JP7429057B2 - Methods and compositions for sequences that guide CAS9 targeting - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Published on the following website on October 16, 2014: http://www. cell. com http://www. cell. com/molecular-cell/fulltext/S1097-2765(14)00751-5

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

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

The CRISPR-Cas system consists of an array of short DNA repeats separated by hypervariable sequences and flanked by cas genes that create adaptive immunity against invasive genetic agents such as phages and plasmids through sequence-specific targeting and interference. Provide (Barrangou et al. 2007. Science. 315: 1709-1712; Browns et al. 2008. Science 321: 960-4; Horvath and Barrangou. 2010. Science. 327: 16 7-70; Marraffini and Sontheimer. 2008. Science. 322:1843-1845; Bhaya et al. 2011. Annu. Rev. Genet. 45: 273-297; Terns and Terns. 2011. Curr. Opin. Microbiol. 14: 321-327; Westra et al.2012. Annu. Rev. Genet. 46:311-339; Barrangou R. 2013. RNA. 4:267-278). Typically, invasive DNA sequences are acquired as new "spacers" (Barrangou et al. 2007. Science. 315:1709-1712), each paired with a CRISPR repeat, creating a new repeat at the CRISPR locus. Inserted as a spacer unit. The repeat-spacer array is then transcribed as long pre-CRISPR RNA (pre-crRNA) (Browns et al. 2008. Science 321:960-4) and small interfering CRISPR RNA (pre-crRNA) that drives sequence-specific recognition. crRNA). Specifically, crRNA guides nucleases toward complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Garneau et al. 2010. Nature. 468:67-71; Haurwitz et al. al.2010.Science.329:1355-1358;Sapranauskas et al.2011.Nucleic Acid Res.39:9275-9282;Jinek et al.2012.Science.337:816-821;Ga siunas et al. 2012. Proc. 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 nearly half of bacteria (about 46%) and the majority of archaea (about 90%). They identify three main CRISPR-Cas proteins based on Cas gene content, organization and diversity in the biochemical processes that drive crRNA biogenesis, and Cas protein complexes that mediate target recognition and cleavage. 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, special Cas endonucleases process the pre-crRNA and then assemble it into large multi-Cas protein complexes that can recognize and cleave nucleic acids complementary to the crRNA. A different process relates to Type II CRISPR-Cas systems. Here, pre-CRNA is processed by a mechanism in which transactivating 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 searches for a target dsDNA sequence (the "protospacer" sequence) that is complementary to the spacer sequence within the complex and cleaves both strands. Recognition and cleavage of the target by the complex in the type II system not only requires complementary sequences between the spacer sequence on the crRNA-tracrRNA complex and the target "protospacer" sequence, but also A protospacer adjacent motif (PAM) sequence located at the 3' end is also required. The exact PAM arrangement required may differ between different Type II systems.

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

One aspect of the invention provides an optional anti-zipper sequence comprising, in the 5' to 3' direction, at least about 3 nucleotides; a bulge sequence comprising at least about 3 nucleotides; - 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 TCAAACAAAGCTTCAGC nucleotides and a hairpin sequence comprising a nucleotide sequence with at least one hairpin (e.g., tracrRNA, tracrDNA), wherein the hairpin has at least three containing matching base pairs,
where 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 the nexus sequence is located immediately upstream of the hairpin sequence.

A second aspect of the invention provides an optional zipper sequence comprising, in the 3' to 5' direction, at least about 3 nucleotides (which hybridizes to the anti-zipper of the tracrRNA if present), at least 2 a bulge sequence comprising a nucleotide sequence of (for example a (-NN-) nucleotide sequence), a stitch sequence comprising a nucleotide sequence of NNTNN (or NNUNN) which hybridizes to the anti-stitch of the tracrRNA, G _R1 comprising the nucleotide G or GTT, and a synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) construct, 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 G _R1 . and G _R1 is located immediately upstream of the spacer sequence.

A third aspect of the invention provides a synthetic CRISPR nucleic acid array comprising nucleotide sequences encoding two or more CRISPR nucleic acid constructs of the invention, wherein the two or more CRISPR nucleic acid constructs are mutually exclusive on said nucleotide sequences. located immediately adjacent to each other, the zipper sequences of said two or more CRISPR nucleic acid constructs are identical, if present, the stitch sequences of said two or more CRISPR nucleic acid constructs are identical, and the spacer sequences of said two or more CRISPR nucleic acid constructs are identical; Sequences may be 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 at least about 70% complementary to and hybridizes with the anti-zipper sequence of the synthetic tracr nucleic acid construct, and the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and hybridizes with 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 provides for contacting 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 resulting in hybridization of the spacer sequence to the target DNA. A method for site-specific cleavage of double-stranded target DNA is provided, the method comprising the step of causing site-specific cleavage of the target DNA within a defined region.

A sixth aspect of the invention provides site-specific cleavage of double-stranded target DNA comprising contacting a transcoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of Cas9 nuclease. provide a method for
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; nucleotides of NNANN; Anti-stitch sequences comprising the sequences; 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), a nexus sequence comprising a nucleotide sequence of GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and a hairpin sequence comprising a nucleotide sequence having at least two hairpins. each hairpin contains at least three matching base pairs, 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; and (b) the CRISPR nucleic acid molecule is located 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 (e.g. a (-NN-) nucleotide sequence), a nucleotide sequence of NNTNN (or NNUNN) a stitch sequence, G _R1 comprising the nucleotides G or GTT, and a spacer sequence having a 5' end and a 3' end, at least 7 nucleotides having 100% identity with the target DNA at the 3'end; 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 G _R1 . , and G _R1 is located immediately upstream of the spacer sequence, and
Further herein, the anti-zipper sequence and the zipper sequence, if present, 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 hybridizes to at least one portion of the target DNA. (e.g., at least about 7 contiguous nucleotides (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) of the target DNA, and any range or variation therein) (adjacent to the protospacer adjacent motif (PAM) on the target DNA) that is at least about 80% complementary to and hybridizes with the CRISPR nucleic acid molecule to the target DNA. Site-specific cleavage of the target DNA occurs within the region defined by the complementary binding of the spacer sequence.Thus, in a typical embodiment, the spacer sequence of the CRISPR nucleic acid molecule is located within the region of the target DNA sequence adjacent to the PAM. The target sequence can comprise, consist essentially of, or consist of 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 contacting the double-stranded target DNA with a chimeric nucleic acid comprising:
(a) an optional anti-zipper sequence comprising at least about 3 nucleotides in the 5' to 3'direction; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising a 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 nexus sequence comprising a nucleotide sequence of (A/G) (A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and a first nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin ( The hairpin comprises at least three 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) an optional zipper sequence comprising at least about 3 nucleotides in the 3' to 5' direction (which, if present, hybridizes to the anti-zipper sequence of the first nucleotide sequence); at least 2; a bulge sequence comprising a nucleotide sequence having nucleotides _of a second nucleotide sequence (the zipper sequence is present If G R1 is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of G _R1 , and G _R1 is located immediately upstream of the spacer sequence. located); and
(c) a third nucleotide sequence encoding an amino acid sequence having at least 80% identity with an amino acid sequence encoding Cas9 nuclease;
wherein the anti-zipper and zipper sequences, if present, 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 one of the target DNAs. hybridizes with a portion (e.g., at least about 7 contiguous nucleotides, preferably up to about 20 contiguous nucleotides) of said target DNA (adjacent to a protospacer adjacent motif (PAM) on the target DNA); Thereby, site-specific cleavage of the target DNA occurs within the region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA.

An eighth aspect of the invention includes a method of site-specific targeting of a polypeptide of interest to a double-stranded (ds) target DNA, wherein a chimeric nucleic acid construct of the present disclosure or an expression cassette comprising said chimeric nucleic acid construct is targeted to contacting the DNA, thereby targeting a polypeptide of interest fused to Cas9 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 includes a method for site-specific targeting of a polypeptide of interest to a double-stranded (ds) target DNA,
contacting a transcoding CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with 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 sequence; 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 TCAAACAAAGCTTCAGC; and by a nucleotide sequence comprising a hairpin sequence comprising a nucleotide sequence having at least one hairpin the hairpin contains at least three 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. 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 an optional zipper sequence comprising at least about 3 nucleotides that hybridizes to an anti-zipper sequence in the 3' to 5' direction, a nucleotide sequence having at least 2 nucleotides, e.g. a bulge sequence comprising a nucleotide sequence of (-NN-)), a stitch sequence comprising a nucleotide sequence of NNTNN, G _R1 comprising a nucleotide G or GTT, and a spacer sequence having a 5' end and a 3'end;'encoded by a nucleotide sequence containing a spacer sequence containing at least 7 nucleotides with 100% identity to the target DNA at the end; the zipper sequence, if present, is located immediately upstream of the bulge sequence; the sequence is located immediately upstream of the stitching sequence, the stitching sequence is located immediately upstream of G _R1 , and G _R1 is located immediately upstream of the spacer sequence, and
Further herein, the Cas9 nuclease contains a mutation in the HNH active site motif, a mutation in the RuvC active site motif, is fused to a 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 a protospacer adjacent motif (PAM) on the target DNA, thereby Site-specific targeting of the polypeptide of interest to the target DNA occurs within the region defined by hybridization of the spacer sequences of the CRISPR nucleic acid molecule.

Further provided herein are expression cassettes, cells and kits comprising 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.

