EP3097212A2 - Méthodes et compositions pour des séquences guidant le ciblage de cas9 - Google Patents

Méthodes et compositions pour des séquences guidant le ciblage de cas9

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Publication number
EP3097212A2
EP3097212A2 EP15740979.8A EP15740979A EP3097212A2 EP 3097212 A2 EP3097212 A2 EP 3097212A2 EP 15740979 A EP15740979 A EP 15740979A EP 3097212 A2 EP3097212 A2 EP 3097212A2
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EP
European Patent Office
Prior art keywords
sequence
nucleic acid
crispr
target dna
acid construct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP15740979.8A
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German (de)
English (en)
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EP3097212A4 (fr
Inventor
Rodolphe Barrangou
Kurt M. SELLE
Alexandra E. BRINER
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North Carolina State University
University of California
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North Carolina State University
University of California
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Application filed by North Carolina State University, University of California filed Critical North Carolina State University
Publication of EP3097212A2 publication Critical patent/EP3097212A2/fr
Publication of EP3097212A4 publication Critical patent/EP3097212A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention relates to a synthetic CRISPR-cas system and methods of use thereof for genome editing.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and phages, as novel "spacers.”
  • CRISPR-Cas systems consist of arrays of short DNA repeats interspaced by hypervariable sequences, flanked by cas genes, that provide adaptive immunity against invasive genetic elements such as phage and plasmids, through sequence-specific targeting and interference (Barrangou et al. 2007. Science. 315:1709-1712; Brouns et al. 2008. Science 321 :960-4; Horvath and Barrangou. 2010. Science. 327: 167-70; Marraffmi and Sontheimer. 2008. Science. 322:1843-1845; Bhaya et al. 2011. Annu. Rev. Genet. 45:273-297; Terns and Terns. 2011. Curr. Opin. Microbiol. 14:321-327; Westra et al. 2012. Annu. Rev. Genet.
  • RNA. 4:267-278 Typically, invasive DNA sequences are acquired as novel "spacers" (Barrangou et al. 2007. Science. 315:1709-1712), each paired with a CRISPR repeat and inserted as a novel repeat-spacer unit in the CRISPR locus.
  • the repeat-spacer array is transcribed as a long pre-CRISPR RNA (pre-crRNA) (Brouns et al. 2008. Science 321 :960-4), which is processed into small interfering CRISPR RNAs (crRNAs) that drive sequence-specific recognition.
  • crRNAs guide nucleases towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Garneau et al. 2010. Nature. 468:67-71; Haurwitz et al. 2010. Science. 329: 1355-1358; Sapranauskas et al. 2011. Nucleic Acid Res. 39:9275-9282; Jinek et al. 2012. Science.
  • Nucleic Acid Res. 41 :4360-4377 based on the cas gene content, organization and variation in the biochemical processes that drive crRNA biogenesis, and Cas protein complexes that mediate target recognition and cleavage.
  • the specialized Cas endonucleases process the pre-crRNAs, which then assemble into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acids complementary to the crRNA.
  • a different process is involved in Type II CRISPR-Cas systems.
  • the pre-CRNAs are processed by a mechanism in which a trans-activating crRNA (tracrRNA) hybridizes to repeat regions of the crRNA.
  • the hybridized crRNA-tracrRNA are cleaved by RNase III and following a second event that removes the 5' end of each spacer, mature crRNAs are produced that remain associated with the both the tracrRN A and Cas9.
  • the mature complex locates a target dsDNA sequence ('protospacer' sequence) that is complementary to the spacer sequence in the complex and cuts both strands.
  • Target recognition and cleavage by the complex in the type II system not only requires a sequence that is complementary between the spacer sequence on the crRNA-tracrRNA complex and the target 'protospacer' sequence but also requires a protospacer adjacent motif (PAM) sequence located at the 3' end of the
  • PAM protospacer adjacent motif
  • PAM sequence The exact PAM sequence that is required can vary between different type II systems.
  • the present disclosure provides methods and compositions for increasing the efficiency and specificity of synthetic type II CRISPR-Cas systems that improve efficiency and specificity for genome editing and other uses.
  • One aspect of the invention provides a synthetic trans-encoded CRlSPR(tracr ) nucleic acid (e.g., tracrRNA, tracrDNA) construct comprising from 5' to 3',
  • an optional anti-zipper sequence comprising at least about three- nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising a nucleotide sequence of T(A/C)A(A/G)(G/A)C (or U(A/C)A(A/G)(G/A)C)), TCAAAC, (or UCAAAC), TAAGGC (or UAAGGC), GATAAGG (or GAUAAGG), GATAAGGCTT (or GAUAAGGCUU), TCAAG (or UCAAG), TCAAGCAA (or UCAAGCAA), T(C/A)AA(A/C)(C/A)(A/G)(A/T) (or U(C/A)AA(A/C)(C/A)(A/G)(A/U)), GATAAGGCCATGCC, TA
  • the anti-zipper sequence when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence.
  • a second aspect of the invention provides a synthetic C RISPR nucleic acid (e.g., crRNA, crDNA) construct comprising, from 3' to 5', an optional zipper sequence comprising at least about three-nucleotides that when present hybridizes to the anti-zipper of a tracrRNA, a bulge sequence comprising at least two nucleotides (e.g., the nucleotide sequence of (-NN-)).
  • a stitch sequence comprising a nucleotide sequence of N TNN (or N UNN) that hybridizes to the anti-stitch of a tracrRNA.
  • a GRI comprising a nucleotide G or G IT.
  • the zipper sequence when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GRI, and the GRI is located immediately upstream of the spacer sequence.
  • a third aspect of the invention provides a synthetic CRISPR nucleic acid array comprising, a nucleotide sequence encoding two or more CRISPR nucleic acid constructs of this invention, wherein the two or more CRISPR nucleic acid constructs are located immediately adjacent to one another on said nucleotide sequence and the zipper sequences of said two or more CRISPR nucleic acid constructs, when present, are identical, the stitch sequences of said two or more CRISPR nucleic acid constructs arc identical, and the spacer sequences of said two or more CRISPR nucleic acid constructs are identical or non-identical.
  • a fourth aspect of the invention provides a chimeric nucleic acid construct comprising the synthetic tracr nucleic acid construct of the invention and the synthetic CRISPR nucleic acid construct of the invention, wherein, when present, the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 70% complementary to and is hybridized to the anti -zipper sequence of said synthetic tracr nucleic acid construct, the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and hybridizes to the anti-stitch sequence of said synthetic tracr nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary.
  • a fifth aspect of the invention provides a method for site-specific cleavage of a double stranded target DNA, comprising: contacting a chimeric nucleic acid construct of this disclosure or an expression cassette comprising said chimeric nucleic acid construct with the target DNA in the presence of a Cas9 nuclease, thereby producing a site-specific cleavage of the target DNA in a region defined by hybridization of the spacer sequence to the target DNA.
  • a sixth aspect of the invention provides a method for site-specific cleavage of a double stranded target DNA. comprising:
  • the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5' to 3', an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNA ; a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC,
  • GATAAGG GATAAGGCTT.
  • TCAAG TCAAG
  • TCAAGCAA T(C/A)AA(A/C)(C/A)(A/G)(A/T)
  • GATAAGGCCATGCC GATAAGGCTAGTCC, T C A A G C A A G C , or
  • a hairpin sequence comprising a nucleotide sequence having at least two hairpins, each hairpin comprising at least three matched base pairs, and the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti -stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and
  • the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3' to 5', an optional zipper sequence comprising at least about 9three nucleotides, a bulge sequence comprising a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of NNTNN (or NNUNN), a GR I comprising a nucleotide G or GTT, and a spacer sequence having a 5' end and a 3' end and comprising at least seven nucleotides at its 3' end having 100% identity to a target DNA. and the zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GRI, and the GRI is located immediately upstream of the spacer sequence, and
  • the anti-zipper sequence and the zipper sequence hybridize to one another, and the anti-stitch sequence and the stitch sequence hybridize to one another, and the spacer sequence of the CRISPR nucleic acid molecule is at least about 80% complementary to and hybridizes to at least a portion of the target DNA (e.g.. at least about 7_consecufive nucleotides of said target DNA (e.g..).
  • the spacer sequence of the CRISPR nucleic acid molecule hybridizes to a portion of a target DNA sequence that is adjacent to a PAM, wherein the target sequence can comprise, consist essentially of, or consist of about 7 to about 20 consecutive nucleotides of the target DNA sequence.
  • a seventh aspect of the invention provides a method for site-specific cleavage of a double stranded target DNA. comprising:
  • contacting the double stranded target DNA with a chimeric nucleic acid comprising,
  • NNANN a nexus sequence comprising a nucleotide sequence of TNANNC
  • TAAGGC GATAAGG.
  • GATAAGGCTT TCAAG, TCAAGCAA.
