JP2022106883A - Methods and compositions for sequences guiding cas9 targeting - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods and compositions for increasing the efficiency and specificity of synthetic type II CRISPR-Cas systems that improve efficiency and specificity for genome editing and other uses.

SOLUTION: A synthetic trans-encoded CRISPR(tracr) nucleic acid (e.g. tracrRNA. tracrDNA) construct comprises, in a direction from 5' to 3', an optional anti-zipper sequence, a bulge sequence, an anti-stitch sequence, a nexus sequence, and a hairpin sequence, the hairpin comprising at least three matched base pairs, wherein the anti-zipper sequence, when present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

Application for application of Article 30, Paragraph 2 of the Patent Law has been submitted. 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

[Priority statement]
This application claims the interests of US Provisional Application No. 61 / 931,515 filed on January 24, 2014 under US Code Vol. 35, Section 119 (e), the entire content of which is in its entirety. , Incorporated herein by reference.
[Field of the present invention]
The present invention relates to a synthetic CRISPR-cas system and its use for genomic organization.

Crustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in combination with related sequences (cas), constitute the CRISPR-Cas system and impart adaptive immunity in many bacteria. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic factors such as plasmids and phages as novel "spacers".

The CRISPR-Cas system consists of an array of short DNA repeats separated by hypervariable sequences, flanked by the cas gene, and adaptive immunity to invasive genetic factors such as phages and plasmids through sequence-specific targeting and interference. Provided (Barrangou et al. 2007. Science. 315: 1709-1712; Browns et al. 2008. Science 321: 960-4; Harbor and Barrangou. 2010. System. 327: 167-70; Marraffin. System. 322: 1843-1845; Baya et al. 2011. Annu. Rev. Genet. 45: 273-297; Turns and Turns. 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 and a new repeat at the CRISPR locus. Inserted as a spacer unit. The repeat-spacer array is subsequently transcribed as a long pre-CRISPR RNA (pre-crRNA) (Browns et al. 2008. Science 321: 960-4), which drives sequence-specific recognition with low molecular weight interfering CRISPR RNA (Browns et al. 2008. Science 321: 960-4). It is processed into crRNA). Specifically, crRNA guides the nuclease towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Garneau et al. 2010. Nature. 468: 67-71; Haurwitz et. al. 2010. Science. 329: 1355 to 1358; Saplanauskas et al. 2011. Nucleic Acid Res. 39: 9275-928; Jinek et al. 2012. Science. 337: 816-821; Gasinus et al. Natl. Acad. Sci. 109: E2579-E2586; Magadan et al. 2012. PLoS One. 7: e40913; Karvelis et al. 2013. RNA Biol. 10: 841-851). These extensive systems occur in nearly half of the bacteria (about 46%) and the majority of archaea (about 90%). They are based on the cas gene content, the organization and diversity in the biochemical processes that drive crRNA biosynthesis, and the Cas protein complex that mediates target recognition and cleavage, and they are the three major CRISPR-Cas. It is classified into the type of system (Makarova et al. 2011. Nature Rev. Microbiol. 9: 467-477; Makarova et al. 2013. Nuclear Acid Res. 41: 4360-4377). In types I and II, a special Cas endonuclease treats the pre-crRNA and then assembles into a large multi-Cas protein complex capable of recognizing and cleaving a nucleic acid complementary to the crRNA. A different process relates to the Type II CRISPR-Cas system. Here, the pre-CRNA is processed by a mechanism by which a transactivated crRNA (tracrRNA) hybridizes to the repeat region of the crRNA. The hybridized crRNA-tracrRNA is cleaved by RNase III, followed by a second event that removes the 5'end of each spacer, producing a mature crRNA that remains bound to both tracrRNA and Cas9. The mature complex then cleaves both strands in search of the target dsDNA sequence (“protospacer” sequence) complementary to the spacer sequence within the complex. Target recognition and cleavage by the complex in a type II system not only requires a complementary sequence between the spacer sequence on the crRNA-tracrRNA complex and the target "protospacer" sequence, but also of the protospacer sequence. It also requires a protospacer flanking motif (PAM) sequence located at the 3'end. The exact PAM sequence required can 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 genomic organization and other uses.

One aspect of the invention is an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; an anti-zipper sequence containing a nucleotide sequence of NNANN. -Stitch sequence; 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 GAUAAGGCCUU), TCAAG (or UCAAG), TCAAGCAA (or UCAAGCAA), T (C / A) AA (A / C) (C / A) ( A / G) (A / T) (or U (C / A) AA (A / C) (C / A) (A / G) (A / U)), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nucleotides A synthetic transcoded CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct comprising a nexus sequence comprising a sequence; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin is provided, wherein the hairpin comprises at least 3 Contains matching base pairs
Here, the anti-zipper sequence, if present, is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the anti-stitch sequence, and the anti-stitch sequence is just upstream of the nexus sequence. It is located and the nexus sequence is located just upstream of the hairpin sequence.

A second aspect of the invention is an optional zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction (hybrid to the anti-zipper of tracrRNA, if any), at least two. A bulge sequence containing a nucleotide (eg, a (-NN-) nucleotide sequence), a stitch sequence containing a NNTN (or NNUNN) nucleotide sequence that hybridizes to the anti-stitch of _tracrRNA , a GR1 containing a nucleotide G or GTT, And a synthetic CRISPR nucleic acid (eg,) comprising a spacer sequence having 5'ends and 3'ends, the 3'end containing at least 7 nucleotides having 100% identity with the target DNA. crRNA, _crDNA ) constructs, the zipper sequence, if present, is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is just upstream of GR1. It is located and _GR1 is located just upstream of the spacer sequence.

A third aspect of the invention provides a synthetic CRISPR nucleic acid array comprising a nucleotide sequence encoding two or more CRISPR nucleic acid constructs of the invention, wherein the two or more CRISPR nucleic acid constructs are on each other on the nucleotide sequence. Located immediately next to each other, the zipper sequences of the two or more CRISPR nucleic acid constructs are the same if present, the stitch sequences of the two or more CRISPR nucleic acid constructs are the same, and the spacers of the two or more CRISPR nucleic acid constructs. The sequences are identical or non-identical.

A fourth aspect of the invention provides a chimeric nucleic acid construct comprising the synthetic tracr nucleic acid construct of the invention and the synthetic CRISPR nucleic acid construct of the invention, wherein, if present, the zipper sequence of the synthetic CRISPR nucleic acid construct. , At least about 70% complementary and hybridized to 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 and hybridized to the anti-stitch sequence of the synthetic tracr nucleic acid construct. And, the bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary.

A fifth aspect of the invention is to contact the chimeric nucleic acid construct of the present disclosure or an expression cassette containing the chimeric nucleic acid construct with the target DNA in the presence of Cas9 nuclease, thereby by hybridization of the spacer sequence to the target DNA. Provided is a method for site-specific cleavage of a double-stranded target DNA, which comprises a step of causing site-specific cleavage of the target DNA within a defined region.

A sixth aspect of the invention involves 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. Providing a way for
Here, (a) the tracr nucleic acid molecule is an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; NNANN nucleotides. Anti-stitch sequence including sequence; TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A) / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAGCAAAGC, or a nexus sequence containing a nucleotide sequence of TCAAACAAAGCTTCAGC; and a hairpin sequence containing a nucleotide sequence having at least two hairpins. Encoded by the nucleotide sequence, each hairpin contains at least three matching base pairs, the anti-zipper sequence is located just upstream of the bulge sequence, and the bulge sequence is just upstream of the anti-stitch sequence. Located, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and (b) CRISPR nucleic acid molecules are in the 3'to 5'direction. , An optional zipper sequence containing at least about 3 nucleotides, a bulge sequence containing a nucleotide sequence having at least 2 nucleotides (eg, a (-NN-) nucleotide sequence), a NNTNN (or NNUNN) nucleotide sequence. A stitch sequence, GR1 containing nucleotides G or GTT, and a spacer sequence having 5'and _3'ends , at least 7 nucleotides having 100% identity with the target DNA at the 3'end. Encoded by a nucleotide sequence, including a spacer sequence containing, the zipper sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is located just upstream of _GR1 . , And _GR1 is located just upstream of the spacer sequence, and
Further here, if anti-zipper sequences and zipper sequences are present, they will hybridize with each other, the anti-stitch and stitch sequences will hybridize with each other, and the spacer sequence of the CRISPR nucleic acid molecule will be at least one of the target DNAs. Part (eg, at least about 7 consecutive nucleotides of the target DNA (eg, about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) And any range or variation within it), which is at least about 80% complementary and hybridizes with the protospacer flanking motif (PAM) on the target DNA, thereby the CRISPR nucleic acid molecule to the target DNA. Within the region defined by the complementary binding of the spacer sequence, site-specific cleavage of the target DNA occurs. Therefore, in a typical embodiment, the spacer sequence of the CRISPR nucleic acid molecule is one of the target DNA sequences flanking the PAM. Hybridized in portions, the target sequence may consist essentially of, or may consist of, containing from about 7 to about 20 contiguous nucleotides of the target DNA sequence.

A seventh aspect of the invention provides a method for site-specific cleavage of a double-stranded target DNA, comprising contacting the double-stranded target DNA with a chimeric nucleic acid comprising:
(A) Voluntary anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; bulge sequence containing at least about 3 nucleotides; anti-stitch sequence containing NNANN nucleotide sequence; TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A / C) (C / A) A first nucleotide sequence comprising (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 one hairpin. The hairpin contains at least three matching base pairs, and if present, the anti-zipper sequence is located just upstream of the bulge sequence and the bulge sequence is located just upstream of the anti-stitch sequence. , The anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence);
(B) Optional zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction (hybrid to the anti-zipper sequence of the first nucleotide sequence, if any), at least 2 A bulge sequence containing a nucleotide sequence having a nucleotide of (eg, (-NN-)), a stitch sequence containing a nucleotide sequence of NNTNN (or _NNUNN ), GR1 containing a nucleotide G or GTT, and a 5'end and A second nucleotide sequence (the zipper sequence is present) that comprises a spacer sequence having a 3'end, the 3'end containing a spacer sequence containing at least 7 nucleotides having 100% complementarity with the target DNA. If so, it is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, the stitch sequence is located just upstream of _GR1 , and _GR1 is just upstream of the spacer sequence. Located); and
(C) A third nucleotide sequence encoding an amino acid sequence having at least 80% identity with the amino acid sequence encoding the Cas9 nuclease,
Here, if an anti-zipper sequence and a zipper sequence are present, they hybridize with each other, the anti-stitch sequence hybridizes to the stitch sequence, and the spacer sequence of the second nucleotide sequence is at least the target DNA. Hybridize with a portion (eg, at least about 7 contiguous nucleotides of the target DNA, preferably up to about 20 contiguous nucleotides) (adjacent to the protospacer flanking motif (PAM) on the target DNA). This results in site-specific cleavage of the target DNA within the region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA.

An eighth aspect of the present invention comprises a method of site-specific targeting of a target polypeptide against a double-stranded (ds) target DNA, targeting the chimeric nucleic acid construct of the present disclosure or an expression cassette containing the chimeric nucleic acid construct. It comprises the step of contacting the DNA and thereby targeting the target polypeptide fused to Cas9 to a specific site on the target DNA, the site being defined by hybridization of a spacer sequence to the target DNA.

A ninth aspect of the present invention comprises a method of site-specific targeting of a target polypeptide on a double-stranded (ds) target DNA.
Including the step of contacting a transcoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with a target DNA in the presence of Cas9 nuclease, wherein
(A) The tracr nucleic acid molecule comprises an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing 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 TCAAGCAAAGC, or a nexus sequence containing a nucleotide sequence of TCAAACAAAGCTTCAGC; Coded, the hairpin contains at least three matching base pairs, the anti-zipper sequence, if present, is located just upstream of the bulge sequence, and the bulge sequence is just upstream of the anti-stitch sequence. Located, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and
(B) The CRISPR nucleic acid molecule is an optional zipper sequence containing at least about 3 nucleotides that hybridize to the anti-zipper sequence in the 3'to 5'direction, a nucleotide sequence having at least 2 nucleotides (eg,). A bulge sequence containing (-NN-) nucleotide sequence), a stitch sequence containing a nucleotide sequence of NNTNN, GR1 containing nucleotide G or GTT, and a spacer sequence having 5'ends and _3'ends , wherein 3 'Encoded by a nucleotide sequence, including a spacer sequence containing at least 7 nucleotides having 100% identity with the target DNA at the end, the zipper sequence, if present, is located just upstream of the bulge sequence and is bulge. The sequence is located just upstream of the stitch sequence, the stitch sequence is located just upstream of _GR1 , and _GR1 is located just upstream of the spacer sequence, and
Further, here, Cas9 nuclease contains a mutation within the HNH active site motif, a mutation within the RuvC active site motif, is fused to the polypeptide of interest, and the anti-zipper and zipper sequences hybridize to each other, if present. The anti-stitch sequence hybridizes to the stitch sequence, and the spacer sequence hybridizes to at least a portion of the target DNA flanking the protospacer flanking motif (PAM) on the target DNA, thereby relative to the target DNA. Within the region defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule, site-specific targeting of the target polypeptide to the target DNA occurs.

Also provided herein are expression cassettes, cells and kits containing 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 described in detail in the description of the invention below.