Figure 3 shows multiple sequence alignments for the Nexus module. Figure 3 shows a maximum likelihood tree for the nexus module. Figures 3A-3D show consensus sequences for the Nexus module. Figure 3A shows the consensus sequence for the Sth Cr1 group, Figure 3B shows the consensus sequence for the Sth Cr3 group, Figure 3C shows the consensus sequence for the Lrh group, and Figure 3D shows the consensus sequence for the Lbu group. Maximum likelihood tree for Cas9 nuclease is shown. Figure 3 shows a multiple sequence alignment for the anti-stitch module. Figures 6A-6D show the consensus sequence for the anti-stitch module. Figure 6A shows the consensus sequence for the Sth Cr1 group, Figure 6B shows the consensus sequence for the Sth Cr3 group, Figure 6C shows the consensus sequence for the Lrh group, and Figure 6D shows the consensus sequence for the Lbu group. Figure 3 shows a multiple sequence alignment for the bulge module. Figures 8A-8D show the consensus sequence for the bulge module. Figure 8A shows the consensus sequence for the Sth Cr1 group, Figure 8B shows the consensus sequence for the Sth Cr3 group, Figure 8C shows the consensus sequence for the Lrh group, and Figure 8D shows the consensus sequence for the Lbu group. Figure 3 shows a multiple sequence alignment for the zipper module. Figure 3 shows a maximum likelihood tree for the zipper module. Multiple sequence alignments for bulge, anti-stitch and nexus modules are shown. Sequence and structural details of the CRISPR-Cas system element of Streptococcus thermophilus CR3 are shown, representing the Sth CR1 group. Figure 2 shows the sequence and structure details of the CRISPR-Cas system factors of Lactobacillus buchneri and represents the Lbu group. Sequence and structural details of the CRISPR-Cas system element of Streptococcus thermophilus CR1 are shown, representing the Sth CR1 group. Sequence and structural details of the CRISPR-Cas system element of Streptococcus pyrogenes M1 GAS are shown, representing the Sth CR3 group. Figure 2 shows the sequence and structure details of the CRISPR-Cas system elements of Lactobacillus rhamnosus and represents the Lrh group. Figure 2 shows sequence and structure details of the CRISPR-Cas system factors of Lactobacillus animalis and represents the Lan group. Sequence and structural details of the CRISPR-Cas system factors of Lactobacillus casei are shown and represent the Lca group. Sequence and structural details of the CRISPR-Cas system factors of Lactobacillus gasseri are shown and represent the Lga group. Figure 2 shows sequence and structure details of the CRISPR-Cas system factors of Lactobacillus jensenii and represents the Lje group. Sequence and structural details of the Lactobacillus pentosus CRISPR-Cas system factors are shown and represent the Lpe group. Figure 2 shows the sequence and structure details of the CRISPR-Cas system element of Streptococcus pyrogenes M1 GAS. Congruence between tracrRNA (left), CRISPR repeats (middle) and Cas9 (right) sequence clustering is shown. A consistent grouping into three families is seen across the three sequence-based phylogenetic trees. Figures 24A-24B depict the Cas9:sgRNA family. Figure 24A shows a phylogenetic tree based on Cas9 protein sequences from various Streptococcus and Lactobacillus species. Sequences were clustered into three families in blue, orange and green. Figure 24B shows the predicted guide RNA consensus sequence and secondary structure for each family. Each consensus RNA consists of crRNA (left) base-paired with tracrRNA. Completely conserved bases are represented in color, variable bases are represented in black (2 possible bases) or black dots (at least 3 possible bases), and are always present. Base positions that do not have a specific meaning are circled. Circles between positions indicate base pairing that is present only in some family members. CRISPR repeat sequence alignment is shown. For each cluster, CRISPR repeat sequence alignments are shown with conserved consensus nucleotides designated under each family: Sth3 (top), Sth1 (middle) and Lb (bottom) families. tracrRNA sequence alignment is shown. For each cluster, experimentally determined or computationally predicted tracrRNA sequence alignments are shown, with conserved consensus nucleotides designated under each family: Sth3 (top), Sth1 (middle) and Lb (bottom) families. . sgRNA nexus sequence alignment is shown. Universally conserved residues are colored red. 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. Figures 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 psgRNA plasmids (Figure 28B) and used in combination with each of the desired Cas9s described in Figure 3. Figures 29A-29C show sgRNA orthogonality. Figure 29A shows Streptococcus thermophilus CRISPR3-Cas9 (top, blue) and S. thermophilus CRISPR3-Cas9 (top, blue). The sgRNA sequence of C. thermophilus CRISPR1-Cas9 (bottom, orange) is shown. Figure 29B shows the protospacer-targeting scheme. The predicted PAM for each sgRNA is shown. Triangles indicate putative cleavage sites for each Cas9. FIG. 29C shows E. Cas9 in E. coli: Chimera-sgRNA orthogonality. Chimeric sgRNA. E. coli expressing SthCRISPR3 Cas9 (blue) and/or SthCRISPR1 Cas9 (orange). Each sgRNA (left) was subjected to transformation assay (right) in E. coli. Low transformation efficiency is due to E. Figure 2 shows a functional Cas9:sgRNA pair via lethal self-targeting of the E. coli genome. Values are S. of three independent experiments. E. M. and reflect the geometric mean. Figure 3 shows CRISPR interference with complementary DNA in Lactobacillus gasseri as an inability to initially transform a plasmid containing a protospacer sequence matching the 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. Figure 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 3 shows CRISPR interference with complementary DNA in Lactobacillus rhamnosus 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 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. The description is not intended to be a detailed list of all the different ways in which the invention may be implemented or all the features that may be added to the invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Accordingly, the present invention anticipates that in some embodiments of the invention, any feature or combination of features presented herein may be excluded or omitted. Additionally, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure and do not depart from the invention. Therefore, the following description is intended to describe some particular embodiments of the invention and is not intended to exhaustively identify all permutations, combinations, and modifications thereof.

Unless otherwise defined, 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 herein to describe the invention 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 citations herein are incorporated by reference in their entirety for the teachings associated with the sentence and/or paragraph in which the citation is cited.

It is expressly intended that the various features of the invention described herein can be used in any combination, unless the context dictates otherwise. Additionally, the present invention anticipates that any feature or combination of features presented herein may be excluded or omitted in some embodiments of the invention. To illustrate, when the specification states that the composition includes components A, B, and C, any of A, B, or C, or combinations thereof, alone or in any combination, may be omitted and discarded. It is clearly intended that it be possible to do so.

As used in the description of the invention and 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.

In addition, "and/or" as used herein includes any and all possible combinations of one or more of the related listed items, and when interpreted selectively ("or"), Mention and include lack of combination.

As used herein, the term "about" when referring to a measurable value such as a dose or a period of time is ±20%, ±10%, ±5%, ±1%, ±0.5% of a specified amount. , or even refer to a variation of ±0.1%.

As used herein, phrases such as "between X and Y" and "between about X and Y" should be construed to include X and Y. be. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and "between about X and about Y"; Phrases such as "from about X to Y" mean "from about X to about Y."

As used herein, "bulge sequence" refers to non-complementary (non-hybridizing) nucleotide sequences included 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-identical) to the corresponding bulge sequence in the synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array. , about 3 nucleotides to about 6 nucleotides (e.g., about 3, 4, 5, 6 nucleotides; e.g., about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, about 3 to about 6 nucleotides; (approximately 4 nucleotides). The bulge sequence of a synthetic CRISPR nucleic acid construct/CRISPR nucleic acid array is located between the zipper and stitch sequences and between the zipper and stitch sequences and has at least two nucleotides (e.g., a (-NN-) nucleotide sequence) ( For example, about 2, 3, 4, 5, 6 nucleotides; eg, about 2 to about 6 nucleotides, about 2 to about 5 nucleotides, about 2 to about 4 nucleotides; about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, etc.), consisting essentially of, or consisting of. The nucleotide composition of the bulge sequence may be any series of at least two (synthetic CRISPR nucleic acid constructs/CRISPR nucleic acid arrays) or three, as long as they are not complementary (e.g., 100% non-identical) and therefore do not hybridize to each other. (synthetic tracr nucleic acid construct). As a result of the non-complementarity of the bulge sequences on synthetic tracr nucleic acid constructs and synthetic CRISPR nucleic acid constructs/CRISPR nucleic acid arrays, when anti-zipper to zipper sequences and anti-stitch to stitch sequences line up and hybridize (in chimeric nucleic acid constructs) ), a protrusion or bulge is formed on the synthetic tracr nucleic acid construct side 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 functioning of the CRISPR-Cas system.

As used herein, the terms "comprise," "comprises," and "comprising" refer to the presence of the described feature, integer, step, operation, factor, and/or component. Although specified, this does not preclude the presence or addition of one or more other features, integers, steps, operations, factors, components, and/or groups thereof.

As used herein, the provisional phrase "consisting essentially of" means that the scope of the claim includes the particular materials or steps recited in the claim and the essential novelty of the claimed invention. is meant to be construed as including those that do not materially affect the feature(s) of. Accordingly, the term "consisting essentially of" when used in the claims of the present invention is not intended to be construed as 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, eg, WO2013/176772; WO/2013/188638). The domains for catalyzing dsDNA cleavage are the RuvC domain and the HNH domain. The RuvC domain has the role of nicking the (-) chain, and the HNH domain has the role of nicking the (+) chain (for example, see 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 are obtained from different sources (eg, different organisms, different coding regions).

"Complementary" as used herein can mean 100% complementarity or identity with a comparator nucleotide sequence, or less than 100% complementarity (e.g., about 70%, 71%, 72% %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

The term "complementarity" or "complementarity" as used herein refers to the natural association 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," where only a portion of the nucleotides are linked, or it can be complete, where 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, "contact," "contacting," "contacted," and grammatical variations thereof are used to describe the desired reaction (e.g., site-specific targeting of a polypeptide of interest). Refers to the bringing together of the components of a desired reaction under appropriate conditions to carry out targeting, integration, transformation, site-specific cleavage (nicking, 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; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Har bor, NY).

A "fragment" or "portion" of a nucleotide sequence of the invention is one that has a reduced length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, is understood to mean a nucleotide sequence (shortened by 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) and is identical to the reference nucleic acid or nucleotide sequence or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) Comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides. Such a nucleic acid fragment or portion according to the invention may, if appropriate, be included in a larger polynucleotide of which it is a constituent. Thus, for example, hybridizing to (or hybridize to, and other grammatical variations thereof) to at least a portion of the target DNA is defined as or refers to hybridization to substantially identical nucleotide sequences.

As used herein, “G _R1 ” is a single nucleotide (G) or short three nucleotide sequence (GTT) contained in a repeat portion of a synthetic CRISPER nucleic acid construct or crRNA. G _R1 does not hybridize with the synthetic tracr nucleic acid constructs of the present disclosure or the tracrRNA anti-repeat. However, in a non-standard Watson-Crick base pairing scheme, G _R1 can wobble base pair with a U at the end of the anti-CRISPR repeat portion of the tracrRNA.

The term "gene" as used herein refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMOs), and the like. A gene may or may not be capable of being used to produce a functional protein or gene product. A gene may include both coding and non-coding regions (eg, introns, regulatory elements, 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 from components normally found associated with the nucleic acid in its natural state. Such components include other cellular materials, culture media from recombinant production, and/or various chemicals used in the chemical synthesis of nucleic acids.

A "hairpin sequence" as used herein is a nucleotide sequence that includes a hairpin. A hairpin (eg, stem-loop, feedback) refers to a nucleic acid molecule having a secondary structure that includes a region of nucleotides forming a double strand further flanked by a single-stranded region on either side. Such structures are well known in the art. As is known in the art, double-stranded regions may contain some mismatches in base pairing or may be completely complementary. In some embodiments of the present 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 the hairpin may be involved in Cas9 binding to the crRNA-tracrRNA complex (e.g., synthetic CRISPR nucleic acid constructs - synthetic

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

Different nucleic acids or proteins that have homology are referred to herein as "homologues." The term homolog 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. Accordingly, the compositions and methods of the invention further include homologs to the nucleotide and polypeptide sequences of the invention. "Orthologous" as used herein refers to homologous nucleotide and/or amino acid sequences in different species that arise from a common ancestral gene during speciation. Homologs of the nucleotide sequences of the invention are those that have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%) with the nucleotide sequences of the invention. , 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 or higher to any one of the Cas9 sequences provided herein.