  • GATAAGGCCATGCC GATAAGGCCATGCC
  • TCAAGCAAAGC, or TC A AAC A A AGCTTC AGC and a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, and, when present, the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence;
  • a second nucleotide sequence comprising from 3 * to 5 * , an optional zipper sequence comprising at least about three nucleotides, which, when present, hybridizes to the anti-zipper sequence of the first nucleotide sequence, a bulge sequence comprising a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of N TNN (or N U N), a GR I comprising a nucleotide G or GTT, and a spacer sequence having a 5 " end and a 3' end and comprising at least seven nucleotides at its 3' end that have 100% complementarity to a target DNA, and the zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the G R1 , and the GRI is located
  • the anti-stitch sequence hybridizes to the stitch sequence and the spacer sequence of the second nucleotide sequence hybridizes to at least a portion of the target DNA (e.g., at least about 7 consecutive nucleotides of said target DNA, preferably up to about 20 consecutive nucleotides ) and adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby resulting in a site-specific cleavage of the target DNA in a region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA
  • the target DNA e.g., at least about 7 consecutive nucleotides of said target DNA, preferably up to about 20 consecutive nucleotides
  • PAM protospacer adjacent motif
  • An eighth aspect of the invention comprises a method of site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA, comprising contacting the chimeric nucleic acid construct of this disclosure or an expression cassette comprising said chimeric nucleic acid construct with the target DNA, thereby targeting the polypeptide of interest fused to the Cas9 to a specific site on the target DNA, said site defined by
  • a ninth aspect of the invention comprises a method of site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA, comprising
  • the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5 " to 3 " , an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising a nucleotide sequence of T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC. GATAAGG, GATAAGGCTT.
  • a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence;
  • the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3 ' to 5', an optional zipper sequence comprising at least about three nucleotides that hybridize to the anti-zipper sequence, a bulge sequence comprising a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)).
  • a stitch sequence comprising a nucleotide sequence of NNTNN, a GR I comprising a nucleotide G or GTT, and a spacer sequence having a 5' end and a 3 ' end and comprising at least seven nucleotides at its 3 ' end having 1 00% identity to a target DNA, and the zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GRI, and the GRI is located immediately upstream of the spacer sequence, and
  • the Cas9 nuclease comprises a mutation in a HNH active site motif, a mutation in a RuvC active site motif, and is fused to a polypeptide of interest, the anti-zipper sequence and the zipper sequence, when present, hybridize to one another, the anti-stitch sequence hybridizes to the stitch sequence, and the spacer sequence hybridizes to at least a portion of the target DNA adjacent to a protospacer adjacent motif (PAM) on the target DNA. thereby resulting in a site-specific targeting of the polypeptide of interest to the target DNA in a region defined by the hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA.
  • PAM protospacer adjacent motif
  • expression cassettes, cells and kits comprising the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules and/or nucleotide sequences of the invention.
  • Fig. 1 shows a multiple sequence alignment for the nexus module.
  • Fig. 2 shows a maximum likelihood tree for the nexus module.
  • Fig. 3A-3D show consensus sequences for the nexus module.
  • Fig. 3A shows the consensus sequence for the Sth Crl group.
  • Fig. 3B shows the consensus sequence for the Sth Cr3 group.
  • Fig. 3C shows the consensus sequence for the Lrh group and
  • Fig. 3D shows the consensus sequence for the Lbu group.
  • Fig. 4 shows a maximum likelihood tree for Cas9 nucleases.
  • Fig. 5 shows a multiple sequence alignment for the anti-stitch module.
  • Fig. 6A-6D show consensus sequences for the anti-stitch module.
  • Fig. 6 A shows the consensus sequence for the Sth Crl group.
  • Fig. 6B shows the consensus sequence for the Sth Cr3 group.
  • Fig. 6C shows the consensus sequence for the Lrh group and
  • Fig. 6D shows the consensus sequence for the Lbu group.
  • Fig. 7 shows a multiple sequence alignment for the bulge module.
  • Fig. 8A-8D show consensus sequences for the bulge module.
  • Fig. 8A shows the consensus sequence for the Sth Crl group.
  • Fig. 8B shows the consensus sequence for the Sth Cr3 group.
  • Fig. 8C shows the consensus sequence for the Lrh group and
  • Fig. 8D shows the consensus sequence for the Lbu group.
  • Fig. 9 shows a multiple sequence alignment for the zipper module.
  • Fig. 10 shows a maximum likelihood tree for the zipper module.
  • Fig. 11 shows a multiple sequence alignment for the bulge, anti-stitch and nexus modules.
  • Fig. 12 shows sequence and structural details for CRISPR-Cas system elements for Streptococcus thermophilus CR3, representing the Sth CR1 group.
  • Fig. 13 shows sequence and structural details for CRISPR-Cas system elements for Lactobacillus buchneri, representing the Lbu group.
  • Fig. 14 shows sequence and structural details for CRISPR-Cas system elements for Streptococcus thermophilus CR1, representing the Sth CR1 group.
  • Fig. 15 shows sequence and structural details for CRISPR-Cas system elements for the Streptococcus pyrogenes M l GAS, representing the Sth CR3 group.
  • Fig. 16 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus rhamnosus, representing the Lrh group.
  • Fig. 17 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus animalis, representing the Lan group.
  • Fig. 18 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus casei, representing the Lea group.
  • Fig. 19 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus gasseri, representing the Lga group.
  • Fig. 20 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus jensenii, representing the Lje group.
  • Fig. 21 shows sequence and structural details for CRISPR-Cas system elements for the Lactobacillus pentosus, representing the Lpe group.
  • Fig. 22 shows sequence and structural details for CRISPR-Cas system elements for Streptococcus pyrogenes Ml GAS.
  • Fig. 23 shows congruence between tracrRNA (left), CRISPR repeat (middle) and Cas9 (right) sequence clustering. Consistent grouping is observed across the three sequence- based phylogenetic trees, into three families.
  • Fig. 24A-24B shows the Cas9:sgRNA families.
  • Fig. 24A shows a phylogenetic tree based on Cas9 protein sequences from various Streptococcus and Lactobacillus species. The sequences clustered into three families in blue, orange and green.
  • Fig. 24B shows a consensus sequence and secondary structure of the predicted guide RNA for each family. Each consensus RNA is composed of the crRNA (left) base-paired with the tracrRNA.
  • Fig. 25 shows CRISPR repeats sequence alignment. For each cluster, CRISPR repeat sequence alignments are shown, with conserved and consensus nucleotides speci ied at the bottom of each family, with Sth3 (top), Sthl (middle) and Lb (bottom) families.
  • Fig. 26 shows tracrRNA sequence alignment. For each cluster, the experimentally determined, or computationally predicted tracrR A sequence alignments are shown, with conserved and consensus nucleotides specified at the bottom of each family, with Sth3 (top), Sthl (middle) and Lb (bottom) families.
  • Fig. 27 shows sgRNA nexus sequence alignment. Universally conserved residues are colored in red. Complementary nucleotides that constitute the nexus stem as summarized in Fig. 24B are underlined. Nucleotides that constitute the nexus loop are centered in the gap.
  • Fig. 28A-B shows a self targeting assay scheme.
  • Orthogonal Cas9 proteins were provided through the pCas9 plasm id (Fig. 28 A ), and used as described in Fig. 3.
  • Various sgRNA chimera were provided through the psgRNA pi asm id (Fig. 28B), and used in combination with each desired Cas9 as described in Fig. 3.
  • Fig. 29A-C shows sgRNA orthogonality.
  • Fig. 29A shows sgRNA sequences for the Streptococcus thermophilics CRISPR3-Cas9 (top, blue) and the S. thermophilus CRISPR 1- Cas9 (bottom, orange).
  • Fig. 29B shows protospacer-targeting scheme. The predicted PAM for each sgRNA is shown.
  • Triangles designate the putative cut sites for each Cas9.
  • Fig. 29C Cas9:chimeric-sgRNA orthogonality in E. coli. Chimeric sgRNAs. Each sgRNA (left) was subjected to the transformation assay (right) in E.
  • Fig 30 shows CRISPR interference against complementary ON A. as inability to transform plasmids that contain protospacer sequences that match the first wild type CRISPR spacer sequence, in Lactobacillus gasseri.
  • the bold sequence, flanked by a PAM (light grey, italicized nucleotides) and variants thereof (single nucleotide polymorphisms (SNPs); black underlined nucleotides) is the protospacer.
  • Low transform ant counts represent an active Lga CRISPR systems which precludes transformation of complementary target DNA.
  • Fig. 31 shows CRISPR interference against complementary' DNA. as inability to transform plasmids that contain protospacer sequences that match the first wild type CRISPR spacer sequence, in Lactobacillus casei.
  • the bold sequence, flanked by a PAM (light grey, italicized nucleotides) and variants thereof (SNPs, black underlined nucleotides) is the protospacer.
  • Low transform ant counts represent an active Lea CRISPR systems which precludes transformation of complementary target DN A.
  • Fig. 32 shows CRISPR interference against complementary DNA. as inability to transform plasmids that contain protospacer sequences that match the first wild type CRISPR spacer sequence, in Lactobacillus rhamnosus.