The multi-sequence alignment for the nexus module is shown. Shows the maximum likelihood tree for the nexus module. 3A-3D show a consensus sequence for the nexus module. FIG. 3A shows a consensus sequence for the Sth Cr1 group, FIG. 3B shows a consensus sequence for the Sth Cr3 group, FIG. 3C shows a consensus sequence for the Lrh group, and FIG. 3D shows a consensus sequence for the Lbu group. The maximum likelihood tree for Cas9 nuclease is shown. The multi-sequence alignment for the anti-stitch module is shown. 6A-6D show a consensus sequence for anti-stitch modules. FIG. 6A shows a consensus sequence for the Sth Cr1 group, FIG. 6B shows a consensus sequence for the Sth Cr3 group, FIG. 6C shows a consensus sequence for the Lrh group, and FIG. 6D shows a consensus sequence for the Lbu group. The multi-sequence alignment for the bulge module is shown. 8A-8D show a consensus sequence for the bulge module. FIG. 8A shows a consensus sequence for the Sth Cr1 group, FIG. 8B shows a consensus sequence for the Sth Cr3 group, FIG. 8C shows a consensus sequence for the Lrh group, and FIG. 8D shows a consensus sequence for the Lbu group. The multi-sequence alignment for the zipper module is shown. Shows the maximum likelihood tree for the zipper module. Multiple sequence alignments for bulge, anti-stitch and nexus modules are shown. Details of the sequence and structure of the CRISPR-Cas system factor of Streptococcus thermophilus CR3 are shown and represent the Sth CR1 group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus buchneri are shown and represent the Lbu group. Details of the sequence and structure of the CRISPR-Cas system factor of Streptococcus thermophilus CR1 are shown and represent the Sth CR1 group. Details of the sequence and structure of the CRISPR-Cas system factor of Streptococcus pyrogenes M1 GAS are shown and represent the Sth CR3 group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus rhhamnosus are shown and represent the Lrh group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus animalis are shown and represent the Lan group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus casei are shown and represent the Lca group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus gasseri are shown and represent the Lga group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus jensenii are shown and represent the Lje group. Details of the sequence and structure of the CRISPR-Cas system factor of Lactobacillus pentosus are shown and represent the Lpe group. Details of the sequence and structure of the CRISPR-Cas system factor of Streptococcus pyrogenes M1 GAS are shown. The confluence between tracrRNA (left), CRISPR repeat (center) and Cas9 (right) sequence clustering is shown. Consistent grouping into the three families is seen across the three phylogenetic trees based on the sequence. 24A-24B show the Cas9: sgRNA family. FIG. 24A shows a phylogenetic tree based on Cas9 protein sequences from various Streptococcus and Lactobacillus species. The sequences were clustered into three families of blue, orange and green. FIG. 24B shows the consensus sequence and secondary structure of predictive guide RNAs for each family. Each consensus RNA consists of crRNA (left) base paired with tracrRNA. Fully conserved bases are in color, variable bases are black (two possible bases), or are represented by black dots (at least three possible bases), and are always present. The base positions that are not the same are circled. The circles between the positions indicate base pairs that are present only in some family members. Shows CRISPR repeat sequence alignment. For each cluster, CRISPR repeat sequence alignment is shown, along with conserved consensus nucleotides designated under each family of the Sth3 (top), Sth1 (center) and Lb (bottom) families. The tracrRNA sequence alignment is shown. For each cluster, experimentally determined or computer-predicted tracrRNA sequence alignment is shown, along with conserved consensus nucleotides designated under each family of the Sth3 (top), Sth1 (center) and Lb (bottom) families. .. The sgRNA nexus sequence alignment is shown. The universally conserved residues are colored red. The complementary nucleotides that make up the nexus stem summarized in FIG. 24B are underlined. The nucleotides that make up the nexus loop are in the middle of the gap. 28A-28B show self-targeting assay schemes. Orthogonal Cas9 protein was provided via the pCas9 plasmid (FIG. 28A) and used as described in FIG. Various sgRNA chimeras were provided via the psgRNA plasmid (FIG. 28B) and used in combination with each of the desired Cas9s described in FIG. 29A-29C show sgRNA orthogonality. FIG. 29A shows Streptococcus thermophilus CRISPR3-Cas9 (top, blue) and S. cerevisiae. The sgRNA sequence of thermophilus CRISPR1-Cas9 (bottom, orange) is shown. FIG. 29B shows a protospacer-targeting scheme. The expected PAM for each sgRNA is shown. The triangle indicates the estimated cutting site of each Cas9. FIG. 29C shows E.I. Cas9 in colli: chimeric-sgRNA orthogonality. Chimeric sgRNA. E. cerevisiae expressing SthCRISPR3 Cas9 (blue) and / or SthCRISPR1 Cas9 (orange). In colli, each sgRNA (left) was subjected to a transformation assay (right). The low transformation efficiency is E. coli. It shows a functional Cas9: sgRNA pair via lethal self-targeting of the colli genome. Values are from S. cerevisiae of three independent experiments. E. M. And reflect the geometric mean. Shows CRISPR interference with complementary DNA as the inability to transform a plasmid containing a protospacer sequence that matches the first wild-type CRISPR spacer sequence in Lactobacillus gasseri. Bold sequences flanked by PAMs (light gray italic nucleotides) and variants thereof (single nucleotide polymorphisms (SNPs); black underlined nucleotides) are protospacers. The low number of transformants represents the Lga CRISPR system of activity that interferes with the transformation of complementary target DNA. It shows CRISPR interference with complementary DNA as an inability to transform a plasmid containing a protospacer sequence that matches the first wild-type CRISPR spacer sequence in Lactobacillus casei. Bold sequences flanked by PAMs (light gray italic nucleotides) and variants thereof (SNPs, black underlined nucleotides) are protospacers. The low number of transformants represents the Lca CRISPR system of activity that interferes with the transformation of complementary target DNA. It shows CRISPR interference with complementary DNA as an inability to transform a plasmid containing a protospacer sequence that matches the first wild-type CRISPR spacer sequence in Lactobacillus rhamnosus. Bold sequences flanked by PAMs (light gray italic nucleotides) and variants thereof (SNPs, black underlined nucleotides) are protospacers. The low number of transformants represents the Lra CRISPR system of activity that interferes with the transformation of complementary target DNA.

Here, the present invention will be described below with reference to the accompanying drawings and examples showing embodiments of the present invention. The description is not intended to be a detailed list of all the different methods in which the invention can be practiced or all the features that can be added to the invention. For example, the features described for one embodiment may be incorporated into another embodiment and the features described for a particular embodiment may be eliminated from that embodiment. Therefore, the present invention expects that in some embodiments of the present invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous changes and additions to the various embodiments suggested herein are apparent to those skilled in the art in the light of the present disclosure and do not deviate from the present invention. Therefore, the following description is intended to illustrate some particular embodiments of the invention, not to thoroughly identify all sorts, combinations and modifications thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the field to which the invention belongs. The terminology used in the description of the present invention herein is for the sole purpose of describing a particular embodiment and is not intended to limit the invention.

All publications, patent applications, patent documents and other references herein are incorporated by reference in their entirety with respect to the teachings associated with the sentences and / or paragraphs in which the references are indicated.

It is expressly intended that the various features of the invention described herein can be used in any combination, unless the context provides otherwise. Furthermore, it is also expected that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. For illustration purposes, where the specification states that the composition comprises components A, B and C, either A, B or C, or a combination thereof, may be omitted and abandoned alone or in any combination. It is clearly intended to be able to.

The singular forms "a", "an" and "the" as used in the description of the present invention and the accompanying claims are intended to include the plural forms as well, unless the context explicitly states otherwise.

Also, as used herein, "and / or" is any and all possible combinations of one or more related listed matters and, when interpreted selectively ("or"). Mention and include lack of combination.

The term "about" as used herein refers to a measurable value such as dose or duration of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5% of a defined amount. , Or indeed ± 0.1% variation.

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

As used herein, "bulge sequence" refers to a non-complementary (non-hybridizing) nucleotide sequence contained in a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct / CRISPR nucleic acid array. In synthetic tracr nucleic acid constructs, the bulge sequence is located between the anti-zipper and anti-stitch sequences and is non-complementary (100% non-identical) with the corresponding bulge sequence in the synthetic CRISPR nucleic acid construct / CRISPR nucleic acid array. , About 3 nucleotides to about 6 nucleotides (eg, about 3, 4, 5, 6 nucleotides; for example, about 3 to about 6 nucleotides, about 3 to about 5 nucleotides, about 3 to It consists of about 4 nucleotides, etc.). The bulge sequence of the synthetic CRISPR nucleic acid construct / CRISPR nucleic acid array is located between the zipper and the stitch sequence and between the zipper and the stitch sequence and contains at least two nucleotides (eg, (-NN-) nucleotide sequence) ( For example, about 2, 3, 4, 5, 6 nucleotides; for example, about 2 to about 6 nucleotides, about 2 to about 5 nucleotides, about 2 to about 4 nucleotides; about 3 to about 6 Containing (such as 1 nucleotide, about 3 to about 5 nucleotides, etc.), and essentially consisting of, or consisting of. Nucleotide compositions of bulge sequences are at least two (synthetic CRISPR nucleic acid constructs / CRISPR nucleic acid arrays) or 3 in any series, unless they are complementary (eg, 100% non-identical) and thereby hybridize with each other. It may be more than one (synthetic tracr nucleic acid construct) nucleotides. When anti-zipper and zipper sequences and anti-stitch and stitch sequences are aligned and hybridized as a result of non-complementary bulge sequences on synthetic tracr nucleic acid constructs and synthetic CRISPR nucleic acid constructs / CRISPR nucleic acid arrays (in chimeric nucleic acid constructs). As), protrusions or bulges are formed on the synthetic tracr nucleic acid construct side of the chimeric nucleic acid construct (see, eg, FIGS. 12-16). Although not 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 described features, integers, steps, operations, factors, and / or components. Identifies, but does not preclude the existence or addition of one or more other features, integers, steps, operations, factors, components, and / or groups thereof.

As used herein, the tentative phrase "becomes essential" is the basic and novel of the invention in which the scope of the claims is the particular substance or step set forth in the claims and the claims. It means that it is interpreted as including those that do not substantially affect the characteristics (s) of. Therefore, the term "becomes essential from" is not intended to be construed as equivalent to "comprising" as used in the claims of the present invention.

"Cas9 nuclease" refers to a large group of endonucleases that catalyze double-stranded DNA cleavage in the CRISPR Cas system. These polypeptides are well known in the art and many of their structures (sequences) have been characterized (see, eg, WO 2013/176772; WO / 2013/188638). The domains for catalyzing the cleavage of dsDNA are the RuvC domain and the HNH domain. The RuvC domain has the role of nicking the (-) strand, and the HNH domain has the role of nicking the (+) strand (eg, Gasinasetal.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 derived from different origins (eg, different organisms, different coding regions).

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

As used herein, the term "complementarity" or "complementarity" refers to the natural binding of a polynucleotide under acceptable 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" to which only a portion of the nucleotides bind, or can be complete if there is total complementarity between the single-stranded molecules. .. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

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

A "fragment" or "part" of the nucleotide sequence of the invention is shorter in length with respect to the reference nucleic acid or nucleotide sequence (eg, 1, 2, 3, 4, 5, 6, 7, 8, It is understood to mean a nucleotide sequence (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides shortened) and is identical to the reference nucleic acid or nucleotide sequence. Or almost identical (eg, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% same) Containing the nucleotide sequence of contiguous nucleotides, consisting essentially of, and / or consisting of. Such nucleic acid fragments or portions according to the present invention may, where appropriate, be contained in a larger polynucleotide of which it is a component. Thus, for example, hybridizing to at least a portion of the target DNA (or hybridizing to, and other grammatical changes thereof) is identical to the length of contiguous nucleotides in the target DNA. Alternatively, it refers to hybridization to substantially the same nucleotide sequence.

As used herein, "GR1" is a single nucleotide (G) or three short nucleotide sequences (GTT) contained in the repeat portion of a synthetic CRISPR nucleic acid construct or _crRNA . GR1 does not hybridize to the anti-repeat of synthetic tracr nucleic acid constructs or _tracrRNAs of the present disclosure. However, in a non-standard Watson-Crick base pairing scheme, GR1 can form wobble base pairs with U at the ends of the anti-CRISPR repeat portion of _tracrRNA .

As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO), and the like. The gene may or may not be used to produce a functional protein or gene product. The gene may contain both coding and non-coding regions (eg, introns, regulators, promoters, enhancers, termination sequences and / or 5'and 3'untranslated regions). A gene can be "isolated", thereby meaning a nucleic acid that is substantially or essentially free from the components normally found in binding to the nucleic acid in its natural state. Such components include other cellular materials, culture media by recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acids.

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

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 comprises a non-naturally occurring multiple copy of the naturally occurring nucleotide sequence.

Different nucleic acids or proteins with homology are referred to herein as "homologs". 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 a percentage of position identity (ie sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the invention further include homologues to the nucleotide and polypeptide sequences of the invention. As used herein, "orthologous" refers to a homologous nucleotide sequence and / or amino acid sequence in a different species that arises from a common ancestral gene during speciation. The homologue of the nucleotide sequence of the present invention has substantial sequence identity (eg, at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%) with the nucleotide sequence of the present 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, the homologue of the Cas9 polypeptide useful in the present invention may be about 70% homologous or higher than any one of the Cas9 sequences provided herein.

As used herein, hybridization, hybridization, hybridization, and their grammatical changes are two fully complementary nucleotide sequences or some mismatched bases. Refers to the binding of substantially complementary sequences in which a pair can 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, the conditions of hybridization may be high stringency, or they may be medium stringency or low stringency, depending on the length of the sequence to be hybridized and the amount of complementarity. May be. The conditions that make up low, medium and high stringency with respect to the purpose of hybridization between nucleotide sequences are well known in the art (eg, Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109). : E2579-E2586; MR Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Y

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

The terms "invasive exogenous genetic factor", "invasive exogenous nucleic acid" or "invasive exogenous DNA" are DNAs that are exogenous to the bacterium (eg, but not limited to viruses, bacteriophages, and / or plasmids). It means a genetic factor derived from a pathogen including.