As used herein, hybridization, hybridize, hybridizing, and grammatical variations thereof refer to the combination of two completely complementary nucleotide sequences or several mismatched bases. Refers to the binding of substantially complementary sequences in which pairs may exist. Hybridization conditions are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some embodiments, hybridization conditions may be of high stringency, or they may be of medium or low stringency, depending on the length of the sequences being hybridized and the amount of complementarity. It may be. Conditions that constitute low, medium, and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109). : E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Pres. s, Cold Spring Harbor, NY).

As used herein, the terms "increase", "increase", "increase", "enhance", "enhance", "enhancing" ” and “enhancement” (and their grammatical changes) are at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% compared to the control. represents an increase of % or more.

The term "invasive exogenous genetic element," "invasive exogenous nucleic acid," or "invasive exogenous DNA" refers to DNA that is exogenous to bacteria, such as, but not limited to, viruses, bacteriophages, and/or plasmids. pathogen-derived genetic factors).

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

As used herein, "nexus sequence" refers to the nucleotide sequence located immediately downstream of the "anti-stitch sequence" within a synthetic tracr nucleic acid construct. The nexus is approximately 6-10 nucleotides in length and contains the highly conserved sequence: TNANNC. In some implementations, 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)). Without being bound to any particular theory, it is believed that based on sequence conservation, nexus may be important in Cas9 orthogonality and recognition.

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

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

The term "nucleotide sequence" as used herein refers to a heteropolymer of nucleotides or a sequence of these nucleotides at the 5' to 3' end of a nucleic acid molecule, including DNA or RNA molecules, cDNA, DNA fragments or portions, Includes genomic DNA, synthetic (eg, chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any 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 to also refer to a heteropolymer of nucleotides. Unless otherwise indicated, nucleic acid molecules and/or nucleotide sequences provided herein are presented herein from left to right in a 5' to 3' direction and are presented according to the United States Sequence Rules. (37 Code of Federal Regulations, Sections 1.821-1.825) and World Intellectual Property Organization (WIPO) Standards ST. 25 using standard codes to represent the nucleotide characteristics. As used herein, "5' region" may refer to the region of a polynucleotide closest to the 5' end. Thus, for example, an agent within the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to a nucleotide located halfway through the polynucleotide. As used herein, "3' region" may refer to the region of a polynucleotide closest to the 3' end. Thus, for example, an agent within the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to a nucleotide located halfway through the polynucleotide.

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

"Protospacer sequence" refers to target double-stranded DNA, and specifically refers to the portion of target DNA that is completely or substantially complementary to (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 " "decrease" (and grammatical changes thereof) by, for example, at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, Indicates a reduction of 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In certain embodiments, the reduction may not result in no or essentially no detectable activity or amount (ie, by an insignificant amount, such as less than about 10% or even less than 5%). Thus, in some embodiments, mutations in Cas9 nuclease increase the nuclease activity of Cas9 by at least about 5%, 10%, 15%, 20%, 25%, as compared to a control (e.g., wild-type Cas9). It may be reduced by 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

As used herein, "repeat sequences" refer to repeat sequences of, for example, wild-type CRISPR loci or synthetic CRISPR nucleic acid constructs separated by "spacer sequences." The repeat sequences are complementary (e.g., 100% base pair match), or substantially complementary, e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher).

"Repeat sequences" of the synthetic CRISPR nucleic acid constructs of the present disclosure include optional "zipper sequences,""bulgesequences,""stitchsequences," and "spacer sequences." In some embodiments, a synthetic CRISPR nucleic acid construct can include G _R1 (in other embodiments included in a stitch sequence).

As used herein, "zipper sequence" refers to an optional portion of a repeat sequence located 3' or immediately upstream (in a 3' to 5' direction) of a bulge sequence within a synthetic CRISPR nucleic acid construct, and at least Approximately 3 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides, or any range or value therein), consisting of, or consisting essentially of. In some embodiments, the zipper arrangement may be referred to as an "upper stem." The "zipper sequence" shares sufficient complementarity with the corresponding and optional "anti-zipper sequence" located on the synthetic tracr nucleic acid construct, and thus, when present, the zipper sequence and the anti-zipper sequence. can hybridize to each other when they come into contact, thereby binding the two nucleic acid constructs together. In some embodiments, the zipper/anti-zipper arrangement can be referred to as an "upper stem." The zipper sequences are fully complementary (e.g., 100% base pair match) or substantially complementary, e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher). Thus, the anti-zipper sequences of the synthetic tracr nucleic acid constructs of the invention must contain at least about 3 nucleotides ( 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). The anti-zipper sequence is an RNase III binding site and therefore includes nucleotide sequences well known in the art to be involved in RNase III binding (e.g., Pertzev and Nicholson, Nucleic Acids Res. 34(13): 3708). -3721 (2006)).

As used herein, "sequence identity" refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant over a window of alignment of constituents (eg, nucleotides or amino acids). "Identity" is not limited to: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and d Genome Projects (Smith, D.W., ed. ) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Pr ess, New York (1991). It can be easily calculated using the following method.

As used herein, a "spacer sequence" is a nucleotide sequence that is complementary to target DNA (eg, a "protospacer sequence"). The spacer sequence is completely complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or higher)). In a typical 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, so that the overall complementarity of the spacer sequence to the target DNA 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 linked to the target DNA. 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 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. are substantially complementary (eg, at least about 70% complementary). In other embodiments, the first 7-10 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA. (eg, at least about 70% complementary). In an exemplary embodiment, the first seven 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 may be substantially complementary to the target DNA ( for example, at least about 70% complementary).

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

As used herein, an "anti-stitch sequence" is a nucleotide sequence that is fully complementary and hybridizes to a stitch sequence (e.g., NNANN, ACAAA, TAAAA, (T/C) (A/G) T (A/G ) (A/G), TAAAA, TGAAA). The anti-stitch sequence is located (in the 5' to 3' direction) immediately downstream of the bulge sequence and immediately upstream of the "nexus sequence" on the synthetic tracr nucleic acid construct. 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 involves reconstructive base pairing after the "bulge sequence." It is thought that then. In some embodiments, 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 molecules, nucleotide sequences or protein sequences, when compared and aligned for maximum correspondence, refers to the following sequence comparisons: At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, as determined by measurement using one of the algorithms or by visual inspection. , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, and/or 100% nucleotide or amino acid residue identity. In some embodiments of the invention, substantial identity exists over a region of the sequences that is at least about 50 residues to about 150 residues in length. Thus, in some embodiments of the invention, substantial identity is at least about 3 to about 15 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 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 exists over a region of the sequence that is about 140, about 150, or more residues in length, and any range therein. In representative embodiments, the sequences may be substantially identical over at least about 22 nucleotides. In certain embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, sequences of the invention may be about 70% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In some embodiments, sequences of the invention may be about 75% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention may be about 80% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention may be about 80% to about 100% identical over at least about 7 nucleotides to about 25 nucleotides. In some embodiments, sequences of the invention may be about 70% identical over at least about 18 nucleotides. In other embodiments, the sequences may be about 85% identical over about 22 nucleotides. In yet other embodiments, the sequences may be 100% homologous over about 16 nucleotides. In further embodiments, the sequences are substantially identical over the length of the coding region. Furthermore, in representative embodiments, substantially identical nucleotide or protein sequences perform substantially the same function (eg, Cas9 HNH and/or RuvC nickase activity).

For sequence comparisons, typically one sequence serves 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 identity of the test sequence(s) to the reference sequence based on the specified program parameters.

Optimal sequence alignments for aligning comparison windows are well known to those skilled in the art and can be performed using tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and Optionally, this can be done by computer implementation of algorithms such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). . The "fractional identity" of an aligned section of a test and reference sequence is the number of identical components that two aligned sequences share, compared to a reference sequence segment (i.e., the entire reference sequence or a smaller portion of the reference sequence). This is the number divided by the total number of components in the specified part). Percent sequence identity is expressed as the fraction of identity multiplied by 100. Comparisons of one or more polynucleotide sequences can be made to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. 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, when aligned with words of the same length in the database array, match or satisfy some positive value threshold score T. The method includes first identifying high scoring sequence pairs (HSPs). T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. Word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (benefit score for pairs of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of word hits in each direction stops if the cumulative alignment score decreases by an amount The cumulative score becomes 0 or less due to reaching . BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989) ).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873 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 would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the minimum sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the minimum sum probability in a comparison of 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 when the two sequences hybridize to each other under stringent conditions. In some exemplary embodiments, two nucleotide sequences that are considered substantially complementary hybridize to each other under high stringency conditions.

"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridizations, are sequence-dependent and differ under different environmental parameters. A comprehensive guide to nucleic acid hybridization is available in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probe Part I Chapter 2 “Overview of principles of hybridization and the strategy of nuclear acid probe assays” Elsevier, New York ( 1993) seen in Generally, high stringency hybridization and wash conditions are selected to be about 5° C. lower than the melting point (T _m ) for the 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. Highly stringent conditions are selected to equal the T _m for the specific probe. Examples of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues in filters in Southern or Northern blots are 50% formamide (42° C.) with 1 mg heparin; Hybridization is carried out overnight. An example of high stringency wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook (below) for a description of SSC buffer). A low stringency wash is often performed before a high stringency wash to remove background probe signal. For example, for duplexes of greater than 100 nucleotides, an example of a moderate stringency wash is 1×SSC, 45° C., 15 minutes. For example, for duplexes of greater than 100 nucleotides, an example of a low stringency wash is 4-6×SSC, 40° C., 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a Na ion salt concentration of less than about 1.0M, typically about 0.01-1.0M. including a Na ion concentration (or other salt) (pH 7.0-8.3) and a temperature typically at least about 30°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Generally, in a particular hybridization assay, a signal-to-noise ratio that is twice (or higher) than that seen for an unrelated probe indicates the 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 copies of a nucleotide sequence are made using the maximum codon degeneracy allowed by the genetic code.