  • the bold sequence, flanked by a PAM light grey, italicized nucleotides ) and variants thereof (SNPs, black underlined nucleotides) is the protospacer.
  • Low transformant counts represent an active Lra CRISPR systems which precludes transformation of complementary target DNA.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • the "bulge sequence” as used herein refers to non-complementary (non-hybridizing) nucleotide sequences comprised in a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array.
  • the bulge sequence is located between the anti-zipper and the anti-stitch sequences is comprised of about three nucleotides to about six nucleotides (e.g., about 3, 4, 5, 6 nucleotides; e.g., about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, about 3 to about 4 nucleotides, and the like) that are non-complementary (100% non-identity) to a corresponding bulge sequence in the synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array.
  • construe t/C R I S R nucleic acid array is located between the zipper and the stitch sequences and between the zipper and the stitch sequences and comprises, consists essentially of, or consists of at least two nucleotides (e.g.. the nucleotide sequence of (-NN-)) (e.g., about 2, 3. 4, 5. 6 nucleotides; e.g.. about 2 to about 6 nucleotides, about 2 to about 5 nucleotides, about 2 to about 4 nucleotides; about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, and the like).
  • nucleotides e.g. the nucleotide sequence of (-NN-)
  • nucleotides e.g. the nucleotide sequence of (-NN-)
  • nucleotides e.g. the nucleotide sequence of (-NN-)
  • nucleotides e.g. the nucleotide sequence of
  • the nucleotide composition of the bulge sequences can be any series of at least two (synthetic CRISP nucleic acid construct/CRISPR nucleic acid array) or three or more (synthetic tracr nucleic acid construct) nucleotides as long as they are not complementary (e.g., 100% non-identity) and therefore do not hybridize to one another.
  • Cas9 nuclease refers to a large group of endonucleases that catalyze the double stranded DNA cleavage in the CRISPR Cas system. These polypeptides are well known in the art and many of their structures (sequences) are characterized (See, e.g.,
  • the domains for catalyzing the cleavage of the ds DNA are the RuvC domain and the HNH domain.
  • the RuvC domain is responsible for nicking the (-) strand and the HNH domain is responsible for nicking the (+) strand (See, e.g., Gasiunas et al. PNAS 109(36):E2579-E2586 (September 4, 2012)).
  • chimeric refers to a nucleic acid molecule or a polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).
  • “Complement” as used herein can mean 100% complementarity or identity with the comparator nucleotide sequence or it can mean less than 100% complementarity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A.”
  • Complementarity between two single-stranded molecules may be "partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • contact refers to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., integration, transformation, site-specific cleavage (nicking, cleaving), amplifying, site specific targeting of a polypeptide of interest and the like).
  • suitable for carrying out the desired reaction e.g., integration, transformation, site-specific cleavage (nicking, cleaving), amplifying, site specific targeting of a polypeptide of interest and the like.
  • the methods and conditions for carrying out such reactions are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Y).
  • a “fragment " or "portion " of a nucleotide sequence of the invention will be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • nucleic acid fragment or portion according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • hybridizing to (or hybridizes to, and other grammatical variations thereof), for example, at least a portion of a target DNA refers to hybridization to a nucleotide sequence that is identical or substantially identical to a length of contiguous nucleotides of the target DNA.
  • G is single nucleotide, G, or a short three nucleotide sequence
  • GTT comprised on the repeat portion of crRNA or a synthetic CRISPER nucleic acid construct.
  • the GFU does not hybridize with the anti-repeat of the tracrRNA or the synthetic tracr nucleic acid construct of this disclosure.
  • the GRI may, however, form a wobble base-pair with a U at the end of the anti- CRISPR repeat portion of the tracrRNA.
  • gene refers to a nucleic acid molecule capable of being used to produce inRNA, antisense RNA, miRNA. anti-mieroRNA anti sense
  • AMO oligodeoxyribonucleotide
  • Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions).
  • a gene may be "isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • a hairpin sequence is a nucleotide sequence comprising hairpins.
  • a hairpin e.g., stem-loop, fold-back
  • a hairpin sequence of the nucleic acid constructs is located at the 3' end of a synthetic tracr nucleic acid construct and immediately downstream of a "nexus sequence " . Wi thout being bound by any particular theory, it is believed that hairpins may be involved in Cas9 binding to a crRNA-tracrRNA complex (e.g., the synthetic CRISPR nucleic acid construct-synthetic
  • a “heterologous” or a “recombinant” nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non- naturall occurring multiple copies of a naturally occurring nucleotide sequence.
  • hornologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • Homology refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity).
  • compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention.
  • hornologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of the invention.
  • a hornologue of a Cas9 polypeptide useful with this invention can be about 70% homologous or more to any one of the Cas9 sequences provided herein.
  • hybridization refers to the binding of two fully complementary nucleotide sequences or substantially complementary sequences in which some mismatched base pairs may be present.
  • the conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some embodiments, the conditions of hybridization can be high stringency, or they can be medium stringency or low stringency depending on the amount of complementarity and the length of the sequences to be hybridized. The conditions that constitute low, medium and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (See, e.g..
  • the terms “increase,” “increasing, *'' “increased,” “enhance. “ “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • invasive foreign genetic element means DNA that is foreign to the bacteria (e.g., genetic elements from, for example, pathogens including, but not limited to, viruses, bacteriophages, and/or plasmids).
  • a “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence.
  • a “wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the organism.
  • a “homologous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.
  • Neexus sequence refers to a nucleotide sequence located immediately downstream of the "anti-stitch sequence " in a synthetic tracr nucleic acid construct.
  • the nexus is about six to ten nucleotides in length comprising a highly conserved sequence: TNANNC.
  • the nexus can be a nucleotide sequence of
  • T(A/C)A(A/G)(G/A)C (or U(A/C)A(A/G)(G/A)C)), GATAAGGC IT (or GAUAAGGCUU), TCAAGCAA (or UCAAGCAA). or T(C/A)AA(A/C)(C/A)(A/G)(A/T) (or
  • nucleic acid refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyl adenine, hypoxanthine and others can also be used for antisense. dsRNA, and ribozyme pairing.
  • nucleic acid constructs o the present disclosure can be DNA or RNA. but are preferably DNA.
  • the nucleic acid constructs of this invention may be described and used in the form of DNA, depending on the intended use, they may also be described and used in the form of RNA.
  • a "synthetic" nucleic acid or nucleotide sequence refers to a nucleic acid or nucleotide sequence that is not found in nature but is constructed by the hand of man and as a consequence is not a product of nature.
  • nucleotide sequence refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded.
  • nucleic acid sequence “nucleic acid, "* "nucleic acid molecule, " “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides. Except as otherwise indicated, nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5 ' to 3 " direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821 - 1.825 and the World Intellectual Property
  • a "5' region” as used herein can mean the region of a polynucleotide that is nearest the 5' end.
  • an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • a "3' region” as used herein can mean the region of a polynucleotide that is nearest the 3' end.
  • an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference
  • polynucleotide molecule or its complementary strand as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence.
  • protospacer sequence refers to the target double stranded DNA and specifically to the portion of the target DN A that is fully or substantially complementary (and hybridizes) to the spacer sequence of the synthetic CRISPR nucleic acid construct.
  • the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” “suppress,” and “decrease “ describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%o, or 100% as compared to a control.
  • the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • a mutation in a Cas9 nuclease can reduce the nuclease activity of the Cas9 by at least about 5%. 10%, 15%. 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control (e.g., a wild-type Cas9).
  • a “repeat sequence” as used herein refers, for example, to the repeat sequences of wild-type CRISPR loci or of the synthetic CRISPR nucleic ac id constructs that are separated by "spacer sequences.”
  • a repeat sequence can complementary (e.g., a 100% base pair match) to or substantially complementary, e.g., at least 70% complementary (e.g., 70%, 71%), 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more), to a corresponding anti-repeat sequence.
  • a “repeat sequence” of a synthetic CRISPR nucleic acid construct of this disclosure comprises an optional “zipper sequence,” a “bulge sequence,” a “stitch sequence,” and a “spacer sequence.”
  • a synthetic CRISPR nucleic acid construct can comprise a GRI that in other embodiments is comprised in the stitch sequence.
  • a "zipper sequence,” as used herein, refers to an optional portion of the repeat sequence that is located 3' or immediately upstream (3' to 5') of the bulge sequence in a synthetic CRISPR nucleic acid construct and comprises, consists of, or consists essentially of at least about three nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides, or any range or value therein).
  • a zipper sequence can be referred to as the "upper stem.”
  • a “zipper sequence” shares sufficient complementarity with a corresponding and also optional “anti- zipper sequence” located on a synthetic tracr nucleic acid construct such that upon contact the zipper sequence and the anti-zipper sequence, when present, and can hybridize to one another, thereby binding the two nucleic acid constructs together.
  • the zipper/anti-zipper sequence can be referred to as an "upper stem.”
  • a zipper sequence can be fully complementary (e.g., a 100% base pair match) to or substantially complementary, e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the corresponding anti-zipper sequence.