A "natural" or "wild" 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 the 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 a nucleotide sequence located just downstream of an "anti-stitch sequence" within a synthetic tracr nucleic acid construct. Nexus is about 6-10 nucleotides in length and contains the highly conserved sequence: TNANNC. In some embodiments, the nexus is a 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) It may be a nucleotide sequence of (A / C) (C / A) (A / G) (A / U)). Without being bound by any particular theory, based on sequence conservation, it is believed that nexus can be important in Cas9 orthogonality and recognition.

Also, as used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "nucleotide sequence," and "polynucleotide" are linear or branched single- or double-stranded RNAs or Refers to DNA, or a hybrid thereof. The term also includes RNA / DNA hybrids. If dsRNA is produced synthetically, uncommon bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others may also be used for antisense, dsRNA, and ribozyme pairings. can. For example, polynucleotides containing C-5 propin analogs of cytidine and uridine have been shown to bind RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications, such as phosphodiester main chains, or modifications to 2'-hydroxy within the ribose glycosyl group of RNA can also be made. The 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 may be described and used in the form of RNA depending on the purpose of use.

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 hand (as a result, not a natural product).

As used herein, the term "nucleotide sequence" refers to a sequence of these nucleotides at the 5'to 3'end of a heteropolymer or nucleic acid molecule of a nucleotide, comprising a DNA or RNA molecule, including a cDNA, DNA fragment or portion. It comprises 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 and also refer to a heteropolymer of nucleotides. Unless otherwise indicated, the nucleic acid molecules and / or nucleotide sequences provided herein are shown herein from left to right in the 5'to 3'direction and are US sequence rules. (Federal Regulations Code, Vol. 37, Sections 1.821 to 1.825) and World Intellectual Property Organization (WIPO) Standard ST. It is represented using a standard code for characterizing the nucleotides shown in 25. As used herein, the "5'region" can mean the region of the polynucleotide closest to the 5'end. Thus, for example, a factor within the 5'region of a polynucleotide can be located somewhere from the first nucleotide located at the 5'end of the polynucleotide to the nucleotide located in the middle of the polynucleotide. As used herein, the "3'region" can mean the region of the polynucleotide closest to the 3'end. Thus, for example, a factor within the 3'region of a polynucleotide can be located somewhere from the first nucleotide located at the 3'end of the polynucleotide to the nucleotide located in the middle of the polynucleotide.

As used herein, the terms "percent sequence identity" or "percent identity" are compared to the test ("subject") polynucleotide molecule (or complementary strand thereof) when the two sequences are optimally aligned. Refers to the percentage of identical nucleotides in the linear polynucleotide sequence of the referenced (“query”) polynucleotide molecule (or its complementary strand). In some embodiments, "percent identity" can refer to the percentage of the same amino acid in the amino acid sequence.

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

As used herein, the terms "reduced", "reduced", "reduced", "reducion", "dimish", "suppress" and "suppress". "Decrease" (and their grammatical changes) are, for example, at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, compared to controls. It shows a decrease of 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In certain embodiments, the reduction cannot or essentially result in a detectable activity or amount (ie, a trivial amount, eg, less than about 10% or even less than 5%). Thus, in some embodiments, mutations in Cas9 nucleases increase Cas9 nuclease activity by at least about 5%, 10%, 15%, 20%, 25%, as compared to controls (eg, wild-type Cas9). It can be reduced by 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

As used herein, "repeat sequence" refers to, for example, the repeat sequence of a wild-type CRISPR locus or synthetic CRISPR nucleic acid construct separated by a "spacer sequence". The repeat sequence is complementary (eg, 100% base pair match) or substantially complementary, eg, at least 70% complementary (eg, 70%, 71%, 72%) to the corresponding anti-repeat sequence. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher).

The "repeat sequence" of the synthetic CRISPR nucleic acid construct of the present disclosure includes an optional "zipper sequence", "bulge sequence", "stitch sequence", and "spacer sequence". In some embodiments, the synthetic CRISPR nucleic acid construct may comprise _GR1 (in other embodiments, included in the stitch sequence).

As used herein, "zipper sequence" refers to any portion of the repeat sequence located immediately upstream of the bulge sequence 3'or (in the direction of 3'to 5') within the synthetic CRISPR nucleic acid construct, at least. Approximately 3 nucleotides (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, Containing, consisting of, or essentially consisting of 24, 25, 26, 27, 28, 29, 30 or more nucleotides, or any range or value within them. 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, if present, the zipper sequence and the anti-zipper sequence. Can hybridize to each other upon contact, thereby binding the two nucleic acid constructs together. In some embodiments, the zipper / anti-zipper arrangement can be referred to as the "upper stem". The zipper sequence is completely complementary (eg, 100% base pair match) or substantially complementary to the corresponding anti-zipper sequence, eg, at least 70% complementary (eg, 70%, 71%, 72%, 73%, etc.). 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 sequence of the synthetic tracr nucleic acid construct of the present invention is at least about 3 nucleotides that are completely or substantially complementary to the synthetic CRISPR nucleic acid construct or the corresponding zipper sequence within the synthetic CRISPR nucleic acid array. For example, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. , 27, 28, 29, 30 or more nucleotides, or any range or value within it). The anti-zipper sequence is the RNase III binding site and thus comprises a nucleotide sequence well known in the art to be involved in the RNase III binding (eg, Pertzev and 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 throughout the window of component (eg, nucleotide or amino acid) alignment. "Identity" is not limited: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome. ) Academic Press, New York (1993); Computer Identity of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Genome Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Identity Primer (Gribskov, M. and Deverex, J., eds.) Known as (Gribskov, M. and Deverex, J., eds.) It can be easily calculated by the method of.

As used herein, the "spacer sequence" is a nucleotide sequence complementary to the target DNA (eg, "protospacer sequence"). The spacer sequence is completely or substantially complementary to the target DNA (eg, at least 70% complementary (eg, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%). 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or higher)). In 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 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 nucleotides (seed sequences) in the 3'region of the 20 nucleotide spacer sequences will be the target DNA. It can 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 parenchyma with the target DNA. 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. (For example, at least about 70% complementary). In a typical embodiment, the first 7 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5'region of the spacer sequence are substantially complementary to the target DNA ( For example, at least about 70% complementary).

As used herein, "stitch sequence" refers to a nucleotide sequence that comprises, consists of, or consists of about 5 nucleotides in length and has a consensus nucleotide sequence of NNTNN. The "stitch sequence" is located on the synthetic CRISPR nucleic acid construct (in the direction of 5'to 3') just upstream of the "bulge sequence" and downstream of " _GR1 ". The "stitch sequence" tends to have a high AT content and hybridizes to the "anti-stitch sequence" located within the synthetic tracr nucleic acid construct. In some particular embodiments, the stitch sequence is NNTNN, TTTGT, TTTTA, (T / C) (T / C) T (T / C) (T / G) (in the 5'to 3'direction). , TTTTA, contains the nucleotide sequence of TTTCA, consists of, or consists of.

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

As used herein, the terms "substantially identical" or "substantially identical" in the association of two nucleic acid molecules, nucleotide sequences or protein sequences are the following sequence comparisons when compared and aligned for maximum match. At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% by measurement or visual inspection using one of the algorithms. , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Refers to two or more sequences or subsequences having%, 99%, and / or 100% nucleotide or amino acid residue identity. In some embodiments of the invention, substantial identity exists over a region of the sequence that is at least about 50 to about 150 residues in length. Thus, in some embodiments of the invention, the substantial identity is at least about 3 to about 15 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13. Length of 14, 15 residues, etc., or any value or range within it), at least about 5 to about 30, at least about 10 to about 30, at least about 16 to about 30, at least about 18 to at least about 25, at least about 18, at least about 22, at least about 25, at least about 30, at least about 40, at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, It is present over the length of about 140, about 150, or more residues, and the region of the sequence which is any range within it. In a representative embodiment, the sequence can be substantially identical over at least about 22 nucleotides. In some particular embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences of the invention can be about 70% to about 100% identical over at least about 16 to about 25 nucleotides. In some embodiments, the sequences of the invention can be about 75% to about 100% identical over at least about 16 to about 25 nucleotides. In a further embodiment, the sequences of the invention can be about 80% to about 100% identical over at least about 16 to about 25 nucleotides. In a further embodiment, the sequences of the invention can be about 80% to about 100% identical over at least about 7 nucleotides to about 25 nucleotides. In some embodiments, the sequences of the invention can be about 70% identical over at least about 18 nucleotides. In other embodiments, the sequences can be about 85% identical over about 22 nucleotides. In yet another embodiment, the sequences can be 100% homologous over about 16 nucleotides. In a further embodiment, the sequences are substantially identical over the length of the coding region. Moreover, in typical embodiments, substantially identical nucleotide or protein sequences perform substantially identical functions (eg, Cas9 HNH and / or RuvC nickase activity).

For sequence comparison, typically one sequence serves as a reference sequence to which the test sequences are compared. When using the sequence comparison algorithm, the test and comparison sequences are entered into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. 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 alignment for arranging comparison windows is widely known to those skilled in the art by tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, and Needleman and Lipman's similarity search method. In some cases, this can be done by computer implementation of algorithms such as GAP, BESTFIT, FASTA, and TFASTA, which are available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). .. The "identity fraction" of the aligned compartments of the test and reference sequences is the number of identical components shared by the two arranged sequences, that is, the entire reference sequence or the smaller of the reference sequence. It is the number divided by the total number of components in the specified part). Percent sequence identity is expressed by multiplying the identity fraction by 100. Comparison 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 the purposes of the present invention, "percent identity" may be determined using BLASTX version 2.0 for the translated nucleotide sequence and BLASTN version 2.0 for the polynucleotide sequence.

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

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

Also, the two nucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. In some typical embodiments, the two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

In the context of nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and differ under different environmental parameters.核酸のハイブリダイゼーションに対する広範なガイドは、Ｔｉｊｓｓｅｎ Ｌａｂｏｒａｔｏｒｙ Ｔｅｃｈｎｉｑｕｅｓ ｉｎ Ｂｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ－Ｈｙｂｒｉｄｉｚａｔｉｏｎ ｗｉｔｈ Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｐｒｏｂｅｓ ｐａｒｔ Ｉ ｃｈａｐｔｅｒ ２“Ｏｖｅｒｖｉｅｗ ｏｆ ｐｒｉｎｃｉｐｌｅｓ ｏｆ ｈｙｂｒｉｄｉｚａｔｉｏｎ ａｎｄ ｔｈｅ ｓｔｒａｔｅｇｙ ｏｆ ｎｕｃｌｅｉｃ ａｃｉｄ ｐｒｏｂｅ ａｓｓａｙｓ”Ｅｌｓｅｖｉｅｒ，Ｎｅｗ Ｙｏｒｋ（１９９３） Seen in. In general, high stringent hybridization and washing conditions are selected to be about 5 ° C. below the melting point ( _Tm ) for specific sequences at defined ionic strength and pH.

T _m is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equal to T _m for the specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues in a filter on a Southern or Northern blot is 50% formamide (42 ° C.) containing 1 mg heparin. Hybridization is performed overnight. An example of high stringent cleaning conditions is 0.1 5M NaCl, 72 ° C., about 15 minutes. Examples of stringent wash conditions are 0.2 × SSC wash, 65 ° C., 15 minutes (see Sambrook (below) for a description of SSC buffers). Low stringency lavage is often performed before high stringency lavage to eliminate background probe signals. For example, an example of medium stringency washing for a duplex of more than 100 nucleotides is 1 x SSC, 45 ° C., 15 minutes. For example, an example of a low stringency wash for a duplex of more than 100 nucleotides is 4-6 × SSC, 40 ° C., 15 minutes. For short probes (eg, about 10-50 nucleotides), stringent conditions are typically less than about 1.0 M salt concentration of Na ions, typically about 0.01-1.0 M. It contains Na ion concentration (or other salt) (pH 7.0-8.3) and the temperature is typically at least about 30 ° C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. In general, in a particular hybridization assay, a signal-to-noise ratio that is twice (or higher) than that found for unrelated probes indicates detection of specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This can occur, for example, when a copy of the nucleotide sequence is made with the maximum codon degeneracy allowed by the genetic code.

The following is an example of a set of hybridization / washing conditions that can be used to clone a homologous nucleotide sequence that is substantially identical to the reference nucleotide sequence of the present invention. In one embodiment, the reference nucleotide sequence is washed with 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50 ° C.), 2 × SSC, 0.1% SDS (50 ° C.). Hybridize with the "test" nucleotide sequence. In another embodiment, the reference nucleotide sequence is washed with 1 × SSC, 0.1% SDS (50 ° C.) in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50 ° C.). , Or, washed with 0.5 × SSC, 0.1% SDS (50 ° C.) in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA (50 ° C.), “test” nucleotide sequence. Hybridizes with. In a further embodiment, the reference nucleotide sequence is washed with 0.1 × SSC, 0.1% SDS in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50 ° C.) (50). "Test" at (° C.) or with a 0.1 × SSC, 0.1% SDS wash (65 ° C.) in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA (50 ° C.). It 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 species of interest. Codon optimization is widely known in the art and involves modification of nucleotide sequences for codon usage bias using species-specific codon usage frequencies. Codon usage is based on sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequence is expressed in the nucleus, codon usage is based on sequence analysis of the highly expressed nuclear gene for the species of interest. Nucleotide sequence modifications are determined by comparing species-specific codon usage with codons present in the native polynucleotide sequence. As will be appreciated in the art, nucleotide sequence codon optimization is less than 100% identical to the native nucleotide sequence (eg, 70%, 71%, 72%, 73%, 74%, 75%, 76). %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.), but still encode a polypeptide having the same function as that encoded by the original native nucleotide sequence. Give rise to 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, the recombinant nucleic acid molecules, nucleotide sequences and polypeptides of the invention are "separated". A "separated" nucleic acid molecule, a "separated" nucleotide sequence or a "separated" polypeptide is a nucleic acid molecule, nucleotide sequence that exists by hand away from its natural environment and is therefore not a natural product. Or a polypeptide. The isolated nucleic acid molecule, nucleotide sequence or polynucleotide is associated with at least some of the other components of an organism or virus of natural origin, such as the structural components of a cell or virus or other polypeptide or polynucleotide. It may be present in a purified form, at least partially separated from the nucleic acids commonly found. In a typical embodiment, the separated nucleic acid molecule, the separated nucleotide sequence and / or the separated polypeptide are 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 can be present in an unnatural environment (eg, a recombinant host cell). Thus, for example with respect to nucleotide sequences, the term "isolated" means that it is detached from naturally occurring chromosomes and / or cells. Also, a polynucleotide is separated from its naturally occurring chromosomes and / or cells, and then genetic conditions, chromosomes and / or cells in which it does not occur naturally, chromosomes and / or cells (eg, different host cells, different regulatory sequences, and / /). Or when inserted at a position in the genome that is different from that found naturally), it is also isolated. Thus, recombinant nucleic acid molecules, nucleotide sequences and their encoded polypeptides are "separated" by human hand in that they exist apart from their natural environment and are therefore not natural products. 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 regulators for expression in various biological cells. Can be done. Thus, in a representative embodiment, the recombinant nucleic acid of the invention can further comprise one or more promoters operably linked to one or more nucleotide sequences.