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

Any nucleotide sequence and/or recombinant nucleic acid molecule of the invention can be codon-optimized for expression in any desired species. Codon optimization is widely known in the art and involves modifying nucleotide sequences for codon usage bias using species-specific codon usage. Codon usage is generated 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 generated 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 understood in the art, codon optimization of a nucleotide sequence is defined as codon optimization of a nucleotide sequence with less than 100% identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%). %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.), but still has the same function as that encoded by the original natural nucleotide sequence. Generate a nucleotide sequence. Thus, in a representative embodiment of the invention, the nucleotide sequences and/or recombinant nucleic acid molecules of the invention 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 "separated." An “isolated” nucleic acid molecule, nucleotide sequence, or “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence that exists apart from its natural environment in the hands of man and is therefore not a product of nature. or a polypeptide. The isolated nucleic acid molecule, nucleotide sequence or polypeptide is associated with at least a portion of other components of an organism or virus of natural origin, such as structural components of a cell or virus or other polypeptides or polynucleotides. may exist in purified form, at least partially separated from the nucleic acids commonly found as In representative embodiments, the isolated nucleic acid molecules, isolated nucleotide sequences and/or isolated polypeptides have 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 present in a non-natural environment (eg, a recombinant host cell). Thus, for example, with respect to a nucleotide sequence, the term "isolated" means that it is separated from the naturally occurring chromosome and/or cell. Also, a polynucleotide can be separated from the chromosome and/or cell in which it naturally occurs and then placed in a genetic context, chromosome and/or cell in which it does not naturally occur (e.g., a different host cell, a different regulatory sequence, and/or or at a location 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 exist apart from their natural environment in the hands of man and are therefore not products of nature; In some embodiments, it can be introduced and present within a recombinant host cell.

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

By "operably linked" or "operably associated" as used herein, it is meant that the indicated factors are functionally related to each other, and generally also physically related. Thus, the terms "operably linked" or "operably related" as used herein refer 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 a situation in which 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 transcription or expression of the nucleotide sequence. Those skilled in the art will appreciate that a control sequence (eg, a promoter) need not be contiguous with the nucleotide sequence with which it is operably associated, so long as the control sequence functions to direct its expression. Thus, for example, an intervening untranslated but still transcribed sequence may be present between the promoter and the nucleotide sequence and the promoter can still be considered "operably linked" to the nucleotide sequence.

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

Promoters include, for example, constitutive, inducible, temporally regulated, developmentally regulated, Chemically controlled, tissue-friendly and/or tissue-specific promoters may be included. These various types of promoters are known in the art.

The choice of promoter will vary depending on the temporal and spatial requirements for expression and will also vary depending on the host cell being transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge existing in the art, an appropriate promoter can be selected for a particular host organism of interest. Thus, for example, much is known about upstream promoters of highly constitutively expressed genes in model organisms, and such knowledge can be easily utilized and implemented in other systems, if desired. can.

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

An expression cassette containing a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette may also be of natural origin but obtained recombinantly, which is useful for heterologous expression.

The expression cassette may also optionally include a transcription and/or translation termination region (ie, a termination region) that is functional in the selected host cell. A variety of transcription termination factors are available for use in expression cassettes and are responsible for terminating transcription beyond the heterologous nucleotide sequence of interest and modifying polyadenylation of the mRNA. The termination region can be native to the transcription initiation region, native to the nucleotide sequence to which it is operably linked, native to the host cell, or another may be derived from a source (ie, exogenous or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).

The expression cassette can also include nucleotide sequences for selectable markers that can be used to select transformed host cells. As used herein, a "selectable marker", when expressed, confers on a host cell expressing the marker a distinguishable phenotype, thus distinguishing such traits from those without the marker. means a nucleotide sequence that allows transformed cells to be distinguished. Such nucleotide sequences determine whether the marker confers a trait that can be selected for by chemical means, e.g. by using a selective agent (e.g. an antibiotic, etc.), or whether the marker simply confers a trait that can be selected for, e.g. by screening (e.g. Depending on whether the trait can be identified through observation or testing (by fluorescence), it may encode either a selectable marker or a screenable marker. 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 conjunction with vectors. The term "vector" refers to a composition for transferring, delivering, or introducing nucleic acid(s) into a cell. Vectors include nucleic acid molecules that contain 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, viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmids, of double-stranded or single-stranded linear or circular form. Vectors, including bacteriophages, artificial chromosomes, or Agrobacterium binary vectors, may or may not be capable of self-transmission or recruitment. A vector as defined herein can transform a prokaryotic or eukaryotic host by integration into the cellular genome, or it can be extrachromosomal (e.g., a self-replicating plasmid with an origin of replication). ). A DNA vehicle that is capable of replication in two different host organisms, which may be selected from actinobacteria and related species, bacteria and eukaryotes (e.g. higher plants, mammals, yeast or fungal cells), naturally or by design. Further included are shuttle vectors, meaning . In some exemplary embodiments, the nucleic acid within the vector is under the control of and operably linked to a suitable promoter or other control element for transcription in the host cell. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other control elements; in the case of cDNA, it is under the control of a suitable promoter or other control element for expression in the host cell. It can be. Accordingly, the nucleic acid molecules and/or expression cassettes of the invention may be contained within vectors as described herein and known in the art.

"Introducing," "introducing," "introducing" (and grammatical variations thereof) in the context of a polynucleotide of interest means that the nucleotide sequence of interest is transferred to a host organism or It means to present a nucleotide sequence to a cell of the organism (eg, a host cell) in such a way that the nucleotide sequence is accessible inside 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 polynucleotide or nucleic acid constructs, and can be identical or Can be located on different expression constructs or transformation vectors. Thus, these polynucleotides can be introduced into cells in a single transformation event, in separate transformation/transfection events, or they can be taken up into an organism by conventional breeding protocols, for example. . Thus, in some aspects of the invention, one or more nucleic acid constructs of the invention (e.g., synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, synthetic CRISPR arrays, chimeric nucleic acid constructs; nucleotide sequences encoding a polypeptide of interest) , a nucleotide sequence encoding a cas9 nuclease, etc.) can be introduced into a host organism or a cell 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, a host cell or host organism is stably transformed with a nucleic acid molecule of the invention. In other embodiments, a host cell or host organism is transiently transformed with a recombinant nucleic acid molecule of the invention.

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

"Stable introducing" or "stablely introduced" in the context of polynucleotides introduced into cells means that the introduced polynucleotide is stably introduced into the genome of the cell. It is contemplated that the polynucleotide will be taken up and thus the cells will be stably transformed with the polynucleotide.

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

Transient transformation can be performed by enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into the organism. It can be detected by, for example. Stable transformation of a cell involves transfection of a cell's genomic DNA with a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of a transgene introduced into an organism (e.g., a plant, mammal, insect, archaea, bacterium, etc.). can be detected by Southern blot hybridization assay or the like. Stable transformation of a cell can be detected, such as by Northern blot hybridization assay of the RNA of the cell, using a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of the transgene introduced into the plant or other organism. Can be done. Stable transformation of cells can also be performed using, for example, polymerase chain reaction (PCR) or methods well known in the art, using specific primer sequences that hybridize to the target sequence(s) of the transgene. Detection can also be performed 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 can be expressed transiently and/or they can be stably integrated into the genome of the host organism.

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

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

Thus, nucleotide sequences can be introduced into a host organism or cells thereof by any number of methods well known in the art. The methods of the invention are not dependent on the particular method by which the nucleotide sequence or sequences are introduced into the organism, as long as they have access to the interior of at least one cell of the organism. If more than one nucleotide sequence is introduced, they can be assembled as part of a single nucleic acid construct or as separate nucleic acid constructs and can be located on the same or different nucleic acid constructs. can. Thus, the nucleotide sequence can be introduced into the cells of interest in a single transformation event or in separate transformation events, or, if relevant, the nucleotide sequence can be introduced as part of a breeding protocol. Can be taken into plants.

The present invention provides compositions with increased efficiency and increased specificity for site-specific nicking, cleavage and/or modification of target DNA and for site-specific targeting of polypeptides of interest to target DNA. and on methods.

Nucleic acid constructs and nucleotide sequences of the invention may be represented in alternative ways that do not affect the overall structure or function of the construct or sequence. Thus, for example, in some cases, a synthetic CRISPR nucleic acid (crRNA) can include a wobble base G _R1 sequence (G/U) and a stitch sequence; It may contain a stitched sequence and therefore does not further represent the G _R1 sequence. In some cases, GR1 does not match (ie, G/A is mismatched with Lje, FIG. 20). Additional equivalences include those shown in the equivalence table (Table 1) provided below (see also Figure 22).

Accordingly, in one aspect of the invention, a synthetic transcoded CRISPR (tracr) nucleic acid (e.g., tracrRNA, tracrDNA) construct is provided, 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 a 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) a nexus sequence comprising the nucleotide sequence of AA (A/C) (C/A) (A/G) (A/U)), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and has at least one hairpin comprises, consists essentially of, or consists of a hairpin sequence comprising a nucleotide sequence, said hairpin comprising at least three matching base pairs, wherein the anti-zipper sequence is 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. In some embodiments, the anti-stitch sequence comprises the nucleotide sequence NNANN, ACAAA, TAAAA, (T/C) (A/G) T (A/G) (A/G), TAAAA, TGAAA. , consisting essentially of or consisting 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 TCAAACAAAGCTTCAGC nucleotides a synthetic transcoded CRISPR comprising, consisting essentially of, or consisting of a nexus 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 three matching base pairs, wherein the anti-zipper sequence, if present, 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 located immediately upstream of the nexus sequence, and the nexus sequence located 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 other embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of 5 nucleotides. In other embodiments, the hairpin sequence of the synthetic tracr nucleic acid construct comprises, consists essentially of, or consists of at least two hairpins, each hairpin comprising at least three matching base pairs.

In a further aspect, the invention provides an optional zipper sequence comprising, consisting essentially of, or consisting of, in the 3' to 5' direction, at least about 3 nucleotides, a nucleotide sequence having at least 2 nucleotides. (e.g., a nucleotide sequence of (-NN-)); a stitched sequence comprising, consisting essentially of, or consisting of a nucleotide sequence of NNTNN (or NNUNN); G R1 comprising, consisting essentially of, or consisting of the nucleotide G _or GTT, and a spacer sequence having a 5' end and a 3' end, the 3' end of which has 100% identity with the target DNA. Provided are synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) constructs 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; is located immediately upstream of the bulge sequence, if present, the bulge sequence is located immediately upstream of the stitching sequence, the stitching sequence is located immediately upstream of G _R1 , and G _R1 is the spacer sequence Located just upstream of.

In a further embodiment, 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 bulge comprising a nucleotide sequence of at least about 2 nucleotides; sequence, a stitched sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end, the 3' end of which comprises at least 7 nucleotides with 100% identity to the target DNA. Synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) constructs are provided that include, consist essentially of, or consist of a spacer sequence, the zipper sequence, if present, being located immediately upstream of the bulge sequence; is located immediately upstream of the stitch array, which is located immediately upstream of the spacer array.

In some embodiments, a synthetic CRISPR nucleic acid array is provided, said synthetic CRISPR nucleic acid array comprising 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 next to each other on the nucleotide sequence, the stitch sequences of the two or more CRISPR nucleic acid constructs are identical, the spacer sequences of the two or more CRISPR nucleic acid constructs are identical or non-identical, and, if present, The zipper sequences of the two or more CRISPR nucleic acid constructs are identical.