  • 70% complementary e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%
  • an anti-zipper sequence of a synthetic tracr nucleic acid construct of this invention comprises, consists of, or consists essentially of at least about three nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides, or any range or value therein) that are fully complementary to or substantially complementary to the corresponding zipper sequence in a synthetic CRISPR nucleic acid construct or a synthetic CRISPR nucleic acid array.
  • nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides, or any range or value therein
  • the anti-zipper sequence is the site of RNase III binding and as such comprises the nucleotide sequences that are well known in the art to be involved in RNase III binding (See, e.g., Pertzev and Nicholson, Nucleic Acids Res. 34(13):3708-3721 (2006)).
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin. A. M., and Griffin, H.
  • a “spacer sequence” as used herein is a nucleotide sequence that is complementary to a target DNA (e.g., the "protospacer sequence”).
  • the spacer sequence can be fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target DNA.
  • the spacer sequence has 100% complementarity to the target DNA.
  • the complementarity of the 3' region of the spacer sequence to the target DNA is 100% but is less than 100% in the 5' region of the spacer and therefore the overall complementarity of the spacer sequence to the target DNA is less than 100%.
  • the first 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, and the like, nucleotides in the 3' region of a 20 nucleotide spacer sequence (seed sequence) can be 100%) complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 7 to 12 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 7 to 10 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. .
  • the first 7 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5 " region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • a “stitch sequence” as used herein refers to a nucleotide sequence comprising, consisting essentially of. or consisting of about 5 nucleotides in length and having the consensus nucleotide sequence of NNTNN.
  • the “stitch sequence” is located (5' to 3') on a synthetic CRISPR nucleic acid construct immediately upstream of the "bulge sequence” and downstream of the "G R i " ⁇
  • the “stitch sequence” tends to have a high AT content and hybridizes to the "anti-stitch sequence” located in the synthetic tracr nucleic acid construct.
  • the stitch sequence comprises, consists essentially of, or consists of the nucleotide sequence of (5 ' to 3 ') NNTNN, TTTGT, TTTTA,
  • TTTTA TTTCA
  • an "anti-stitch sequence” as used herein refers to a nucleotide sequence that is fully complementary to and hybridizes to the stitch sequence (e.g., NNANN, ACAAA, TAAAA, (T/C)(A/G)T(A/G)(A/G), TAAAA, TGAAA).
  • the anti-stitch sequence is located on a synthetic tracr nucleic acid construct immediately downstream (5' to 3') of the bulge sequence and immediately upstream of the "nexus sequence.” Without wishing to be bound by any particular theory, it is believed that the hybridization of the stitch sequence of the synthetic crRNA construct with the anti-stitch sequence of synthetic tracrRNA construct is involved in re-establishing base-pairing after the "bulge sequence.” In some embodiments, the stitch/anti- stitch can be referred to as the "lower stem.”
  • the phrase "substantially identical,” or “substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues to about 150 residues in length.
  • the substantial identity exists over a region of the sequences that is at least about 3 to about 15 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 residues in length and the like or any value or any range therein) , at least about 5 to about 30 , at least about 10 to about 30, at least about 16 to about 30, at least about 18 to at least about 25, at least about 18, at least about 22, at least about 25, at least about 30, at least about 40, at least about 50, about 60, about 70, about 80, about 90, about 100, about 1 10, about 120, about 130, about 140, about 150, or more residues in length, and any range therein.
  • sequences of the sequences can be substantially identical over at least about 22 nucleotides. In some particular embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, sequences of the invention can be about 70% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In some embodiments, sequences of the invention can be about 75% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention can be about 80% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention can be about 80% to about 100% identical over at least about 7 nucleotides to about 25 nucleotides. In some
  • sequences of the invention can be about 70% identical over at least about 18 nucleotides. In other embodiments, the sequences can be about 85% identical over about 22 nucleotides. In still other embodiments, the sequences can be 100% homologous over about 16 nucleotides. In a further embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, in representative embodiments,
  • substantially identical nucleotide or protein sequences perform substantially the same function (e.g., Cas9 HNH and/or RuvC nickase activities).
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of eedleman and Wunsch. the search for similarity method o Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and IF AST A available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlengfh
  • E expectation
  • BLOSUM62 scoring matri see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karl in & Altschul, Proc. Natl Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001.
  • the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
  • Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of a medium stringency wash for a duplex of, e.g.. more than 100 nucleotides, is l x SSC at 45°C for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3.
  • nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence hybridizes to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC-4, 1 niM EDTA at 50°C with washing in 2X SSC. 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1 % SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM ED T A at 50°C with washing in 0.5X SSC. 0.1% SDS at 50°C.
  • the reference nucleotide sequence hybridizes to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS).
  • nucleotide sequence and/or recombinant nucleic acid molecule of this invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. As is understood in the art.
  • nucleotide sequence and/or recombinant nucleic acid molecule of this invention can be codon optimized for expression in the particular species of interest.
  • the recombinant nucleic acids molecules, nucleotide sequences and polypeptides of the invention are "'isolated. "
  • An “isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand o man. exists apart from its native environment and is therefore not a product of nature.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell.
  • a non-native environment such as, for example, a recombinant host cell.
  • isolated means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences. and/or different position in the genome than as found in nature). Accordingly, the recombinant nucleic acid molecules, nucleotide sequences and their encoded polypeptides are "isolated" in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.
  • nucleotide sequences and/or recombinant nucleic acid molecules of the invention can be operatively associated with a variety of promoters and other regulatory elements for expression in various organisms cells.
  • a recombinant nucleic acid of this invention can further comprise one or more promoters operably linked to one or more nucleotide sequences.
  • operably linked or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • operably linked refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.
  • a “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase 11 and directs the initiation of transcription.
  • promoters are found 5', or upstream. relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e., "chimeric genes” or “chimeric polynucleotides.” These various types of promoters are known in the art.
  • promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
  • a nucleic acid construct of the invention can be an "expression cassette” or can be comprised within an expression cassette.
  • expression cassette means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleic acid constructs of the invention (e.g., a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; a nucleotide sequence encoding a polypeptide of interest, a nucleotide sequence encoding a cas9 nuclease)), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette also can optionally include a transcriptional and/or
  • translational termination region i. e. , termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e. , foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
  • An expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g. , an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g. , fluorescence).
  • vector refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide scqucncc(s) to be transferred, delivered or introduced.
  • V ectors for use in transformation of host organisms are well known in the art.
  • Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial
  • chromosome or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • a vector as defined herein can transform prokaryotic or eukaryotic host cither by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell. Accordingly, the nucleic acid molecules of this invention and/or expression cassettes can be comprised in vectors as described herein and as known in the art.
  • "Introducing,” “introduce,” “introduced” (and grammatical variations thereof) in the context of a polynucleotide of interest means presenting the nucleotide sequence of interest to the host organism or cell of said organism (e.g., host cell) in such a manner that the nucleotide sequence gains access to the interior of a cell.
  • these nucleotide sequences can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different expression constructs or
  • nucleic acid constructs of this invention e.g., a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; a nucleotide sequence encoding a polypeptide of interest, a nucleotide sequence encoding a cas9 nuclease, and the like
  • a host organism or a cell of said host organism e.g., a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; a nucleotide sequence encoding a polypeptide of interest, a nucleotide sequence encoding a cas9 nuclease, and the like
  • transformation refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient.
  • a host cell or host organism is stably transformed with a nucleic acid molecule of the invention.
  • a host cell or host organism is transiently transformed with a recombinant nucleic acid molecule of the invention.
  • polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • stably introducing or “stably introduced” in the context of a polynucleotide introduced into a cell is intended that the introduced polynucleotide is stably incorporated into the genome o the cell, and thus the cell is stably transformed with the polynucleotide.
  • “Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
  • “Genome” as used herein also includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast or mitochondrial genome. Stable transformation as used herein can also refer to a transgene that is maintained
  • extrachromasomally for example, as a minichromosome or a plasmid.
  • Transient transformation may be detected by. for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • Stable transformation o a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g.. a plant, a mammal, an insect, an archaea, a bacterium, and the like).
  • Stable transformation of a cell can be detected by.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • PCR polymerase chain reaction
  • the nucleotide sequences, constructs, expression cassettes can be expressed transiently and/or they can be stably incorporated into the genome of the host organism.
  • a recombinant nucleic acid molecule/polynucleotide of the invention can be introduced into a cell by any method known to those of skill in the art.
  • transformation of a cell comprises nuclear transformation.
  • transformation of a cell comprises plastid transformation (e.g., chloroplast transformation).
  • the recombinant nucleic acid comprises nuclear transformation.
  • molecule/polynucleotide of the invention can be introduced into a cell via conventional breeding techniques.
  • a nucleotide sequence therefore can be introduced into a host organism or its cell in any number of ways that are well known in the art.
  • the methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into the organism, only that they gain access to the interior of at least one cell of the organism.
  • nucleotide sequences can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, or in separate
  • nucleotide sequence can be incorporated into a plant, as part of a breeding protocol.
