JP2020103295A - Methods and compositions related to sequences that guide cas9-targeting - Google Patents

Abstract

To provide methods and compositions for DNA targeting and genome editing of proteins.SOLUTION: The invention provides a chimeric nucleic acid construct comprising: (a) a synthetic trans-encoded CRISPR(tracr) nucleic acid construct; and (b) a synthetic CRISPR nucleic acid construct. Also provided is a non-medical method for the site specific cleavage of a double stranded target DNA comprising contacting the chimeric nucleic acid construct with a target DNA in the presence of a Cas9 nuclease to cause hybridization of the spacer sequence of the synthetic CRISPR nucleic acid construct to a portion of the target DNA flanking a protospacer adjacent motif (PAM) on the target DNA, thereby causing site specific cleavage of the target DNA in a region defined by complementary hybridization of the spacer sequence to the target DNA.SELECTED DRAWING: None

Description

[Statement of priority]
This application claims the benefit of U.S. Provisional Application No. 61/931,515, filed January 24, 2014, under 35 USC 119(e), the entire contents of which are hereby incorporated by reference. , Incorporated herein by reference.
[Field of the Invention]
The present invention relates to a synthetic CRISPR-cas system and its use for genomic organization.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in combination with related sequences (cas), make up the CRISPR-Cas system and provide adaptive immunity in many bacteria. CRISPR-mediated immunization occurs through the incorporation of DNA from invasive genetic elements such as plasmids and phage as a novel "spacer".

The CRISPR-Cas system consists of an array of short DNA repeats separated by hypervariable sequences, flanked by cas genes, and through sequence-specific targeting and interference, adaptive immunity to invasive genetic elements such as phage and plasmids. Provided (Barrangou et al. 2007. Science. 315: 1709-1712; Brouns et al. 2008. Science 321: 960-4; Horvath and Barrangou. 2010. Science. 327: 167-70. Marraffin. 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. 46:311-339; Barrangou R. 2013. RNA. 4:267-278). Typically, invasive DNA sequences have been acquired as novel "spacers" (Barrangou et al. 2007. Science. 315:1709-1712), each paired with a CRISPR repeat and a novel repeat at the CRISPR locus. It is inserted as a spacer unit. Subsequently, the repeat-spacer array is transcribed as long pre-CRISPR RNA (pre-crRNA) (Browns et al. 2008. Science 321:960-4), which drives the small interfering CRISPR RNA (sequence-specific recognition). crRNA). Specifically, crRNA guides the nuclease towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonuclease (Garnau 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.337:816-821; Gasiunas. Natl. Acad. Sci. 109: E2579-E2586; Magadan et al. 2012. PLoS One. 7: e40913; Karvelis et al. 2013. RNA Biol. 10: 841-851). These widespread systems occur in almost half of the bacteria (about 46%) and the majority of archaea (about 90%). They are based on cas gene content, organization and diversity in the biochemical processes that drive crRNA biosynthesis, and the Cas protein complex that mediates target recognition and cleavage, the three major CRISPR-Cas. System type (Makarova et al. 2011. Nature Rev. Microbiol. 9: 467-477; Makarova et al. 2013. Nucleic Acid Res. 41: 4360-4377). In types I and II, a specialized Cas endonuclease processes pre-crRNA, which then assembles into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acid complementary to crRNA. A different process relates to the Type II CRISPR-Cas system. Here, pre-CRNA is processed by a mechanism in which trans-activated crRNA (tracrRNA) hybridizes to the repeat region of crRNA. The hybridized crRNA-tracrRNA is cleaved by RNaseIII, followed by a second event that removes the 5'end of each spacer, producing mature crRNA that remains bound to both tracrRNA and Cas9. The mature complex then cleaves both strands looking for a target dsDNA sequence ("protospacer" sequence) complementary to the spacer sequence in the complex. Recognition and cleavage of the target by the complex in the type II system not only requires a complementary sequence between the spacer sequence on the crRNA-tracrRNA complex and the target "protospacer" sequence, but also that of the protospacer sequence. It also requires a protospacer flanking motif (PAM) sequence located at the 3'end. The exact PAM sequence required may differ between different type II systems.

The present disclosure provides methods and compositions that enhance the efficiency and specificity of the synthetic Type II CRISPR-Cas system and improve the efficiency and specificity of genomic organization and other applications.

One aspect of the invention is, in the 5'to 3'direction, an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-zipper sequence comprising a nucleotide sequence of NNANN. -Stitch arrangement; 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 TCAAACAAAAGCTTCAGC nucleotides A synthetic transcoding CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct comprising a nexus sequence comprising a sequence; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least 3 Containing matching base pairs,
Here, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, and the anti-stitch sequence is immediately upstream of the nexus sequence. And the nexus sequence is located immediately upstream of the hairpin sequence.

A second aspect of the invention is an optional zipper sequence comprising at least about 3 nucleotides in the 3'to 5'direction (which hybridizes to the anti-zipper of tracrRNA, if present), at least 2 nucleotides (e.g., (- NN- nucleotide sequence of)) bulge sequence comprising, anti TracrRNA - stitching sequence comprising a nucleotide sequence hybridizing NNTNN (or NNUNN) the stitch, _{G R1} containing nucleotide G or GTT, And a synthetic CRISPR nucleic acid (eg, a spacer sequence having a 5′ end and a 3′ end, the 3′ end of which includes at least 7 nucleotides having 100% identity to the target DNA). CrRNA, providing CrDNA) construct, zipper sequences, if present, situated immediately upstream of the bulge sequence, bulge sequence located immediately upstream of the stitch sequence, stitch sequence immediately upstream of the G _R1 And _GR1 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 the invention, wherein the two or more CRISPR nucleic acid constructs are separated from each other on said nucleotide sequence. Located immediately adjacent, the zipper sequences of the two or more CRISPR nucleic acid constructs are the same if present, the stitch sequences of the two or more CRISPR nucleic acid constructs are the same, and the spacers of the two or more CRISPR nucleic acid constructs are the same. The sequences are identical or non-identical.

A fourth aspect of the invention provides a chimeric nucleic acid construct comprising a synthetic tracr nucleic acid construct of the invention and a synthetic CRISPR nucleic acid construct of the invention, wherein the zipper sequence of the synthetic CRISPR nucleic acid construct, if present, is Hybridizing to the anti-zipper sequence of the synthetic tracr nucleic acid construct at least about 70% complementary and hybridizing, wherein the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and hybridizing to the anti-stitch sequence of the 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 is to contact a chimeric nucleic acid construct of the present disclosure or an expression cassette comprising said chimeric nucleic acid construct with a target DNA in the presence of Cas9 nuclease, thereby hybridizing the spacer sequence to the target DNA. Provided is a method for site-specific cleavage of double-stranded target DNA comprising the step of causing site-specific cleavage of target DNA within a defined region.

A sixth aspect of the invention provides for the site-specific cleavage of double-stranded target DNA comprising contacting the transcoded CRISPR(tracr) nucleic acid molecule and the CRISPR nucleic acid molecule with the target DNA in the presence of Cas9 nuclease. Provide a way to
Wherein (a) a tracr nucleic acid molecule is in the 5'to 3'direction, an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide of NNANN. Anti-stitch sequences including sequences; TNANC, 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), GATAAGGGCCATGCC, TAAGGCCATTCC, TCAAGCAAAGC, or TCAAAACAAAGCTTCAGC; and a hairpin sequence comprising a nucleotide sequence having at least two hairpins. Each hairpin comprises at least 3 matching base pairs, the anti-zipper sequence is located immediately upstream of the bulge sequence, and the bulge sequence is immediately upstream of the anti-stitch sequence. Located, 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 in the 3'to 5'direction. , An optional zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising a nucleotide sequence having at least 2 nucleotides (eg (-NN-) nucleotide sequence), a NNTNN (or NNUNN) nucleotide sequence. stitching sequence, G _R1 comprises a nucleotide G or _GTT, and 5 a spacer sequence having 'and 3' ends, at least seven nucleotides with the target DNA and 100% identity to the 3 'end comprising comprising a spacer sequence is encoded by the nucleotide sequence, zipper sequence is located immediately upstream of the bulge sequence, bulge sequence located immediately upstream of the stitch sequence, stitching sequence is located immediately upstream of the G _R1 , And _GR1 are located immediately upstream of the spacer sequence, and
Furthermore, where the anti-zipper sequence and the zipper sequence are present, they hybridize to each other, the anti-stitch sequence and the stitch sequence hybridize to each other, and the spacer sequence of the CRISPR nucleic acid molecule comprises at least one of the target DNAs. Parts (eg, at least about 7 consecutive nucleotides of the target DNA (eg, about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., And any range or variation therein (adjacent to the protospacer flanking motif (PAM) on the target DNA) is at least about 80% complementary and thereby hybridizes, whereby the CRISPR nucleic acid molecule to the target DNA Within the region defined by the complementary binding of the spacer sequence, site-specific cleavage of the target DNA occurs, thus, in an exemplary embodiment, the spacer sequence of the CRISPR nucleic acid molecule is one of the target DNA sequences flanking the PAM. Hybridized to a region, the target sequence can comprise, consist essentially of, or consist of from about 7 to about 20 contiguous nucleotides of the target DNA sequence.

A seventh aspect of the invention provides a method for site-specific cleavage of double stranded target DNA comprising the step of contacting double stranded target DNA with a chimeric nucleic acid comprising:
(A) in the 5'to 3'direction, an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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), GATAAGGCCATCGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAGAGTCTCAGC nexus sequence comprising a nucleotide sequence; and a first nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin ( The hairpin contains at least 3 matching base pairs and, if present, the anti-zipper sequence is located immediately upstream of the bulge sequence and 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) at least 2 in the 3'to 5'direction, an optional zipper sequence comprising at least about 3 nucleotides (which, if present, hybridizes to the anti-zipper sequence of the first nucleotide sequence). bulge sequence comprising (nucleotide sequence e.g. (-Nn)) a nucleotide sequence having a nucleotide, stitching sequence comprising the nucleotide sequence of NNTNN (or NNUNN), _{G R1} comprises a nucleotide G or GTT, and the 5 'end and A second nucleotide sequence comprising a spacer sequence having a 3′ end, the spacer sequence comprising at least 7 nucleotides having 100% complementarity with the target DNA at the 3′ end (the zipper sequence is present) If, located immediately upstream of the bulge sequence, bulge sequence located immediately upstream of the stitch sequence, stitching sequence is located immediately upstream of the G _R1, and, G _R1 is immediately upstream of a spacer sequence Located); and
(C) a third nucleotide sequence encoding an amino acid sequence having at least 80% identity to the amino acid sequence encoding Cas9 nuclease,
Here, if the anti-zipper sequence and the zipper sequence are present, they hybridize to each other, the anti-stitch sequence hybridizes to the stitch sequence, and the spacer sequence of the second nucleotide sequence is at least the target DNA. Hybridize to a portion (eg, at least about 7 contiguous nucleotides of said target DNA, preferably up to about 20 contiguous nucleotides) (adjacent to the protospacer flanking motif (PAM) on the target DNA), This causes a site-specific cleavage of the target DNA within the region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA.

The eighth aspect of the present invention includes a method of site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, wherein the chimeric nucleic acid construct of the present disclosure or an expression cassette containing the chimeric nucleic acid construct is targeted. The step of contacting with the DNA, whereby the polypeptide of interest fused to Cas9, is targeted to a specific site on the target DNA, said site being defined by hybridization of a spacer sequence to the target DNA.

A ninth aspect of the invention comprises a method of site-specific targeting of a polypeptide of interest to double stranded (ds) target DNA,
Contacting the transcoded CRISPR(tracr) nucleic acid molecule and the CRISPR nucleic acid molecule with a target DNA in the presence of Cas9 nuclease, wherein:
(A) a tracr nucleic acid molecule comprises in the 5'to 3'direction an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch arrangement; 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, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nexus sequence; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin, Encoded, the hairpin comprises at least 3 matching base pairs, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, and the bulge sequence is immediately upstream of the anti-stitch sequence. Located, 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) a CRISPR nucleic acid molecule is an optional zipper sequence comprising in the 3'to 5'direction at least about 3 nucleotides that hybridizes to an anti-zipper sequence, a nucleotide sequence having at least 2 nucleotides (eg, (-Nn) nucleotide sequence) bulges sequence comprising the stitch sequence comprising the nucleotide sequence of NNTNN, a spacer sequence having a _{G R1,} and 5 'and 3' ends containing nucleotide G or GTT, Part 3 The zipper sequence, if present, is located immediately upstream of the bulge sequence, if present, and comprises a spacer sequence comprising at least 7 nucleotides having at least 7 nucleotides with 100% identity to the target DNA at the end. sequence is located immediately upstream of the stitch sequence, stitching sequence is located immediately upstream of the G _R1 and G _R1 is located immediately upstream of a spacer sequence, and,
Further herein, the Cas9 nuclease comprises a mutation within the HNH active site motif, a mutation within the RuvC active site motif, fused to the polypeptide of interest, and the anti-zipper and zipper sequences, if present, hybridize to each other. And 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 the protospacer flanking motif (PAM) on the target DNA, and thereby to the target DNA. Site-specific targeting of the polypeptide of interest to the target DNA occurs within a region defined by the hybridization of the spacer sequence of the CRISPR nucleic acid molecule.

Further provided herein are expression cassettes, cells and kits that include the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules and/or nucleotide sequences of the invention.

These and other aspects of the invention are set forth in detail in the description of the invention below.