In other aspects, 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 invention, wherein the zipper sequence of the synthetic CRISPR nucleic acid construct is anti-zipper sequence and at least about 70% (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% identical to and hybridizes with the anti-stitch sequence of the synthetic tracr nucleic acid construct, and 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 other embodiments, a chimeric nucleic acid construct is provided comprising, consisting essentially of, or consisting of a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct of the invention, wherein NNUNN of the stitched sequence of the synthetic CRISPR nucleic acid construct is , is 100% complementary to and hybridizes with the anti-stitch sequence NNANN of the synthetic tracr nucleic acid construct, and (G) of the stitch sequence forms a wobble base pairing with the U of the anti-stitch sequence, and the 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 if a zipper sequence and an anti-zipper sequence are present, the zipper sequence of the synthetic CRISPR nucleic acid construct is non-complementary. Hybridizes to the zipper array.

In some embodiments, the chimeric nucleic acid construct optionally further comprises nucleotides at the ends of the hybridized sequences (distal to the bulge sequence) that link the hybridized zipper and anti-zipper sequences. can. In a further embodiment, in the absence of a zipper and anti-zipper, the chimeric nucleic acid construct can optionally further include nucleotides that link the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid. The connecting nucleotides can be any nucleotides (e.g., T, A, G, C), and the number of nucleotides connecting the zipper sequence and the anti-zipper or bulge sequence is from about 3 to about 7. could be.

In a further aspect, a 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 (e.g., about 70% identity) 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.); , an amino acid sequence having at least 70% identity to an amino acid sequence encoding Cas9 nuclease. 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 contain a HNH motif and a RuvC motif (see, eg, WO2013/176772; WO/2013/188638). In some embodiments, the HNH motif or RuvC motif may contain mutations that reduce or eliminate their activity compared to wild-type Cas9 nuclease. In some embodiments, only one motif (eg, either the HNH motif or the RuvC motif) is mutated. In other embodiments, both motifs are mutated so that both activities are reduced or eliminated. Any type of mutation can be used to reduce or eliminate the activity of the HNH motif and/or RuvC motif in Cas9 nuclease, including missense mutations, nonsense mutations, frameshift mutations, etc.

This disclosure identifies various CRISPR-Cas systems and groupings of Cas9 nucleases. These groupings are: Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, Cas9 nucleases Lactobacillus buchneri CD034 (Lb) group of, and Lactobacillus rhamnosus GG of Cas9 nuclease (Lrh) group. Non-limiting examples of Sth CR1 group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 1-9 and 51. Non-limiting examples of Sth CR3 group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 10-23. Non-limiting examples of Lb group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 28, 30-33, 35, 43, 44, 47, 50 and 52. Non-limiting examples of Lrh group Cas9 nucleases include the Cas9 nucleases encoded by the polypeptide sequences of SEQ ID NOs: 24-27, 29, 34, 36-42, 45 and 53. Additional Cas9 nucleases include, but are not limited to, those of Lactobacillus curvatus CRL 705. Additional Cas9 nucleases useful in the invention include, but are not limited to, Cas9 from Lactobacillus animalis KCTC 3501, and Lactobacillus farciminis WP 010018949.1.

Thus, in some embodiments, the Cas9 nuclease is a Cas9 from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, a Streptococcus thermophilus CRISPR 3 of Cas9 nucleases (Sth CR3) group-derived Cas9, Lactobacillus buchneri of 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. In further embodiments, 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 comprises any one of SEQ ID NO: 1 to SEQ ID NO: 53. comprises, consists essentially of, or consists of a nucleotide sequence encoding an amino acid sequence having at least 70% identity with the amino acid sequence.

Additionally, 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 other embodiments, the Cas9 nuclease may include at least one nuclear localization sequence.

The inventors have surprisingly discovered a functional pairing between a specific 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; If GGCTAGTCC, The Cas9 nuclease is derived from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases; if the nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is derived from the Lactobacillus nuclease. is derived from the S. illus buchneri CD034 (Lb) group; or , when the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, the Cas9 nuclease is from the Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.

As described herein, Cas9 nucleases useful in the invention may include mutations in the HNH motif and/or the RuvC motif, thereby reducing or eliminating the activity of the respective motif. As is known in the art, mutations within the HNH motif reduce/eliminate site-specific nicks on the (+) strand of double-stranded target DNA, and mutations in the RucV active site reduce/eliminate site-specific nicks on the (+) strand of double-stranded target DNA. Reduce/eliminate site-specific nicks in the (-) strand of DNA. Mutation of both active sites reduces/eliminates DNA cleavage (ie, reduces/eliminates site-specific cleavage of target DNA). Thus, in some embodiments, synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure include a Cas9 nuclease with a mutation within the RuvC active site motif. In other embodiments, synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the present disclosure include a Cas9 nuclease with a mutation in the HNH active site motif. In still further 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 include a Cas9 nuclease with mutations within the HNH active site motif and within the RuvC motif.

In yet a further embodiment, the Cas9 nuclease having mutations within the HNH and RuvC motifs, thereby reducing or eliminating nuclease activity, further comprises a polypeptide of interest fused to the Cas9 nuclease. Such a Cas9-polypeptide of interest fusion protein can be used to direct or target a polypeptide of interest to a specific 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. Accordingly, in some embodiments, a chimeric nucleic acid construct of the present disclosure or an expression cassette comprising a chimeric nucleic acid construct of the present disclosure is contacted with target DNA in the presence of a Cas9 nuclease (e.g., SEQ ID NO: 1-53), provides a method for site-specific cleavage of double-stranded target DNA, comprising producing site-specific cleavage of 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, and the Cas9 nuclease contains a mutation within the RuvC active site motif, thereby , cleavage of the (+) strand of the double-stranded target to generate a site-specific nick in the (+) strand of the double-stranded target DNA. In other embodiments, the site-specific cleavage is a site-specific nick of the (-) strand of double-stranded target DNA, and the Cas9 nuclease contains a point mutation within the HNH active site motif, thereby 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: transcoding a CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with a Cas9 nuclease (e.g., SEQ ID NO: 1). ~53), wherein: (a) the tracr nucleic acid molecule comprises an anti-zipper sequence comprising at least about 3 nucleotides in the 5' to 3'direction; bulge sequence containing the nucleotide sequence of NNANN; TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG; and at least one the anti-zipper sequence is encoded by a nucleotide sequence comprising, consisting essentially of, or consisting of two hairpins, the hairpin comprising at least three matching base pairs, and the anti-zipper sequence comprising: The bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence. and (b) the CRISPR nucleic acid molecule comprises, in the 3' to 5' direction, a zipper sequence comprising at least about 3 nucleotides, a bulge sequence comprising a nucleotide sequence having at least 2 nucleotides, NNTNN ( or NNUNN), a G _R1 containing the nucleotide G or GTT, and a spacer sequence having a 5' end and a 3' end, the 3' end of which has 100% identity with the target DNA. 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 and G _R1 is located immediately upstream of the spacer sequence, and further wherein the anti-zipper and anti _- stitch sequences of the tracr nucleic acid molecule, respectively, sequence and the stitch sequence, and the spacer sequence of the CRISPR nucleic acid molecule is at least about 80% complementary to and hybridizes with a portion of the target DNA (adjacent to the protospacer adjacent motif (PAM) on the target DNA). % complementarity and hybridization, thereby resulting in site-specific cleavage of the target DNA within the region defined by the complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA.

In other embodiments, a method is provided for site-specific cleavage of double-stranded target DNA, comprising contacting the double-stranded target DNA with a chimeric nucleic acid comprising: (a) an 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 nexus sequence comprising a nucleotide sequence of (A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and a first nucleotide sequence comprising a nucleotide sequence comprising at least one hairpin (the hairpin being , 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 located immediately upstream of the nexus sequence. and the nexus sequence is located immediately upstream of the hairpin sequence); (b) a zipper sequence comprising at least about 3 nucleotides in the 3' to 5'direction; a bulge sequence comprising a nucleotide sequence of NNTNN (or NNUNN); a stitch sequence comprising a nucleotide sequence of _NNTNN (or NNUNN); a second 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 ₍ c) a Cas9 nuclease (e.g., SEQ ID NOS: 1-53 ₎ ; ), wherein the anti-zipper and anti-stitch sequences of the first nucleotide sequence encode an amino acid sequence having at least 80% identity with an amino acid sequence encoding a second nucleotide sequence; hybridizes to the zipper and stitch sequences of the nucleotide sequence, and the spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to a protospacer adjacent motif (PAM) on the target DNA), which resulting in site-specific cleavage of the target DNA within a region defined by the 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 a transcoding CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule are injected with a Cas9 nuclease (e.g. 1-53), wherein (a) the tracr nucleic acid molecule comprises an anti-zipper sequence comprising at least about 3 nucleotides in the 5' to 3'direction; Bulge sequence comprising at least about 3 nucleotides; anti-stitch sequence comprising the nucleotide sequence 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 TCAAACAAAGCTTCAGC; and , a nucleotide sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising at least three matching base pairs, and an 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) The 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., a (-NN-) nucleotide sequence), a nucleotide sequence of NNTNN, a stitch sequence comprising a nucleotide sequence, a G _R1 comprising the nucleotide G or GTT, and a spacer sequence having a 5' end and a 3' end, the spacer sequence having 100% identity with the target DNA at its 3'end; 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 G _R1 . and G _R1 is located immediately upstream of the spacer sequence, and further wherein the Cas9 nuclease contains a mutation in the HNH active site motif, a mutation in the RuvC active site motif, and a polynucleotide of interest. Fused to the peptide, the anti-zipper sequence is about 70% complementary to and hybridizes to the zipper sequence, the stitching sequence is 100% complementary to and hybridizing to the stitching sequence, and the spacer sequence is a protospacer on the target DNA. is approximately 80% complementary to and hybridizes with the target DNA adjacent to the adjacent motif (PAM), thereby binding the target polypeptide to the target DNA in a region defined by the complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. Provides site-specific targeting.

In exemplary embodiments, for synthetic tracr nucleic acid constructs, as described herein, the bulge sequence or first nucleotide sequence of the synthetic tracr nucleic acid molecule comprises about 3, 4 or 5 nucleotides. can derive from, consist essentially of, or consist of. In other embodiments, the bulge sequence may include, consist essentially of, or consist of 5 nucleotides, and the hairpin sequence may include at least 2 hairpins, and the hairpin sequence may include at least 2 hairpins. The hairpin can consist essentially of, or can consist of, each hairpin containing at least three matching base pairs.