  • the present invention is directed to compositions and methods having increased efficiency and increased specificity for site-specific nicking, cleaving and/or modification of target DNA and for site-specific targeting of polypeptides of interest to target DNA.
  • the synthetic CRISPR nucleic acid (crRNA) can comprise a wobble base GRI sequence (G U) and a stitch sequence or alternatively, the erRNA can comprise a stitch sequence that comprises the (G/U) wobble base and therefore does not further describe a GRI sequence.
  • the GR1 does not pair (i.e. G/A mismatch with Lje, Fig. 20).
  • Further equivalencies include those as shown in the equivalency table (Table 1) provided below (see also. Fig. 22).
  • a synthetic trans-encoded CRISPR (tracr) nucleic acid e.g., tracrRNA, tracrDNA
  • said construct comprising, consisting essentially of, or consisting of from 5' to 3', an anti -zipper sequence comprising, consisting essentially of or consisting of at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C (or U(A C)A(A/G)(G/A)C)), TCAAAC, (or UCAAAC), TAAGGC (or UAAGGC), GATAAGG (or GAUAAGG), GATAAGGCTT (or GAUAAGGCUU), TCAAG (or UCA
  • the anti-stitch sequence an comprise, consist essentially of, or consist of a nucleotide sequence of NNANN, AC AAA, TAAAA, (T/C)(A/G)T(A/G)(A/G), TAAAA, TGAAA.
  • a synthetic trans-encoded CRISPR(tracr) nucleic acid construct comprising, consisting essentially of, or consisting of from 5' to 3', an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising consisting essentially of, or consisting of a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C (or U(A/C)A(A/G)(G/A)C)), TCAAAC, (or UCAAAC), TAAGGC (or UAAGGC), GATAAGG (or GAUAAGG), GATAAGGCTT (or GAUAAGGCUU), TCAAG (or UCAAG), TCAAGCAA (or
  • UCAAGCAA T(C/A)AA(A/C)(C/A)(A/G)(A/T) (or U(C/A)AA(A/C)(C/A)(A/G)(A/U)), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or
  • a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, wherein the anti-zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence.
  • the bulge sequence of a synthetic tracr nucleic acid construct comprises, consists essentially of or consists of at least about three nucleotides. In some embodiments, the bulge sequence of a synthetic tracr nucleic acid construct comprises, consists essentially of or consists of at least about four nucleotides. In other embodiments, the bulge sequence of a synthetic tracr nucleic acid construct comprises, consists essentially of or consists of five nucleotides. In other embodiments, the hairpin sequence of a synthetic tracr nucleic acid construct comprises, consists essentially of or consists of at least two hairpins, wherein each hairpin comprises at least three matched base pairs.
  • the present invention provides a synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) construct comprising, consisting essentially of, or consisting of from 3' to 5', an optional zipper sequence comprising, consisting essentially of, or consisting of at least about three nucleotides, a bulge sequence that comprises, consists essentially of, or consists of a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)).
  • a synthetic CRISPR nucleic acid e.g., crRNA, crDNA construct comprising, consisting essentially of, or consisting of from 3' to 5', an optional zipper sequence comprising, consisting essentially of, or consisting of at least about three nucleotides, a bulge sequence that comprises, consists essentially of, or consists of a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)
  • a stitch sequence comprising, consisting essentially of, or consisting of a nucleotide sequence of NNTNN (or NNUN ), a GRI comprising, consisting essentially of, or consisting of a nucleotide G or GTT, and a spacer sequence having a 5' end and a 3 ' end and comprising, consisting essentially of, or consisting of at least seven nucleotides at its 3 ' end that have 100% identity to a target DNA, and the zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GRI, and the GR I is located immediately upstream of the spacer sequence.
  • synthetic CRISPR nucleic acid e.g., crRNA, crDNA
  • synthetic CRISPR nucleic acid construct comprising, consisting essentially of, or consisting of, from 3 ' to 5', an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti-zipper, a bulge sequence that comprises the nucleotide sequence of at least about two nucleotides, a stitch sequence comprising a nucleotide sequence of NNUNN (, and a spacer sequence having a 5 ' end and a 3 ' end and comprising at least seven nucleotides at its 3 ' end having 100% identity to a target DNA, and the zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence.
  • an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti
  • a synthetic CRISPR nucleic acid array comprising, a nucleotide sequence encoding two or more CRISPR nucleic acid constructs of this invention, wherein the two or more CRISPR nucleic acid constructs are located immediately adjacent to one another on said nucleotide sequence, the stitch sequences of said two or more CRISPR nucleic acid constructs are identical, the spacer sequences of said two or more CRISPR nucleic acid constructs are identical or non-identical, and, when present, the zipper sequences of said two or more CRISPR nucleic acid constructs are identical.
  • a chimeric nucleic acid construct (or guide nucleic acid construct) comprising a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct of this invention, wherein the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 70% (e.g., about 70%, 71 %, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%. 97%, 98%.
  • 70% e.g., about 70%, 71 %, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91 %, 92%, 93%, 94%, 95%
  • the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% identical to and hybridized to the anli-stitch sequence of said synthetic tracr nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary.
  • a chimeric nucleic acid construct comprising, consisting essentially of. or consisting of. a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct of this invention, wherein the UNN of the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and hybridizes to the ⁇ o the anti-stitch sequence of said synthetic tracr nucleic acid construct and the (G) of said stitch sequence forms a wobble base pair with the U of said anti- stitch sequence, the bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRIS PR nucleic acid construct are non-complementary and, when the zipper sequence and anti-zipper sequence are present, the zipper sequence of the synthetic CRISPR nucleic acid construct is hybridized to the anti-zipper sequence of said synthetic tracr nucleic acid construct.
  • a chimeric nucleic acid construct can optionally further comprise nucleotides linking the hybridized zipper and the anti-zipper sequence at the end of the hybridized sequences that is distal to the bulge sequences.
  • the chimeric nucleic acid construct can optionally further comprise nucleotides linking the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid.
  • a linking nucleotide can be any nucleotide (e.g., T, A, G, C) and the number of nucleotides linking the zipper sequence and anti-zipper sequence or the bulge sequence can be about three to about seven.
  • a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct of the invention can further comprise a Cas9 nuclease, nucleotide sequence encoding an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%. 97%, 98%, 99%.
  • a Cas9 nuclease nucleotide sequence encoding an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • Cas9 nucleases useful with this invention can be any Cas9 nuclease known to catalyze DNA cleavage in a CRISPR-Cas system. As known in the art. such Cas9 nucleases comprise a HNH motif and a RuvC motif (See. e.g.,
  • the HNH motif or the RuvC motif can comprise mutations that reduce or eliminate their activity as compared to wild-type Cas9 nucleases.
  • just one motif is mutated (e.g., either the HNH motif or the RuvC motif).
  • both motifs are mutated such that both activities are reduced or eliminated. Any type of mutation including missense mutations, nonsense mutations, frameshift mutations, and the like, can be used to reduce or eliminate the activity of the HNH motif and/or the RuvC motif in a Cas9 nuclease.
  • the present disclosure identifies several CRISPR-Cas systems and groupings of Cas9 nucleases. These groupings include a Streptococcus thermophilus CRISPR 1 (Sth CRl ) group of Cas9 nucleases, a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, and a
  • Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases Non-limiting examples of Sth CRl group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 1-9 and 51.
  • Non-limiting examples of Sth CR3 group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 10-23.
  • Non-limiting examples of Lb group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs:28, 30-33, 35, 43, 44, 47, 50 and 52.
  • Non- limiting examples of Lrh group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs:24-27, 29, 34, 36-42, 45 and 53. Additional Cas9 nucleases include, but are not limited to, those of Lactobacillus curvatus CRL 705. Still further Cas9 nucleases useful with this invention include, but are not limited to, a Cas9 from Lactobacillus animalis KCTC 3501, and Lactobacillus farciminis WP 010018949.1.
  • the Cas9 nuclease may comprise, consist essentially of, or consist of a Cas9 from a Streptococcus thermophilus CRIS R 1 (Sth CRl ) group of Cas9 nucleases, a Cas9 from Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, a Cas9 nuclease from a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, and/or a Cas9 nuclease from a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.
  • an amino acid sequence encoding a Cas9 nuclease can be an amino acid sequence of any one of SEQ ID NO:l to SEQ ID NO:53.
  • a Cas9 nuclease useful with a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR nucleic acid array, and/or a chimeric nucleic acid construct of this disclosure comprises, consists essentially of, or consists of a nucleotide sequence encoding an amino acid sequence having at least 70% identity to an amino acid sequence of any one of SEQ ID NO:l to SEQ ID NO:53.
  • the Cas9 nuclease can be encoded by a nucleotide sequence that is codon optimized for an organism comprising the target DNA.
  • the Cas9 nuclease can comprise at least one nuclear localization sequence.