By "operably linked" or "operably associated" as used herein, it means that the factors indicated are functionally and generally physically related to each other. Thus, as used herein, the term "operably linked" or "operably associated" refers to a nucleotide sequence on a single nucleic acid molecule that is functionally related. Thus, the first nucleotide sequence operably linked to the second nucleotide sequence means a situation in which the first nucleotide sequence is functionally located in a functional relationship with the second nucleotide sequence. For example, when a promoter is responsible for transcription or expression of a nucleotide sequence, the promoter is operably associated with said nucleotide sequence. One of skill in the art will appreciate that a control sequence (eg, a promoter) does not need to be flanked by a nucleotide sequence associated with its operability, as long as the control sequence functions to direct its expression. Thus, for example, an intervening untranslated sequence that is still transcribed may be present between the promoter and the nucleotide sequence, and the promoter can still be considered to be "operably linked" to the nucleotide sequence.

A "promoter" is a nucleotide sequence (ie, a coding sequence) that controls or controls transcription of a nucleotide sequence that is operably associated with a promoter. The coding sequence can encode a polypeptide and / or a functional RNA. Typically, a "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and controls the initiation of transcription. Generally, the promoter is found upstream of the start of the coding region of the 5'or corresponding coding sequence. The promoter region may contain other factors that act as regulators of gene expression. These include TATA box consensus sequences and often include 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.), Polypropylene Press, plenum Press, p. 227).

Promoters are, for example, constitutive, inducible, temporally controlled, developmentally controlled, for use in the preparation of recombinant nucleic acid molecules (ie, "chimeric genes" or "chimeric polynucleotides"). It may include chemically controlled, tissue-suitable and / or tissue-specific promoters. These various types of promoters are known in the art.

Promoter selection varies according to the temporal and spatial requirements for expression and also depends on the host cell being transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge that exists in the art, suitable promoters 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 readily utilized and implemented in other systems as needed. can.

In some embodiments, the nucleic acid constructs of the invention may be "expression cassettes" or may be contained within expression cassettes. As used herein, an "expression cassette" encodes a nucleotide sequence of interest (eg, a nucleic acid construct of the invention (eg, a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; the polypeptide of interest). Means 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). Therefore, some aspects of the invention provide an expression cassette designed to express the nucleotide sequences of the invention.

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

The expression cassette may also optionally contain functional transcriptional and / or translational termination regions (ie, termination regions) within selected host cells. A variety of transcription termination factors are available for use in expression cassettes, are involved in transcription termination beyond the heterologous nucleotide sequence of interest, and modify mRNA polyadenylation. The termination region can be natural to the transcription initiation region, to the operably linked nucleotide sequence of interest, to the host cell, or to another. It can be derived from the origin (ie, to the promoter, to the nucleotide sequence of interest, to the host, exogenous or heterologous, or any combination thereof).

The expression cassette can also contain nucleotide sequences for selectable markers that can be used to select transformed host cells. As used herein, a "selectable marker", when expressed, gives the host cell expressing the marker a distinguishable phenotype, and thus such traits from those without the marker. Means a nucleotide sequence that allows transformed cells to be distinguished. Such nucleotide sequences indicate whether the marker provides a trait that can be selected by chemical means, eg, by using a selective agent (eg, antibiotic), or whether the marker simply, eg, screens (eg, eg, screening). Either a selectable marker or a screenable marker can be encoded, depending on whether the trait can be identified via observation or testing by fluorescence). 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 the expression cassette, the nucleic acid molecules and nucleotide sequences described herein can be used in the context of vectors. The term "vector" refers to a composition for transferring, delivering or introducing a nucleic acid (s) (or nucleic acids) into a cell. The vector comprises a nucleic acid molecule containing the nucleotide sequence (s) to be transferred, delivered or introduced. Vectors for use in the transformation of host organisms are well known in the art. Non-limiting examples of the general class of vectors are, but are not limited to, double-stranded or single-stranded linear or cyclic viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, phosmids. Includes vectors, bacteriophages, artificial chromosomes, or agrobacterium binary vectors, which may or may not be self-transmitting or mobilizing. The vectors defined herein can transform a prokaryotic or eukaryotic host by integration into the cellular genome or can be extrachromosomal (eg, a self-replicating plasmid with an origin of replication). ). A DNA vehicle that is capable of replication in two different host organisms that can be selected naturally or by design from actinomycetes and related species, bacteria and eukaryotes (eg, more advanced plants, mammals, yeast or fungal cells). The shuttle vector, which means, is further included. In some typical embodiments, the nucleic acids in the vector are under the control of a suitable promoter or other regulator for transcription in the host cell and are operably linked. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may include its own promoter or other regulator, and in the case of cDNA, this may be under the control of a suitable promoter or other regulator for expression in the host cell. Can be. Thus, the nucleic acid molecules and / or expression cassettes of the invention can be included within the vector as described herein and known in the art.

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

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

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

“Stabilly introduced” or “stable introduced” in the context of a polynucleotide introduced into a cell means that the introduced polynucleotide is stably introduced into the cell's genome. It is intended to be taken up and therefore cells are stably transformed with polynucleotides.

As used herein, "stable transformation" or "stable transformation" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, more specifically by progeny of multiple subsequent generations. As used herein, "genome" also includes nuclear and plastid genomes, and thus includes integration of nucleic acids into, for example, chloroplast or mitochondrial genomes. Stable transformations as used herein can also refer to transgenes that are maintained extrachromosomally, for example, as minichromosomes or plasmids.

Transient transformation can detect the presence of a peptide or polypeptide encoded by one or more transgenes introduced into an organism, enzyme-linked immunosorbent assay (ELISA) or Western blot. It can be detected by such as. Stable transformation of cells is the genomic DNA of cells using nucleic acid sequences that specifically hybridize to the nucleotide sequences of the transgene introduced into an organism (eg, plants, mammals, insects, paleontology, bacteria, etc.). It can be detected by a Southern blot hybridization assay or the like. Stable transformation of cells should be detected by a Northern blot hybridization assay of cellular RNA using a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of the transgene introduced into a plant or other organism. Can be done. Stable transformation of cells is also well known in the art for polymerase chain reaction (PCR), for example, with specific primer sequences that hybridize to the target sequence (s) of the transgene. It can also be detected by other amplification reactions that result in amplification of the transgene sequence and can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

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

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

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

Thus, the nucleotide sequence can be introduced into the host organism or its cells by any number of methods well known in the art. The methods of the invention do not depend on any particular method for the introduction of one or more nucleotide sequences into an organism as long as they are close 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 cell of interest in a single transformation event or in a separate transformation event, or if relevant, the nucleotide sequence is part of a reproductive protocol. It can be incorporated into plants.

The present invention is a composition with increased efficiency and increased specificity for site-specific nicking, cleavage and / or modification of the target DNA and for site-specific targeting of the target polypeptide to the target DNA. And how.

Nucleic acid constructs and nucleotide sequences of the invention can 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, the synthetic CRISPR nucleic acid ( _crRNA ) can contain a fluctuating base GR1 sequence (G / U) and a stitch sequence, or the crRNA contains a (G / U) fluctuating base. It can include stitch sequences and therefore does not further represent the _GR1 sequence. In some cases, GR1 does not pair (ie, G / A is mismatched with Lje, FIG. 20). Further equivalence includes those shown in the equivalence table provided below (Table 1) (see also FIG. 22).

Thus, in one aspect of the invention, a synthetic transcoded CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct is provided, said construct containing at least about 3 nucleotides in the 5'to 3'direction. Anti-zipper sequence containing, consisting of, or consisting of; bulge sequence containing at least about 3 nucleotides; anti-stitch sequence containing nucleotide sequence of NNANN; TNANNC, T (A / C) A (A /) G) (G / A) C (or U (A / C) A (A / G) (G / A) C)), TCAAAC, (or UCAAAC), TAAGGC (or UAAGGC), GATAAGG (or GAUAAGG), GATAAGGCTT (or GAUAAGGCUU), TCAAG (or UCAAG), TCAAGCAA (or UCAAGCAA), T (C / A) AA (A / C) (C / A) (A / G) (A / T) (or U (C) / A) AA (A / C) (C / A) (A / G) (A / U)), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or a nexus sequence containing the nucleotide sequence of TCAAACAAAGCTTCAGC; and having at least one hairpin. Containing, consisting of, or consisting of a hairpin sequence comprising a nucleotide sequence, said hairpin comprises at least three matching base pairs, where the anti-zipper sequence is immediately upstream of the bulge sequence. Located, the bulge sequence is located just upstream of the anti-stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence. In some embodiments, the anti-stitch sequence comprises the nucleotide sequences of NNANN, ACAAA, TAAAA, (T / C) (A / G) T (A / G) (A / G), TAAAA, TGAAA. , From, or consists of.

In a further embodiment, an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; an anti-stitch containing the nucleotide sequence of NNANN. Sequence; TNANC, 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 GAUAAGGCCUU), TCAAG (or UCAAG), TCAAGCAA (or UCAAGCAA), T (C / A) AA (A / C) (C / A) ( A / G) (A / T) (or U (C / A) AA (A / C) (C / A) (A / G) (A / U)), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC nucleotides Synthetic transcoded CRISPR containing, consisting of, or consisting of a nexus sequence and a hairpin sequence containing, consisting of, and a nucleotide sequence having at least one hairpin. A (tracr) nucleic acid construct is provided, wherein the hairpin contains at least three matching base pairs, where the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is , The anti-stitch sequence is located just upstream of the anti-stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence.

In some embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, or consists of, at least about 3 nucleotides. In some embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, or consists of, at least about 4 nucleotides. In other embodiments, the bulge sequence of the synthetic tracr nucleic acid construct comprises, consists of, or consists of 5 nucleotides. In other embodiments, the hairpin sequence of the synthetic tracr nucleic acid construct comprises, consists of, or consists of at least two hairpins, and each hairpin comprises at least three matching base pairs.

In a further aspect, the invention is a nucleotide sequence having at least 2 nucleotides, an optional zipper sequence consisting of, or consisting essentially of, containing at least about 3 nucleotides in the 3'to 5'direction. A bulge sequence consisting of or consisting essentially of (eg, a nucleotide sequence of (-NN-)), a stitch sequence consisting of or consisting of a nucleotide sequence of NNTNN (or NUNN), A spacer sequence having GR1 consisting of, or consisting essentially of, nucleotide G or GTT, and 5'and _3'ends , with 100% identity with the target DNA at its 3'end. Provided is a synthetic CRISPR nucleic acid (eg, crRNA, crDNA) construct comprising, consisting of, or consisting of a spacer sequence comprising, consisting of, or consisting of at least 7 nucleotides having, and a zipper sequence. Is located just upstream of the bulge sequence, if present, the bulge sequence is located just upstream of the stitch sequence, the stitch sequence is located just upstream of _GR1 , and _GR1 is the spacer sequence. Located just upstream of.

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

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

In another aspect, a chimeric nucleic acid construct (or guide nucleic acid construct) comprising the synthetic tracr nucleic acid construct and the synthetic CRISPR nucleic acid construct of the present invention is provided, wherein the zipper sequence of the synthetic CRISPR nucleic acid construct is the synthetic tracr nucleic acid construct. Anti-zipper sequence and at least about 70% (eg, about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82) %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Complementary and hybridized (99%, etc.), the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% identical and hybridized to the anti-stitch sequence of the synthetic tracr nucleic acid construct, and the bulge sequence and synthetic CRISPR of the synthetic CRISPR nucleic acid construct. The bulge sequence of the nucleic acid construct is non-complementary.

In another embodiment, a chimeric nucleic acid construct comprising, including, or consisting of a synthetic tracr nucleic acid construct and a synthetic CRISPR nucleic acid construct of the present invention is provided, wherein the NNUNN of the stitch sequence of the synthetic CRISPR nucleic acid construct is provided. , 100% complementary and hybridized with NNANN of the anti-stitch sequence of the synthetic tracr nucleic acid construct, the (G) of the stitch sequence forms a fluctuating base pairing with U of the anti-stitch sequence, and 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 the anti-zipper sequence of the synthetic tracr nucleic acid construct. Hybridizes to a zipper sequence.

In some embodiments, the chimeric nucleic acid construct may optionally further comprise a nucleotide linking the hybridized zipper and anti-zipper sequence at the end of the hybridized sequence (distal to the bulge sequence). can. In a further embodiment, in the absence of a zipper and anti-zipper, the chimeric nucleic acid construct can optionally further comprise a nucleotide linking the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid. The nucleotide to be linked may be any nucleotide (eg, T, A, G, C), and the number of nucleotides linking the zipper sequence and the anti-zipper sequence or the bulge sequence is about 3 to about 7. could be.