3 shows a multiple sequence alignment for the Nexus module. 3 shows a maximum likelihood tree for a Nexus module. 3A to 3D show consensus arrangements for Nexus modules. 3A shows the consensus sequence for the Sth Cr1 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. The maximum likelihood tree for Cas9 nuclease is shown. 6 shows a multiple sequence alignment for an anti-stitch module. 6A-6D show a consensus arrangement for an anti-stitch module. 6A shows the consensus sequence for the Sth Cr1 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. 3 shows a multiple sequence alignment for the bulge module. 8A-8D show the consensus arrangement for the bulge module. 8A shows a consensus sequence for the Sth Cr1 group, FIG. 8B shows a consensus sequence for the Sth Cr3 group, FIG. 8C shows a consensus sequence for the Lrh group, and FIG. 8D shows a consensus sequence for the Lbu group. 3 shows a multiple sequence alignment for zipper modules. 6 shows a maximum likelihood tree for a zipper module. 3 shows multiple sequence alignments for bulge, anti-stitch and nexus modules. The sequence and structure details of the CRISPR-Cas system factor of Streptococcus thermophilus CR3 are shown and represent the Sth CR1 group. Figure 3 shows the sequence and structure details of the CRISPR-Cas system factor of Lactobacillus buchneri and represents the Lbu group. The sequence and structure details of the CRISPR-Cas system factor of Streptococcus thermophilus CR1 are shown and represent the Sth CR1 group. The sequence and structure details of the CRISPR-Cas system factor of Streptococcus pyrogenes M1 GAS are shown and represent the Sth CR3 group. The sequence and structure details of the CRISPR-Cas system factor of Lactobacillus rhamnosus are shown and represent the Lrh group. Figure 3 shows the sequence and structural details of the CRISPR-Cas system factor of Lactobacillus animalis and represents the Lan group. Figure 2 shows the sequence and structure details of the CRISPR-Cas system factor of Lactobacillus casei and represents the Lca group. Figure 3 shows the sequence and structural details of the CRISPR-Cas system factor of Lactobacillus gasseri and represents the Lga group. Figure 3 shows the sequence and structural details of the CRISPR-Cas system factor of Lactobacillus jensenii and represents the Lje group. Figure 5 shows the sequence and structural details of the CRISPR-Cas system factor of Lactobacillus pentosus and represents the Lpe group. 1 shows the details of the sequence and structure of the CRISPR-Cas system factor of Streptococcus pyrogenes M1 GAS. Congrence between tracrRNA (left), CRISPR repeats (middle) and Cas9 (right) sequence clustering is shown. A consistent grouping into three families is found across the three sequence-based phylogenetic trees. 24A-24B show the Cas9:sgRNA family. Figure 24A shows a phylogenetic tree based on the Cas9 protein sequence from various Streptococcus and Lactobacillus species. The sequences were clustered in 3 families, blue, orange and green. FIG. 24B shows the consensus sequence and secondary structure of the predicted guide RNAs for each family. Each consensus RNA consists of crRNA (left) base-paired with tracrRNA. Fully conserved bases are colored, variable bases are black (2 potential bases) or black dots (at least 3 potential bases) and are always present The base positions that do not mean are circled. Circles between positions indicate base pairing that is present only in some family members. 3 shows a CRISPR repeat sequence alignment. For each cluster, a CRISPR repeat sequence alignment is shown, with conserved consensus nucleotides designated under each of the Sth3 (top), Sth1 (middle) and Lb (bottom) families. 3 shows a tracrRNA sequence alignment. For each cluster, an experimentally determined or computationally predicted tracrRNA sequence alignment is shown, along with conserved consensus nucleotides designated under each of the Sth3 (top), Sth1 (middle) and Lb (bottom) families. .. 3 shows an sgRNA nexus sequence alignment. Universally conserved residues are colored in red. The complementary nucleotides that make up the Nexus Stem, summarized in Figure 24B, are underlined. The nucleotides that make up the nexus loop are in the center of the gap. 28A-28B show a self-targeting assay scheme. Orthogonal Cas9 protein was provided via the pCas9 plasmid (Figure 28A) and used as described in Figure 3. Various sgRNA chimeras were provided via the psgRNA plasmid (Figure 28B) and used in combination with each of the desired Cas9 described in Figure 3. 29A-29C show sgRNA orthogonality. Figure 29A shows Streptococcus thermophilus CRISPR3-Cas9 (top, blue) and S. The sgRNA sequence of thermophilus CRISPR1-Cas9 (lower, orange) is shown. FIG. 29B shows a protospacer-targeting scheme. The expected PAM for each sgRNA is shown. Triangles indicate putative cleavage sites of each Cas9. FIG. 29C shows that E. Cas9 in E. coli: chimera-sgRNA orthogonality. Chimeric sgRNA. E. coli expressing Sth CRISPR3 Cas9 (blue) and/or Sth CRISPR1 Cas9 (orange). Each sgRNA (left) was subjected to a transformation assay (right) in E. coli. Low transformation efficiency is due to E. 2 shows a functional Cas9:sgRNA pair via lethal self-targeting of the E. coli genome. Values are the S.E. of three independent experiments. E. M. And reflect the geometric mean. Figure 7 shows CRISPR interference on complementary DNA as an inability to transform a plasmid containing a proto-spacer sequence in Lactobacillus gasseri that matches the first wild-type CRISPR spacer sequence. The bold sequence flanked by PAM (light gray italicized nucleotides) and its variants (single nucleotide polymorphisms (SNPs); black underlined nucleotides) is the protospacer. A low number of transformants represents an active Lga CRISPR system that prevents transformation of complementary target DNA. 3 shows CRISPR interference with complementary DNA in Lactobacillus casei as an inability to transform a plasmid containing a protospacer sequence that matches the original wild-type CRISPR spacer sequence. The bold sequence flanked by PAM (light gray italicized nucleotides) and its variants (SNPs, black underlined nucleotides) is the protospacer. A low number of transformants represents an active Lca CRISPR system that prevents transformation of complementary target DNA. Figure 7 shows CRISPR interference on complementary DNA as an inability to transform a plasmid containing a protospacer sequence in Lactobacillus rhamnosus that matches the original wild type CRISPR spacer sequence. The bold sequence flanked by PAM (light gray italicized nucleotides) and its variants (SNPs, black underlined nucleotides) is the protospacer. A low number of transformants represents an active Lra CRISPR system that prevents transformation of complementary target DNA.

The invention will now be described with reference to the accompanying drawings and examples in which embodiments of the invention are shown. This description is not intended to be a detailed listing of all different ways in which the invention may be practiced or all features that may be added to the invention. For example, features described with respect to one embodiment can be incorporated into another embodiment, and features described with respect to a particular embodiment can be eliminated from that embodiment. Accordingly, the invention anticipates that, in some embodiments of the invention, any feature or combination of features shown herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure and do not depart from the invention. Therefore, the following description is intended to describe some specific embodiments of the present invention, but not to exhaustively identify all permutations, combinations and modifications thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

All publications, patent applications, patent documents, and other references mentioned herein are incorporated by reference in their entirety for the teachings associated with the sentence and/or paragraph in which the reference is cited.

It is expressly contemplated that the various features of the invention described herein can be used in any combination, unless context dictates otherwise. Furthermore, the present invention also anticipates that, in some embodiments of the present invention, any feature or combination of features shown herein may be excluded or omitted. For purposes of explanation, if the specification states that the composition comprises components A, B, and C, either A, B, or C, or combinations thereof, may be omitted and disclaimed, alone or in any combination. It is expressly intended that this can be done.

As used in the description of the invention and in the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

Also, as used herein, "and/or" means any and all possible combinations of one or more of the associated listed items, and, when construed selectively ("or"), It refers to and includes the lack of combination.

As used herein, the term “about” when referring to a measurable value, such as dose or duration, is ±20%, ±10%, ±5%, ±1%, ±0.5% of the stated amount. , Or indeed ±0.1% variation.

As used herein, terms such as “between X and Y” and “between about X and Y” should be construed to include X and Y. is there. As used herein, phrases such as "between about X and about Y" mean "between about X and about Y" and "from about X to about X". A phrase such as "from about X to Y" means "from about X to about Y".

As used herein, "bulge sequence" refers to non-complementary (non-hybridizing) nucleotide sequences contained in synthetic tracr nucleic acid constructs and synthetic CRISPR nucleic acid constructs/ CRISPR nucleic acid arrays. In the synthetic tracr nucleic acid construct, the bulge sequence is located between the anti-zipper and anti-stitch sequences and is non-complementary (100% non-identity) with the corresponding bulge sequence in the synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array. , About 3 nucleotides to about 6 nucleotides (eg, about 3, 4, 5, 6 nucleotides; eg, about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, about 3 to Approximately 4 nucleotides). The bulge sequence of the synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array is located between the zipper and the stitch sequence and between the zipper and the stitch sequence and has at least two nucleotides (eg, a nucleotide sequence of (-NN-)) ( For example, about 2, 3, 4, 5, 6 nucleotides; for example, about 2 to about 6 nucleotides, about 2 to about 5 nucleotides, about 2 to about 4 nucleotides; about 3 to about 6 Nucleotides, such as about 3 to about 5 nucleotides), consists essentially of, or consists of. The nucleotide composition of the bulge sequences may be any series of at least 2 (synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array) or 3 as long as they are not complementary (eg, 100% non-identity) and thereby hybridize to each other. It may be one or more nucleotides (synthetic tracr nucleic acid construct). Synthetic tracr nucleic acid constructs and synthetic CRISPR nucleic acid constructs/When anti-zippers and zipper sequences and anti-stitch and stitch sequences align and hybridize as a result of non-complementarity of the bulge sequences on the array (in chimeric nucleic acid constructs ), an overhang or bulge is formed on the side of the synthetic tracr nucleic acid construct of the chimeric nucleic acid construct (see, eg, FIGS. 12-16). Without wishing to be bound by any particular theory, it is believed that the bulge structure may be involved in the function of the CRISPR-Cas system.

As used herein, the terms “comprise”, “comprises” and “comprising” refer to the presence of stated features, integers, steps, operations, factors, and/or components. Although specified, it does not preclude the presence or addition of one or more other features, integers, steps, operations, factors, components, and/or groups thereof.

As used in this specification, the provisional term "consisting essentially of" means that the scope of the claim is either a particular substance or step recited in the claim and the basic and novel invention(s) recited in the claim. Is meant to include those that do not materially affect the characteristic(s) of. Thus, the term "consisting essentially of" as used in the claims of the present invention is not intended to be interpreted as being equivalent to "comprising."

"Cas9 nuclease" refers to a large group of endonucleases that catalyze double-stranded DNA breaks in the CRISPR Cas system. These polypeptides are well known in the art and many of their structures (sequences) have been characterized (see, for example, WO 2013/176772; WO/2013/188638). The domains for catalyzing the cleavage of dsDNA are the RuvC domain and the HNH domain. The RuvC domain has a role to put a nick in the (−) chain, and the HNH domain has a role to put a nick in the (+) chain (for example, Gasiunasetal.PNAS 109(36):E2579-E2586 (September 4, 2012). reference).

As used herein, "chimera" refers to a nucleic acid molecule or polypeptide in which at least two components have been obtained from different sources (eg, different organisms, different coding regions).

As used herein, “complementarity” can mean 100% complementarity or identity with a comparator nucleotide sequence, or less than 100% complementarity (eg, 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%, etc.).

The terms "complementarity" or "complementarity" as used herein refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence "AGT" binds to the complementary sequence "TCA". Complementarity between two single-stranded molecules can be "partial," in which only some of the nucleotides are joined, or can be complete if there is total complementarity between the single-stranded molecules. .. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, "contacting," "contacting," "contacted," and their grammatical changes refer to the desired reaction (eg, site-specific to the polypeptide of interest). Refers to co-locating the components of the desired reaction under suitable conditions to effect targeting, integration, transformation, site-specific cleavage (nick, cleavage), amplification, etc.). Methods and conditions for carrying out such reactions are well known in the art (eg, Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; MR. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

A "fragment" or "portion" of a nucleotide sequence of the invention has a reduced length with respect to a reference nucleic acid or nucleotide sequence (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides (shortened by nucleotides) and is identical to the reference nucleic acid or nucleotide sequence. Or almost the same (eg, 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) It comprises, consists essentially of, and/or consists of a nucleotide sequence of contiguous nucleotides. Such nucleic acid fragments or parts according to the invention may, where appropriate, be comprised in larger polynucleotides of which they are a constituent. Thus, for example, hybridizing to (or hybridizing to, and other grammatical changes thereof) to at least a portion of the target DNA is identical to the length of contiguous nucleotides of the target DNA. Alternatively, it refers to hybridization to substantially identical nucleotide sequences.

_{"G R1"} as used herein includes the repeat portion of the synthetic CRISPER nucleic acid construct or CrRNA, a single nucleotide (G) or short 3 nucleotide sequence (GTT). G _R1 is anti synthetic tracr nucleic acid construct or tracrRNA of the present disclosure - not repeat hybridize. However, the non-standard Watson-Crick base pairing _{scheme, G R1} may form a U and wobble base pairs at the end of the anti--CRISPR repeat portion of TracrRNA.

As used herein, the term “gene” refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMO) and the like. A gene may or may not be used to produce a functional protein or gene product. A gene may include both coding and non-coding regions (eg, introns, regulators, promoters, enhancers, termination sequences and/or 5'and 3'untranslated regions). A gene can be "isolated", by which is meant a nucleic acid that is substantially or essentially free of its naturally occurring components that normally associate with it. Such components include other cellular materials, recombinantly produced culture media, and/or various chemicals used in the chemical synthesis of nucleic acids.

As used herein, a "hairpin sequence" is a nucleotide sequence that contains a hairpin. A hairpin (eg, stem-loop, feed-back) refers to a nucleic acid molecule having a secondary structure that includes a region of nucleotides that form a duplex that is flanked on either side by a single-stranded region. Such structures are well known in the art. As is known in the art, double-stranded regions may contain some mismatches within base pairing or may be perfectly complementary. In some embodiments of the disclosure, the hairpin sequence of the nucleic acid construct is located immediately downstream of the "nexus sequence" at the 3'end of the synthetic tracr nucleic acid construct. Without being bound to any particular theory, it is believed that hairpins may be involved in Cas9 binding to the crRNA-tracrRNA complex (eg, synthetic CRISPR nucleic acid construct-synthetic).

A "heterologous" or "recombinant" nucleotide sequence is a nucleotide sequence that is not naturally associated with the host cell into which it is introduced and includes non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.

Different nucleic acids or proteins with homology are referred to herein as "homologues." The term homologue 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 positional identity (ie, sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the compositions and methods of the invention further include homologues to the nucleotide and polypeptide sequences of the invention. As used herein, “orthologous” refers to homologous nucleotide and/or amino acid sequences in different species that arise from a common ancestral gene during speciation. A homologue of a nucleotide sequence of the invention has substantial sequence identity with the nucleotide sequence of the invention (eg, 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%). Thus, for example, a homologue of a Cas9 polypeptide useful in the invention may be about 70% homologous to or higher than any one of the Cas9 sequences provided herein.

As used herein, hybridization, hybridize, hybridize, and their grammatical alterations are two perfectly complementary nucleotide sequences or some mismatched bases. Refers to the binding of substantially complementary sequences in which a pair may be present. Hybridization conditions are well known in the art and will 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 may be high stringency, or they may be of moderate or low stringency, depending on the length of the hybridized sequences and the amount of complementarity. May be Conditions that constitute low, medium and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (eg, Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109). : E2579-E2586; MR Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual 4th Ed., Cold Spring Harbor Laboratory, N. Harbour).

As used herein, the terms “increase”, “increasing”, “increased”, “enhanced”, “enhanced”, “enhancing”. "And "enhancement" (and their grammatical changes) are at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500 compared to controls. Represents a% or more increase.

The terms “invasive exogenous genetic factor”, “invasive exogenous nucleic acid” or “invasive exogenous DNA” refer to DNA that is exogenous to a bacterium (eg, but is not limited to viruses, bacteriophages, and/or plasmids. Genetic factors derived from pathogens including).

“Natural” 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. Thus, for example, a "wild type mRNA" is an mRNA that is naturally occurring or endogenous in the organism. A "homologous" nucleic acid sequence is a nucleotide sequence naturally associated with the host cell into which it is introduced.

As used herein, "nexus sequence" refers to a nucleotide sequence located immediately downstream of an "anti-stitch sequence" within a synthetic tracr nucleic acid construct. The nexus is about 6-10 nucleotides in length and contains the highly conserved sequence: TNANNC. In some embodiments, the nexus is T(A/C)A(A/G)(G/A)C (or U(A/C)A(A/G)(G/A)C)). , GATAAGGCTT (or GAUAAGGCUU), TCAAGCAA (or UCAAGCAA), or 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)) nucleotide sequence. Without being bound to any particular theory, based on sequence conservation, it is believed that the nexus may be important in Cas9 orthogonality and recognition.