  • tracrRNA a synthetic tracr nucleic acid construct
  • Cas9 nucleases a functional pairing between the nexus sequence of a synthetic tracr nucleic acid construct (tracrRNA, tracrDNA) with particular groups of Cas9 nucleases.
  • tracrRNA a synthetic tracr nucleic acid construct
  • Cas9 nucleases a group of Cas9 nucleases.
  • the nexus sequence is GATAAGGC or G A 1 A AGGCC A TGC C .
  • the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases; when the nexus sequence is TAAGGC or TAAGGCTAGTCC, the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases; when the nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is lrom a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases; or when the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC and the Cas9 nuclease is from a Lactobacillus rhamnosus GG (Lrh ) group of Cas9 nucleases.
  • STh CR1 Streptococcus thermophilus CRISPR 1
  • a Cas9 nuclease useful with this invention can comprise a mutation in a HNH motif and/or a RuvC motif, thereby reducing or eliminating the activity of the respective motif.
  • a mutation in the HNH motif reduces/eliminates site-specific nicking of the (+) strand a double stranded target DNA and a mutation in the RucV active site reduces/eliminates site-specific nicking of the (— ) strand of the double stranded target DNA.
  • a mutation in both active sites reduces/eliminates cleavage of the
  • a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, and/or a chimeric nucleic acid construct of this disclosure comprises a Cas9 nuclease having a mutation in the RuvC active site motif.
  • a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, and/or a chimeric nucleic acid construct of this disclosure comprises a Cas9 nuclease having a mutation in the HNH active site motif.
  • a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, and/or a chimeric nucleic acid construct of this disclosure comprises a Cas9 nuclease having a mutation in the HNH active site motif and in the RuvC motif.
  • a Cas9 nuclease having a mutation in the HNH and RuvC motifs, thereby having reduced or eliminated nuclease activity further comprises a polypeptide of interest fused to the Cas9 nuclease.
  • a Cas9-polypeptide of interest fusion protein can be used to direct or target the polypeptide of interest to a particular target DNA.
  • a method for site-specific cleavage of a double stranded target DNA comprising: contacting a chimeric nucleic acid construct of this disclosure or an expression cassette comprising a chimeric nucleic acid construct of this disclosure with the target DNA in the presence of a Cas9 nuclease (e.g., SEQ ID NOs:l-53), thereby producing a site-specific cleavage of the target DNA in a region defined by complementary hybridization of the spacer sequence to the target DNA.
  • a Cas9 nuclease e.g., SEQ ID NOs:l-53
  • the site-specific cleavage can be a site- specific nicking of a (+) strand of the double stranded target DNA and said Cas9 nuclease comprises a mutation in a RuvC active site motif, thereby cleaving the (+) strand of the double stranded target and producing a site-specific nick in said (+) strand the double stranded target DNA.
  • the site-specific cleavage is a site-specific nicking of the (-) strand of the double stranded target DNA and said Cas9 nuclease comprises a point mutation in a I IN I I active site motif, thereby cleaving the (-) strand of the double stranded target DNA and producing a site-specific nick in said (-) strand the double stranded target DNA.
  • a method for site-specific cleavage of a double stranded target DNA comprising: contacting a trans-encoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of a Cas9 nuclease (e.g., SEQ ID NOs:l-53), wherein (a) the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5' to 3', an anti -zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about three nucleotides; an anti- stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC,
  • a Cas9 nuclease e.g.,
  • GATAAGG GATAAGGCTT, TCAAG, TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TC A AG C A AAGC .
  • a hairpin sequence comprising a nucleotide sequence comprising, consisting essentially of, or consisting of at least one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and (b) the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3' to 5', a zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising a nucleotide sequence having at least two nucleotides , a stitch sequence comprising a nucleotide sequence of NNTNN (or NNUNN), a G R1 comprising a nucleot
  • the zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GRI, and the G R1 is located immediately upstream of the spacer sequence, and further wherein the anti-zipper sequence and anti-stitch sequence of the tracr nucleic acid molecule are at least about 70% complementary to and hybridize to the zipper sequence and the stitch sequence of the CRISPR nucleic acid molecule, respectively, and the spacer sequence of the CRISPR nucleic acid molecule is at least about 80% complementary to and hybridizes to a portion of the target DNA and adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby resulting in a site-specific cleavage of the target DNA in a region defined by the complementary binding of the spacer sequence of the CRISPR nucle
  • PAM protospacer adjacent motif
  • a method for site-specific cleavage of a double stranded target DNA comprising: contacting the double stranded target DNA with a chimeric nucleic acid comprising, (a) a first nucleotide sequence comprising from 5' to 3', an anti- zipper sequence comprising at least about 9 nucleotides; a bulge sequence comprising at least about 4 nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN; a nexus sequence comprising a nucleotide sequence of TNANNC, ' I ( A/C ) A( A/G )( G/ A )C .
  • TCAAAC TAAGGC, GATAAGG. GATAAGGCTT. TCAAG. TCAAGCAA.
  • a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; (b) a second nucleotide sequence comprising from 3' to 5', a zipper sequence comprising at least about 3 nucleotides, a bulge sequence that comprises a nucleotide sequence having at least two nucleotides, a stitch sequence comprising a nucleotide sequence of NNTNN (or NNUNN), a GR !
  • nucleotide G or GTT comprising a nucleotide G or GTT, and a spacer sequence having a 5' end and a 3' end and comprising at least seven nucleotides at its 3' end that have 100% identity to a target DNA
  • the zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the GJU, and the G R 1 is located immediately upstream of the spacer sequence
  • a third nucleotide sequence encoding an amino acid sequence having at least 80% identity to an amino acid sequence encoding a Cas9 nuclease (e.g., SEQ ID NOs:l-53), wherein the anti-zipper sequence and the anti-stitch sequence of the first nucleotide sequence hybridize to the zipper sequence and stitch sequence of the second nucleotide sequence and the spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA and adjacent to
  • a method of site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA comprising contacting a trans- encoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of a Cas9 nuclease (e.g., SEQ ID NOs:l-53), wherein (a) the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5' to 3', an anti- zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of
  • NNANN a nexus sequence comprising a nucleotide sequence of TNANNC
  • T(A/C)A(A/G)(G/A)C T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCC,
  • a hairpin sequence comprising a nucleotide sequence having at one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and (b) the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3' to 5', a zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising at least two nucleotides (e.g., the nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of NNTNN, a G R1 comprising a nucleo
  • the bulge sequence of a synthetic tracr nucleic acid molecule or a first nucleotide sequence can comprise, consist essentially of. or consist of about three, four or five nucleotides.
  • the bulge sequence can comprises, consists essentially of or consists of five nucleotides
  • the hairpin sequence can comprise, consist essentially of or consist of at least two hairpins, wherein each hairpin comprises at least three matched base pairs.
  • the present invention provides a method for site-specific cleavage of a double stranded target DNA, comprising: contacting a trans-encoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of a Cas9 nuclease, wherein (a) the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5' to 3', an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of N AN , a nexus sequence comprising a nucleotide sequence of TNANNC.
  • T(A/C)A(A/G)(G/A)C, TCAAAC
  • a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream o the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence;
  • the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3' to 5', an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti-zipper, a bulge sequence that comprises the nucleotide sequence of (-NN-), a stitch sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end and comprising at least seven nucleotides at its 3' end having 100% identity to a target DNA, and
  • the zipper sequence when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence, and
  • the anti-zipper sequence hybridizes to the zipper sequence
  • the NNANN of anti-stitch sequence is complementary to and hybridizes to the NNUNN of the stitch sequence
  • the spacer sequence of the CRISPR nucleic acid molecule hybridizes to a portion of the target DNA and adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby resulting in a site-specific cleavage of the target DNA in a region defined by the hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA.
  • PAM protospacer adjacent motif
  • a method for site-specific cleavage of a double stranded target DNA comprising: contacting the double stranded target DNA with a chimeric nucleic acid comprising, (a) a first nucleotide sequence comprising from 5' to 3', an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN, a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCA
  • the anti-zipper sequence when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence;
  • a second nucleotide sequence comprising from 3' to 5', an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti-zipper, a bulge sequence comprising a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end and comprising at least seven nucleotides at its 3' end having 100% identity to a target DNA, and the zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence; and
  • the zipper sequence hybridizes to the anti -zipper sequence
  • the NNANN of anti-stitch sequence is complementary to and hybridizes to the NNUNN of the stitch sequence
  • the spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA and adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby resulting in a site-specific cleavage of the target DNA in a region defined by the hybridization of the spacer sequence of the second nucleotide sequence to the target DNA.