In a further aspect, the synthetic tracr nucleic acid construct, synthetic CRISPR nucleic acid construct, CRISPR nucleic acid array, or chimeric nucleic acid construct of the present invention is at least 70% identical (eg, about 70%) to the amino acid sequence encoding the Cas9 nuclease, 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 with the amino acid sequence encoding Cas9 nuclease can be further included. The Cas9 nuclease useful in the present invention may be any Cas9 nuclease known to catalyze DNA cleavage in the CRISPR-Cas system. As is known in the art, such Cas9 nucleases include HNH motifs and RuvC motifs (see, eg, WO 2013/176772; WO / 2013/188638). In some embodiments, the HNH or RuvC motifs may contain mutations that reduce or eliminate their activity compared to wild-type Cas9 nucleases. 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, including missense mutations, nonsense mutations, frameshift mutations, etc., can be used to reduce or eliminate the activity of HNH and / or RuvC motifs in Cas9 nucleases.

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

Thus, in some embodiments, the Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, Cas9 from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, and Cas9 from the Cas9 nuclease CRISPR 3 (Sth CR3) group. It may or may consist essentially of a Cas9 nuclease from the CD034 (Lb) group and / or a Cas9 nuclease from the Cas9 nuclease Lactobacillus rhhamnosus GG (Lrh) group. In a further embodiment, the amino acid sequence encoding Cas9 nuclease may be any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 53. In a further embodiment, the Cas9 nuclease useful in the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, synthetic CRISPR nucleic acid arrays, and / or chimeric nucleic acid constructs of the present disclosure is any one of SEQ ID NOs: 1 to SEQ ID NO: 53. Containing, consisting of, or consisting of a nucleotide sequence encoding an amino acid sequence having at least 70% identity with the amino acid sequence.

Moreover, in certain embodiments, the Cas9 nuclease can be encoded by a codon-optimized nucleotide sequence for the organism containing the target DNA. In yet another embodiment, the Cas9 nuclease may contain at least one nuclear localization sequence.

The inventor has surprisingly discovered a functional pairing between a particular group of Cas9 nucleases and the nexus sequences of synthetic tracr nucleic acid constructs (tracrRNA, tracrDNA). Thus, in some embodiments, if the nexus sequence is GATAAGGC or GATAAGGCCATGCC, the Cas9 nuclease is from the Cas9 nuclease Streptococcus thermophilus CRISPR 1 (STh CR1) group; if the nexus sequence is TAAGGC or TAAGGCTAGTCC. The Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases; if the nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is from the Lactobacillus buchneri group (03) of the Cas9 nuclease. If the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, the Cas9 CRISPR is from the Cas9 nuclease Lactobacillus rhhamnosus GG (Lrh) group.

As described herein, Cas9 nucleases useful in the present invention may contain mutations in the HNH and / or RuvC motifs, thereby reducing or eliminating the activity of each motif. As is known in the art, mutations within the HNH motif reduce / eliminate site-specific nicks in the (+) strand of the double-stranded target DNA, and mutations in the LucV active site are double-stranded targets. Reduces / eliminates site-specific nicks in the (-) strand of DNA. Mutations in both active sites reduce / eliminate DNA cleavage (ie, reduce / eliminate site-specific cleavage of target DNA). Thus, in some embodiments, the synthetic tracr nucleic acid constructs, CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and / or chimeric nucleic acid constructs of the present disclosure comprise a Cas9 nuclease with a mutation within the RuvC active site motif. In other embodiments, the synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and / or chimeric nucleic acid constructs of the present disclosure comprise a Cas9 nuclease with a mutation within the HNH active site motif. In a further embodiment, the synthetic tracr nucleic acid constructs, CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, and / or chimeric nucleic acid constructs of the present disclosure contain Cas9 nucleases with mutations within the HNH active site motif and within the RuvC motif.

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

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

In a further embodiment, methods for site-specific cleavage of double-stranded target DNA are provided, including: transcoding CRISPR (tracr) nucleic acid molecules and CRISPR nucleic acid molecules, Cas9 nuclease (eg, SEQ ID NO: 1). The step of contacting with the target DNA in the presence of ~ 53), where (a) the tracr nucleic acid molecule is an anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; at least about 3 Barge sequence containing nucleotides; Anti-stitch sequence containing nucleotide sequence of NNANN; TNANNC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, Nexus sequence comprising the nucleotide sequence of TCAAGCAA, T (C / A) AA (A / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and at least 1. Encoded by a nucleotide sequence, including, consisting of, or consisting of, a hairpin sequence comprising a nucleotide sequence, said hairpin containing at least three matching base pairs and an anti-zipper sequence. The bulge sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the anti-stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence. And (b) the CRISPR nucleic acid molecule is a zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction, a bulge sequence containing a nucleotide sequence having at least 2 nucleotides, NNTNN ( Or a stitch sequence containing a nucleotide sequence of NNUNN), GR1 containing a nucleotide G or GTT, and a spacer sequence having 5'ends and _3'ends , 100% identity with the target DNA at the 3'end. Encoded by a nucleotide sequence, including a spacer sequence containing at least 7 nucleotides having, the zipper sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is G. It is located just upstream of _R1 and GR1 is located just upstream of the spacer sequence, and further here, the anti-zipper sequence and anti-nucleotide of the _tracr nucleic acid molecule. The Tetch sequence is at least about 70% complementary and hybrid to the zipper and stitch sequences of the CRISPR nucleic acid molecule, respectively, and the spacer sequence of the CRISPR nucleic acid molecule is part of the target DNA (protospacer flanking motif on the target DNA). (Adjacent to (PAM)) is at least about 80% complementary and hybridizes, thereby causing 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. Bring.

In another embodiment, a method for site-specific cleavage of a double-stranded target DNA is provided, comprising contacting the double-stranded target DNA with a chimeric nucleic acid comprising: (A) Anti-zipper sequence containing at least about 9 nucleotides in the direction from 5'to 3'; bulge sequence containing at least about 4 nucleotides; anti-stitch sequence containing NNANN nucleotide 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 / A first nucleotide sequence (the hairpin comprises a nexus sequence comprising a nucleotide sequence of G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin. , The anti-zipper sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the anti-stitch sequence, and the anti-stitch sequence is the nexus sequence. The nexus sequence is located just upstream of the hairpin sequence); (b) a zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction, at least 2 nucleotides. A bulge sequence containing a nucleotide sequence having, a stitch sequence containing a nucleotide sequence of NNTNN (or NNUNN), GR1 containing a nucleotide G or GTT, and a spacer sequence having 5'ends and _3'ends thereof, 3'. A second nucleotide sequence containing a spacer sequence containing at least 7 nucleotides having 100% identity with the target DNA at the end (the zipper sequence is located just upstream of the bulge sequence and the bulge sequence is of the stitch sequence. Located just upstream, the stitch sequence is located just upstream of GR1, and _GR1 is located just upstream of the spacer sequence); and (c) _Cas9nuclease (eg, SEQ ID NOs: 1-53). A third nucleotide sequence encoding an amino acid sequence having at least 80% identity with the amino acid sequence encoding) (where the anti-zipper and anti-stitch sequences of the first nucleotide sequence are second. Hybridizing to the zipper and stitch sequences of the nucleotide sequence, the spacer sequence of the second nucleotide sequence becomes part of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA). It hybridizes, thereby resulting in site-specific cleavage of the target DNA within the region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA).

In a further embodiment, a method of site-specific targeting of the target polypeptide against a double-stranded (ds) target DNA is provided, transcoded CRISPR (tracr) nucleic acid molecules and CRISPR nucleic acid molecules, with a Cas9 nuclease (eg, sequence). Including the step of contacting with the target DNA in the presence of numbers 1-53), where (a) the tracr nucleic acid molecule is an anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; A bulge sequence containing at least about 3 nucleotides; an anti-stitch sequence containing the nucleotide sequence of NNANN; TNANNC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT , TCAAG, TCAAGCAA, T (C / A) AA (A / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence comprising the nucleotide sequence of TCAAACAAAGCTTCAGC; , Encoded by a nucleotide sequence, comprising a nucleotide sequence comprising a nucleotide sequence having at least one hairpin, said hairpin containing at least 3 matching base pairs and an anti-zipper sequence located just upstream of the bulge sequence. However, the bulge sequence is located just upstream of the anti-stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and ( b) The CRISPR nucleic acid molecule is a zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction, a bulge sequence containing at least 2 nucleotides (eg, a (-NN-) nucleotide sequence), NNTNN. A stitch sequence containing a nucleotide sequence, GR1 containing a nucleotide G or GTT, and a spacer sequence having 5'ends and _3'ends , at least 7 having 100% identity with the target DNA at the 3'end. Encoded by a nucleotide sequence, including a spacer sequence containing nucleotides, the zipper sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is just upstream of _GR1 . And GR1 is located just upstream of the spacer sequence, and further here _Cas9 nuclease is a variation within the HNH active site motif. Differently, it contains mutations within the RuvC active site motif and is fused to the polypeptide of interest, the anti-zipper sequence is approximately 70% complementary and hybridized with the zipper sequence, and the stitch sequence is 100% complementary to the stitch sequence. And hybridizes, the spacer sequence is approximately 80% complementary and hybridizes to the target DNA flanking the protospacer flanking motif (PAM) on the target DNA, whereby the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. It results in site-specific targeting of the target polypeptide to the target DNA in the region defined by complementary binding.

In a typical embodiment, as described herein, for a synthetic tracr nucleic acid construct, the bulge sequence or first nucleotide sequence of the synthetic tracr nucleic acid molecule comprises about 3, 4, or 5 nucleotides. Can and can be essentially, or can consist of. In other embodiments, the bulge sequence can contain, from, or can consist of 5 nucleotides, and the hairpin sequence can contain at least 2 hairpins, and from. It can be or consist of essentially, where each hairpin contains at least three matching base pairs.

In a further embodiment, the invention is 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 Cas9 nuclease. Provides a way to, here,
(A) The tracr nucleic acid molecule comprises an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch arrangement, TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAAGCTTCAGC, encoded by a nucleotide sequence comprising a nucleotide sequence comprising a nucleotide sequence having at least one hairpin. The hairpin contains at least three matching nucleotide pairs, and the anti-zipper sequence, if present, is located just upstream of the bulge sequence and the bulge sequence is just upstream of the anti-stitch sequence. Located, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and (b) CRISPR nucleic acid molecules are in the 3'to 5'direction. , An optional zipper sequence containing a nucleotide sequence having at least 3 nucleotides that hybridizes to an anti-zipper, a bulge sequence containing a (-NN-) nucleotide sequence, a stitch sequence containing a NNUNN nucleotide sequence, and 5'. A spacer sequence having a terminal and a 3'end is encoded by a nucleotide sequence comprising a spacer sequence at the 3'end containing at least 7 nucleotides having 100% identity with the target DNA, and the zipper sequence. If present, it is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, the stitch sequence is located just upstream of the spacer sequence, and,
Further here, if an anti-zipper and zipper sequence is present, the anti-zipper sequence hybridizes to the zipper sequence, and the anti-stitch sequence NNANN is complementary and hybridizes to the stitch sequence NNUNN and hybridizes to the CRISPR nucleic acid. The spacer sequence of the molecule hybridizes to a portion of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA), thereby being defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. Within the region, it results in site-specific cleavage of the target DNA.

In a further embodiment, a method for site-specific cleavage of the double-stranded target DNA is provided, which method comprises contacting the double-stranded target DNA with a chimeric nucleic acid comprising:
(A) Voluntary anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; bulge sequence containing at least about 3 nucleotides; anti-stitch sequence containing NNANN nucleotide sequence, TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or a first nucleotide sequence comprising a nexus sequence comprising a nucleotide sequence of TCAAACAAAGCTTCAGC, a hairpin sequence comprising a nucleotide sequence having at least one hairpin (the hairpin said). Contains at least 3 matching base pairs),
Here, the anti-zipper sequence, if present, is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the anti-stitch sequence, and the anti-stitch sequence is just upstream of the nexus sequence. Located and the nexus sequence is located just upstream of the hairpin sequence;
(B) An optional zipper sequence containing a nucleotide sequence having at least 3 nucleotides that hybridizes to the anti-zipper in the 3'to 5'direction, a nucleotide sequence having at least 2 nucleotides (eg, (-). A bulge sequence containing NN-) nucleotide sequence), a stitch sequence containing NNUNN nucleotide sequence, and a spacer sequence having 5'ends and 3'ends, 100% identity with the target DNA at the 3'end. The second nucleotide sequence, including the spacer sequence containing at least 7 nucleotides, and the zipper sequence, if present, are located just upstream of the bulge sequence and the bulge sequence is located just upstream of the stitch sequence. However, the stitch sequence is located immediately upstream of the spacer sequence; and (c) encodes an amino acid sequence having at least 80% identity with the amino acid sequence encoding Cas9 nuclease (eg, SEQ ID NOs: 1-53). Third nucleotide sequence,
Here, if a zipper sequence and an anti-zipper sequence are present, the zipper sequence hybridizes to the anti-zipper sequence, and the anti-stitch sequence NNAN is complementary to and hybridizes to the stitch sequence NNUNN. The spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA), whereby the spacer sequence of the second nucleotide sequence to the target DNA Within the region defined by hybridization, it results in site-specific cleavage of the target DNA.