Also, as used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "nucleotide sequence," and "polynucleotide" refer to linear or branched single-stranded or double-stranded RNA or Refers to DNA or hybrids thereof. The term also includes RNA/DNA hybrids. When dsRNA is produced synthetically, unusual bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others may also be used for antisense, dsRNA, and ribozyme pairing. it can. For example, polynucleotides containing C-5 propyne analogs of cytidine and uridine have been shown to bind RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications can also be made, for example to the phosphodiester backbone or 2'-hydroxy within the ribose sugar group of RNA. The nucleic acid construct of the present disclosure may be DNA or RNA, but is preferably DNA. Therefore, the nucleic acid construct of the present invention can be described and used in the form of DNA, but may be described and used in the form of RNA depending on the purpose of use.

A "synthetic" nucleic acid or nucleotide sequence, as used herein, refers to a nucleic acid or nucleotide sequence that is not found in nature, but that is constructed by the hand of man (and is consequently not a natural product).

The term "nucleotide sequence" as used herein refers to a sequence of these nucleotides from the heteropolymer of nucleotides or the 5'to 3'ends of a nucleic acid molecule, including DNA or RNA molecules, cDNA, DNA fragments or portions, It includes genomic DNA, synthetic (eg, chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, either of which may be single-stranded or double-stranded. The terms "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably herein and also refer to a heteropolymer of nucleotides. Unless otherwise indicated, nucleic acid molecules and/or nucleotide sequences provided herein are set forth herein left to right in the 5'to 3'direction and refer to the US sequence rules. (The Code of Federal Regulations, Volume 37, Sections 1.821 to 1.825) and the World Intellectual Property Organization (WIPO) Standard ST. It is represented using a standard code for characterizing the nucleotides shown in 25. As used herein, "5' region" may mean the region of the polynucleotide closest to the 5'end. Thus, for example, the factor within the 5'region of the polynucleotide may be located anywhere from the first nucleotide located at the 5'end of the polynucleotide to the nucleotide located in the middle of the polynucleotide. As used herein, a "3' region" can mean the region of a polynucleotide that is closest to the 3'end. Thus, for example, the factor within the 3'region of the polynucleotide may be located anywhere from the first nucleotide located at the 3'end of the polynucleotide to the nucleotide located in the middle of the polynucleotide.

As used herein, the term “percent sequence identity” or “percent identity” is compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. Refers to the percentage of identical nucleotides within the linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complement). In some embodiments, "percent identity" can refer to the percentage of identical amino acids within an amino acid sequence.

“Protospacer sequence” refers to a target double-stranded DNA, specifically a portion of target DNA that is completely or substantially complementary (and hybridizes) to the spacer sequence of a synthetic CRISPR nucleic acid construct.

As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," "suppress," and "reduce." "Decrease" (and their grammatical changes) is, for example, at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, compared to a control. It shows a reduction of 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In certain embodiments, the reduction may or may not essentially result in a detectable activity or amount (ie, a trivial amount, eg, less than about 10% or even less than 5%). Thus, in some embodiments, the Cas9 nuclease mutation causes the nuclease activity of Cas9 to be at least about 5%, 10%, 15%, 20%, 25% compared to a control (eg, wild-type Cas9). It may be reduced by 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

As used herein, “repeat sequence” refers to the repeat sequence of a wild-type CRISPR locus or synthetic CRISPR nucleic acid construct separated by, for example, a “spacer sequence”. A repeat sequence is complementary (eg, 100% base pair match) or substantially complementary to the corresponding anti-repeat sequence, eg, at least 70% complementary (eg, 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 higher).

The "repeat sequence" of the synthetic CRISPR nucleic acid constructs of the present disclosure includes the optional "zipper sequences", "bulge sequences", "stitch sequences", and "spacer sequences". In some embodiments, the synthetic CRISPR nucleic acid construct may comprise a G _{R1 (in} other embodiments included in the stitch sequence).

As used herein, "zipper sequence" refers to an optional portion of the repeat sequence located 3'or immediately upstream (in the 3'to 5'direction) of the bulge sequence within the synthetic CRISPR nucleic acid construct, and at least About 3 nucleotides (eg 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), consisting of, or consisting essentially of. In some embodiments, the zipper sequence may be referred to as the "upper stem." A "zipper sequence" shares sufficient complementarity with the corresponding and optionally "anti-zipper sequence" located on the synthetic tracr nucleic acid construct, and thus, if present, the zipper sequence and the anti-zipper sequence. When they come into contact, they can hybridize to each other, thereby joining the two nucleic acid constructs together. In some embodiments, the zipper/anti-zipper sequence can be referred to as the "upper stem". A zipper sequence is completely complementary (eg, 100% base pair match) or substantially complementary to the corresponding anti-zipper sequence, eg, at least 70% complementary (eg, 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 higher). Accordingly, the anti-zipper sequences of the synthetic tracr nucleic acid constructs of the present invention are at least about 3 nucleotides (complementary or substantially complementary to a corresponding zipper sequence in a synthetic CRISPR nucleic acid construct or synthetic CRISPR nucleic acid array ( For example, 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), consisting, or consisting essentially of. The anti-zipper sequence is the RNase III binding site and thus includes nucleotide sequences well known in the art to be involved in RNase III binding (eg, Pertzev and Nicholson, Nucleic Acids Res. 34(13):3708. -3721 (2006)).

As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the window of alignment of the constituents (eg, nucleotides or amino acids). "Identity" includes, but is not limited to: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and J. Gen. ) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press Jr. Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., 19th, Stockton Yw. It can be easily calculated by the method.

As used herein, a "spacer sequence" is a nucleotide sequence complementary to a target DNA (eg, "protospacer sequence"). The spacer sequence may be fully or substantially complementary to the target DNA (eg, at least 70% complementary (eg, 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 higher)). In an exemplary embodiment, the spacer sequence has 100% complementarity with the target DNA. In a further embodiment, the complementarity of the 3'region of the spacer sequence to the target DNA is 100%, but less than 100% in the 5'region of the spacer, thus the overall complementarity of the spacer sequence to the target DNA is It is less than 100%. Thus, for example, the first 7,8,9,10,11,12,13,14,15,16, etc. nucleotides (seed sequence) in the 3'region of a 20 nucleotide spacer sequence are It may be 100% complementary, while the remaining nucleotides in the 5'region of the spacer sequence are substantially complementary (eg, at least about 70% complementary) to the target DNA. In some embodiments, the first 7-12 nucleotides of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5'region of the spacer sequence are substantially complementary to the target DNA. Are complementary (eg, at least about 70% complementary). In another embodiment, the first 7-10 nucleotides of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5'region of the spacer sequence are substantially complementary to the target DNA. (Eg, at least about 70% complementary). In an exemplary embodiment, 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 to the target DNA ( For example, at least about 70% complementary).

As used herein, a "stitch sequence" refers to a nucleotide sequence comprising, consisting essentially of, or consisting of a nucleotide of about 5 lengths and having the consensus nucleotide sequence of NNTNN. "Stitch sequence" is a synthetic CRISPR nucleic acid construct Butsujo, located downstream of (in the 5 'to 3' direction) immediately upstream of the "bulge sequence" and "G _R1". The "stitch sequence" tends to have a high AT content and hybridizes to the "anti-stitch sequence" located within the synthetic tracr nucleic acid construct. In some particular embodiments, the stitch arrangement is NNTNN, TTTGT, TTTTA, (T/C)(T/C)T(T/C)(T/G) (in the 5'to 3'direction). , TTTTA, comprising the nucleotide sequence of TTTCA, consisting essentially of or consisting of.

As used herein, an "anti-stitch sequence" is a nucleotide sequence that perfectly complements and hybridizes with a stitch sequence (eg, NNANN, ACAAA, TAAAA, (T/C)(A/G)T(A/G ) (A/G), TAAAA, TGAAA). The anti-stitch sequence is located on the synthetic tracr nucleic acid construct immediately downstream (in the 5'to 3'direction) of the bulge sequence and immediately upstream of the "nexus sequence". Without wishing to be bound by any particular theory, hybridization of the stitch sequence of the synthetic crRNA construct with the anti-stitch sequence of the synthetic tracrRNA construct is involved in the reconstructed base pairing after the "bulge sequence". It is thought that. In some implementations, the stitch/anti-stitch can be referred to as a "lower stem."

As used herein, the phrase "substantially identical" or "substantial identity" in the context of two nucleic acid molecule, nucleotide or protein sequences, when compared and aligned for maximal match, refers to the following sequence comparison: At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% by measurement or visual inspection using one of the algorithms , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Refers to two or more sequences or subsequences having %, 99%, and/or 100% nucleotide or amino acid residue identity. In some embodiments of the invention, substantial identity exists over regions of the sequence that are at least about 50 residues and about 150 residues in length. Thus, in some embodiments of the present invention, substantial identity is at least about 3 to about 15 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 14, 15 residues in length, or any value or 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 110, about 120, about 130, It is present over a region of sequence of about 140, about 150, or more residues in length, and any range therein. In an exemplary embodiment, 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, the sequences of the present invention can be about 70% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In some embodiments, the sequences of the present invention can be about 75% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In a further embodiment, the sequences of the present invention can be about 80% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In a further embodiment, the sequences of the present invention can be about 80% to about 100% identical over at least about 7 nucleotides to about 25 nucleotides. In some embodiments, the sequences of the present invention can be about 70% identical over at least about 18 nucleotides. In another embodiment, the sequences can be about 85% identical over about 22 nucleotides. In yet another embodiment, the sequences may be 100% homologous over about 16 nucleotides. In a further embodiment, the sequences are substantially identical over the length of the coding region. Furthermore, in an exemplary embodiment, substantially identical nucleotide or protein sequences perform substantially identical function (eg, Cas9 HNH and/or RuvC nickase activity).

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and comparison sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal sequence alignments for aligning comparison windows are well known to those of skill in the art, with tools such as Smith and Waterman local homology algorithms, Needleman and Wunsch homology alignment algorithms, Pearson and Lipman similarity search methods, and Optionally, it may be done by computer implementation of algorithms such as GAP, BESTFIT, FASTA, and TFASTA, which are available as part of GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). .. The “fraction of identity” of an aligned segment of a test sequence and a reference sequence is defined as the number of identical components shared by two aligned sequences by the reference sequence segment (ie, the entire reference sequence or a lesser of the reference sequence). It is the number divided by the total number of components in the specified part. Percent sequence identity is expressed as the fraction of identity times 100. Comparisons of one or more polynucleotide sequences can be made to the full length polynucleotide sequence or a portion thereof, or to longer polynucleotide sequences. For purposes of the present invention, "percent identity" may be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. This algorithm identifies short words of length W in the query array that match or meet some positive threshold score T when aligned with words of the same length in the database array. According to the first step of identifying a high score sequence pair (HSP). T is called the adjacent word score threshold (Altschul et al., 1990). These first adjacent word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits then extend in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (beneficial score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score drops by an amount X below its maximum achieved value, accumulation of one or more negative score residue alignments, or the end of either sequence. The cumulative score becomes 0 or less due to the arrival at. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (nucleotide sequences) uses word length (W) 11, expected value (E) 10, cutoff 100, M=5, N=-4, and a comparison of both strands by default. For amino acid sequences, the BLASTP program uses word length (W) 3, expectation (E) 10, and BLOSUM62 score matrix as defaults (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989). See).

In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of similarities between two sequences (see, eg, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873 5787 (1993)). .. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a test nucleic acid sequence is considered to be similar to a reference sequence if the minimum sum probability in comparing the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the minimum sum probability in comparing a test nucleotide sequence to a reference nucleotide sequence is less than about 0.001.

Also, two nucleotide sequences can be considered to be substantially complementary if the two sequences hybridize to each other under stringent conditions. In some exemplary embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under conditions of high stringency.

In the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence dependent and are different under different environmental parameters. Extensive guide to the hybridization of nucleic acids, 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) Seen in. Generally, high stringency hybridization and wash conditions are selected to be about 5° C. below the melting point (T _m ) for a specific sequence at a defined ionic strength and pH.

The T _m is the temperature (at 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 _Tm for the specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues in filters on Southern or Northern blots is 50% formamide (42°C) containing 1 mg heparin, Hybridization is performed overnight. An example of highly stringent wash conditions is 0.15M NaCl, 72°C, about 15 minutes. An example of stringent wash conditions is 0.2 x SSC wash, 65°C, 15 minutes (see Sambrook (below) for a description of SSC buffer). High stringency washes are often preceded by low stringency washes to remove background probe signal. An example of a moderate stringency wash, for example for a duplex of more than 100 nucleotides, is 1×SSC, 45° C., 15 minutes. An example of a low stringency wash, eg, for a duplex of more than 100 nucleotides is 4-6×SSC, 40° C., 15 minutes. For short probes (eg, about 10-50 nucleotides), stringent conditions are typically at a salt concentration of Na ion less than about 1.0 M, typically about 0.01-1.0 M. Including Na ion concentration (or other salt) (pH 7.0-8.3), the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal-to-noise ratio that is twice (or higher) than that seen for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is made with the maximum codon degeneracy permitted by the genetic code.

The following is an example of a set of hybridization/wash conditions that can be used to clone a homologous nucleotide sequence that is substantially identical to a reference nucleotide sequence of the invention. In one embodiment, the reference nucleotide sequence is 2×SSC in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA (50° C.), washed with 0.1% SDS (50° C.), Hybridizes with the "test" nucleotide sequence. In another embodiment, the reference nucleotide sequence is washed with 1×SSC, 0.1% SDS (50° C.) in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50° C.). , Or a "test" nucleotide sequence in 0.5% SSC, 0.1% SDS wash (50°C) in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA (50°C). Hybridize with. In yet a further embodiment, the reference nucleotide sequence is 0.1% SSC, 0.1% SDS in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50° C.) (50 C.) or with 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50° C.), 0.1×SSC, 0.1% SDS wash (65° C.), “test”. Hybridizes with the nucleotide sequence.

Any nucleotide sequence and/or recombinant nucleic acid molecule of the present invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of the nucleotide sequence for codon usage bias with species-specific codon usage. Codon usage is made based on sequence analysis of the most highly expressed genes for the species of interest. If the nucleotide sequence is expressed in the nucleus, codon usage is made based on sequence analysis of highly expressed nuclear genes for the species of interest. Nucleotide sequence modifications are determined by comparing species-specific codon usage to codons present in the native polynucleotide sequence. As will be appreciated in the art, codon optimization of a nucleotide sequence is less than 100% identical to the native nucleotide sequence (eg, 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%, etc.) but still encode a polypeptide having the same function as encoded by the original native nucleotide sequence. Gives a nucleotide sequence. Thus, in an exemplary embodiment of the invention, the nucleotide sequences of the invention and/or recombinant nucleic acid molecules may be codon optimized for expression in a particular species of interest.