In a further embodiment, the invention provides a method for site-specific cleavage of double-stranded target DNA comprising contacting a transcoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of a Cas9 nuclease. Here, we provide a method for
(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 sequence, 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 TCAAACAAAGCTTCAGC; encoded by a nucleotide sequence that includes a hairpin sequence that includes a nucleotide sequence that has at least one hairpin; the hairpin comprises at least three matching base pairs, and the anti-zipper sequence, if present, 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; and (b) the CRISPR nucleic acid molecule is located in the 3' to 5' direction. , an optional zipper sequence comprising a nucleotide sequence having at least three nucleotides that hybridize to the anti-zipper, a bulge sequence comprising a nucleotide sequence of (-NN-), a stitch sequence comprising a nucleotide sequence of NNUNN, and a 5' a spacer sequence having a terminus and a 3' end, the spacer sequence comprising at its 3' end at least 7 nucleotides with 100% identity to the target DNA, and a zipper sequence comprising: If present, is located immediately upstream of the bulge array, the bulge array is located immediately upstream of the stitched array, the stitched array is located immediately upstream of the spacer array, and
Further herein, if an anti-zipper and a zipper sequence are present, the anti-zipper sequence hybridizes to the zipper sequence, NNANN of the anti-stitch sequence is complementary to and hybridizes to NNUNN of the stitch sequence, and the CRISPR nucleic acid The spacer sequence of the molecule hybridizes to a portion of the target DNA (adjacent to the protospacer adjacent motif (PAM) on the target DNA) and is thereby defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. within the region, resulting 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 contacting double-stranded target DNA with a chimeric nucleic acid comprising:
(a) an optional anti-zipper sequence comprising at least about 3 nucleotides in the 5' to 3'direction; a bulge sequence comprising at least about 3 nucleotides; an anti-stitch sequence comprising a 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), a nexus sequence comprising a nucleotide sequence of (A/G) (A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC, a hairpin sequence comprising a nucleotide sequence having at least one hairpin (the hairpin contains at least 3 matching base pairs),
where 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 the nexus sequence is located immediately upstream of the hairpin sequence;
(b) an optional zipper sequence comprising, in the 3' to 5' direction, a nucleotide sequence having at least 3 nucleotides that hybridizes to the anti-zipper, a nucleotide sequence having at least 2 nucleotides, such as (- a bulge sequence comprising a nucleotide sequence of NN-), a stitch sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end, the 3' end of which has 100% identity to the target DNA. and a zipper sequence, if present, located immediately upstream of the bulge sequence, and the bulge sequence located immediately upstream of the stitching sequence. and (c) encodes an amino acid sequence having at least 80% identity with an amino acid sequence encoding a Cas9 nuclease (e.g., SEQ ID NOs: 1-53); a third nucleotide sequence,
Here, if a zipper sequence and an anti-zipper sequence are present, the zipper sequence hybridizes to the anti-zipper sequence, NNANN of the anti-stitch sequence is complementary to and hybridizes to NNUNN of the stitch sequence, and the The spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to the protospacer adjacent motif (PAM) on the target DNA), thereby causing the spacer sequence of the second nucleotide sequence to It results in site-specific cleavage of 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 a transcoding CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule are injected with a Cas9 nuclease (e.g. 1 to 53) with the target DNA,
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; nucleotides of NNANN; anti-stitch sequences, including sequences 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), a nexus sequence comprising a nucleotide sequence of GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC, a hairpin sequence comprising a nucleotide sequence having at least one hairpin; encoded by a nucleotide sequence, said hairpin comprising at least three matching base pairs, and an anti-zipper sequence, if present, located immediately upstream of a bulge sequence, the bulge sequence being an anti-stitch sequence. the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and (b) the CRISPR nucleic acid molecule is an optional zipper sequence comprising a nucleotide sequence having at least 3 nucleotides that hybridizes to the anti-zipper in the ' direction, a nucleotide sequence having at least 2 nucleotides (e.g., a (-NN-) nucleotide sequence) a bulge sequence comprising a nucleotide sequence of NNUNN, a stitch sequence comprising a nucleotide sequence of NNUNN, and a spacer sequence having a 5' end and a 3' end, the 3' end of which at least 7 nucleotides having 100% identity with the target DNA. 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 sequence, 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 has a zipper sequence and an anti-zipper sequence. If present, the zipper sequence hybridizes to the anti-zipper sequence, the NNANN of the anti-stitch sequence is complementary to and hybridizes to the NNUNN of the stitch sequence, and the spacer sequence hybridizes to the protospacer flanking motif ( PAM), thereby resulting in site-specific targeting of the polypeptide of interest to the target DNA within a region defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the 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 the bulge sequence. Additional nucleotides can be further included at the end of the hybridized sequence that is distal to the end of the hybridized sequence, thereby linking the hybridized zipper and anti-zipper sequences. In a further embodiment, in the absence of a zipper and anti-zipper, the chimeric nucleic acid construct can optionally further include nucleotides that link the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid. The connecting nucleotides can be any nucleotides (eg, T, A, G, C), and the number of nucleotides connecting the zipper and anti-zipper sequences 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 that includes at least one nuclear localization sequence described herein, is suitable for use in the present invention. It can be used in any method.

Further provided herein are expression cassettes and vectors that include the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules and/or nucleotide sequences of the invention and 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 a host organism. Any cell/host organism for which the present 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 includes, but is not limited to, Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces po mbe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum, or Arabidopsis thaliana. It's fine. In further embodiments, cells useful in the invention include, but are not limited to, stem cells, somatic cells, reproductive 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 other embodiments, cells useful in the invention include, but are not limited to, Homo sapiens, Drosophila melanogaster, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae, Glycine max, Zeae maydis, Gossypium hirsutum, or Contains cells derived from Arabidopsis thaliana.

In a further aspect of the invention, the polypeptide of interest includes, but is not limited to, helicases, nucleases, methyltransferases, gyrase, demethylases, kinases, dismutases, integrases, transposases, telomerase, recombinases, acetyltransferases, deacetylases, polymerases, phosphatases, May include ligases, ubixin ligases, photolyases or glycosylases. In another aspect of the invention, the polypeptide of interest has depurination activity, oxidation activity, pyrimidine dimer formation activity, alkylation activity, DNA repair activity, DNA damage activity, deubiquitination activity, adenylation activity, deadenylation activity. It has SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, telomere repair activity, or deamination activity. In representative embodiments, the polypeptide of interest exhibits kinase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, phosphatase activity, ubixin ligase activity, deubiquitination activity, or telomere repair activity. It may be a polypeptide that has

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

Accordingly, 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 invention. In another aspect, a kit is provided for site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, which kit comprises a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array of the invention. or chimeric nucleic acid constructs. In some aspects, kits may include synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and/or chimeric nucleic acid constructs of the invention contained in one or more expression cassettes. In yet a further aspect, the kit further comprises a Cas9 nuclease (e.g., 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 include a primer, said primer comprising a portion of a CRISPR repeat sequence in both directions. In other embodiments, the kit may include primers designed to extend through CRISPR repeat sequences in both directions, including the boundaries of the CRISPR array (i.e., one leader end and one trailer end). .
In further embodiments, the kit may further include instructions for use.

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

Example 1. Evaluation of the functional role of modules identified within guide sequences Clustered regularly interspaced short palindromic repeat (CRISPR) and associated Cas proteins confer adaptive immunity against invasive genetic elements in Bacteria and Archaea ¹ . In the type II CRISPR-Cas system, the signature RNA-guided endonuclease Cas9 specifically targets sequences complementary to the CRISPR spacer, resulting in double-stranded DNA breaks (DSB) using two nickase domains. Nuclei c. 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) ). Any DNA sequence can be targeted as long as it is flanked by Cas9-specific protospacer-adjacent motifs (PAMs). Garneau, J. E. et al. Nature 468, 67-71 (2010); Sapranauskas, 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 transactivating crRNA (tracrRNA) ⁸ . This natural complex can be replaced by a synthetic single guide RNA (sgRNA) chimera that mimics the crRNA:tracrRNA duplex (Jinek, M. et al., Science 337, 816-821 (2012)). sgRNA in combination with Cas9 makes a convenient, compact, and portable sequence-specific targeting system suitable for engineering and heterologous transfer to a variety of model systems of industrial and translational interest.

Therefore, Cas9:sgRNA technology, which provides a compact and practical means to generate double-strand breaks (DSBs), has a revolutionary genome organization (Mali, P. et al., Science 339, 823 -826 (2013); Cong, L. et al. Science 339, 819-823 (2013); Jiang, W. et al. Nat. Biotechnol. 31, 233-239 (2013); Sander, J. D. & Joung , J.K. Nature Biotechnol. 32, 347-355. (2014)), opened new avenues for high-throughput genome-wide genetic screens ^13,14 and expanded the transcriptional regulation toolbox (Qi, L.S. .et al. Cell 152, 1173-1183 (2013); Gilbert, L.A. et al. Cell 154, 442-451 (2013)). Furthermore, the absence of interaction between the evolutionarily distant Cas9:sgRNAs (Chylinski, K. et al. RNA biology 10, 726-737 (2013); Fonfara, I. et al. Nucleic Acids Res (2013) ); Esvelt, K.M. et al. Nature Methods (2013)), when coexisting functional type II CRISPR-Cas systems function simultaneously, multiplex-independent targeting can be achieved within cells. (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, critical properties of sgRNA guides and their involvement in defining functionally orthologous Cas9 endonucleases remain to be characterized. Indeed, early attention in Cas9 targeting and cleavage focused on spacer:target complementarity and PAM sequence sensitivity, while the factors driving Cas9:sgRNA interaction and the orthogonality between the type II CRISPR-Cas system Information defining factors that indicate sex remains lacking. We therefore set out to identify and characterize properties within sgRNA that confer Cas9 targeting and cleavage specificity to open new engineering avenues for CRISPR technology.

Accordingly, the functional roles and implications of the various modules identified within the guide sequences described herein (e.g., synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, chimeric nucleic acid constructs (tracr nucleic acids-synthetic CRISPR nucleic acid constructs)) In order to evaluate the implication), we designed mutants of the guide mutations containing modifications or deletions of each of the aforementioned functional modules. We used the SthCRISPR3 system as a representative functional model. We first established a positive functional control (wild type, WT) using a selected and natural guide sequence. We then tested a "stitch" mutant in which the stitch was deleted and observed loss of function. We then tested a ``bulge'' mutant, in which the bulge was deleted, and observed a loss of function. We then tested a ``nexus'' mutant, in which the nexus was deleted, and observed a loss of function. We then tested "hairpin" mutants in which the first hairpin was deleted and observed loss of function. We subsequently tested sequence specificity and variability sensitivity and in the mutant constructs We established that the sequence of the nexus is specific, while allowing for variability with respect to other functional modules, especially zippers, bulges, hairpins and stitches. The results of these experiments are shown in Figures 1-21. .