In a further embodiment, a method of site-specific targeting of the target polypeptide against double-stranded (ds) target DNA is provided, transcoded CRISPR (tracr) nucleic acid molecules and CRISPR nucleic acid molecules, with Cas9 nuclease (eg, sequence). Including the step of contacting with the target DNA in the presence of numbers 1-53).
Here, (a) the tracr nucleic acid molecule is an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; NNANN nucleotides. Anti-stitch sequences including sequences, TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A) / C) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence containing the nucleotide sequence of TCAAACAAAGCTTCAGC, including a hairpin sequence containing a nucleotide sequence having at least one hairpin. Encoded by the nucleotide sequence, the hairpin contains 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 the anti-stitch sequence. The anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and (b) CRISPR nucleic acid molecules are 3'to 5 An optional zipper sequence containing a nucleotide sequence having at least 3 nucleotides that hybridizes to the anti-zipper in the direction of', a nucleotide sequence having at least 2 nucleotides (eg, a nucleotide sequence of (-NN-)). A bulge sequence containing, a stitch sequence containing a nucleotide sequence of NNUNN, and a spacer sequence having 5'ends and 3'ends, at least 7 nucleotides having 100% identity with the target DNA at the 3'end. It is encoded by a nucleotide sequence, including a spacer sequence containing, and the zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is a spacer. Located just upstream of the sequence, and further here, Cas9 nuclease contains mutations within the HNH active site motif, mutations within the RuvC active site motif, fused to the polypeptide of interest, and the zipper and anti-zipper sequences If present, the zipper sequence hybridizes to the anti-zipper sequence and the anti-stitch sequence NNAN is complementary to the stitch sequence NNUNN and hive. Ridized and the spacer sequence hybridizes to the target DNA flanking the protospacer flanking motif (PAM) on the target DNA, thereby within the region defined by hybridization of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA. , Provides site-specific targeting of the target polypeptide to the target DNA.

In some embodiments, if the anti-zipper and zipper sequences of the tracr nucleic acid molecule and the aCRISPR nucleic acid molecule or the first and second nucleotide sequences hybridize, the hybridized sequence may optionally be a bulge sequence. Additional nucleotides can be included at the end of the hybridized sequence, which is distal to, 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 comprise a nucleotide linking the bulge sequence of the synthetic trac nucleic acid sequence to the bulge sequence of the synthetic CRISPR nucleic acid. The linking nucleotide can be any nucleotide (eg, T, A, G, C) and the number of nucleotides linking the zipper and anti-zipper sequence or bulge sequence 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 any containing at least one nuclear localization sequence described herein is of the invention. Can be used in a method.

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

In a further aspect, the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and / or nucleotide sequences of the invention can be introduced into cells of the host organism. Any cell / host organism for which the 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, spores, humans, or non-human primates.特定の実施態様では、宿主生物は、制限されないが、Ｈｏｍｏ ｓａｐｉｅｎｓ、Ｄｒｏｓｏｐｈｉｌａ ｍｅｌａｎｏｇａｓｔｅｒ、Ｍｕｓ ｍｕｓｃｕｌｕｓ、Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ、Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｇｌｙｃｉｎｅ ｍａｘ、Ｚｅａｅ ｍａｙｄｉｓ、Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ、または、Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａであっIt's okay. In a further embodiment, the cells useful in the present invention are, but are not limited to, stem cells, somatic cells, germ cells, plant cells, animal cells, bacterial cells, paleobacterial cells, fungal cells, mammalian cells, insect cells, avian cells. , Fish cells, amphibian cells, spore animal cells, human cells, or non-human primate cells.他の実施態様では、本発明で有用な細胞は、制限されないが、Ｈｏｍｏ ｓａｐｉｅｎｓ、Ｄｒｏｓｏｐｈｉｌａ ｍｅｌａｎｏｇａｓｔｅｒ、Ｍｕｓ ｍｕｓｃｕｌｕｓ、Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ、Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｇｌｙｃｉｎｅ ｍａｘ、Ｚｅａｅ ｍａｙｄｉｓ、Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ、または、 Includes cells from Arabidopsis thaliana.

In a further aspect of the invention, the polypeptide of interest is, but is not limited to, helicase, nuclease, methyltransferase, gyrace, demethylase, kinase, dismutase, integrase, transposase, telomerase, recombinase, acetyltransferase, deacetylase, polymerase, phosphatase, It may include ligase, ubixin ligase, phototriase or glycosylase. In another aspect of the invention, the polypeptide of interest comprises depurining activity, oxidizing activity, pyrimidine dimer forming activity, alkylating activity, DNA repair activity, DNA damaging activity, deubiquitination activity, adenylation activity, deadenylation activity. It has SUMOylation activity, deSUMOlation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity or telomere repair activity, or deaminoization activity. In a typical embodiment, the polypeptide of interest exhibits kinase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, or telomere repair activity. It may be a polypeptide having.

Kits containing the nucleic acid constructs, nucleic acid molecules, and / or nucleotide sequences of the present invention are further provided herein.

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

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

Here, the present invention will be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims to the present invention, but are intended to be exemplary of certain embodiments. Any modification in the illustrated method conceived by one of ordinary skill in the art is intended to be within the scope of the present invention.

Example 1. Assessment of the functional role of the modules identified within the guide sequence Crusted regularly interspaced short palindromic repeat (CRISPR) and related Cas proteins provide adaptive immunity to invasive genetic factors in bacteria and archaea ¹ . In the CRISPR-Cas system type II, the signature RNA-guided endonuclease Cas9 specifically targets the sequence complementary to the CRISPR spacer, resulting in double-stranded DNA cleavage (DSB) using two nickase domains. (Makarova, K.S. et al. Nat Rev Microbiol 9,467-477 (2011); Garneau, J.E. et al. Nature 468, 67-71 (2010); Sapranaskas, R. et al. Nucleic. Acids Res 39,9275-9282 (2011); Gasinuas, G. et al. Proc Natl Acad Sci USA 109, E2579-E2586 (2012); Jinek, M. et al., Science 337,816-821 (2012) ). Any DNA sequence can be targeted as long as the Cas9-specific protospacer-adjacent motif (PAM) is flanking. Garneau, J. et al. E. et al. Nature 468, 67-71 (2010); Sapranaskas, R. et al. et al. Nucleic Acids Res 39, 9275-9282 (2011); Gasinuas, G. et al. et al. Proc Natl Acad Sci US A 109, E2579-E2586 (2012); Jinek, M. et al. et al. , Science 337, 816-821 (2012); Sternberg, S. et al. 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 transactivated 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)). The sgRNA in combination with Cas9 creates a convenient, compact, and portable, sequence-specific targeting system suitable for heterogeneous migration to a variety of engineering and industrial and translationally interesting model systems.

Therefore, the Cas9: sgRNA technique, which provides a compact and practical means for producing double-strand breaks (DSBs), has a revolutionary genomic organization (Mali, P. et al., Science 339, 823). -826 (2013); Kong, L. et al. Science 339, 819-823 (2013); Jiang, W. et al. Nat. Biotechnology. 31,233-239 (2013); Sander, JD & Jung , JK Nature Biotechnology. 32, 347-355. (2014)), paving the way for high-throughput genome-wide gene screening ¹³ , 14, expanding the toolbox for transcriptional regulation (Qi, LS). . Et al. Cell 152, 1173-1183 (2013); Gilbert, LA et al. Cell 154,442-451 (2013)). In addition, the absence of interactions between evolutionarily distant Cas9: sgRNAs (Chylinski, K. et al. RNA biology 10,726-737 (2013); Fonfara, I. et al. Nucleic Acids Res (2013). ); Esvelt, KM et al. Nature Methods (2013)), allowing multiple independent targeting to be achieved intracellularly when coexisting functional type II CRISPR-Cas systems function simultaneously. (Barrangou, R. et al. Science 315, 1709-1712 (2007); Horvas, P. et al. J Bacteriol. 190, 1401-1412 (2008)). Despite the widespread use of these molecular mechanisms, the critical properties of sgRNA guides and their involvement in the definition of the functionally orthologous Cas9 endonuclease have not yet been characterized. In fact, early attention to Cas9 targeting and cleavage focuses on spacers: target complementarity and PAM sequence sensitivity, while the factors driving Cas9: sgRNA interactions and the orthogonality between type II CRISPR-Cas systems. Information that defines the factors that direct sex remains lacking. Therefore, we set out to identify and characterize properties within sgRNAs that confer Cas9 targeting and cleavage specificity in order to pave the way for new engineering on CRISPR technology.

Therefore, the functional roles and implications of the various modules identified within the guide sequences described herein (eg, synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, chimeric nucleic acid constructs (tracr nucleic acid-synthetic CRISPR nucleic acid constructs)). To evaluate nucleic acids), we designed variants of the guide mutations, including modifications or deletions of each of the functional modules described above. We used the SthCRISPR3 system as a representative functional model. We selected and first established positive functional controls (wild type, WT) using natural guide sequences. Then we tested "stitch" mutants lacking stitches and observed loss of function. Then we tested the bulge-deficient "bulge" mutant and observed the loss of function. Then we tested the nexus-deficient "nexus" mutant and observed the loss of function. We then tested a "hairpin" variant lacking the first hairpin and observed a loss of function. Subsequently, we tested sequence specificity and variability susceptibility and in the construct of the mutation, We have established that the nexus sequence is specific, while being variable tolerant for other functional modules, especially zippers, bulges, hairpins and stitches. The results of these experiments are shown in Figures 1-21. ..

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

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

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

The multi-sequence alignment for the zipper module is shown in FIG. 9 and FIG. 10 shows the maximum likelihood tree for the zipper module.

FIG. 11 shows multi-sequence alignment for bulge, anti-stitch and nexus modules. FIG. 4 shows the maximum likelihood tree for Cas9 nuclease.

FIGS. 12-21 show guide sequences and targeting for various cRNA: tracRNA constructs, representing the Sth CR1 group, Streptococcus thermophilus CR3 (FIG. 12); Lactobacillus buchneri representing the Lbu group (FIG. 13); Sth CR1 group. Streptococcus thermophilus CR1 (FIG. 14); Streptococcus thermogenes M1 GAS (FIG. 15) representing the Sth CR3 group; Lactobacillus ramnosus (FIG. 16); Lactobacillus lamnosus (FIG. 16); (FIG. 18); Lactobacillus gasseri (FIG. 19) representing the Lga group; Lactobacillus jenseni (FIG. 20) representing the Lje group; and Lactobacillus pentosus representing the Lpe group (FIG. 21). The underside of each of FIGS. 12-21 represents a target dsDNA containing a target sequence (open structure) and an adjacent (3') PAM. The upper part of each figure represents a CRISPR RNA (crRNA) consisting of a 5'part complementary to the target sequence and a 3'part derived from a CRISPR repeat; and also a tracrRNA consisting of an anti-CRISPR repeat portion and a nexus. And 3'hairpins are also shown. As shown in each of FIGS. 12 to 21, the complementary portions of the crRNA: tracrRNA duplex are the lower stem (lower complementary portion), the bulge (herniated mismatch) and the upper stem (upper complementary portion). Consists of.

Example 2. Determining the Properties of Guide RNA Sequences in Various Further Type II Systems The findings described in Example 1 establish an important module in sgRNA required to support Streptococcus pyrogenes Cas9 (SpyCas9) activity. However, although widely used for genomic organization, SpyCas9 is just one of many naturally found Cas9 orthologs (Chylinski, K. et al. RNA biology 10,726-737 (2013)). Genome, I. et al. Nucleic Acids Res (2013)). Therefore, we then investigated whether the same sgRNA sequence properties also occur in other type II CRISPR-Cas systems. We sampled 41 Cas9 sequences from the Streptococcus and Lactobacillus genomes (type II system preferentially occurs ² ), identified their corresponding CRISPR repeats, and predicted tracrRNA sequences. The Cas9 protein sequence was clustered into three major sequence groups (Fig. 23). As expected, similar grouping was seen when clustering was performed using either CRISPR-repeat or predicted tracrRNA sequences (FIGS. 24A, 23, 25, 26) and anti-anti-intracrRNA. The presence of CRISPR repeats and the close molecular relationships between Cas9 and crRNA: tracrRNA pairs were given (Makarova, K.S. et al. Nat Rev Microbiol 9,467-477 (2011); Deltcheva, E. et. al. Nature 471,602-607 (2011); Fonfara, I. et al. Nucleic Acids Res (2013)). Within the tracrRNA sequence, we consistently observed the functional modules identified for SpyCas9 (Fig. 24B), and the overall sgRNA / crRNA: tracrRNA structure between the families was conserved and at high levels within the cluster. The array was stored in.

The presence of a bulge with a directional twist between the lower stem (ie stitch / anti-stitch) and the upper stem (ie zipper / anti-zipper) was consistently seen throughout the variety of systems. The length of the inferior stem was highly conserved within the family and was variable between families. Interestingly, the highest level of conservation was seen with respect to the nexus sequence (Fig. 24B, Fig. 27). The general nexus shape, with 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, some bases in the nexus were strictly conserved even among different families, including A52 and C55 (FIGS. 24A-B), further emphasizing the critical role of this module. In fact, A52 interacts with the backbone of residues 1103-1107 near the 5'end of the target chain in the crystal structure of SpyCas9, and protein backbone-nexus interactions may be required for PAM binding. Suggest.

