Gene transfer with synthetic cationic amphiphiles: Prospects for gene therapy”, Bioconjuage Chem 5:382-89. 
         5. Blaese et al. (1995) “Vectors in cancer therapy: how will they deliver”, Cancer Gene Ther. 2:291-97. 
         6. Blaese et al. (1995) “T lympocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years”, Science 270(5235):475-80. 
         7. Briner et al. (2014) “Guide RNA functional modules direct Cas9 activity and orthognality”, Molecular Cell 56:333-39. 
         8. Buchschacher and Panganiban (1992) “Human immunodeficiency virus vectors for inducible expression of foreign genes”, J. Virol. 66:2731-39. 
         9. Burstein et al. (2017) “New CRISPR-Cas systems from uncultivated microbes”, Nature 542:237-41. 
         10. Canver et al., (2015) “BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis”, Nature Vol. 527, Pgs. 192-214. 
         11 Chang and Wilson (1987) “Modification of DNA ends can decrease end-joining relative to homologous recombination in mammalian cells”, Proc. Natl. Acad. Sci. USA 84:4959-4963. 
         12. Charlesworth et al. (2019) “Identification of preexisting adaptive immunity to Cas9 proteins in humans”, Nature Medicine, 25(2), 249. 
         13. Chung et al. (2006) “ Agrobacterium  is not alone: gene transfer to plants by viruses and other bacteria”, Trends Plant Sci. 11(1):1-4. 
         14. Coelho et al. (2013) “Safety and efficacy of RNAi therapy for transthyretin amyloidosis” N. Engl. J. Med. 369, 819-829. 
         15. Crystal (1995) “Transfer of genes to humans: early lessons and obstacles to success”, Science 270(5235):404-10. 
         16. Dillon (1993) “Regulation gene expression in gene therapy” Trends in Biotechnology 11(5):167-173. 
         17. Dranoff et al. (1997) “A phase I study of vaccination with autologous, irradiated melanoma cells engineered to secrete human granulocyte macrophage colony stimulating factor”, Hum. Gene Ther. 8(1):111-23. 
         18. Dunbar et al. (1995) “Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation”, Blood 85:3048-57. 
         19. Ellem et al. (1997) “A case report: immune responses and clinical course of the first human use of ganulocyte/macrophage-colony-stimulating-factor-tranduced autologous melanoma cells for immunotherapy”, Cancer Immunol Immunother 44:10-20. 
         20. Gao and Huang (1995) “Cationic liposome-mediated gene transfer” Gene Ther. 2(10):710-22. 
         21. Haddada et al. (1995) “Gene Therapy Using Adenovirus Vectors”, in: The Molecular 
       
    
     Repertoire of Adenoviruses III: Biology and Pathogenesis, ed. Doerfler and Bohm, pp. 297-306.
     22. Han et al. (1995) “Ligand-directed retro-viral targeting of human breast cancer cells”, Proc. Natl. Acad. Sci. USA 92(21):9747-51.   23. Humbert et al., (2019) “Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates”, Sci. Trans. Med., Vol. 11, Pgs. 1-13.   24. Inaba et al. (1992) “Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor”, J Exp Med. 176(6):1693-702.   25. Jinek et al. (2012) “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science 337(6096):816-21.   26. Johan et al. (1992) “GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of  Neurospora crassa  and is expressed at high levels in the brain and thymus”, J Virol 66(3):1635-40.   27. Judge et al. (2006) “Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo”, Mol Ther. 13(3):494-505.   28. Kohn et al. (1995) “Engraftment of gene-modified umbilical cord blood cells in neonates with adnosine deaminase deficiency”, Nature Medicine 1:1017-23.   29. Kremer and Perricaudet (1995) “Adenovirus and adeno-associated virus mediated gene transfer”, Br. Med. Bull. 51(1):31-44.   30. Macdiarmid et al. (2009) “Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug”, Nat Biotehcnol. 27(7):643-51.   31. Malech et al. (1997) “Prolonged production of NADPH oxidase-corrected granulocyes after gene therapy of chronic granulomatous disease”, PNAS 94(22):12133-38.   32. Maxwell et al. (2018) “A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer adjacent motifs”, Methods 14348-57   33. Miller et al. (1991) “Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus”, J Virol. 65(5):2220-24.   34. Miller (1992) “Human gene therapy comes of age”, Nature 357:455-60.   35. Mitani and Caskey (1993) “Delivering therapeutic genes—matching approach and application”, Trends in Biotechnology 11(5):162-66.   36. Nabel and Felgner (1993) “Direct gene transfer for immunotherapy and immunization”, Trends in Biotechnology 11(5):211-15.   37. Nehls et al. (1996) “Two genetically separable steps in the differentiation of thymic epithelium” Science 272:886-889.   38. Remy et al. (1994) “Gene Transfer with a Series of Lipphilic DNA-Binding Molecules”, Bioconjugate Chem. 5(6):647-54.   39. Sentmanat et al. (2018) “A Survey of Validation Strategies for CRISPR-Cas9 Editing”, Scientific Reports 8:888, doi:10.1038/s41598-018-19441-8.   40. Sommerfelt et al. (1990) “Localization of the receptor gene for type D simian retroviruses on human chromosome 19”, J. Virol. 64(12):6214-20.   41. Van Brunt (1988) “Molecular framing: transgenic animals as bioactors” Biotechnology 6:1149-54.   42. Vigne et al. (1995) “Third-generation adenovectors for gene therapy”, Restorative Neurology and Neuroscience 8(1,2): 35-36.   43. Wagner et al. (2019) “High prevalence of  Streptococcus pyogenes  Cas9-reactive T cells within the adult human population” Nature Medicine, 25(2), 242   44. Wilson et al. (1989) “Formation of infectious hybrid virion with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus”, J. Virol. 63:2374-78.   45. Yu et al. (1994) “Progress towards gene therapy for HIV infection”, Gene Ther. 1(1):13-26.   46. Zetsche et al. (2015) “Cpf1 is a single RNA-guided endonuclease of a class 2 CRIPSR-Cas system” Cell 163(3):759-71.   47. Zuris et al. (2015) “Cationic lipid-mediated delivery of proteins enables efficient protein based genome editing in vitro and in vivo” Nat Biotechnol. 33(1):73-80.