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

Figure 5 shows multiple sequence alignments for the anti-stitch module, while Figures 6A-6D show the Sth Cr1 group (Figure 6A), Sth Cr3 group (Figure 6B), Lrh group (Figure 6C) and Lbu group. (Figure 6D) shows the consensus sequence for the anti-stitch module.

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

Figure 11 shows multiple sequence alignments for the bulge, anti-stitch and nexus modules. Figure 4 shows the maximum likelihood tree for Cas9 nuclease.

Figures 12-21 show guide sequences and targeting for various cRNA:tracRNA constructs, including Streptococcus thermophilus CR3 representing the Sth CR1 group (Figure 12); Lactobacillus buchneri representing the Lbu group (Figure 13); represent Streptococcus thermophilus CR1 (Figure 14); Streptococcus pyrogenes M1 GAS (Figure 15) representing the Sth CR3 group; Lactobacillus rhamnosus (Figure 16) representing the Lrh group; Lactobacillus animalis (Figure 17); Lactobacillus casei representing the Lca group (Figure 18); Lactobacillus gasseri representing the Lga group (Figure 19); Lactobacillus jensenii representing the Lje group (Figure 20); and Lactobacillus pentosus representing the Lpe group (Figure 21). The lower side of each of Figures 12-21 represents the target dsDNA, including the target sequence (open structure) and adjacent (3') PAM. The top of each figure represents the CRISPR RNA (crRNA), consisting of the 5' part complementary to the target sequence, and the 3' part derived from the CRISPR repeat; and also the tracrRNA, consisting of the anti-CRISPR repeat part, and the nexus and 3' hairpin are also shown. As shown in each of FIGS. 12-21, the complementary portions of the crRNA:tracrRNA duplex include the lower stem (lower complementary portion), the bulge (herniated mismatch), and the upper stem (upper complementary portion). Consisting of

Example 2. Characterization of Guide RNA Sequences in Various Additional Type II Systems The findings described in Example 1 establish the key modules in sgRNA required 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 properties also occur in other type II CRISPR-Cas systems. We sampled 41 Cas9 sequences from Streptococcus and Lactobacillus genomes (where type II systems preferentially occur ⁾ , identified their corresponding CRISPR repeats, and predicted tracrRNA sequences. Cas9 protein sequences clustered into three main sequence groups (Figure 23). As expected, similar grouping was seen when clustering was performed using either CRISPR-repeat or predicted tracrRNA sequences (Figure 24A, Figure 23, Figure 25, Figure 26), with anti- The presence of CRISPR repeats and the relevant molecular relationship between Cas9 and crRNA:tracrRNA pairs (Makarova, K.S. et al. Nat Rev Microbiol 9, 467-477 (2011); Deltcheva, E. et al. al. Nature 471, 602-607 (2011); Fonfara, I. et al. Nucleic Acids Res (2013)). Within the tracrRNA sequences, we consistently observed functional modules identified for SpyCas9 (Figure 24B), with the overall sgRNA/crRNA:tracrRNA structure between families conserved and high levels within the cluster. The array was stored in .

The presence of a bulge with directional twist between the lower stem (ie, stitch/anti-stitch) and the upper stem (ie, zipper/anti-zipper) was consistently seen across the variety of systems. Lower stem length was highly conserved within and variable between families. Interestingly, the highest level of conservation was found for 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, a unique double-stem nexus (FIGS. 24A-B) was unique and ubiquitous in the Lactobacillus system. Surprisingly, several bases within the nexus were strictly conserved even between different families, including A52 and C55 (Figures 24A-B), further highlighting the critical role of this module. Indeed, A52 interacts with the backbone at residues 1103-1107 near the 5' end of the target strand in the crystal structure of SpyCas9, suggesting that interaction of the nexus with the protein backbone may be required for PAM binding. suggests.

Determination of the relationship of guide RNA structure to Cas9 protein orthogonality. The findings described herein suggest a potential relationship between sgRNA sequence and structure and the diversity of Cas9 proteins. This observation prompted us to determine the sgRNA modules that define the group of Cas9 orthologs. Therefore, we found that two naturally coexisting orthologous S. thermophilus type II system, i.e., Sth1 Cas9 and Sth3 Cas9 (Horvath, P. et al. I investigated the relationship. A series of experiments was designed based on self-targeting activity in Escherichia coli (Figures 28A-B) and demonstrated that specific mutations within the sgRNA promote cleavage activity at previously orthologous Cas9. I tested it to see if it could be done. We have identified an E. coli that contains overlapping Cas9 target sites for the Sth1Cas9 and Sth3Cas9 systems. We identified a region within the E. coli genome and confirmed that the cleavage occurred at a single nucleotide (Figure 29B) and that the PAM sequences overlapped favorably. We produced chimeric versions of the two sgRNA backbones, replacing the spacer, lower stem (i.e. stitch/anti-stitch)-bulge-upper stem (i.e. zipper/anti-zipper), nexus and hairpin (Figure 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 are indeed orthogonal in this assay system and that each guide alone drives targeting at its cognate Cas9 (Figure 29C). Next, we showed that replacement of the spacer sequence resulted in an sgRNA with a CRISPR3 spacer and a CRISPR1 backbone capable of supporting Sth1Cas9 cleavage activity. However, the converse is not true for Sth3Cas9 activity. sgRNA containing a CRISPR1 spacer and CRISPR3 backbone does not support Sth3Cas9 activity (Figure 29C). We hypothesize that this unidirectional interfunctionality is due to the flexibility of the spatial requirements between the protospacer and PAM in the SthCRISPR1 system (Chen et al. J. Biol. Chem. doi: 10. 1074/jbc.M113.539726. (2014)) (Figure 29C, top panel). We then showed that the functionality between sgRNA and Cas9 can be changed simply by swapping the nexus-hairpin combinations between the two orthogonal systems. The main consequence of sgRNA reprogramming is that its ability to guide the original Cas9 is lost in the process (Figure 29C, bottom panel). This is in contrast to the standard idea that CRISPR repeat sequences play an important role in defining orthologous CRISPR-Cas systems. However, these results demonstrate that chimeric sgRNAs with altered nexus sequences can reprogram orthogonality in a predictable and unidirectional manner, further redirecting orthogonal Cas9 proteins associated with different PAMs. (Esvelt, K.M. 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-bound and protein/RNA/DNA complexes show that upon binding guide RNA, Cas9 undergoes a dramatic conformational change to promote target DNA binding and cleavage conformation. (Jinek, M. et al., Science 343, 6176 (2014). The crystal structure is consistent with the electron microscopy image, and the SpyCas9:sgRNA:DNA:complex and apo-SpyCas9 are We show that substantial rearrangement of the RNA- and DNA-binding domains occurs between the two structures, occupying significantly different structures (Jinek, M. et al., Science 343, 6176 (2014); Nishimasu, H. et al. Cell 156, 935 (2014)). The nexus occupies a critical position in the SpyCas9-sgRNA:DNA complex, aligning many critical components of proteins and sgRNAs, and coordinating both proteins and RNAs. is properly positioned to receive the target DNA duplex for cleavage. Upon binding sgRNA:DNA, the arginine-rich bridge helix binds to the base and lower stem of the nexus. In addition, the nexus , two small regions from the two lobes of SpyCas9, which we propose to establish 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 . NIR2 also interacts directly with the lower stem. The interacting, nexus-opposite face of the binding site lies in close proximity to the 3' end of the target strand, suggesting that interaction with the nexus may be necessary to prime the PAM recognition site. In particular, in the Actinomyces naeslundii Cas9 (AnaCas9) apo-structure (Jinek, M. et al., Science 343, 6176 (2014)), NIR2 is trimmed and contains an insertion of about 50 amino acids. There is motivation to believe that large nexuses (and possibly with PAM arrays) can be recognized.

However, these results reveal six distinct features within the guide RNA and establish the bulge and nexus as structure- and sequence-specific properties that guide Cas9 targeting and cleavage. This is the basis for the optimization of sgRNA composition, and contains smaller regions of double-stranded RNA that potentially triggers an innate immune response, making it more suitable for packaging such as adeno-associated viruses. This provides a design opportunity to design short minimal guide RNAs. This understanding of type II CRISPR-Cas systems was developed by Briner et al. (Mol. Cell 56:333-339 (2014)) and Nishimasu et al. (Cell 156:935-949 (2014)), sequence modification has been shown to affect the ability of the modified sequence to guide Cas9. Notably, these studies We confirm that nexus sequences within the guide are critical for guiding Cas9 toward 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 that have the potential to exploit the diversity of natural Cas9 orthologs. , including short Cas9 variants for convenient packaging and delivery. In addition, the ability to reprogram Cas9 using chimeric mgRNAs offers flexibility in controlling target frequency (shorter PAMs occur frequently) and specificity through reduced off-target cleavage (longer PAMs rarely occur). will enable increased use of a variety of PAMs for This also opens up multiplexing opportunities by using a single Cas9 with different chimeric guides or by using orthogonal systems simultaneously with different combinations of standard or chimeric sgRNAs. Taken together, our findings open new avenues for Cas9-dependent DNA targeting and set the stage for the development of next generation CRISPR-based technologies.

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

Plasmid construction. To construct the Cas9-expression plasmid, the Cas9 genes from the CRISPR1 locus (Sth1-Cas9) and the CRISPR3 locus (Sth3-Cas9) were cloned into S. PCR amplification was performed from genomic DNA extracted from thermophilus LMD-9. Each PCR product was combined with the PCR-amplified backbone of pwtCas9-bacteria (Addgene #44250) (Qi, L.S. et al. Cell 152, 1173-1183 (2013)) by Gibson assembly. To construct the sgRNA-expression plasmid, the SpeI restriction site in the pdCas9-bacterial plasmid (Addgene #44249) (Id.) was removed by digesting the plasmid with SpeI, blunt-ended, and religated to create the pdCas9ΔSpeI plasmid. was produced. Separately, S. gBlock (IDT) encoding a zraP-targeting sgRNA based on the CRISPR1 (C1) or CRISPR3 (C3) locus in P. thermophilus LMD-9 was generated by Gibson assembly into a pgRNA-bacterial plasmid (Addgene #44251) (Id.) in combination with the PCR-amplified backbone of the original S. 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 and ligated to the SpeI/KpnI or KpnI/HindIII sites of the psgRNA-C1 or psgRNA-C3 plasmids. All plasmid modifications were confirmed by sequencing.