Determining the relationship of guide RNA structure to Cas9 protein orthogonality. The findings described herein suggest a potential relationship between the sequence and structure of sgRNAs and the diversity of Cas9 proteins. This observation prompted us to determine the sgRNA module that defines the Cas9 orthologous group. Therefore, we have two naturally coexisting orthologous S. cerevisiae. A thermophilus type II system, ie, endonucleases from Sth1 Cas9 and Sth3 Cas9, was selected (Horverse, P. et al. J Bacteriol. 190, 1401-1412 (2008)) and between the sgRNA composition and Cas9 orthogonality. I investigated the relationship. A series of experiments were designed based on self-targeting activity in Escherichia coli (FIGS. 28A-B), in which specific mutations in the sgRNA promoted cleavage activity in Cas9, which was prevousry orthologous. I tested if I could do it. We include Cas9 target sites that overlap with respect to the Sth1Cas9 and Sth3Cas9 systems. Regions within the colli genome were identified and it was confirmed that cleavage occurred at 1 nucleotide (FIG. 29B) and that the PAM sequences preferably overlap. We produced chimeric versions of the two sgRNA backbones and replaced the spacer, lower stem (ie stitch / anti-stitch) -bulge-upper stem (ie zipper / anti-zipper), nexus and hairpin (Fig. 29C). ), And drive self-targeting by either Sth1Cas9 or Sth3Cas9 (Gomaa, A.A. et al. MBio. 5, e00928-13 (2014)) and tested their ability. Initially, we confirmed that these two systems were actually orthogonal in this assay system, and that each guide alone drives targeting at its cognate Cas9 (Fig. 29C). We then showed that swapping spacer sequences results in sgRNAs with CRISPR3 spacers and CRISPR1 backbones that can support Sth1Cas9 cleavage activity. However, the opposite is not true for Sth3Cas9 activity. The sgRNA containing the CRISPR1 spacer and the CRISPR3 backbone does not support Sth3Cas9 activity (Fig. 29C). We hypothesize that this one-way reciprocity 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.M113.359726. (2014)) (Fig. 29C, upper panel). We then showed that the functionality between sgRNA and Cas9 can be altered simply by exchanging nexus-hairpin combinations between the two orthogonal systems. The main result of sgRNA reprogramming is the loss of the ability to guide the original Cas9 in the process (Fig. 29C, lower panel). This is in contrast to the standard notion that the CRISPR repeat sequence plays an important role in the definition of the CRISPR-Cas system for orthologous. However, these results show that chimeric sgRNAs with altered nexus sequences can reprogram orthogonality in a predictable and unidirectional way, further adding orthogonal Cas9 proteins associated with different PAMs. It is critical to use (Esvelt, KM et al. Nature Methods (2013)).

Recent structural and biochemical data have begun to shed light on the mechanism of DNA recognition and cleavage by Cas9 (Jinek, M. et al., Science 337, 816-821 (2012); Jinek, M. et al. , Science 343,6176 (2014); Nishimasu, H. et al. Cell 156,935 (2014)). Electron micrographs of apo-, RNA-binding and protein / RNA / DNA complexes show that when binding to guide RNA, Cas9 undergoes dramatic conformational changes to promote target DNA binding and cleavage structures. (Jinek, M. et al., Science 343,6176 (2014). The crystal structure is consistent with the image taken by the electron microscope, and SpyCas9: sgRNA: DNA: complex and apo-SpyCas9 Substantial rearrangement of RNA- and DNA-binding domains has been performed between the two structures, indicating that they occupy significantly different structures (Jinek, M. et al., Science 343, 6176 (2014); Nishimasu, H. et al. Cell 156,935 (2014)). Nexus occupies a critical position in the SpyCas9-sgRNA: DNA complex, aligns many important components of protein and sgRNA, both protein and RNA. The arginine-rich bridge helix binds to the nexus base and lower stem upon binding to sgRNA: DNA. In addition, the nexus receives a target DNA duplex for cleavage. , Two small regions from two lobes of SpyCas9 (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, among other things, two tryptophan residues identified as important in PAM recognition ^21. NIR2 is also directly with the inferior stem. The surface that interacts and opposes the nexus-binding site is very close to the 3'end of the target chain, suggesting that interaction with the nexus may be necessary to align the PAM recognition site. In particular, in the Actinomyces naeslundii Cas9 (AnaCas9) apo-structure (Jinek, M. et al., Science 343, 6176 (2014)), NIR2 is arranged and contains the insertion of about 50 amino acids. Motivation to think that large nexus (and possibly with PAM sequences) can be recognized There is.

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

The ability to reprogram Cas9 orthogonality using chimeric sgRNAs with modified nexus sequences paves the way for the development of new Cas9 proteins that have the potential to harness the diversity of natural Cas9 orthologs. Includes a short Cas9 variant for convenient packaging and delivery. In addition, the ability to reprogram Cas9 with chimeric mgRNA is a flexible control of target frequency (frequent short PAMs) and specificity due to reduced off-target cleavage (longer PAMs are rare). Will allow for increased use of various PAMs for (occur). It also opens up multiple opportunities by using a single Cas9 with various chimeric guides, or by using an orthogonal system simultaneously with different combinations of standard or chimeric sgRNAs. In summary, our findings pave the way for Cas9-dependent DNA targeting and set the stage for the development of next-generation CRISPR-based technologies.

Example 3.
Cas9 genes from the CRISPR1 and CRISPR3 loci were presented in S. cerevisiae. PCR amplification was performed from genomic DNA derived from thermus thermophilus LMD-9 and cloned into pwtCas9-bacterium (Addgene # 44250)) (Qi, L.S. et al. Cell 152, 1173-1183 (2013)). To construct an sgRNA-expression plasmid, remove the Spei restriction site in the pdCas9-bacterial plasmid (Addgene # 44249) (Id.) And use gBlock (IDT) encoding a zraP-targeting sgRNA based on the CRISPR1 or CRISPR3 locus. , PgRNA-Bacterial plasmid (Addgene # 44251) (Id.) In combination with PCR-amplified main strand. For transformation analysis E. Using colli K-12, the transformation efficiency was as previously explained (Gomaa, A.A. et al. MBio. 5, e00928-13 (2014)), transformation for the sgRNA plasmid tested. The number of bodies was calculated by dividing by the number of transformants for the psgRNA-C1-T4 control plasmid.

Plasmid construction. To construct a Cas9-expression plasmid, the Cas9 gene from the CRISPR1 locus (Sth1-Cas9) and the CRISPR3 locus (Sth3-Cas9) was subjected to S. cerevisiae. 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 an sgRNA-expression plasmid, the SpeI restriction site in the pdCas9-bacterial plasmid (Addgene # 44249) (Id.) Was removed, blunt-ended, religated and pdCas9ΔSpeI plasmid by digesting the plasmid with SpeI. Produced. Separately, S.M. A gBlock (IDT) encoding a zraP-targeting sgRNA based on the CRISPR1 (C1) or CRISPR3 (C3) locus in thermophylus LMD-9, pgRNA-bacterial plasmid (Addgene # 44251) (Id.) By Gibson assembly. In combination with the PCR-amplified backbone of the original S. cerevisiae. The pyogenes sgRNA sequence was replaced with the designed sgRNA sequence. The resulting sgRNA plasmid and pdCas9ΔSpeI backbone were then digested with AatII and XhoI and the gel-extracted fragments of each sgRNA plasmid and pdCas9ΔSpeI plasmid were then ligated together to form psgRNA-SthC1 and psgRNA-SthC3. To modify the sgRNA sequence, 5'phosphorylated oligonucleotides were ligated by annealing to the SpI / KpnI or KpnI / HindIII sites of the psgRNA-C1 or psgRNA-C3 plasmid. All plasmid modifications were confirmed by sequencing.

Strain and growth conditions. E. colli K-12 subst. MG1655 (genotype: E. coli K-12 F λ-ilvG-rfb-5C rf-1) was used for all transformation analyzes. Strains were aerobically grown 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. Medium was supplemented with antibiotics (34 μg / ml chloramphenicol, 50 μg / ml ampicillin) as needed.

Transformation assay. Frozen stock of cells carrying the indicated Cas9-expression plasmid was streaked for isolation and individual colonies were inoculated into 3 ml LB medium and cultured overnight. The resulting culture was back-diluted in 45 ml of LB medium, measured with a Nanodrop 2000c spectrophotometer (Thermo Scientific) and grown until ABS ₆₀₀ reached 0.6-0.8. The cultures were then pipeted and washed twice with ice-cold 10% glycerol before resuspending in 200-400 μl of 10% glycerol. Suspended cells (50 μl) were transformed with 25 ng of the indicated sgRNA-expression plasmid using a MicroPulser Electroporator (BioRad) and recovered in 300 μl SOC medium (Quality Biological) for 1 hour. After recovery, 200 μl cultures containing different amounts of LB medium were plated on LB agar containing 100 ng / ml anhydrotetracycline. The transformation efficiency is as previously explained (Gomaa, A.A. et al. MBio. 5, e00928-13 (2014)), which indicates the number of transformants for the DgRNA plasmid tested psgRNA-C1-. Calculated by dividing by the number of transformants for the T4 control plasmid. To reduce variability between experiments in transformation efficiency, the tested sgRNA and control plasmids were transformed into the same batch of electrocompetent cells. Similarly, for FIGS. 30-32, transformation analysis was used to test the ability of the plasmid to be electroporated into a Lactobacillus strain carrying an active CRISPR-Cas system (Lbu-Lactobacillus buchneri, FIG. 30; Lrh). -Lactobacillus rhamnosus, FIG. 32; Lca-Lactobacillus casei, FIG. 31. The plasmid was designed to contain the same protospacer sequence as the first spacer sequence within the host's CRISPR locus. Adjacent to the protospacer at full PAM. As such, various plasmids were designed (NTAAC for Lga; NNGAA for Lca; NGAAA for Lrh; PAM regions are the nucleotides underlined in FIGS. 30-32, and their mutated variants (efficiency tested). The nucleotides obtained are italic). Also, the experiments were performed with the control's non-targeting sequence (the penultimate entry for each experiment shown in FIGS. 30-32, and a control plasmid without a target sequence). (The last entry for each experiment shown in FIGS. 30-32 was also included. The ability of the natural CRISPR-Cas system to prevent plasmid uptake by DNA targeting is the ability of the test sequence and the transformation efficiency between the two aforementioned controls. Measured as a difference.

Claims (50)