  • PAM protospacer adjacent motif
  • a method of site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA comprising contacting a trans- encoded CRJSPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of a Cas9 nuclease (e.g., SEQ ID NOs:l-53),
  • the tracr nucleic acid molecule is encoded by a nucleotide sequence comprising from 5' to 3', an optional anti-zipper sequence comprising at least about three nucleotides; a bulge sequence comprising at least about three nucleotides; an anti-stitch sequence comprising a nucleotide sequence of NNANN, a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC,
  • GATAAGG GATAAGGCTT, TCAAG, TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCG TAAGGCTAGTCC. TCAAGCAAAGC. or
  • a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matched base pairs, and the anti-zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence;
  • the CRISPR nucleic acid molecule is encoded by a nucleotide sequence comprising from 3' to 5', an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti-zipper, a bulge sequence comprising a nucleotide sequence having at least two nucleotides (e.g., the nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end and comprising at least seven nucleotides at its 3' end having 100% identity to a target DNA, and
  • the zipper sequence when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence, and
  • the Cas9 nuclease comprises a mutation in a HNH active site motif, a mutation in a RuvC active site motif, and is fused to a polypeptide of interest
  • the zipper sequence and anti-zipper sequence are present, the zipper sequence hybridizes to the anti-zipper sequence, the NNANN of anti-stitch sequence is complementary to and hybridizes to the NNUNN of the stitch sequence, and the spacer sequence hybridizes to the target DNA adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby resulting in a site-specific targeting of the polypeptide of interest to the target DNA in a region defined by the hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA.
  • PAM protospacer adjacent motif
  • the hybridized sequences can optionally further comprise additional nucleotides at the end of the of the hybridized sequences that is distal to the bulge sequences, thereby linking the hybridized zipper and the anti-zipper sequence.
  • the chimeric nucleic acid construct can optionally further comprise nucleotides linking the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid.
  • a linking nucleotide can be any nucleotide (e.g.. T, A, G, C) and the number of nucleotides linking the zipper and anti-zipper sequences or the bulge sequences can be about three to about seven.
  • Any wild-type, mutated, codon-optimized Cas9 nuclease or those comprising at least one nuclear localization sequence as described herein can be used with the methods of the invention including but not limited to SEQ ID NOs:l-53.
  • expression cassettes and vectors comprising the nucleic acid constructs, the nucleic acid arrays, nucleic acid molecules and/or the nucleotide sequences of this invention, which can be used with the methods of this disclosure.
  • nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and/or nucleotide sequences of this invention can be introduced into a cell of a host organism.
  • Any cell/host organism for which this invention is useful with can be used.
  • Exemplary host organisms include, but are not limited to, a plant, bacteria, archaeon, fungus, animal, mammal, insect, bird, fish, amphibian, cnidarian, human, or non-human primate.
  • a host organism can be, but is not limited to Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum, or Arabidopsis thaliana.
  • a cell useful with this invention can be, but is not limited to a stem cell, somatic cell, germ cell, plant cell, animal cell, bacterial cell, archaeon cell, fungal cell, mammalian cell, insect cell, bird cell, fish cell, amphibian cell, cnidarian cell, human cell, r non-human primate cell.
  • a cell useful with this invention includes but is not limited to a cell from Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum, or Arabidopsis thaliana.
  • a polypeptide of interest can include but is not limited to a helicase, a nuclease, a methyltransferase, a gyrase, a demethylase, a kinase, a dismutase, an integrase, a transposase, a telomerase, a recombinase, an acetyltransferase, a deacetylase, a polymerase, a phosphatase, a ligase, a ubiquitin ligase, a photolyase or a glycosylase.
  • a polypeptide of interest comprises depurination activity, oxidation activity, pyrimidine dimer forming activity, alkylation activity, DNA repair activity, DNA damage activity, deubiquitinating activity, adenylation activity, deadenylation activity SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity or telomere repair activity, or deamination activity.
  • a polypeptide of interest can be a polypeptide having kinase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, or telomere repair activity.
  • kits comprising the nucleic acid constructs, nucleic acid molecules, and/or nucleotide sequences of this invention.
  • kits for site-specific cleavage of double stranded DNA comprising a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array or a chimeric nucleic acid construct of this invention.
  • a kit for site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA is provided, the kit comprising a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array or a chimeric nucleic acid construct of this invention.
  • the kit can comprise the synthetic tracr nucleic acid construct, the synthetic CRISPR nucleic acid construct, the CRISPR nucleic acid array and/or the chimeric nucleic acid construct of this invention comprised in one or more expression cassettes.
  • a kit can further comprise a Cas9 nuclease (e.g., SEQ ID NOs:l-53) for use with the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and/or nucleotide sequences of this invention described herein.
  • kits can comprise primers said primers comprising portions of CRISPR repeat sequences in both directions.
  • a kit can comprise primers designed to comprise the boundaries of a CRISPR array (namely the leader end on one side, and the trailer end on the other), and extend through the CRISPR repeat sequence in both directions.
  • kits can further comprise instructions for use.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Cas proteins provide adaptive immunity against invasive genetic elements in bacteria and archaca 1 .
  • CRISPR-Cas systems the signature RNA-guided endonuclease Cas9 specifically targets sequences complementary to CRISPR spacers and generates double- stranded DNA breaks (DSBs) using two nickase domains (Makarova, K. S. et al. Nat Rev Microbiol 9, 467-477 (201 1); Garneau, J. E. et al. Nature 468, 67-71 (2010); Sapranauskas, R. et al. Nucleic Acids Res 39.
  • RNA duplex consisting of CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) 8 .
  • This native complex can be replaced by a synthetic single guide RNA (sgRNA) chimera which mimics the
  • crRNA:tracrRNA duplex Jinek, M. et al, Science 337, 816-821 (2012).
  • sgRNAs in combination with Cas9 make convenient, compact, and portable sequence-specific targeting systems that are amenable to engineering and heterologous transfer into a variety of model systems of industrial and translational interest.
  • the Cas9:sgRNA technology which provides a compact and practical means to generate double strand breaks (DSBs), has revolutionized genome editing (Mali, P. et al, Science 339, 823-826 (2013); Cong. L. et al. Science 339, 819-823 (2013); Jiang, W. et al. Nat. Biotechnol. 31, 233-239 (2013); Sander, J. D. & Joung, J. K. Nature Biotechnol. 32, 347-355. (2014)), opened new avenues for high-throughput genome-wide genetic screens 13 ' 14 , and expanded the toolbox for transcriptional control (Qi. L. S. et al Cell 152, 1173-1 183 (2013); Gilbert, L. A.
  • Fig. 1 shows a multiple sequence alignment for the nexus module
  • Fig. 2 provides a maximum likelihood tree for the nexus module developed through this research.
  • Fig. 3A-3D show consensus sequences for the nexus module for the Sth Crl group (Fig. 3A) the Sth Cr3 group (Fig. 3B), for the Lrh group Fig. 3C and for the Lbu group (Fig. 3D).
  • Fig. 5 shows a multiple sequence alignment for the anti-stitch module
  • Fig. 6A- 6D show consensus sequences for the anti -stitch module for the Sth Crl group (Fig. 6 A), for the Sth Cr3 group (Fig. 6B), for the Lrh group (Fig. 6C) and for the Lbu group (Fig. 6D).
  • Fig. 7 a multiple sequence alignment for the bulge module is provided in Fig. 7 with the consensus sequences for the bulge module for the for the Sth Crl group is shown in Fig. 8A.
  • Fig. 8B shows the consensus sequence for the bulge module for the Sth Cr3 group.
  • Fig. 8C shows the consensus sequence for the Lrh group and
  • Fig. 8D shows the consensus sequence for bulge module for the Lbu group.
  • a multiple sequence alignment for the zipper module is provided in Fig. 9 with Fig. 10 showing a maximum likelihood tree for the zipper module.
  • Fig. 11 shows a multiple sequence alignment for the bulge, anti-stitch and nexus modules.
  • Fig. 4 shows a maximum likelihood tree for Cas9 nucleases.
  • Figs. 12-21 show guide sequences and targeting for various cRNA:tracRNA constructs including Streptococcus thermophilus CR3, representing the Sth CR1 group (Fig. 12); Lactobacillus buchneri, representing the Lbu group(Fig. 13); Streptococcus
  • thermophilus CR1 representing the Sth CR1 group (Fig. 14); Streptococcus pyrogenes Ml GAS, representing the Sth CR3 group (Fig. 15); Lactobacillus rhamnosus, representing the Lrh group (Fig. 16); Lactobacillus animalis, representing the Lan group (Fig. 17);
  • Lactobacillus casei representing the Lea group (Fig. 18); Lactobacillus gasseri, representing the Lga group (Fig. 19); Lactobacillus jensenii, representing the Lje group (Fig. 20); and Lactobacillus pentosus, representing the Lpe group (Fig. 21).
  • the lower portion of each of Figs. 12-21 represents target dsDNA, including the target sequence (open structure) and the flanking (3') PAM.
  • each figure represents the CRISPR RNA (crRNA), which consists of a 5' portion complementary to the target sequence, as well as a 3' portion derived from the CRISPR repeat; and also represents the tracrRNA, which consists of an anti- CRISPR repeat portion, as well as the nexus and 3' hairpins.
  • crRNA CRISPR RNA
  • tracrRNA an anti- CRISPR repeat portion
  • the complementary portion of the crRNA :tracrRN A duplex consists of the lower stem (bottom complementary portion), a bulge (herniated mismatch) and upper stem (top complementary portion).