In some embodiments, recombinant nucleic acid molecules, nucleotide sequences and polypeptides of the invention are "isolated." An "isolated" nucleic acid molecule, "isolated" nucleotide sequence or "isolated" polypeptide is a nucleic acid molecule, a nucleotide sequence, that exists by its own hand apart from its natural environment and is therefore not a natural product. Or it is a polypeptide. An isolated nucleic acid molecule, nucleotide sequence or polypeptide is related to at least a portion of other components of a naturally occurring organism or virus, such as a structural component of a cell or virus or other polypeptide or polynucleotide. It may exist in a purified form, at least partially cleaved from the nucleic acids commonly found in. In an exemplary embodiment, the isolated nucleic acid molecule, isolated nucleotide sequence and/or isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%. , 60%, 70%, 80%, 90%, 95%, or higher purity.

In other embodiments, the isolated nucleic acid molecule, nucleotide sequence or polypeptide may be in a non-natural environment (eg, a recombinant host cell). Thus, for example, with respect to nucleotide sequences, the term "isolated" means that it is separated from the naturally occurring chromosome and/or cell. Also, a polynucleotide is separated from the chromosome and/or cell in which it naturally occurs, and then from the genetic context, chromosomes and/or cells in which it does not naturally occur (eg, different host cells, different regulatory sequences, and/or Or a position in the genome that differs from that found in nature). Thus, recombinant nucleic acid molecules, nucleotide sequences, and their encoded polypeptides, are "separated" in that they are separated by the hand of man from the natural environment and thus are not naturally occurring products. In some embodiments, it can be introduced and present in a recombinant host cell.

In any of the embodiments described herein, the nucleotide sequences of the invention and/or recombinant nucleic acid molecules are operably associated with various promoters and other regulatory elements for expression in various biological cells. You can Thus, in an exemplary embodiment, the recombinant nucleic acids of the invention can further include one or more promoters operably linked to one or more nucleotide sequences.

By "operably linked" or "operably linked" as used herein is meant that the indicated factors are functionally related to each other, and generally also physically related. Thus, the term “operably linked” or “operably related” as used herein refers to nucleotide sequences on a single nucleic acid molecule that are functionally related. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to the situation where the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of the nucleotide sequence. Those skilled in the art will appreciate that the control sequence (eg, promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, so long as the control sequence functions to direct its expression. Thus, for example, an intervening, non-translated, yet transcribed sequence may be present between the promoter and the nucleotide sequence, and the promoter may still be considered "operably linked" to the nucleotide sequence.

A "promoter" is a nucleotide sequence (ie, a coding sequence) that controls or controls the transcription of a nucleotide sequence operably associated with the promoter. The coding sequence may encode a polypeptide and/or a functional RNA. A "promoter" typically refers to the nucleotide sequence containing the binding site for RNA polymerase II and controls the initiation of transcription. Generally, the promoter is found upstream of the 5'or the start of the coding region for the corresponding coding sequence. The promoter region may include other factors that act as regulators of gene expression. These contain the TATA box consensus sequence and often the CAAT box consensus sequence (Breathnach and Chamber, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box can be replaced with an AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, 211-press. 227).

A promoter is, for example, constitutive, inducible, temporally controlled, developmentally controlled, for use in the preparation of a recombinant nucleic acid molecule (ie, “chimeric gene” or “chimeric polynucleotide”). It may comprise a chemically controlled, tissue-suitable and/or tissue-specific promoter. These various types of promoters are known in the art.

The choice of promoter will depend on the temporal and spatial requirements for expression, and also on the transformed host cell. Promoters for many different organisms are well known in the art. Appropriate promoters can be selected for a particular host organism of interest, based on the widespread knowledge existing in the art. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms, and such knowledge may be readily available and implemented in other systems, as needed. it can.

In some embodiments, the nucleic acid construct of the invention may be or be contained within an "expression cassette." As used herein, an "expression cassette" encodes a nucleotide sequence of interest (eg, a nucleic acid construct of the invention (eg, a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; a polypeptide of interest). A recombinant nucleic acid molecule comprising a nucleotide sequence, a nucleotide sequence encoding a cas9 nuclease)), wherein said nucleotide sequence is operatively associated with at least a control sequence (eg a promoter). Thus, some aspects of the invention provide expression cassettes designed to express the nucleotide sequences of the invention.

The expression cassette containing the nucleotide sequence of interest may be chimeric, meaning that at least one of its constituents is heterologous to at least one of its other constituents. The expression cassette may also be of recombinant origin, which is of natural origin but which is useful for heterologous expression.

The expression cassette may also optionally include a transcriptional and/or translational termination region (ie, a termination region) that is functional in the host cell of choice. A variety of transcription terminators are available for use in expression cassettes, involved in termination of transcription beyond the heterologous nucleotide sequence of interest, and correcting mRNA polyadenylation. The termination region can be native to the transcription initiation region, native to the operably linked nucleotide sequence of interest, native to the host cell, or another. It may be derived from origin (ie, for the promoter, for the nucleotide sequence of interest, exogenous or heterologous to the host, or any combination thereof).

The expression cassette can also include a nucleotide sequence for a selectable marker that can be used to select for transformed host cells. As used herein, a "selectable marker", when expressed, confers a distinct phenotype on the host cell expressing the marker, and, therefore, those that do not have that marker from such traits. By a nucleotide sequence that allows transformed cells to be distinguished. Whether such a nucleotide sequence confers a trait that allows the marker to be selected by chemical means, such as by using a selection agent (eg, an antibiotic, etc.), or whether the marker simply provides, for example, screening (eg, Fluorescence) can encode either a selectable or screenable marker, depending on whether it is a trait that can be identified through observation or testing. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

In addition to expression cassettes, the nucleic acid molecules and nucleotide sequences described herein can be used in the context of vectors. The term "vector" refers to a composition for transferring, delivering or introducing nucleic acid(s) (or nucleic acid(s)) into cells. Vectors include nucleic acid molecules that include the nucleotide sequence(s) to be transferred, delivered or introduced. Vectors for use in transforming host organisms are well known in the art. Non-limiting examples of general classes of vectors include, but are not limited to, double-stranded or single-stranded linear or circular forms of viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmids. It may or may not be capable of self-infection or mobilization, including vectors, bacteriophage, artificial chromosomes, or Agrobacterium binary vectors. Vectors as defined herein can transform prokaryotic or eukaryotic hosts by integration into the cell genome or can be extrachromosomal (eg, self-replicating plasmids with origins of replication). ). A DNA vehicle capable of replication in two different host organisms, naturally or by design, which may be selected from actinomycetes and related species, bacteria and eukaryotes (eg, higher plant, mammalian, yeast or fungal cells). Further included is a shuttle vector, which means In some exemplary embodiments, the nucleic acid in the vector is under the control of a suitable promoter or other regulatory element for transcription in the host cell and is operably linked. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may include its own promoter or other regulatory element, and in the case of cDNA, it may be under the control of a suitable promoter or other regulatory element for expression in the host cell. Can be Thus, the nucleic acid molecules and/or expression cassettes of the present invention can be contained within a vector as described herein and known in the art.

"Introducing," "introduce," "introduced" (and grammatical changes thereof) in the context of a polynucleotide of interest refers to the nucleotide sequence of interest as a host organism or It is meant to be presented to the cells of the organism (eg, host cells) in such a way that the nucleotide sequence is accessible to the interior of the cell. If more than one nucleotide sequence is introduced, these nucleotide sequences can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotides or nucleic acid constructs, identical or It can be located on different expression constructs or transformation vectors. Thus, these polynucleotides may be introduced into the cell in a single cell transformation event, in a separate transformation/transfection event, or, for example, they may be taken up by an organism by conventional breeding protocols. .. Thus, in some aspects of the invention, one or more nucleic acid constructs of the invention (eg, synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, synthetic CRISPR arrays, chimeric nucleic acid constructs; nucleotide sequences encoding a polypeptide of interest). , Cas9 nuclease-encoding nucleotide sequences) can be introduced into a host organism or cells of said host organism.

The term "transformation" or "transfection" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of cells can be stable or transient. Thus, in some embodiments, host cells or host organisms are stably transformed with the nucleic acid molecules of the invention. In another embodiment, the host cell or host organism is transiently transformed with the recombinant nucleic acid molecule of the invention.

"Transiently transformed" in the context of polynucleotides means that the polynucleotide is introduced into a cell and does not integrate into the cell's genome.

“Stablely introducing” or “stablely introduced” in the context of a polynucleotide introduced into a cell means that the introduced polynucleotide is stable within the genome of the cell. It is intended to be taken up and thus the cells stably transformed with the polynucleotide.

As used herein, "stable transformation" or "stably transformed" means that the nucleic acid molecule has been introduced intracellularly and integrated within the genome of the cell. Thus, an integrated nucleic acid molecule can be inherited by its progeny, and more specifically, by the progeny of multiple subsequent generations. As used herein, "genome" also includes the nuclear and plastid genomes, and thus includes, for example, the integration of nucleic acids into the chloroplast or mitochondrial genome. Stable transformation as used herein may also refer to a transgene that is maintained extrachromosomally, eg as a minichromosome or a plasmid.

Transient transformation is an enzyme-linked immunosorbent assay (ELISA) or Western blot capable of detecting the presence of a peptide or polypeptide encoded by one or more transgenes introduced into an organism. And the like. Stable transformation of cells involves the use of nucleic acid sequences that specifically hybridize to the nucleotide sequences of the transgene introduced into an organism (eg, plant, mammal, insect, archaea, bacterium, etc.) It can be detected by the Southern blot hybridization assay, etc. Stable transformation of cells should be detected, for example, by Northern blot hybridization assay of RNA of cells using a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of a transgene introduced into a plant or other organism. You can Stable transformation of cells is also well known in the art, for example, using the polymerase chain reaction (PCR) with specific primer sequences that hybridize to the target sequence(s) of the transgene. It can also be detected by other amplification reactions that result in amplification of the transgene sequence and can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.

Thus, in some embodiments, the nucleotide sequences, constructs, expression cassettes may be transiently expressed and/or they may be stably integrated into the genome of the host organism.

The recombinant nucleic acid molecule/polynucleotide of the present invention can be introduced into cells by any method known to those skilled in the art. In some embodiments of the invention, transforming the cells comprises nuclear transformation. In other embodiments, transforming the cells comprises plastid transformation (eg, chloroplast transformation). In yet a further embodiment, the recombinant nucleic acid molecule/polynucleotide of the invention can be introduced into cells via conventional breeding techniques.

In both eukaryotes and prokaryotes, transformation procedures are well known and routine in the art and are described throughout the literature (eg, Jiang et al. 2013. Nat. Biotechnol. 31:233-. 239; Rane et al. Nature Protocols 8:2281-2308 (2013)).

Thus, the nucleotide sequence can be introduced into the host organism or its cells in any number of ways well known in the art. The method of the invention does not depend on a particular method in order for one or more nucleotide sequences to be introduced into an organism, as long as they approach the interior of at least one cell of the organism. If more than one nucleotide sequence is introduced, they can assemble 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. it can. Thus, the nucleotide sequence can be introduced into the cell of interest in a single transformation event or in a separate transformation event, or, where relevant, the nucleotide sequence can be part of a breeding protocol. Can be incorporated into plants.

The present invention provides compositions having increased efficiency and increased specificity for site-specific nicking, cleavage and/or modification of target DNA, and for site-specific targeting of a polypeptide of interest to the target DNA. And on the method.

The nucleic acid constructs and nucleotide sequences of the invention can be represented in alternative ways without affecting the overall structure or function of the construct or sequence. Thus, for example, in some cases, synthetic CRISPR nucleic (crRNA) may include a wobble base _{G R1} sequence (G / U) and stitch sequences, or, CrRNA comprises (G / U) wobble base Stitch arrays can be included and thus do not further represent the _GR1 array. In some cases GR1 is unpaired (ie G/A is mismatched with Lje, FIG. 20). Further equivalences include those shown in the equivalence table (Table 1) provided below (see also Figure 22).

Accordingly, in one aspect of the invention there is provided a synthetic transcoding CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct, said construct comprising at least about 3 nucleotides in the 5'to 3'direction. An anti-zipper sequence comprising; consisting essentially of or consisting of: a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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, TAAGGCCATTCC, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nexus sequence; and at least one hairpin Comprising, consisting essentially of, or consisting of a hairpin sequence comprising a nucleotide sequence, said hairpin comprising at least 3 matching base pairs, wherein the anti-zipper sequence is immediately upstream of the bulge sequence. Located, 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. In some embodiments, the anti-stitch sequence comprises the nucleotide sequence of NNANN, ACAAA, TAAAA, (T/C)(A/G)T(A/G)(A/G), TAAAA, TGAAA. Consists essentially of, or consists of.

In a further embodiment, in the 5'to 3'direction an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch comprising a nucleotide sequence of NNANN. Sequence; 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 TCAAACAAAAGCTTCAGC nucleotides A synthetic transcoding CRISPR comprising, consisting essentially of or consisting of a nexus sequence comprising, consisting essentially of or consisting of a sequence, and a hairpin sequence comprising a nucleotide sequence having at least one hairpin. (Tracr) nucleic acid constructs are provided, wherein the hairpin comprises at least 3 matching base pairs, wherein the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is , Immediately upstream of the anti-stitch sequence, the anti-stitch sequence is immediately upstream of the nexus sequence, and the nexus sequence is immediately upstream of the hairpin sequence.

In some embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of at least about 3 nucleotides. In some embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of at least about 4 nucleotides. In another embodiment, the bulge sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of 5 nucleotides. In another embodiment, the hairpin sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of at least 2 hairpins, each hairpin comprising at least 3 matching base pairs.

In a further aspect, the invention provides an optional zipper sequence comprising, consisting essentially of or consisting of at least about 3 nucleotides in the 3'to 5'direction, a nucleotide sequence having at least 2 nucleotides. A bulge sequence comprising, consisting essentially of, or consisting of (e.g., a nucleotide sequence of (-NN-)), comprising a nucleotide sequence of NNTNN (or NNUNNN), consisting essentially of, or consisting of, comprising a nucleotide G or GTT, consisting essentially of, or consisting of G _R1, and 5 a spacer sequence having 'and 3' ends, the target DNA and 100% identity to the 3 'end A synthetic CRISPR nucleic acid (eg, crRNA, crDNA) construct comprising, consisting essentially of, or consisting of a spacer sequence comprising, consisting essentially of or consisting of at least 7 nucleotides having a zipper sequence. , if present, situated immediately upstream of the bulge sequence, bulge sequence located immediately upstream of the stitch sequence, stitching sequence is located immediately upstream of the G _R1, and, G _R1 is a spacer sequence Located just upstream of.

In a further embodiment, an optional zipper sequence comprising in the 3'to 5'direction a nucleotide sequence having at least 3 nucleotides that hybridize to the anti-zipper, a bulge comprising a nucleotide sequence of at least about 2 nucleotides. A sequence, a stitch sequence comprising the nucleotide sequence of NNUNN (and a spacer sequence having 5'and 3'ends, comprising at least 7 nucleotides at its 3'end with 100% identity to the target DNA) Synthetic CRISPR nucleic acid (eg, crRNA, crDNA) constructs comprising, consisting essentially of, or consisting of a spacer sequence are provided, wherein the zipper sequence, if present, is located immediately upstream of the bulge sequence, Is located immediately upstream of the stitch array and the stitch array is located immediately upstream of the spacer array.