A synthetic transcoded CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) construct.
From 5'to 3',
Optional anti-zipper sequence containing at least about 3 nucleotides; bulge sequence containing at least about 3 nucleotides; anti-stitch sequence containing NNANN nucleotide 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 nexus sequence containing a nucleotide sequence of TCAAACAAAGCTTCAGC; and a hairpin sequence containing a nucleotide sequence having at least one hairpin.
The hairpin contains at least three matching base pairs and contains
Here, the anti-zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, and the anti-stitch sequence is said. It is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence.
Synthetic transcoded CRISPR (tracr) nucleic acid (eg, tracrRNA, tracrDNA) constructs. A synthetic CRISPR nucleic acid (eg, crRNA, crDNA) construct.
In the direction of 3'to 5',
Optional zipper sequence containing at least about 3 nucleotides, bulge sequence containing nucleotide sequence having at least 2 nucleotides, stitch sequence containing nucleotide sequence of _NNTN , GR1 containing nucleotide G or GTT, and 5' A spacer sequence having an end and a 3'end, the 3'end containing a spacer sequence containing at least 7 nucleotides having 100% complementarity with the target DNA.
The zipper sequence, if present, is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is located just upstream of the _GR1 . And the _GR1 is located just upstream of the spacer array.
Synthetic CRISPR nucleic acid construct. Synthetic CRISPR nucleic acid array
Includes a nucleotide sequence encoding two or more CRISPR nucleic acid constructs of claim 2.
Here, the two or more CRISPR nucleic acid constructs are located immediately next to each other on the nucleotide sequence, and
The stitch sequences of the two or more CRISPR nucleic acid constructs are identical and
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.
CRISPR nucleic acid array. A chimeric nucleic acid construct comprising the synthetic tracr nucleic acid construct of claim 1 and the synthetic CRISPR nucleic acid construct of claim 2.
The stitch sequence of the synthetic CRISPR nucleic acid construct hybridizes to the anti-stitch sequence of the synthetic tracr nucleic acid construct 100% complementary.
The bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary.
If present, the zipper sequence of the synthetic CRISPR nucleic acid construct hybridizes to the anti-zipper sequence of the synthetic tracr nucleic acid construct at least about 70% complementary.
Chimeric nucleic acid construct. The synthetic tracr nucleic acid construct of claim 1, the synthetic CRISPR nucleic acid construct of claim 2, the CRISPR nucleic acid array of claim 3, or the chimeric nucleic acid construct of claim 4.
It further comprises a nucleotide sequence encoding an amino acid sequence having at least 70% identity with the amino acid sequence encoding the Cas9 nuclease.
A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct. A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct according to claim 5.
The Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases.
The Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases.
The Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, or
The Cas9 nuclease is a Cas9 nuclease derived from the Lactobacillus rhhamnosus GG (Lrh) group of Cas9 nucleases.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct according to claim 5.
The amino acid sequence encoding the Cas9 nuclease is selected from the group of amino acid sequences of SEQ ID NOs: 1 to 53, or any combination thereof.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. The synthetic tracr nucleic acid construct, the synthetic CRISP nucleic acid construct, the CRISPR nucleic acid array, or the chimeric nucleic acid construct according to claim 5.
The nexus sequence is GATAAGGC or GATAAGGCCATGCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;
The nexus sequence is TAAGGC or TAAGGCTAGTCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;
The nexus sequence is TCAAGC or TCAAGCAAAGC, and the Cas9 nuclease is from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases; or
The nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, and the Cas9 nuclease is from the Lactobacillus rhhamnosus GG (Lrh) group of Cas9 nucleases.
Synthetic tracr nucleic acid constructs, synthetic CRISP nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct according to any one of claims 6 to 8.
The Cas9 nuclease contains a mutation within the RuvC active site motif.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct according to any one of claims 6 to 8.
The Cas9 nuclease contains a mutation within the HNH active site motif.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. A synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a CRISPR nucleic acid array, or a chimeric nucleic acid construct according to any one of claims 6 to 8.
The Cas9 nuclease contains mutations within the HNH active site motif and within the RuvC active site motif and is fused to the polypeptide of interest.
Synthetic tracr nucleic acid constructs, synthetic CRISPR nucleic acid constructs, CRISPR nucleic acid arrays, or chimeric nucleic acid constructs. An expression cassette
The synthetic tracr nucleic acid construct of any one of claims 1 or 5 to 11, the synthetic CRISPR nucleic acid construct of any one of claims 2 or 5 to 11, any one of claims 3 or 5 to 11. The CRISPR nucleic acid array according to claim, or the chimeric nucleic acid construct according to any one of claims 4 to 11.
Expression cassette. It ’s a cell,
The synthetic tracr nucleic acid construct of any one of claims 1 or 5 to 11, the synthetic CRISPR nucleic acid construct of any one of claims 2 or 5 to 11, any one of claims 3 or 5 to 11. The CRISPR nucleic acid array according to claim, or the chimeric nucleic acid construct according to any one of claims 4 to 11, or the expression cassette according to claim 12.
cell. The cell of claim 13.
The cells include stem cells, somatic cells, germ cells, plant cells, animal cells, bacterial cells, paleobacterial cells, fungal cells, mammalian cells, insect cells, avian cells, fish cells, amphibian cells, spore animal cells, and humans. Cells, or non-human primate cells,
cell. A method for site-specific cleavage of double-stranded target DNA.
The step of contacting the chimeric nucleic acid construct according to any one of claims 4 to 10 or the expression cassette according to claim 12 is included.
The expression cassette comprises the chimeric nucleic acid construct of any one of claims 4-10 with the target DNA in the presence of Cas9 nuclease, thereby defined by complementary hybridization of the spacer sequence to the target DNA. In the region, a site-specific cleavage of the target DNA occurs.
Method. The method of claim 15.
The site-specific cleavage is a site-specific nick of the (+) strand of the double-stranded target DNA.
The Cas9 nuclease contains a mutation within the RuvC active site motif, thereby cleaving the (+) strand of the double-stranded target to give a site-specific nick to the (+) of the double-stranded target DNA. Generated in a 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 to give a site-specific nick to the (-) strand of the double-stranded target DNA. Generate in,
Method. A method for site-specific cleavage of double-stranded target DNA.
Including the following steps:
The step of contacting a transcoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target DNA in the presence of Cas9 nuclease, where.
(A) The tracr nucleic acid molecule contains an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch sequence including; TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A / C) ) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAGCAAAGC, a nexus sequence containing a nucleotide sequence; Encoded by the sequence, 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 the anti-zipper sequence. Located just upstream of the stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and
(B) The CRISPR nucleic acid molecule is an optional zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction, a bulge sequence containing a nucleotide sequence having at least about 2 nucleotides, and nucleotides of NNTNN. A stitch sequence containing a sequence, GR1 containing a nucleotide G or _GTT , and a spacer sequence having a 5'end and a 3'end, at least 7 having 100% identity with the target DNA at the 3'end. The zipper sequence is located just upstream of the bulge sequence, the bulge sequence is located just upstream of the stitch sequence, and the stitch sequence is located just upstream of the bulge sequence. It is located just upstream of said _GR1 and said _GR1 is located just upstream of said spacer sequence and
Further, here, when the anti-zipper sequence and the anti-stitch sequence are present, the anti-zipper hybridizes to the zipper sequence, and the stitch sequence hybridizes to the anti-stitch sequence. The spacer sequence of the CRISPR nucleic acid molecule is at least about 80% complementary and hybridizes with a portion of the target DNA (adjacent to the protospacer flanking motif (PAM) on the target DNA), thereby hybridizing the target DNA. Causes 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.
Method. A method for site-specific cleavage of double-stranded target DNA.
The double-stranded target DNA is described below:
(A) Anti-zipper sequence containing at least about 3 nucleotides in the direction from 5'to 3'; bulge sequence containing at least about 3 nucleotides; anti-stitch sequence containing NNANN nucleotide 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 / A first nucleotide sequence (the hairpin comprises a nexus sequence comprising a nucleotide sequence of G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin. The anti-zipper sequence, if present, is located immediately upstream of the bulge sequence and the bulge sequence is located just upstream of the anti-stitch sequence. , The anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence);
(B) A zipper sequence containing at least about 3 nucleotides, a bulge sequence containing a nucleotide sequence having at least 2 nucleotides, a stitch sequence containing a nucleotide sequence of NNTN, a nucleotide G or GTT in the direction from 3'to 5'. GR1 containing, and a spacer sequence having at least 7 nucleotides having 5'ends and _3'ends at the 3'end having 100% identity with the target DNA. Two nucleotide sequences (the zipper sequence, if present, located immediately upstream of the bulge sequence, the bulge sequence located immediately upstream of the stitch sequence, the stitch sequence immediately upstream of the _GR1 ). It is located upstream and said _GR1 is located just upstream of said spacer sequence); and (c) a third encoding an amino acid sequence having at least 80% identity with the amino acid sequence encoding the Cas9 nuclease. Including the step of contacting with a chimeric nucleic acid containing the nucleotide sequence of
Here, if the anti-zipper sequence and the zipper sequence are present, they hybridize with each other, the anti-stitch sequence hybridizes to the stitch sequence, and the spacer sequence of the second nucleotide sequence. Hybridizes to at least a portion of the target DNA, and the spacer sequence of the second nucleotide sequence hybridizes to a portion of the target DNA (adjacent to a protospacer flanking motif (PAM) on the target DNA). Soybeans, thereby causing site-specific cleavage of the target DNA within the region defined by the complementary binding of the spacer sequence of the second nucleotide sequence to the target DNA.
Method. 17 or the method of claim 20.
The site-specific cleavage is a site-specific nick of the (+) strand of the double-stranded target DNA.
The Cas9 nuclease contains a mutation within the RuvC active site motif, thereby cleaving the (+) strand of the double-stranded DNA at a cleavage site located 5 nucleotides upstream of the PAM sequence, site-specific. Generates a nick in the double-stranded target DNA,
Method. 17 or the method of claim 20.
The site-specific cleavage is a site-specific nick of the (-) strand of the double-stranded target DNA.
The Cas9 nuclease contains a mutation within the HNH active site motif, thereby cleaving the (-) strand of the double-stranded DNA at a cleavage site located 5 nucleotides upstream of the PAM sequence, site-specific. Generates a nick in the double-stranded target DNA,
Method. The method according to any one of claims 15 to 20.
The Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases.
The Cas9 nuclease is Cas9 from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases.
The Cas9 nuclease is a Cas9 nuclease from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, or the Cas9 nuclease is a Cas9 nuclease from the Lactobacillus rhhamnosus GG (Lrh) group of Cas9 nucleases.
Method. The method according to any one of claims 15 to 20.
The amino acid sequence encoding the Cas9 nuclease is selected from the group of amino acid sequences of SEQ ID NOs: 1 to 53, or any combination thereof.
Method. The method according to any one of claims 15 to 20.
The nexus sequence is GATAAGGC or GATAAGGCCATGCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;
The nexus sequence is TAAGGC or TAAGGCTAGTCC, and the Cas9 nuclease is from the Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;
The nexus sequence is TCAAGC or TCAAGCAAAGC, the Cas9 nuclease is from the Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases; or the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC, and the Cas9 nuclease is Cas9 nuclease, Cas9. Derived from the rhamnosus GG (Lrh) group,
Method. A method of site-specific targeting of a target polypeptide against a double-stranded (ds) target DNA.
The step of contacting the chimeric nucleic acid construct of claim 8 or the expression cassette of claim 12 with the target DNA is included.
Here, the expression cassette contains the chimeric nucleic acid construct of claim 8, whereby the target polypeptide fused to Cas9 is targeted to a specific site on the target DNA, and the site is , Defined by complementary hybridization of the spacer sequence to the target DNA.
Method. A method of site-specific targeting of a target polypeptide against a double-stranded (ds) target DNA.
A step of contacting a transcoded CRISPR (tracr) nucleic acid molecule and a CRISPR nucleic acid molecule with the target in the presence of Cas9 nuclease DNA, wherein
(A) The tracr nucleic acid molecule contains an optional anti-zipper sequence containing at least about 3 nucleotides in the 5'to 3'direction; a bulge sequence containing at least about 3 nucleotides; a nucleotide sequence of NNANN. Anti-stitch sequence including; TNANC, T (A / C) A (A / G) (G / A) C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T (C / A) AA (A / C) ) (C / A) (A / G) (A / T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or nexus sequence comprising a nucleotide sequence of TCAAACAAAGCTTCAGC; and a hairpin sequence comprising a nucleotide sequence having at least one hairpin. Encoded by a nucleotide sequence, 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 said anti. -The anti-stitch sequence is located just upstream of the stitch sequence, the anti-stitch sequence is located just upstream of the nexus sequence, and the nexus sequence is located just upstream of the hairpin sequence; and (b) the CRISPR. The nucleic acid molecule is an optional zipper sequence containing at least about 3 nucleotides in the 3'to 5'direction, a bulge sequence containing a nucleotide sequence having at least about 2 nucleotides, and a stitch sequence containing a nucleotide sequence of NNTNN. , GR1 containing nucleotide G or GTT, and a spacer sequence having 5'ends and _3'ends , the 3'end containing at least 7 nucleotides having 100% identity with the target DNA. Encoded by a nucleotide sequence, including the sequence, the zipper sequence, if present, is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, and the stitch sequence is located. The _GR1 is located just upstream of the GR1, and the _GR1 is located just upstream of the spacer array, and
Further, here, the Cas9 nuclease contains a mutation in the HNH activity site motif, a mutation in the RuvC activity site motif, is fused to the target polypeptide, and the anti-zipper sequence and the zipper sequence, if any, are present. Hybridize with each other, the stitch sequence is 100% complementary to the stitch sequence and hybridizes, and the spacer sequence hybridizes to the target DNA flanking the protospacer flanking motif (PAM) on the target DNA. Thereby, site-specific targeting of the target polypeptide to the target DNA occurs within the region defined by the complementary binding of the spacer sequence of the CRISPR nucleic acid molecule to the target DNA.
Method. The method according to any one of claims 17 to 23 or 25.
The PAM comprises a nucleotide sequence of GG, GGNG, AGAA, AAAAA or GAA.
Method. The method according to any one of claims 15 to 26.
The Cas9 nuclease is encoded by a codon-optimized nucleotide sequence for the organism containing the target DNA.
Method. The method according to any one of claims 15 to 27.
The Cas9 nuclease contains at least one nuclear localization sequence.
Method. 27 or 28 of the method.
The organism is a plant, bacterium, archaea, fungus, animal, mammal, insect, bird, fish, amphibian, spore animal, human, or non-human primate.
Method. The method according to any one of claims 27 and 28.
前記生物は、Ｈｏｍｏ ｓａｐｉｅｎｓ、Ｄｒｏｓｏｐｈｉｌａ ｍｅｌａｎｏｇａｓｔｅｒ、Ｍｕｓ ｍｕｓｃｕｌｕｓ、Ｒａｔｔｕｓ ｎｏｒｖｅｇｉｃｕｓ、Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｇｌｙｃｉｎｅ ｍａｘ、Ｚｅａｅ ｍａｙｄｉｓ、Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ、または、Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａである、
Method. The method according to any one of claims 24 to 30.
The target polypeptide is helicase, nuclease, methyltransferase, gyrace, demethylase, kinase, dismutase, integrase, transposase, telomerase, recombinase, acetyltransferase, deacetylase, polymerase, phosphatase, ligase, ubixin ligase, phototriase or glycosyllase. Yes or
The target polypeptide has depurine activity, oxidation activity, pyrimidine dimer forming activity, alkylation activity, DNA repair activity, DNA damage activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, and deSUMOlation. Includes activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity or telomere repair activity, or deaminolation activity,
Method. A kit for site-specific cleavage of double-stranded DNA,
The kit comprises the synthetic tracr nucleic acid construct of claim 1.
kit. A kit for site-specific cleavage of double-stranded DNA,
The kit comprises the synthetic CRISPR nucleic acid construct of claim 2.
kit. A kit for site-specific cleavage of double-stranded DNA,
The kit comprises the CRISPR nucleic acid array of claim 3.
kit. A kit for site-specific cleavage of double-stranded DNA,
The kit comprises the chimeric nucleic acid construct of claim 4.
kit. A kit for site-specific targeting of a target polypeptide against double-stranded (ds) target DNA.
The kit comprises the synthetic tracr nucleic acid construct of claim 1.
kit. A kit for site-specific targeting of a target polypeptide against double-stranded (ds) target DNA.
The kit comprises the synthetic CRISPR nucleic acid construct of claim 2.
kit. A kit for site-specific targeting of a target polypeptide against double-stranded (ds) target DNA.
The kit comprises the CRISPR nucleic acid array of claim 3.
kit. A kit for site-specific targeting of a target polypeptide against double-stranded (ds) target DNA.
The kit comprises the chimeric nucleic acid construct of claim 4.
kit. The kit of claim 32 or 36.
Further comprising the synthetic CRISPR nucleic acid construct of claim 2 and / or the CRISPR nucleic acid array of claim 3.
kit. The kit of claim 32 or 36.
The synthetic tracr nucleic acid construct is contained within an expression cassette.
kit. 33 or 37 of the kit.
The synthetic CRISPR nucleic acid construct is contained within an expression cassette.
kit. The kit of claim 34 or 38, wherein
The CRISPR nucleic acid array is contained within an expression cassette.
kit. The kit of claim 35 or 39, wherein
The chimeric nucleic acid construct is contained within the expression cassette.
kit. The kit of claim 40
The synthetic tracr nucleic acid construct, the synthetic CRISPR nucleic acid construct and / or the CRISPR nucleic acid array are contained within one or more expression cassettes.
kit. The kit of claim 32 or 36.
Further contains primers to generate a CRISPR array,
kit. The kit according to any one of claims 32 to 46.
Further containing Cas9 nuclease,
kit. The kit according to any one of claims 32 to 35.
Further comprising the Cas9 nuclease according to any one of claims 5 to 11.
kit. The kit according to any one of claims 36 to 39.
Further comprising the Cas9 nuclease of claim 5 or claim 11.
kit. A kit according to any one of claims 32 to 49, further including instructions for use.
kit.

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