Strains and growth conditions. E. coli K-12 subst. MG1655 (genotype: E. coli K-12 F λ-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 harboring the indicated Cas9-expression plasmid were streaked for isolation and individual colonies were inoculated into 3 ml of LB medium and grown overnight. The resulting culture was back-diluted into 45 ml of LB medium and grown until the ABS ₆₀₀ was 0.6-0.8 as measured on a Nanodrop 2000c spectrophotometer (Thermo Scientific). Cultures were then pipetted and washed twice with ice-cold 10% glycerol before being resuspended in 200-400 μl of 10% glycerol. Suspended cells (50 μl) were transformed with 25 ng of the indicated sgRNA-expression plasmids using a MicroPulser Electroporator (BioRad) and allowed to recover for 1 hour in 300 μl of SOC medium (Quality Biological). After recovery, 200 μl cultures containing different amounts of LB medium were plated on LB agar containing 100 ng/ml anhydrotetracycline. Transformation efficiency was calculated by calculating the number of transformants for the tested DgRNA plasmids as previously described (Gomaa, A.A. et al. MBio. 5, e00928-13 (2014)). Calculated by dividing by the number of transformants for the T4 control plasmid. To reduce inter-experimental variation in transformation efficiency, the tested sgRNA plasmids and control plasmids were transformed into the same batch of electrocompetent cells. Similarly, with respect to Figures 30-32, transformation assays were used to test the ability of the plasmids to be electroporated into Lactobacillus strains harboring 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 within the host's CRISPR locus.Flanking the protospacer with a complete PAM Various plasmids were designed (NTAAC for Lga; NNGAA for Lca; NGAAAA for Lrh; The experiments were also performed using a control non-targeting sequence (the penultimate entry for each experiment shown in Figures 30-32) and a control plasmid lacking the targeting sequence. (A final entry was also included for each experiment shown in Figures 30-32.) The ability of the native CRISPR-Cas system to interfere with plasmid uptake by DNA targeting is due to the difference in transformation efficiency between the test sequences and the two previously described controls. measured as a difference.

Claims (21)

A single guide RNA (sgRNA) comprising: (a) a synthetic transcoding CRISPR (tracr) nucleic acid construct portion; and (b) a synthetic CRISPR nucleic acid construct portion;
The synthetic tracr nucleic acid construct portion comprises, in the 5' to 3' direction, nucleotides 4 to 112 of SEQ ID NO: 69;
Here, the synthetic tracr nucleic acid construct portion is:
an anti-zipper sequence comprising at least 4 nucleotides, wherein said at least 4 nucleotides comprise the nucleotide sequence of ACCA (nucleotides 25-28 of SEQ ID NO: 69);
A bulge sequence comprising the nucleotide sequence of GA (nucleotides 29-30 of SEQ ID NO: 69);
an anti-stitch sequence comprising the nucleotide sequence UUUAAAAU (nucleotides 31-38 of SEQ ID NO: 69);
a nexus sequence comprising the nucleotide sequence CAAGCAAAUGCAUCUUUUUGAUGCAAAGUUUC (nucleotides 39-68 of SEQ ID NO: 69), said nexus forming a hairpin structure comprising a double-stranded double stem; and
a hairpin sequence comprising a nucleotide sequence having two hairpins, each hairpin comprising a stem having at least 5 matching base pairs;
including;
wherein the anti-zipper array is located immediately upstream of the bulge array, the bulge array is located immediately upstream of the anti-stitch array, and the anti-stitch array is located immediately upstream of the nexus array. and the nexus sequence is located immediately upstream of the hairpin sequence; and
The synthetic CRISPR nucleic acid construct portion comprises in the 3' to 5' direction:
a zipper sequence comprising at least 4 nucleotides, wherein said at least 4 nucleotides comprise a nucleotide sequence of UGGU;
a stitched sequence comprising a nucleotide sequence of AGAUUUUG; and
a spacer sequence having a 5' end and a 3' end and comprising at least 7 nucleotides having 100% complementarity with the target DNA at its 3'end;
including;
the zipper arrangement is located immediately upstream of the stitching arrangement, and the stitching arrangement is located immediately upstream of the spacer arrangement;
here,
The stitch sequence of the synthetic CRISPR nucleic acid construct portion is 100% complementary to and hybridizes to the anti-stitch sequence of the synthetic tracr nucleic acid construct portion, and the zipper sequence of the synthetic CRISPR nucleic acid construct portion is hybridized to the anti-stitch sequence of the synthetic tracr nucleic acid construct portion. is at least about 90% complementary to and hybridized to said anti-zipper sequence of a nucleic acid construct portion, said hybridized zipper sequence and said anti-zipper sequence being joined by a nucleotide at a distal end to said bulge sequence; ,
wherein the sgRNA functions with a Cas9 polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 30.
sgRNA. The sgRNA of claim 1,
the sgRNA is capable of forming a nucleic acid-protein complex with a Cas9 polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 30;
sgRNA. The sgRNA according to claim 1 or 2,
The Cas9 polypeptide is a Cas9 nuclease from the Lactobacillus gasseri (Lga) group.
sgRNA. The sgRNA according to any one of claims 1 to 3,
the Cas9 polypeptide comprises a mutation within the RuvC active site motif;
sgRNA. The sgRNA according to any one of claims 1 to 4,
the Cas9 polypeptide comprises a mutation within the HNH active site motif;
sgRNA. The sgRNA according to any one of claims 1 to 5,
The Cas9 polypeptide contains mutations within the HNH active site motif and within the RuvC active site motif, and is fused to the target polypeptide.
sgRNA. encoding the sgRNA of any one of claims 1 to 6;
Expression cassette. A cell (excluding human germ cells, human zygotes, and human embryos) comprising the expression cassette of claim 7. A non-medical method for site-specific cleavage of double-stranded target DNA, the method comprising:
The sgRNA of any one of claims 1 to 6 is contacted with a target DNA in the presence of Cas9 nuclease, wherein the spacer sequence of the synthetic CRISPR nucleic acid construct portion is arranged on the target DNA by a protospacer adjacent motif (PAM). ) hybridizes to a portion of said target DNA adjacent to said target DNA, thereby causing site-specific cleavage of said target DNA within a region defined by complementary hybridization of said spacer sequence to said target DNA;
Methods (excluding application to human germ cells, human fertilized eggs, and human embryos) . 10. The method of claim 9,
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 and creating a site-specific nick in the (+) strand of the double-stranded target DNA. generate within 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 within the HNH active site motif, thereby cleaving the (-) strand of the double-stranded target DNA and creating a site-specific nick in the (-) strand of the double-stranded target DNA. generate within,
Method. A non-medical method of site-specific targeting of a polypeptide of interest to double-stranded (ds) target DNA, the method comprising:
contacting the sgRNA of claim 6, thereby targeting the polypeptide of interest fused to the Cas9 to a specific site on the target DNA, wherein the site is defined by complementary hybridization of spacer sequences,
Methods (excluding application to human germ cells, human fertilized eggs, and human embryos) . 11. The method of claim 9 or 10,
the PAM comprises a CTAA, TAA, or GAA nucleotide sequence;
Method. 13. The method of claim 9, 10 or 12,
the Cas9 nuclease is encoded by a nucleotide sequence that is codon-optimized for the organism containing the target DNA;
Method. 14. The method of claim 9, 10, 12 or 13,
the Cas9 nuclease comprises at least one nuclear localization sequence;
Method. A system for modifying target nucleic acids, the system comprising:
an sgRNA comprising (a) a synthetic transcoding CRISPR (tracr) nucleic acid construct portion; and (b) a synthetic CRISPR nucleic acid construct portion;
The synthetic tracr nucleic acid construct portion comprises, in the 5' to 3' direction, nucleotides 4 to 112 of SEQ ID NO: 69;
Here, the synthetic tracr nucleic acid construct portion is:
an anti-zipper sequence comprising at least 4 nucleotides, wherein said at least 4 nucleotides comprise the nucleotide sequence of ACCA (nucleotides 25-28 of SEQ ID NO: 69);
A bulge sequence comprising the nucleotide sequence of GA (nucleotides 29-30 of SEQ ID NO: 69);
an anti-stitch sequence comprising the nucleotide sequence UUUAAAAU (nucleotides 31-38 of SEQ ID NO: 69);
a nexus sequence comprising the nucleotide sequence CAAGCAAAUGCAUCUUUUUGAUGCAAAGUUUC (nucleotides 39-68 of SEQ ID NO: 69), said nexus forming a hairpin structure comprising a double-stranded double stem; and
a hairpin sequence comprising a nucleotide sequence having two hairpins, each hairpin comprising a stem having at least 5 matching base pairs;
including;
wherein the anti-zipper array is located immediately upstream of the bulge array, the bulge array is located immediately upstream of the anti-stitch array, and the anti-stitch array is located immediately upstream of the nexus array. and the nexus sequence is located immediately upstream of the hairpin sequence; and
The synthetic CRISPR nucleic acid construct portion comprises in the 3' to 5' direction:
a zipper sequence comprising at least 4 nucleotides, wherein said at least 4 nucleotides comprise a nucleotide sequence of UGGU;
a stitched sequence comprising a nucleotide sequence of AGAUUUUG; and
a spacer sequence having a 5' end and a 3' end and comprising at least 7 nucleotides having 100% complementarity with the target DNA at its 3'end;
including;
the zipper arrangement is located immediately upstream of the stitching arrangement, and the stitching arrangement is located immediately upstream of the spacer arrangement;
here,
The stitch sequence of the synthetic CRISPR nucleic acid construct portion is 100% complementary to and hybridizes to the anti-stitch sequence of the synthetic tracr nucleic acid construct portion, and the zipper sequence of the synthetic CRISPR nucleic acid construct portion is hybridized to the anti-stitch sequence of the synthetic tracr nucleic acid construct portion. is at least about 90% complementary to and hybridized to said anti-zipper sequence of a nucleic acid construct portion, said hybridized zipper sequence and said anti-zipper sequence being joined by a nucleotide at a distal end to said bulge sequence; Ru;
and,
(c) a Cas9 nuclease having at least 90% identity with the amino acid sequence of SEQ ID NO: 30;
including;
wherein the Cas9 nuclease is capable of binding and forming a complex with the sgRNA, and wherein the spacer sequence of the synthetic CRISPR nucleic acid construct portion is adjacent to a protospacer adjacent motif (PAM) on the target DNA. hybridizes to a portion of target DNA, thereby guiding the Cas9 to the target nucleic acid and modifying the target nucleic acid;
system. 16. The system of claim 15,
The Cas9 nuclease having at least 90% identity with the amino acid sequence of SEQ ID NO: 30 is Cas9 from the Lactobacillus gasseri (Lga) group of Cas9 nucleases.
system. 17. The system of claim 15 or 16,
The Cas9 nuclease contains a mutation within the RuvC active site motif.
system. The system according to any one of claims 15 to 17,
The Cas9 nuclease contains a mutation within the HNH active site motif.
system. The system according to any one of claims 15 to 18,
the modification of the nucleic acid is site-specific cleavage of double-stranded target DNA;
system. The system according to any one of claims 15 to 19,
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.
system. The system according to any one of claims 15 to 20,
the PAM comprises a CTAA, TAA, or GAA nucleotide sequence;
system.