In some embodiments, a synthetic CRISPR nucleic acid array is provided, wherein the synthetic CRISPR nucleic acid array comprises nucleotide sequences encoding two or more CRISPR nucleic acid constructs of the invention, wherein the two or more CRISPR nucleic acid constructs are: Located immediately adjacent to each other on the nucleotide sequence, the stitch sequences of the two or more CRISPR nucleic acid constructs are the same, the spacer sequences of the two or more CRISPR nucleic acid constructs are the same or non-identical, and if present , The zipper sequences of the two or more CRISPR nucleic acid constructs are the same.

In another aspect, there is provided 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 the present invention, wherein the zipper sequence of the synthetic CRISPR nucleic acid construct is of the synthetic tracr nucleic acid construct. An anti-zipper sequence and at least about 70% (eg, 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%, etc.) complementary and hybridizing, the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% identical to the anti-stitch sequence of the synthetic tracr nucleic acid construct, hybridizing, the bulge sequence of the synthetic CRISPR nucleic acid construct and the synthetic CRISPR The bulge sequences of the nucleic acid construct are non-complementary.

In another embodiment is provided 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 the invention, wherein the NNUNNN of the stitch sequence of the synthetic CRISPR nucleic acid construct is , The synthetic tracr nucleic acid construct is 100% complementary to and hybridizes with the NNANN of the anti-stitch sequence, and the (G) of the stitch sequence forms a wobble base pair with the U of the anti-stitch sequence to form a synthetic CRISPR. The bulge sequence of the nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary, and the zipper sequence and the anti-zipper sequence of the synthetic CRISPR nucleic acid construct, if present, are anti-compounds of the synthetic tracr nucleic acid construct. Hybridizes to the zipper sequence.

In some embodiments, the chimeric nucleic acid construct optionally further comprises a nucleotide joining the hybridized zipper and anti-zipper sequences at the end of the hybridized sequence (distal to the bulge sequence). it can. In a further embodiment, in the absence of the zipper and the anti-zipper, 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. The linking nucleotides can be any nucleotide (eg, T, A, G, C) and the number of nucleotides linking the zipper and anti-zipper or bulge sequences can be from about 3 to about 7. possible.

In a further aspect, the synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of the invention has at least 70% identity (eg, about 70%) to a Cas9 nuclease, an amino acid sequence encoding a Cas9 nuclease. , 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%, etc.), or , A Cas9 nuclease-encoding amino acid sequence having at least 70% identity to the amino acid sequence. The Cas9 nuclease useful in the present invention may be any Cas9 nuclease known to catalyze DNA cleavage in the CRISPR-Cas system. As is known in the art, such Cas9 nucleases include an HNH motif and a RuvC motif (see, for example, WO2013/176772; WO/2013/188638). In some embodiments, the HNH or RuvC motifs may contain mutations that reduce or eliminate their activity as compared to the wild-type Cas9 nuclease. In some embodiments, only one motif is mutated (eg, either the HNH motif or the RuvC motif). In another embodiment, both motifs are mutated so that both activities are reduced or eliminated. Any type of mutation, including missense mutations, nonsense mutations, frameshift mutations, etc. can be used to reduce or eliminate the activity of the HNH and/or RuvC motifs in Cas9 nuclease.

The present disclosure identifies various CRISPR-Cas system and Cas9 nuclease groupings. These are grouped into the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nuclease, the Streptococcus thermophilus CRISPR 3 (Sth laclus glublus glublus and lactobacillus glusula glub) of Cas9 nuclease. (Lrh) group is included. Non-limiting examples of Sth CR1 group Cas9 nucleases include the Cas9 nuclease encoded by the polypeptide sequences of SEQ ID NOs: 1-9 and 51. Non-limiting examples of Sth CR3 group Cas9 nuclease include the Cas9 nuclease encoded by the polypeptide sequences of SEQ ID NOs: 10-23. Non-limiting examples of Lb group Cas9 nucleases include 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 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. Additional Cas9 nucleases useful in the present invention include, but are not limited to, Cas9 from Lactobacillus animalis KCTC 3501, and Lactobacillus farsiminis WP 010018949.1.

Thus, in some embodiments, the Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, the Cas9 nuclease of Streptococcus thermophilus CRISPRb3 (Sth CR3) Cas9 nuclease, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPRb3 (Sth CR3) Cas9 nuclease. It may comprise, consist essentially of, or consist of a Cas9 nuclease from the CD034(Lb) group, and/or a Cas9 nuclease from the Lactobacillus rhamnosus GG(Lrh) group of Cas9 nucleases. In a further embodiment, the amino acid sequence encoding the Cas9 nuclease may be the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO:53. Also, in a further embodiment, the Cas9 nuclease useful in the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, synthetic CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure is one of SEQ ID NO: 1 to SEQ ID NO: 53. It comprises, consists essentially of, or consists of a nucleotide sequence that encodes an amino acid sequence that has at least 70% identity to the amino acid sequence.

Furthermore, in certain embodiments, the Cas9 nuclease can be encoded by a nucleotide sequence that is codon optimized for the organism containing the target DNA. In yet another embodiment, the Cas9 nuclease can include at least one nuclear localization sequence.

The inventors have surprisingly discovered a functional pairing between a particular group of Cas9 nucleases and the nexus sequences of synthetic tracr nucleic acid constructs (tracrRNA, tracrDNA). Thus, in some embodiments, the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases when the nexus sequence is GATAAGGC or GATAAGGCCATGCC; and when the nexus sequence is TAAGGC or TAAGGCTAGTCC. The Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nuclease; when the nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is from Cas9 nuclease Lactobacillus buchnerb group (CD3). When the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, the Cas9 nuclease is derived from the Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.

As described herein, Cas9 nucleases useful in the present invention may include mutations in the HNH and/or RuvC motifs, which reduce or eliminate the activity of each motif. As is known in the art, mutations within the HNH motif reduce/eliminate site-specific nicks in the (+) strand of double-stranded target DNA, and mutations in the RucV active site affect double-stranded target DNA. Reduce/eliminate site-specific nicks on the (-) strand of DNA. Mutations in both active sites reduce/eliminate DNA cleavage (ie, reduce/eliminate site-specific cleavage of target DNA). Thus, in some embodiments, the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure comprise a Cas9 nuclease having a mutation in the RuvC active site motif. In other embodiments, the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure comprise a Cas9 nuclease having a mutation within the HNH active site motif. In yet a further embodiment, the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure comprise a Cas9 nuclease having mutations within the HNH active site motif and within the RuvC motif.

In yet a further embodiment, a Cas9 nuclease having mutations in the HNH and RuvC motifs, thereby reducing or eliminating nuclease activity, further comprises the polypeptide of interest fused to the Cas9 nuclease. Such Cas9-target polypeptide fusion proteins can be used to direct or target the target polypeptide to a particular target DNA.

Further provided herein are methods for using the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure. Thus, in some embodiments, the chimeric nucleic acid construct of the present disclosure or an expression cassette comprising the chimeric nucleic acid construct of the present disclosure is contacted with target DNA in the presence of a Cas9 nuclease (eg, SEQ ID NOs: 1-53), Provide a method for site-specific cleavage of double-stranded target DNA comprising causing site-specific cleavage of the target DNA within a region defined by complementary hybridization of a spacer sequence to the target DNA. .. In some embodiments, the site-specific cleavage may be a site-specific nick of the (+) strand of double-stranded target DNA, wherein the Cas9 nuclease comprises a mutation within the RuvC active site motif, whereby Cleaves the (+) strand of the double-stranded target, creating a site-specific nick in the (+) strand of the double-stranded target DNA. In another embodiment, the site-specific cleavage is a site-specific nick on the (-) strand of the double-stranded target DNA, wherein the Cas9 nuclease comprises a point mutation within the HNH active site motif, whereby The (-) strand of the double-stranded target DNA is cleaved to generate a site-specific nick in the (-) strand of the double-stranded target DNA.

In a further embodiment, a method for site-specific cleavage of double-stranded target DNA is provided, comprising: a transcoding CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule, and a Cas9 nuclease (eg, SEQ ID NO: 1). ~53) in the presence of the target DNA, wherein (a) the tracr nucleic acid molecule comprises in the 5'to 3'direction an anti-zipper sequence comprising at least about 3 nucleotides; at least about 3 A bulge sequence containing N nucleotides; an anti-stitch sequence containing the nucleotide sequence of NNANN; A nexus sequence comprising the nucleotide sequence of TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTTCAGC; and at least 1 Comprising a hairpin sequence comprising a nucleotide sequence comprising, consisting essentially of or consisting of three hairpins, said hairpin comprising at least 3 matching base pairs, and the anti-zipper sequence comprising: 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 immediately upstream of the hairpin sequence. And (b) the CRISPR nucleic acid molecule has, in the 3′ to 5′ direction, a zipper sequence containing at least about 3 nucleotides, a bulge sequence containing a nucleotide sequence having at least 2 nucleotides, NNTNN( or stitch sequence comprising the nucleotide sequence of NNUNN), a spacer sequence having a _{G R1,} and 5 'and 3' ends containing nucleotide G or GTT, the target DNA and 100% identity to the 3 'end A zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, and the stitch sequence is G located immediately upstream of _R1, and, G _R1 is located immediately upstream of a spacer sequence, and further wherein, anti tracr nucleic acid molecule - zipper sequence and anti - scan The tetch sequence is at least about 70% complementary and hybridizes with 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 a part of the target DNA (protospacer flanking motif on the target DNA). (Adjacent to (PAM)) is at least about 80% complementary and hybridizes, thereby site-specific cleavage of the target DNA within the region defined by complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. Bring

In another embodiment, there is provided a method for site-specific cleavage of double stranded target DNA comprising the step of contacting double stranded target DNA with a chimeric nucleic acid comprising: (A) anti-zipper sequence comprising at least about 9 nucleotides in the 5'to 3'direction; a bulge sequence comprising at least about 4 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nexus sequence; and a first nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin (the hairpin is The anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, and the anti-stitch sequence is the nexus sequence. Immediately upstream of the hairpin sequence, and the nexus sequence is located immediately upstream of the hairpin sequence); (b) in the 3'to 5'direction, a zipper sequence comprising at least about 3 nucleotides, at least 2 nucleotides; bulge sequence, stitch sequence comprising the nucleotide sequence of NNTNN (or NNUNN) comprising a nucleotide sequence having, _{G R1} comprises a nucleotide G or GTT, and 5 a spacer sequence having 'and 3' ends, the 3 ' A second nucleotide sequence (a zipper sequence is located immediately upstream of the bulge sequence and a bulge sequence is of the stitch sequence) containing a spacer sequence at the end that contains at least 7 nucleotides with 100% identity to the target DNA. immediately located upstream, stitching sequence _is located immediately upstream of the _{G R1, and, G R1} is located immediately upstream of a spacer sequence); and, (c) Cas9 nuclease (e.g., SEQ ID NO: 1-53 A third nucleotide sequence (wherein the anti-zipper sequence and the anti-stitch sequence of the first nucleotide sequence are combined with the second amino acid sequence having at least 80% identity to the amino acid sequence encoding The spacer sequence of the second nucleotide sequence, which hybridizes to the zipper sequence and the stitch sequence of the nucleotide sequence, is attached to a part of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA). Hybridize, thereby resulting in site-specific cleavage of the target DNA within the region defined by complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA).

In a further embodiment, a method of site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA is provided, wherein the transcoded CRISPR (tracr) nucleic acid molecule and the CRISPR nucleic acid molecule are labeled with a Cas9 nuclease (eg sequence Nos. 1-53) and contacting the target DNA in the presence of (a) a tracr nucleic acid molecule, wherein the anti-zipper sequence comprises in the 5'to 3'direction at least about 3 nucleotides; A bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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, TAAGGCCATGTCC, TCAAGCAAAGC, or a nexus sequence comprising the nucleotide sequence of TCAAACAAAGCTTCAGC; and A hairpin sequence comprising a nucleotide sequence having at least one hairpin, the hairpin comprising at least 3 matching base pairs, the anti-zipper sequence 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) a CRISPR nucleic acid molecule comprises, in the 3'to 5'direction, a zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising at least 2 nucleotides (e.g. (-NN-) nucleotide sequence), an NNTNN. stitch sequence comprising the nucleotide sequence, a spacer sequence having a G _R1, and 5 'and 3' ends containing nucleotide G or GTT, at least seven having the target DNA and 100% identity to the 3 'end comprising a spacer sequence comprising nucleotides encoded by the nucleotide sequence, zipper sequence is located immediately upstream of the bulge sequence, bulge sequence located immediately upstream of the stitch sequence, stitch sequence immediately upstream of the G _R1 located in, _{and, G R1} is located immediately upstream of a spacer sequence, and further wherein, Cas9 nuclease, varying in HNH active site motif Different, containing a mutation in the RuvC active site motif and fused to the polypeptide of interest, the anti-zipper sequence is approximately 70% complementary to and hybridizes to the zipper sequence, and the stitch sequence is 100% complementary to the stitch sequence. And hybridizes, and the spacer sequence is approximately 80% complementary to and hybridizes with the target DNA adjacent to the protospacer flanking motif (PAM) on the target DNA, whereby the spacer sequence of the CRISPR nucleic acid molecule to the target DNA is Regions defined by complementary binding provide for site-specific targeting of the polypeptide of interest to target DNA.

In an exemplary embodiment, as described herein, for a synthetic tracr nucleic acid construct, the bulge sequence or first nucleotide sequence of the synthetic tracr nucleic acid molecule comprises about 3, 4 or 5 nucleotides. It can consist essentially of, or consist of. In another embodiment, the bulge sequence may comprise, consist essentially of, or consist of, 5 nucleotides, and the hairpin sequence may comprise at least 2 hairpins, It can consist essentially of or consist of, wherein each hairpin comprises at least 3 matching base pairs.

In a further embodiment, the present invention provides for site-specific cleavage of double-stranded target DNA comprising the step of contacting a transcoding CRISPR(tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of Cas9 nuclease. Provides a method of where
(A) a tracr nucleic acid molecule comprises in the 5'to 3'direction an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch arrangement, TNANC, 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), GATAAGGCCATGCCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence containing the nucleotide sequence of TCAAACAAAAGCTTCAGC, coded by a nucleotide sequence containing a hairpin sequence having at least one hairpin The hairpin comprises at least 3 matching base pairs, and the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is immediately upstream of the anti-stitch sequence. Located, 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 in the 3'to 5'direction. An optional zipper sequence comprising a nucleotide sequence having at least 3 nucleotides which hybridizes to the anti-zipper, a bulge sequence comprising the nucleotide sequence of (-NN-), a stitch sequence comprising the nucleotide sequence of NNUNNN, and 5'. A zipper sequence is encoded by a nucleotide sequence comprising a spacer sequence having an end and a 3′ end, the spacer sequence comprising at its 3′ end at least 7 nucleotides having 100% identity to the target DNA, and If present, immediately upstream of the bulge array, the bulge array is immediately upstream of the stitch array, the stitch array is immediately upstream of the spacer array, and
Furthermore, where the anti-zipper and zipper sequences are present, the anti-zipper sequences hybridize to the zipper sequences, the anti-stitch sequence NNANN is complementary to and hybridizes to the stitch sequence NNUNN, and the CRISPR nucleic acid The spacer sequence of the molecule hybridizes to a portion of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA), thereby being defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. Within the region, results in site-specific cleavage of the target DNA.