  • Example 2 Determination of guide RNA sequence features in various additional Type II systems.
  • Example 1 The findings described in Example 1 establish important modules in sgRNA that are required to support Streptococcus pyrogenes Cas9 (SpyCas9) activity.
  • SpyCas9 is merely one of many Cas9 orthologs found naturally (Chylinski, K. et al. RNA biology 10, 726-737 (2013); Fonfara, I. et al. Nucleic Acids Res (2013)).
  • Fig. 23 The Cas9 protein sequences clustered into three main sequence groups (Fig. 23). Similar grouping was observed when clustering was carried out using either CRISPR-repeat or predicted tracrRNA sequences (Fig. 24A, Figs. 23, 25, 26), as anticipated, given the presence of an anti-CRISPR repeat within the tracrRNA, and the intimate molecular relationship between Cas9 and crRNA:tracrRNA pairs (Makarova, K. S. et al. Nat Rev Microbiol 9, 467-477 (201 1 );
  • SpyCas9 sgRNA:DNA: complex and apo-SpyCas9 occupy significantly different
  • RNA-and DNA-binding domains taking place between the two structures (Jinek, M. et al, Science 343, 6176 (2014); Nishimasu, H. et al. Cell 156, 935 (2014)).
  • the nexus occupies a critical position in the SpyCas9-sgRNA:DNA complex, coordinating a number of key components of the protein and sgRNA, positioning both protein and RNA appropriately to receive target DNA duplexes for cleavage.
  • sgRNA: DNA Upon binding sgRNA: DNA, the arginine-rich bridge helix binds to the base of the nexus and to the lower stem.
  • the nexus interacts with two small regions (which we propose to establish as Nexus Interacting Region 1 (NIR1) 446-497 and Nexus Interacting Region 2 (NIR2) 1 105-1138) from the two lobes of SpyCas9. Both of these regions are disordered in the apoSpyCas9 structure, and notably contain two tryptophan residues identified as being
  • NIR2 also interacts directly with the lower stem, and the face opposite the nexus-binding site lies in close proximity to the 3 ' end of the target strand, suggesting that interaction with the nexus may be required to order the PAM recognition site.
  • Actinomyces naeslundii Cas9 Actinomyces naeslundii Cas9 (AnaCas9) apo-structure (Jinek, M. et al, Science 343. 6176 (2014)).
  • NIR2 is ordered, and contains an about 50 amino acid insertion. It is plausible to speculate that AnaCas9 may recognize a larger nexus and possibly
  • the ability to reprogram Cas9 orthogonality using chimeric sgRNAs with altered nexus sequences opens new avenues for the exploitation of novel Cas9 proteins, with the potential to harness the diversity of natural Cas9 orthologs, including short Cas9 variants for convenient packaging and delivery. Additionally, the ability to reprogram Cas9 using chimeric mgRNAs will allow for increased use of various PAMs for flexible management of target frequency (short PAMs with frequent occurrence) and specificity by reducing off- target cleavage (longer PAMs with infrequent occurrence). This also expands multiplexing opportunities, by using a single Cas9 with various chimeric guides, or by concurrently using orthogonal systems with different combinations of standard or chimeric sgRNAs.
  • the cas9 genes from the CR1SPR 1 locus and the CRISPR3 locus were PGR amplified from genomic DNA from S. thermophilus LMD-9, and cloned into pwtCas9-Bacteria (Addgene #44250) ) (Qi, L. S. et al. Cell 152, 1 173-1 1 83 (2013 )).
  • the Spel restriction site in the pdCas9-bacteria plasmid (Addgene #44249) (Id.) was removed and a gBlock (IDT) encoding a zraP-targeting sgRNA based on the CRISPR1 or the CRISPR3 locus was combined with the PCR-amplified backbone of the pgRNA-bacteria plasmid (Addgene #44251) (Id).
  • coli K-12 was used for transformation assays, and transformation efficiency was calculated by dividing the number of transformants for the tested sgRNA plasmid by the number of transformants for the psgRNA-Cl-T4 control plasmid. as described previously (Gomaa, A. A. et al. At Bio. 5, e00928-13 (2014)).
  • Plasmid construction To construct the Cas9-expressing plasmids, the Cas9 genes from the CRJSPR1 locus (Sthl-Cas9) and the CRISPR3 locus (Sth3-Cas9) were PCR amplified from genomic DNA extracted from S. thermophilus LMD-9. Each PCR product was combined with the PCR-amplified backbone of pwtCas9-Bacteria (Addgene #44250) (Qi, L. S. et al. Cell 152, 1173-1183 (2013)) by Gibson assembly.
  • the Spel restriction site in the pdCas9-bacteria plasmid (Addgene #44249) (Id.) was removed by digesting the plasmid with Spel, blunt ending, and religating to generate the pdCas9ASpeI plasmid.
  • a gBlock (IDT) encoding a rra/ argeting sgRNA based on the CRISPR1 (CI) locus or the CRISPR3 (C3) locus in S.
  • thermophilus LMD-9 was combined with the PCR-amplified backbone of the pgRNA-bacteria plasmid (Addgene #44251) (Id.) by Gibson assembly, thereby replacing the original S. pyogenes sgRNA sequence with the designed sgRNA sequence.
  • the resulting sgRNA plasmids and the pdCas9ASpeI backbone were then digested with Aatll and Xhol, and the gel-extracted fragment of each sgRNA plasmid and the pdCas9ASpeI plasmid were ligated together, forming psgRN A-SthC 1 and psgRNA-SthC3.
  • 5' phosphorylated oligonucleotides were annealed and ligated into the Spel/Kpnl or
  • E. coli K-12 subst. MG1655 (genotype: E. coli K-12 F ⁇ - ilvG- rfb-5C rph-1) was used for all transformation assays.
  • the strain was grown aerobically in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) at 37°C and 250 RPM unless indicated otherwise.
  • the medium was supplemented with antibiotics (34 ng/ml of chloramphenicol, 50 ng/ml Ampicillin) as appropriate.
  • Transformation assay Freezer stocks of cells harboring the indicated Cas9- expressing plasmid were streaked to isolation and individual colonies were inoculated into 3 ml of LB medium and cultured overnight. The resulting cultures were back-diluted into 45 ml of LB medium and grown to an ABS 0 o of 0.6 - 0.8 as measured on a Nanodrop 2000c spectrophotometer (Thermo Scientific). The cultures were then pelleted and washed with ice-cold 10% glycerol twice before being resuspended in 200-400 ⁇ of 10% glycerol.
  • Suspended cells (50 ⁇ ) were transformed with 25 ng of the indicated sgR A-expressing plasmid using a MicroPulser Electroporator (BioRad) and recovered in 300 ⁇ of SOC medium (Quality Biological) for 1 hour. After recovery, 200 ⁇ of cultures with different amounts of LB medium were plated on LB agar with 100 ng/ml of anhydrotetracycline. The transformation efficiency was calculated by dividing the number of transformants for the tested DgRNA plasmid by the number of transformants for the psgRNA-C 1 -T4 control plasmid, as described previously (Gomaa, A. A. et al. MBio. 5, e00928-13 (2014)). In order to reduce experiment-to-experiment variability in transformation efficiency, the tested sgRNA plasmid and the control plasmid were transformed into the same batch of
  • Figs. 30-32 transformation assays were used to test the ability of a plasmid to be electroporated into Lactobacillus strains that carry active CRISPR- Cas systems (Lbu - Lactobacillus buchneri, Fig. 30; Lrh - Lactobacillus rhamnosus, Fig. 32; Lea - Lactobacillus casei, Fig. 31.
  • the plasmid was engineered as to contain a protospacer sequence identical to the first spacer sequence in the CRISPR locus of the host.
  • plasmids were engineered as to flank the protospacer with a perfect PAM (NTAAC for Lga; NNGAA for Lea; NGAAA for Lrh; the PAM region is the underlined nucleotides in Figs. 30- 32, and mutated variants thereof (the nucleotides being tested for efficiency are italicized).
  • NTAAC perfect PAM
  • mutated variants thereof the nucleotides being tested for efficiency are italicized
  • control non-targeting sequence (next to last entry for each experiment shown in Figs. 30-32, as well as a control plasmid with no target sequence (last entry for each experiment shown in Figs. 30-32.
  • the ability of the native CRISPR-Cas system to interfere with plasmid uptake by DNA targeting is measured as the difference in transformation efficiency between the test sequence and that of the two aforementioned controls.

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Abstract

La présente invention concerne des méthodes et des compositions pour l'édition du génome et le ciblage de l'ADN de protéines.
EP15740979.8A 2014-01-24 2015-01-23 Méthodes et compositions pour des séquences guidant le ciblage de cas9 Pending EP3097212A4 (fr)

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CA2936646A1 (fr) 2015-07-30
CA2936646C (fr) 2024-04-30
JP2022106883A (ja) 2022-07-20
JP7429057B2 (ja) 2024-02-07
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JP2017503514A (ja) 2017-02-02
WO2015112896A9 (fr) 2015-11-26

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