In yet a further embodiment, a method for site-specific cleavage of double-stranded target DNA is provided, the method comprising the step of contacting double-stranded target DNA with a chimeric nucleic acid comprising:
(A) in the 5'to 3'direction an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN, 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, TCAAGCAAAGC, or TCAAACAAGCATTCAGC Nexus sequence comprising a nucleotide sequence, a first nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin (said hairpin Contains at least 3 matching base pairs),
Here, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, and the anti-stitch sequence is immediately upstream of the nexus sequence. And the nexus sequence is located immediately upstream of the hairpin sequence;
(B) in the 3'to 5'direction, an optional zipper sequence comprising a nucleotide sequence having at least 3 nucleotides that hybridizes to the anti-zipper, a nucleotide sequence having at least 2 nucleotides (eg (- A nucleotide sequence of NN-)), a stitch sequence containing the nucleotide sequence of NNUNNN, and a spacer sequence having a 5'end and a 3'end, wherein the 3'end has 100% identity with the target DNA. A second nucleotide sequence, comprising a spacer sequence comprising at least 7 nucleotides having, and a zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is located immediately upstream of the stitch sequence. And the stitch sequence is located immediately upstream of the spacer sequence; and (c) encodes an amino acid sequence having at least 80% identity to the amino acid sequence encoding the Cas9 nuclease (eg, SEQ ID NOs: 1-53), A third nucleotide sequence,
Here, when a zipper sequence and an anti-zipper sequence are present, the zipper sequence hybridizes to the anti-zipper sequence, and the anti-stitch sequence NNANN is complementary to and hybridizes with the stitch sequence NNUNNN. The spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to the proto-spacer flanking motif (PAM) on the target DNA), whereby the spacer sequence of the second nucleotide sequence to the target DNA It results in site-specific cleavage of the target DNA within the region defined by hybridization.

In a further embodiment, a method of site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA is provided, wherein the transcoded CRISPR (tracr) nucleic acid molecule and the CRISPR nucleic acid molecule are labeled with a Cas9 nuclease (eg sequence No. 1-53) and contacting the target DNA in the presence of
Wherein (a) a tracr nucleic acid molecule is in the 5'to 3'direction, an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide of NNANN. Anti-stitch sequences including sequences, TNANC, 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), GATAAGGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence comprising the nucleotide sequence of TCAAACAAAGCTTCAGC, comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin, Encoded by a nucleotide sequence, the hairpin comprises at least 3 matching base pairs, and the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is the anti-stitch sequence. Immediately upstream of, 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 3'to 5 An optional zipper sequence comprising a nucleotide sequence having at least 3 nucleotides that hybridize to the anti-zipper in the'direction, a nucleotide sequence having at least 2 nucleotides (eg, a nucleotide sequence of (-NN-)) A bulge sequence containing NNUNN, a stitch sequence containing a nucleotide sequence of NNUNN, and a spacer sequence having 5′ and 3′ ends, the 3′ end having at least 7 nucleotides having 100% identity with the target DNA. Containing a spacer sequence, encoded by a nucleotide sequence, and the zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, and the stitch sequence is the spacer Located immediately upstream of the sequence, and further here, the Cas9 nuclease comprises a mutation in the HNH active site motif, a mutation in the RuvC active site motif, fused to the polypeptide of interest, and a zipper sequence and an anti-zipper sequence. If present, the zipper sequence hybridizes to the anti-zipper sequence and the anti-stitch NNANN is complementary to the stitch sequence NNUNNN and is not The lysed, spacer sequence hybridizes to the target DNA adjacent to the protospacer flanking motif (PAM) on the target DNA, thereby within the region defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. , Provides site-specific targeting of a polypeptide of interest to target DNA.

In some embodiments, when the anti-zipper and zipper sequences or the first and second nucleotide sequences of the tracr and aCRISPR nucleic acid molecules hybridize, the hybridized sequences optionally include bulge sequences. Additional nucleotides can be further included at the end of the hybridized sequence distal to, thereby joining the hybridized zipper and anti-zipper sequences. In a further embodiment, in the absence of the zipper and the anti-zipper, 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. The linking nucleotides can be any nucleotide (eg, T, A, G, C) and the number of nucleotides linking the zipper and anti-zipper or bulge sequences can be from about 3 to about 7.

Any wild-type, mutant, codon-optimized Cas9 nuclease, including, but not limited to, SEQ ID NOs: 1-53, or those containing at least one nuclear localization sequence described herein, are of the invention. Can be used in the method.

Further provided herein are expression cassettes and vectors comprising the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules and/or nucleotide sequences of the invention, which can be used in the methods of the disclosure.

In a further aspect, the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and/or nucleotide sequences of the invention can be introduced into cells of the host organism. Any cell/host organism for which the invention is useful can be used. Exemplary host organisms include, but are not limited to, plants, bacteria, archaea, fungi, animals, mammals, insects, birds, fish, amphibians, cnidarians, humans, or non-human primates. In certain embodiments, the host organism is not limited, Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum or,, Arabidopsis thaliana met You can In a further embodiment, cells useful in the present invention include, but are not limited to, stem cells, somatic cells, germ cells, plant cells, animal cells, bacterial cells, archaeal cells, fungal cells, mammalian cells, insect cells, avian cells. , Fish cells, amphibian cells, cnidarian cells, human cells, or non-human primate cells. In another embodiment, cells useful in the present invention include, but are not limited, Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum, or It contains cells from Arabidopsis thaliana.

In a further aspect of the invention, the polypeptide of interest includes but is not limited to helicase, nuclease, methyltransferase, gyrase, demethylase, kinase, dismutase, integrase, transposase, telomerase, recombinase, acetyltransferase, deacetylase, polymerase, phosphatase, It may include a ligase, a ubixin ligase, a photolyase or a glycosylase. In another aspect of the present invention, the polypeptide of interest has depurinating activity, oxidizing activity, pyrimidine dimer forming activity, alkylating activity, DNA repair activity, DNA damaging activity, deubiquitinating activity, adenylating activity, deadenylating activity. It has a sumoylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, a demyristoylation activity or a telomere repair activity, or a deamination activity. In an exemplary embodiment, the polypeptide of interest has kinase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, phosphatase activity, ubixin ligase activity, deubiquitination activity, or telomere repair activity. May have a polypeptide.

Further provided herein are kits that include the nucleic acid constructs, nucleic acid molecules, and/or nucleotide sequences of the invention.

Thus, in one aspect, a kit for site-specific cleavage of double-stranded DNA is provided, which kit comprises 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 present invention. In another aspect, a kit for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA is provided, which kit comprises a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array. Or a chimeric nucleic acid construct. In some aspects, the kit may comprise 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 the present invention contained in one or more expression cassettes. In a still further aspect, the kit further comprises a Cas9 nuclease (eg, SEQ ID NOs: 1-53) for use with the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and/or nucleotide sequences of the invention described herein. Good.

In a further aspect, the kit may comprise a primer, said primer comprising a portion of the CRISPR repeat sequence in both directions. In another embodiment, the kit may include primers designed to include the boundaries of the CRISPR array (ie, one leader end and the other trailer end) and extend through the CRISPR repeat sequence in both directions. ..
In a further embodiment, the kit may further comprise instructions for use.

The invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims to the invention, but are intended to be illustrative of the particular embodiments. Any modifications in the exemplified methods that occur to those skilled in the art are intended to be within the scope of the present invention.

Example 1. Assessing the functional role of the modules identified in the guide sequence Clustered regularly interspersed short pa ndrometic repeat (CRISPR) and related Cas proteins confer adaptive immunity to invasive genetic factors in bacteria and archaea ¹ . In the type II CRISPR-Cas system, the signature RNA-guided endonuclease Cas9 specifically targets the sequence complementary to the CRISPR spacer, resulting in a double-stranded DNA break (DSB) with two nickase domains. (Makarova, K.S. et al. Nat Rev Microbiol 9, 467-477 (2011); Garneau, JE et al. Nature 468, 67-71 (2010); Saplanauskas, R. et al. Nucleic. Acids Res 39, 9275-9228 (2011); Gasiunas, G. et al. Proc Natl Acad Sci US A 109, E2579-E2586 (2012); Jinek, M. et al., Science 337, 816-821 (2012). ). Any DNA sequence can be targeted as long as the Cas9-specific protospacer-flanking motif (PAM) is flanked. Garneau, J.; E. et al. Nature 468, 67-71 (2010); Saplanauskas, R.; et al. Nucleic Acids Res 39, 9275-9282 (2011); Gasiunas, G.; et al. Proc Natl Acad Sci US A 109, E2579-E2586 (2012); Jinek, M.; et al. , Science 337, 816-821 (2012); Sternberg, S.; H. et al. Nature 507, 62 (2014)). Targeting and cleavage by the Cas9 system relies on an RNA duplex consisting of CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA) ⁸ . This natural complex can be replaced by a synthetic single guide RNA (sgRNA) chimera that mimics a crRNA:tracrRNA duplex (Jinek, M. et al., Science 337, 816-821 (2012)). The sgRNA in combination with Cas9 creates a convenient, compact, and portable, sequence-specific targeting system suitable for heterologous transfer to various model systems of engineering and industrial and translational interest.

Therefore, the Cas9:sgRNA technology, which provides a compact and practical means for producing double-strand breaks (DSBs), has a revolutionary genomic organization (Mali, P. et al., Science 339,823). Cong, L. et al. Science 339, 819-823 (2013); Jiang, W. et al. Nat. Biotechnol. 31, 233-239 (2013); Sander, JD & Jung. , J. K. Nature Biotechnol. 32, 347-355. (2014), paving a new avenue for high-throughput genome-wide gene screening ^13,14 , and expanding the toolbox of transcription regulation (Qi, LS .. et al. Cell 152, 1173-1183 (2013); Gilbert, LA et al. Cell 154, 442-451 (2013)). Moreover, the absence of an interaction between evolutionarily distant Cas9:sgRNAs (Chylinski, K. et al. RNA biology 10, 726-737 (2013); Fonfara, I. et al. Nucleic Acids Res (2013). ); Esvelt, KM et al. Nature Methods (2013)), when coexistent functional type II CRISPR-Cas systems function simultaneously, multiple independent targeting can be achieved intracellularly (Barrangou, R. et al. Science 315, 1709-1712 (2007); Horvath, P. et al. J Bacteriol. 190, 1401-1412 (2008)). Despite the widespread use of these molecular mechanisms, the critical properties of sgRNA guides and their involvement in the definition of functionally orthologous Cas9 endonucleases have not yet been characterized. Indeed, the initial focus of Cas9 targeting and cleavage focuses on spacer:target complementarity and PAM sequence sensitivity, while the orthogonality between factors driving Cas9:sgRNA interactions and the type II CRISPR-Cas system. There remains a lack of information defining the factors that direct sex. Therefore, we set out to identify and characterize features within sgRNAs that provide Cas9 targeting and cleavage specificity to open new engineering avenues for CRISPR technology.

Thus, the functional roles and implications of various modules identified within the guide sequences described herein (eg, synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, chimeric nucleic acid constructs (tracr nucleic acid-synthetic CRISPR nucleic acid constructs) ( In order to evaluate implication, we designed mutants of the guided mutations, which contain alterations or deletions of each of the aforementioned functional modules. We used the SthCRISPR3 system as a representative functional model. We selected and initially established a positive functional control (wild type, WT) using the native guide sequence, then we tested a "stitch" mutant with deletion of stitches and observed loss of function. We then tested the bulge-deleted "bulge" mutant and observed loss of function, then we tested the nexus-deleted "nexus" mutant and observed loss of function. We then tested the "hairpin" mutant with the deletion of the first hairpin and observed loss of function.We subsequently tested sequence specificity and variability susceptibility to It was established that the nexus sequence is specific, while variably permissive for other functional modules, notably zippers, bulges, hairpins and stitches.The results of these experiments are shown in Figures 1-21. ..

Therefore, FIG. 1 shows a multiple sequence alignment for the Nexus module, and FIG. 2 provides the maximum likelihood tree for the Nexus module developed throughout this study. 3A to 3D show consensus sequences for Nexus modules of the Sth Cr1 group (FIG. 3A), the Sth Cr3 group (FIG. 3B), the Lrh group (FIG. 3C) and the Lbu group (FIG. 3D).

FIG. 5 shows a multiple sequence alignment for the anti-stitch module, while FIGS. 6A-6D show the Sth Cr1 group (FIG. 6A), Sth Cr3 group (FIG. 6B), Lrh group (FIG. 6C) and Lbu group. FIG. 6D shows a consensus arrangement for the anti-stitch module of FIG. 6D.

Similarly, a multiple sequence alignment for the bulge module is provided in Figure 7, and a consensus sequence for the bulge module of the Sth Cr1 group is shown in Figure 8A. FIG. 8B shows the consensus sequence for the bulge module of the Sth Cr3 group. FIG. 8C shows the consensus sequence for the Lrh group, and FIG. 8D shows the consensus sequence for the bulge module in the Lbu group.

The multiple sequence alignment for the zipper module is shown in FIG. 9, and FIG. 10 shows the 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 the maximum likelihood tree for Cas9 nuclease.

12-21 show guide sequences and targeting for various cRNA:tracRNA constructs, Streptococcus thermophilus CR3 (FIG. 12) representing the Sth CR1 group; Lactobacillus bachneri (FIG. 13) representing the Lbu group; Sth CR1 group. Streptococcus thermophilus CR1 (FIG. 14); Streptococcus pyrogenes M1 GAS (FIG. 15) that represents the Sth CR3 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 side of each of Figures 12-21 represents the target dsDNA containing the target sequence (open structure) and flanking (3') PAM. The upper side of each figure represents a CRISPR RNA (crRNA) consisting of a 5'portion complementary to a target sequence and a 3'portion derived from a CRISPR repeat; and also a tracrRNA consisting of an anti-CRISPR repeat portion and a nexus. Also shown is the 3'hairpin. As shown in each of FIGS. 12 to 21, the complementary portion of the crRNA:tracrRNA duplex has a lower stem (lower complementary portion), a bulge (herniated mismatch) and an upper stem (upper complementary portion). Consists of.

Example 2. Characterization of Guide RNA Sequences in Various Additional Type II Systems The findings described in Example 1 establish a key module in sgRNA necessary to support Streptococcus pyrogenes Cas9 (SpyCas9) activity. However, although widely used for genome organization, SpyCas9 is just one of many Cas9 orthologs found in nature (Chylinski, K. et al. RNA biology 10, 726-737 (2013)). Fonfara, I. et al. Nucleic Acids Res (2013)). Therefore, we next investigated whether the same sgRNA sequence characteristics also occurred in other type II CRISPR-Cas systems. We sampled 41 Cas9 sequences from the Streptococcus and Lactobacillus genomes (type II systems preferentially occur ² ), identified their corresponding CRISPR repeats, and predicted tracrRNA sequences. The Cas9 protein sequence was clustered into three main sequence groups (Figure 23). As expected, similar grouping was seen when clustering was performed with either CRISPR-repeats or predicted tracrRNA sequences (Figure 24A, Figure 23, Figure 25, Figure 26) and anti-intra-crRNA. The existence of CRISPR repeats and a relevant molecular relationship between Cas9 and crRNA:tracrRNA pairs were given (Makarova, KS et al. Nat Rev Microbiol 9, 467-477 (2011); Deltacheva, E. et. Al. Nature 471, 602-607 (2011); Fonfara, I. et al. Nucleic Acids Res (2013)). Within the tracrRNA sequence, we consistently observed the functional modules identified for SpyCas9 (Fig. 24B), and the overall sgRNA/crRNA:tracrRNA structure between families was conserved, with high levels within the cluster. The sequence was stored in.

The presence of a bulge with a directional twist between the lower stem (i.e. stitch/anti-stitch) and the upper stem (i.e. zipper/anti-zipper) was consistently seen across system versatility. The length of the lower stem was highly conserved within the family and variable between families. Interestingly, the highest levels of conservation were seen with the nexus sequences (Figure 24B, Figure 27). The general nexus shape with a GC-rich stem and offset uracil was shared between the two Streptococcus families. In contrast, the unique dual-stem nexus (FIGS. 24A-B) was unique and ubiquitous in the Lactobacillus system. Surprisingly, some bases within the nexus were tightly conserved even among different families including A52 and C55 (Fig. 24A-B), further highlighting the critical role of this module. Indeed, A52 interacts with the backbone of residues 1103-1107 near the 5'end of the target strand in the crystal structure of SpyCas9, and that interaction of the protein backbone with nexus may be required for PAM binding. Suggest.

Determining the relationship of guide RNA structure to the Cas9 protein orthogonality. The findings described herein suggest potential relationships between the sequence and structure of sgRNAs and the diversity of Cas9 proteins. This observation prompted us to determine the sgRNA modules that define the Cas9 orthologous group. Therefore, we find that two naturally coexisting orthologous S. A thermophilus type II system, that is, an endonuclease derived from Sth1 Cas9 and Sth3 Cas9 is selected (Horvath, P. et al. J Bacteriol. 190, 1401-1412 (2008)), and between the sgRNA composition and Cas9 orthogonality. I investigated the relationship. A series of experiments were designed based on self-targeting activity in Escherichia coli (FIGS. 28A-B), where specific mutations in sgRNA promote cleavage activity at orthologous orthologoous Cas9. I tested whether I could do it. We have shown that E. coli contains Cas9 target sites that overlap for the Sth1Cas9 and Sth3Cas9 systems. A region within the E. coli genome was identified, confirming that the cleavage occurred at one nucleotide (FIG. 29B) and that the PAM sequences overlap favorably. We produced chimeric versions of the two sgRNA backbones and replaced the spacer, lower stem (ie stitch/anti-stitch)-bulge-upper stem (ie zipper/anti-zipper), nexus and hairpin (FIG. 29C). ), and their ability to drive self-targeting by either Sth1Cas9 or Sth3Cas9 (Gomaa, AA et al. MBio. 5, e00928-13 (2014)). Initially, we confirmed that these two systems were indeed orthogonal in this assay system, and that each guide alone drives targeting at its cognate Cas9 (Figure 29C). We next show that the exchange of spacer sequences results in an sgRNA with a CRISPR3 spacer and a CRISPR1 backbone that is capable of supporting Sth1Cas9 cleavage activity. However, the opposite is not true for Sth3Cas9 activity. The sgRNA containing the CRISPR1 spacer and the CRISPR3 backbone do not support Sth3Cas9 activity (FIG. 29C). We hypothesize that this unidirectional reciprocal functionality is due to the flexibility of the requirement for the space between the protospacer and the PAM in the SthCRISPR system (Chen et al. J. Biol. Chem. doi: 10. 1074/jbc.M113.539726. (2014)) (FIG. 29C, upper panel). We then showed that the functionality between sgRNA and Cas9 could be altered by simply exchanging the nexus-hairpin combination between the two orthogonal systems. The main consequence of sgRNA reprogramming is the loss of the ability to guide the original Cas9 in the process (FIG. 29C, lower panel). This is in contrast to the standard belief that the CRISPR repeat sequence plays an important role in the definition of the orthologous CRISPR-Cas system. However, these results indicate that chimeric sgRNAs with altered nexus sequences can reprogram orthogonality in a predictable and unidirectional manner, further enhancing the orthogonal Cas9 protein associated with different PAMs. Critical to use (Esvelt, KM et al. Nature Methods (2013))).

Recent structural and biochemical data are beginning to shed light on the mechanism of DNA recognition and cleavage by Cas9 (Jinek, M. et al., Science 337, 816-821 (2012); Jinek, M. et al. , Science 343, 6176 (2014); Nishimasu, H. et al. Cell 156, 935 (2014)). Electron micrographs of apo-, RNA-binding and protein/RNA/DNA complexes show that Cas9 undergoes dramatic conformational changes upon binding to guide RNA, promoting the structure of target DNA binding and cleavage. (Jinek, M. et al., Science 343, 6176 (2014). SpyCas9:sgRNA:DNA:complex and apo-SpyCas9 showed that the crystal structure was consistent with the images obtained by electron microscopy. It is shown that substantial rearrangement of RNA- and DNA-binding domains occurs between the two structures, occupying strikingly distinct structures (Jinek, M. et al., Science 343, 6176 (2014); Nishimasu, H. et al. Cell 156, 935 (2014).) Nexus occupies a critical position in the SpyCas9-sgRNA:DNA complex, aligning many important components of proteins and sgRNAs, both protein and RNA. To receive the target DNA duplex for cleavage.When bound to sgRNA:DNA, the arginine-rich bridge helix binds to the base of the nexus and the lower stem. , Two small regions from two lobes of SpyCas9 (we propose to establish them as Nexus Interacting Region 1 (NIR1) 446-497 and Nexus Interacting Region 2 (NIR2) 1105-1138). Both of these regions are disordered within the apoSpyCas9 structure and contain, inter alia, two tryptophan residues identified as important in PAM recognition ^21. NIR2 also directly interacts with the lower stem. The side that interacts and is opposite the nexus-binding site is in close proximity to the 3'end of the target strand, suggesting that interaction with nexus may be necessary to align the PAM recognition site. Notably, in Actinomyces naeslundii Cas9 (AnaCas9) apo-structure (Jinek, M. et al., Science 343, 6176 (2014)), NIR2 is arranged and contains an insertion of about 50 amino acids. Motivation to think that a large nexus (and optionally with PAM sequences) can be recognized There is.

However, these results reveal six distinct features within the guide RNA, establishing bulge and nexus as structure- and sequence-specific properties that guide Cas9 targeting and cleavage. This is more suitable as a basis for optimization of sgRNA compositions and packaging into adeno-related viruses etc. that contain smaller regions of double-stranded RNA that potentially provoke an innate immune response. Provide a design with the opportunity to design a short, minimal guide RNA. This understanding of the type II CRISPR-Cas system is described in Briner et al. (Mol. Cell 56:333-339 (2014)) and Nishimasu et al. (Supported by Cell 156:935-949 (2014), sequence modifications have been shown to affect the ability of the modified sequence to guide Cas9. Notably, these studies show that Confirm that the nexus sequences within the guide are critical for guiding Cas9 towards complementary DNA and subsequent cleavage.

The ability to reprogram Cas9 orthogonality using chimeric sgRNAs with modified nexus sequences opens new avenues for the development of novel Cas9 proteins with the potential to harness the diversity of natural Cas9 orthologs. , Including a short Cas9 variant for convenient packaging and delivery. In addition, the ability to reprogram Cas9 with chimeric mgRNAs provides flexibility in target frequency management (frequent short PAMs) and specificity due to reduced off-target cleavage (rarely longer PAMs). Will occur) and will allow increased use of various PAMs. It also opens up multiple opportunities by using a single Cas9 with various chimera guides, or simultaneously using orthogonal systems with different combinations of standard or chimeric sgRNAs. Taken together, our findings pave the way for new Cas9-dependent DNA targeting and set the stage for the development of next-generation CRISPR-based technologies.

Example 3.
The cas9 gene from the CRISPR1 and CRISPR3 loci was transformed into S. PCR amplified from genomic DNA from thermophilus LMD-9 and cloned into pwtCas9-bacteria (Addgene #44250)) (Qi, LS et al. Cell 152, 1173-1183 (2013)). To construct the sgRNA-expression plasmid, the SpeI restriction site in the pdCas9-bacterial plasmid (Addgene #44249) (Id.) was removed and gBlock(IDT) encoding a zraP-targeting sgRNA based on the CRISPR1 or CRISPR3 locus was added. , PgRNA-bacterial plasmid (Addgene #44251) (Id.) in combination with the PCR-amplified backbone. For transformation analysis E. Transformation efficiency with E. coli K-12 was tested for sgRNA plasmids tested as previously described (Gomaa, AA et al. MBio. 5, e00928-13 (2014)). Body numbers were calculated by dividing by the number of transformants for the psgRNA-C1-T4 control plasmid.

Plasmid construction. To construct the Cas9-expression plasmid, the Cas9 gene from the CRISPR1 locus (Sth1-Cas9) and CRISPR3 locus (Sth3-Cas9) was transformed into S. PCR amplification was performed from the genomic DNA extracted from thermophilus LMD-9. Each PCR product was combined by Gibson assembly with the PCR-amplified backbone of pwtCas9-bacteria (Addgene #44250) (Qi, L.S. et al. Cell 152, 1173-1183 (2013)). To construct an sgRNA-expression plasmid, the SpeI restriction site in pdCas9-bacterial plasmid (Addgene #44249) (Id.) was removed by digesting the plasmid with SpeI, blunt-ended and religated to pdCas9ΔSpeI plasmid. Produced. Separately, S. gBlock(IDT), which encodes a zraP-targeting sgRNA based on the CRISPR1 (C1) locus or CRISPR3 (C3) locus in thermophilus LMD-9, was transformed by gibson assembly into pgRNA-bacterial plasmid (Addgene #44251) (Id.). PCR-amplified backbone of the original S. The pyogenes sgRNA sequence was replaced with the designed sgRNA sequence. The resulting sgRNA plasmid and pdCas9ΔSpeI backbone were then digested with AatII and XhoI and the gel-extracted fragments of each sgRNA plasmid and pdCas9ΔSpeI plasmid were ligated together to form psgRNA-SthC1 and psgRNA-SthC3. To modify the sgRNA sequence, 5'phosphorylated oligonucleotides were annealed to the SpeI/KpnI or KpnI/HindIII sites of the psgRNA-C1 or psgRNA-C3 plasmids and ligated. All plasmid modifications were confirmed by sequencing.

Strains and growth conditions. E. coli K-12 subst. MG1655 (genotype: E. coli K-12Fλ-ilvG-rfb-5C rph-1) was used for all transformation analyses. Strains were 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 otherwise indicated. The medium was supplemented with antibiotics (34 μg/ml chloramphenicol, 50 μg/ml ampicillin) as needed.

Transformation assay. Frozen stocks of cells carrying the indicated Cas9-expression plasmid were streaked for isolation and individual colonies were inoculated into 3 ml LB medium and grown overnight. The resulting culture was back-diluted in 45 ml LB medium and grown to an ABS ₆₀₀ of 0.6-0.8 as measured by a Nanodrop 2000c spectrophotometer (Thermo Scientific). The cultures were then pipetted and washed twice with ice-cold 10% glycerol before resuspending in 200-400 μl of 10% glycerol. Suspended cells (50 μl) were transformed with 25 ng of the indicated sgRNA-expression plasmid using MicroPulser Electroporator (BioRad) and recovered in 300 μl of SOC medium (Quality Biological) for 1 hour. After recovery, 200 μl cultures containing different amounts of LB medium were plated on LB agar containing 100 ng/ml anhydrotetracycline. Transformation efficiency was determined as previously described (Gomaa, AA et al. MBio. 5,e00928-13 (2014)) by comparing the number of transformants for the DgRNA plasmid tested to psgRNA-C1-. Calculated by dividing by the number of transformants for the T4 control plasmid. The tested sgRNA plasmids and control plasmids were transformed into the same batch of electrocompetent cells to reduce inter-experimental variations in transformation efficiency. Similarly, with respect to Figures 30-32, transformation analysis was used to test the ability of the plasmid to be electroporated into a Lactobacillus strain carrying an active CRISPR-Cas system (Lbu-Lactobacillus buchneri, Figure 30; Lrh. -Lactobacillus rhamnosus, Figure 32; Lca-Lactobacillus casei, Figure 31. The plasmid was designed to contain a protospacer sequence identical to the first spacer sequence in the host CRISPR locus, flanked by protospacers in the complete PAM. Various plasmids were designed as follows (NTAAC for Lga; NNGAA for Lca; NGAAA for Lrh; PAM region is the nucleotides underlined in Figures 30-32, and their mutated variants (tested for efficiency). The nucleotides are italicized.) Also, the experiment was performed with the control non-targeting sequence (the penultimate entry for each experiment shown in Figures 30-32, and the control plasmid without the target sequence). (A final entry was also included for each experiment shown in Figures 30-32. The ability of the native CRISPR-Cas system to prevent plasmid uptake by DNA targeting depends on the efficiency of transformation between the test sequence and the two aforementioned controls. Measured as the difference.

Claims (50)

A synthetic transcoding CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct, comprising:
From 5'to 3',
An optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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 , A TAGGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nucleotide sequence; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin,
The hairpin comprises at least 3 matching base pairs,
Wherein the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, and the anti-stitch sequence is Located immediately upstream of the nexus sequence, and said nexus sequence is located immediately upstream of said hairpin sequence,
Synthetic transcoding CRISPR(tracr) nucleic acid (eg, tracrRNA, tracrDNA) constructs. A synthetic CRISPR nucleic acid (eg, crRNA, crDNA) construct, comprising:
From 3'to 5',
Optional zipper sequence comprising at least about 3 nucleotides, bulge sequence comprising a nucleotide sequence having at least two nucleotides, stitching sequence comprising the nucleotide sequence of NNTNN, G _R1 comprises a nucleotide G or _GTT, and 5 ' A spacer sequence having an end and a 3'end, the spacer sequence comprising at its 3'end at least 7 nucleotides having 100% complementarity with the target DNA,
The zipper sequence, if present, situated immediately upstream of the bulge arrangement, the bulge sequence located immediately upstream of the stitch arrangement, the stitching sequence is located immediately upstream of the G _R1, And said _GR1 is located immediately upstream of said spacer sequence,
Synthetic CRISPR nucleic acid constructs. A synthetic CRISPR nucleic acid array comprising:
A nucleotide sequence encoding two or more CRISPR nucleic acid constructs of claim 2,
Wherein the two or more CRISPR nucleic acid constructs are located immediately adjacent to each other on the nucleotide sequence, and
The stitch sequences of the two or more CRISPR nucleic acid constructs are identical, and
Said spacer sequences of said two or more CRISPR nucleic acid constructs are identical or non-identical, and
The zipper sequences of the two or more CRISPR nucleic acid constructs, if present, are identical,
CRISPR nucleic acid array. A chimeric nucleic acid construct comprising the synthetic tracr nucleic acid construct of claim 1 and the synthetic CRISPR nucleic acid construct of claim 2, wherein
The stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and hybridizes to the anti-stitch sequence of the synthetic tracr nucleic acid construct,
The bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary,
If present, the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 70% complementary to and hybridizes to the anti-zipper sequence of the synthetic tracr nucleic acid construct,
Chimeric nucleic acid construct. The synthetic tracr nucleic acid construct of claim 1, the synthetic CRISPR nucleic acid construct of claim 2, the CRISPR nucleic acid array of claim 3, or the chimeric nucleic acid construct of claim 4.
Further comprising a nucleotide sequence encoding an amino acid sequence having at least 70% identity to the amino acid sequence encoding Cas9 nuclease,
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. The synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of claim 5, wherein
The Cas9 nuclease is Cas9 derived from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nuclease,
The Cas9 nuclease is Cas9 derived from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nuclease,
The Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus buchneri CD034(Lb) group of Cas9 nucleases, or
The Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus rhamnosus GG (Lrh) group of Cas9 nuclease,
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. The synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of claim 5, wherein
The amino acid sequence encoding a Cas9 nuclease is selected from the group of amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 53, or any combination thereof.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. The synthetic tracr nucleic acid construct, synthetic CRISP nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of claim 5, wherein
The nexus sequence is GATAAGGC or GATAAGGCCATGCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;
The nexus sequence is TAAGGC or TAAGGCTAGTCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;
The nexus sequence is TCAAGC or TCAAGCAAAGC, and the Cas9 nuclease is from the Lactobacillus buchneri CD034(Lb) group of Cas9 nucleases; or
The nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, and the Cas9 nuclease is derived from the Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases,
Synthetic tracr nucleic acid constructs, synthetic CRISP nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. 9. The synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of any one of claims 6 to 8,
The Cas9 nuclease contains a mutation in the RuvC active site motif,
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. 9. The synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of any one of claims 6 to 8,
The Cas9 nuclease contains a mutation within the HNH active site motif,
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. 9. The synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of any one of claims 6 to 8,
The Cas9 nuclease contains mutations within the HNH active site motif and within the RuvC active site motif and is fused to the polypeptide of interest,
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. An expression cassette,
12. A synthetic tracr nucleic acid construct according to any one of claims 1 or 5 to 11, a synthetic CRISPR nucleic acid construct according to any one of claims 2 or 5 to 11 and any one of claims 3 or 5 to 11. 12. A CRISPR nucleic acid array according to claim 14 or a chimeric nucleic acid construct according to any one of claims 4 to 11,
Expression cassette. A cell,
12. A synthetic tracr nucleic acid construct according to any one of claims 1 or 5 to 11, a synthetic CRISPR nucleic acid construct according to any one of claims 2 or 5 to 11 and any one of claims 3 or 5 to 11. 13. A CRISPR nucleic acid array according to claim 12, or a chimeric nucleic acid construct according to any one of claims 4 to 11, or an expression cassette according to claim 12.
cell. The cell of claim 13, wherein
The cells are stem cells, somatic cells, germ cells, plant cells, animal cells, bacterial cells, archaeal cells, fungal cells, mammalian cells, insect cells, avian cells, fish cells, amphibian cells, cnidarian cells, humans. Cells, or non-human primate cells,
cell. A method for site-specific cleavage of double-stranded target DNA, comprising:
Contacting the chimeric nucleic acid construct of any one of claims 4 to 10 or the expression cassette of claim 12,
The expression cassette comprises the chimeric nucleic acid construct of any one of claims 4 to 10 together with target DNA in the presence of Cas9 nuclease, thereby being defined by complementary hybridization of the spacer sequence to the target DNA. Causing site-specific cleavage of the target DNA within the region,
Method. The method of claim 15, wherein
The site-specific cleavage is a site-specific nick of the (+) strand of the double-stranded target DNA,
The Cas9 nuclease contains a mutation in the RuvC active site motif, thereby cleaving the (+) strand of the double-stranded target, resulting in a site-specific nick to the (+) of the double-stranded target DNA. Generated in the chain, or
The site-specific cleavage is a site-specific nick of the (-) strand of the double-stranded target DNA,
The Cas9 nuclease contains a point mutation in the HNH active site motif, thereby cleaving the (-) strand of the double-stranded target DNA to create a site-specific nick for the double-stranded target DNA (-) strand. To generate within,
Method. A method for site-specific cleavage of double-stranded target DNA, comprising:
Including the following steps:
Contacting a transcoded CRISPR(tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with said target DNA in the presence of Cas9 nuclease, wherein:
(A) the tracr nucleic acid molecule comprises, in the 5′ to 3′ direction, an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch sequences including; TNANC, 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), GATAAGGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence comprising the nucleotide sequence of TCAAACAAAAGCTTCAGC; and a nucleotide comprising a hairpin sequence comprising at least one hairpin. Encoded by a sequence, the hairpin comprises at least 3 matching base pairs, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, and the bulge sequence is the anti-zipper sequence. Located immediately upstream of the stitch array, the anti-stitch array is located immediately upstream of the nexus array, and the nexus array is located immediately upstream of the hairpin array; and
(B) the CRISPR nucleic acid molecule comprises, in the 3'to 5'direction, an optional zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising a nucleotide sequence having at least about 2 nucleotides, the nucleotides of NNTNN. stitch sequence comprising the sequence, G _R1 comprises a nucleotide G or _GTT, and, a spacer sequence having a 5 'and 3' ends, at least 7 having a target DNA and 100% identity to the 3 'end Comprising a spacer sequence comprising nucleotides, encoded by a nucleotide sequence, the zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, and the stitch sequence is located immediately upstream of the G _R1, and the G _R1 is located immediately upstream of said spacer sequences, and,
Furthermore, when the anti-zipper sequence and the anti-stitch sequence are present, the anti-zipper hybridizes to the zipper sequence, the stitch sequence hybridizes to the anti-stitch sequence, 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 (adjacent to a protospacer flanking motif (PAM) on the target DNA), whereby the target DNA is To site-specific cleavage of the target DNA within a region defined by complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to
Method. A method for site-specific cleavage of double-stranded target DNA, comprising:
The double-stranded target DNA is as follows:
(A) in the 5'to 3'direction, an anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising the nucleotide sequence of NNANN; 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, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nexus sequence; and a first nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin (the hairpin is , At least 3 matching base pairs, the anti-zipper sequence is located immediately upstream of the bulge sequence, if present, and 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) in the 3'to 5'direction, a zipper sequence containing at least about 3 nucleotides, a bulge sequence containing a nucleotide sequence having at least 2 nucleotides, a stitch sequence containing the nucleotide sequence of NNTNN, a nucleotide G or GTT. G _{R1 including,} and, a spacer sequence having a 5 'and 3' ends, including a spacer sequence comprising at least seven nucleotides having a target DNA and 100% identity to the 3 'end, the second nucleotide sequence (the zipper sequence, if present, situated immediately upstream of the bulge arrangement, the bulge sequence located immediately upstream of the stitch arrangement, the stitching sequence, immediately the G _R1 Upstream, and said _GR1 is located immediately upstream of said spacer sequence); and (c) a third amino acid sequence encoding an amino acid sequence having at least 80% identity to the amino acid sequence encoding the Cas9 nuclease. Contacting with a chimeric nucleic acid comprising a nucleotide sequence of
Here, if the anti-zipper sequence and the zipper sequence are present, they hybridize to each other, 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, and the spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to a protospacer flanking motif (PAM) on the target DNA). Soybean, thereby causing site-specific cleavage of the target DNA within the region defined by complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA.
Method. A method according to claim 17 or claim 20,
The site-specific cleavage is a site-specific nick of the (+) strand of the double-stranded target DNA,
The Cas9 nuclease contains a mutation in the RuvC active site motif that causes the (+) strand of the double-stranded DNA to be cleaved at a cleavage site located 5 nucleotides upstream of the PAM sequence, resulting in a site-specific Generate a nick in the double-stranded target DNA,
Method. A method according to claim 17 or claim 20,
The site-specific cleavage is a site-specific nick of the (-) strand of the double-stranded target DNA,
The Cas9 nuclease contains a mutation within the HNH active site motif that causes the (-) strand of the double-stranded DNA to cleave at a cleavage site located 5 nucleotides upstream of the PAM sequence, resulting in a site-specific Generate a nick in the double-stranded target DNA,
Method. A method according to any one of claims 15 to 20, wherein
The Cas9 nuclease is Cas9 derived from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nuclease,
The Cas9 nuclease is Cas9 derived from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nuclease,
The Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, or the Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases,
Method. A method according to any one of claims 15 to 20, wherein
The amino acid sequence encoding a Cas9 nuclease is selected from the group of amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 53, or any combination thereof.
Method. The method according to any one of claims 15 to 20, wherein
The nexus sequence is GATAAGGC or GATAAGGCCATGCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;
The nexus sequence is TAAGGC or TAAGGCTAGTCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;
The nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases; or the nexus sequence is TCAAAAC or TCAAAACAAAGCTTCAGC and the Cas9 nuclease is Cas9Lus It is derived from the Rhamnosus GG (Lrh) group,
Method. A method for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, the method comprising:
Contacting the chimeric nucleic acid construct of claim 8 or the expression cassette of claim 12 with the target DNA,
Wherein the expression cassette comprises the chimeric nucleic acid construct of claim 8, whereby the target polypeptide fused to the Cas9 is targeted to a specific site on the target DNA, the site being Defined by complementary hybridization of the spacer sequence to the target DNA,
Method. A method for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, comprising:
Contacting a transcoded CRISPR(tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with said target in the presence of Cas9 nuclease DNA, wherein:
(A) said tracr nucleic acid molecule comprises in the 5'to 3'direction an optional anti-zipper sequence comprising at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch sequences including; TNANC, 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), GATAAGGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAAACAAAGCTTCAGC nexus sequence; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin, Encoded by a nucleotide sequence, the hairpin comprises at least 3 matching base pairs, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, and the bulge sequence is the anti-zipper sequence. -Immediately upstream of the stitch sequence, the anti-stitch sequence is directly upstream of the nexus sequence, and the nexus sequence is immediately upstream of the hairpin sequence; and (b) the CRISPR The nucleic acid molecule comprises, in the 3'to 5'direction, an optional zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising a nucleotide sequence having at least about 2 nucleotides, a stitch sequence comprising the nucleotide sequence of NNTNN. , G _R1 comprising nucleotide G or GTT, and a spacer sequence having 5′ and 3′ ends, the spacer sequence comprising at least 7 nucleotides having 100% identity to the target DNA at its 3′ end. Including a sequence, encoded by a nucleotide sequence, the zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, and the stitch sequence is located immediately upstream of the G _R1, and the G _R1 is located immediately upstream of said spacer sequences, and,
Further, wherein the Cas9 nuclease contains a mutation in the HNH active site motif, a mutation in the RuvC active site motif, is fused to the polypeptide of interest, and if the anti-zipper sequence and the zipper sequence are present, those Hybridize to each other, the stitch sequence is 100% complementary to the stitch sequence and hybridizes, and the spacer sequence hybridizes to the target DNA adjacent to a protospacer flanking motif (PAM) on the target DNA. Thereby causing site-specific targeting of the polypeptide of interest to the target DNA within a region defined by complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA,
Method. 26. A method according to any one of claims 17 to 23 or 25,
The PAM comprises a nucleotide sequence of GG, GGNG, AGAA, AAAA or GAA,
Method. 27. The method of any of claims 15-26, wherein
The Cas9 nuclease is encoded by a nucleotide sequence codon optimized for the organism containing the target DNA.
Method. A method according to any one of claims 15 to 27,
The Cas9 nuclease comprises at least one nuclear localization sequence,
Method. A method according to claim 27 or claim 28, wherein
The organism is a plant, bacterium, archaea, fungus, animal, mammal, insect, bird, fish, amphibian, cnidarian, human, or non-human primate,
Method. 29. The method of any one of claims 27 or 28,
The organisms, Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum or a Arabidopsis thaliana,
Method. 31. A method according to any one of claims 24 to 30,
The target polypeptide is helicase, nuclease, methyltransferase, gyrase, demethylase, kinase, dismutase, integrase, transposase, telomerase, recombinase, acetyltransferase, deacetylase, polymerase, phosphatase, ligase, ubixin ligase, photolyase or glycosylase. Yes, or
The target polypeptide is a depurinating activity, an oxidizing activity, a pyrimidine dimer forming activity, an alkylating activity, a DNA repairing activity, a DNA damaging activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a SUMOylating activity, a desumoylating activity. Activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation or telomere repair activity, or deamination activity,
Method. A kit for site-specific cleavage of double-stranded DNA, comprising:
The kit comprises the synthetic tracr nucleic acid construct of claim 1,
kit. A kit for site-specific cleavage of double-stranded DNA, comprising:
The kit comprises the synthetic CRISPR nucleic acid construct of claim 2,
kit. A kit for site-specific cleavage of double-stranded DNA, comprising:
The kit comprises the CRISPR nucleic acid array of claim 3,
kit. A kit for site-specific cleavage of double-stranded DNA, comprising:
The kit comprises the chimeric nucleic acid construct of claim 4,
kit. A kit for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, comprising:
The kit comprises the synthetic tracr nucleic acid construct of claim 1,
kit. A kit for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, comprising:
The kit comprises the synthetic CRISPR nucleic acid construct of claim 2,
kit. A kit for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, comprising:
The kit comprises the CRISPR nucleic acid array of claim 3,
kit. A kit for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, comprising:
The kit comprises the chimeric nucleic acid construct of claim 4,
kit. A kit according to claim 32 or claim 36, wherein
Further comprising the synthetic CRISPR nucleic acid construct of claim 2 and/or the CRISPR nucleic acid array of claim 3.
kit. A kit according to claim 32 or claim 36, wherein
The synthetic tracr nucleic acid construct is contained within an expression cassette,
kit. The kit of claim 33 or claim 37, wherein
The synthetic CRISPR nucleic acid construct is contained within an expression cassette,
kit. The kit of claim 34 or claim 38, wherein
The CRISPR nucleic acid array is contained within an expression cassette,
kit. 40. The kit of claim 35 or claim 39, wherein
The chimeric nucleic acid construct is contained within an expression cassette,
kit. The kit of claim 40, wherein
The synthetic tracr nucleic acid construct, the synthetic CRISPR nucleic acid construct and/or the CRISPR nucleic acid array is contained within one or more expression cassettes,
kit. A kit according to claim 32 or claim 36, wherein
Further comprising primers for generating a CRISPR array,
kit. The kit according to any one of claims 32 to 46,
Further comprising a Cas9 nuclease,
kit. The kit according to any one of claims 32 to 35,
Further comprising the Cas9 nuclease of any one of claims 5 to 11,
kit. A kit according to any one of claims 36 to 39, wherein
Further comprising the Cas9 nuclease of claim 5 or claim 11,
kit. 50. The kit of any one of claims 32-49, further comprising instructions for use,
kit.