RNA-induced nucleases, their active fragments and variants, and methods of use.

Abstract

Compositions and methods for binding to a target sequence of interest are provided. The compositions are used for cleaving or modifying a target sequence of interest, visualizing the target sequence of interest, and modifying the expression of the sequence of interest. The compositions include an RNA-guided nuclease (RGN) polypeptide, a CRISPR RNA, a trans-activating CRISPR RNA, a guide RNA (RNA), and nucleic acid molecules encoding these. Vectors and host cells containing the nucleic acid molecules are also provided. An RGN system for binding to a target sequence of interest is further provided, the RGN system comprising an RNA-guided nuclease polypeptide and one or more guide RNAs.

Description

[Technical Field]

[0001] Cross-references of related applications This application claims priority to United States Provisional Application No. 63 / 014,970, filed on 24 April 2020, and to United States Provisional Application No. 63 / 077,211, filed on 11 September 2020, each of which is incorporated herein by reference in its entirety.

[0002] Declaration regarding array lists The array listing accompanying this application is provided in ASCII format instead of a paper copy and is incorporated herein by reference. This ASCII copy, named L103438_1200WO_0084_1_SL, is 878,362 bytes in size, was created on April 21, 2021, and filed electronically via EFS-Web. [Background technology]

[0003] This application relates to the fields of molecular biology and gene editing.

[0004] Targeted genome editing or modification is rapidly becoming an important tool for both basic and applied research. Early methods involved manipulating nucleases (such as meganucleases, zinc finger fusion proteins, or TALENs), requiring the creation of chimeric nucleases with programmable, sequence-specific DNA-binding domains specifically manipulated for each particular target sequence. RNA-inducible nucleases (such as CRISPR-associated (Cas) proteins in clustered, regularly spaced short palindromic repeats (CRISPR)-Cas bacterial systems) can target specific sequences by being complexed with guide RNAs that specifically hybridize to those target sequences. Creating target-specific guide RNAs is less costly and more efficient than creating chimeric nucleases for each target sequence. Using such RNA-inducible nucleases, it is possible to edit the genome by introducing sequence-specific double-strand breaks, which are repaired via error-prone non-homologous end joins (NHEJs), thus introducing mutations at specific genomic locations. Alternatively, heterologous recombination repair can introduce heterologous DNA into the genomic region. RNA-induced nucleases (RGNs) can also be used for base editing when fused with deaminases. [Overview of the project] [Problems that the invention aims to solve]

[0005] Compositions and methods for binding to target sequences of interest are provided. The compositions are used for cleaving or modifying target sequences of interest, detecting target sequences of interest, and modifying the expression of target sequences of interest. The compositions include RNA-inducible nuclease (RGN) polypeptides, CRISPR RNA (crRNA), trans-activated CRISPR RNA (tracrRNA), guide RNA (gRNA), nucleic acid molecules encoding these, and vectors and host cells containing these nucleic acid molecules. RGN systems for binding to target sequences of interest are also provided, which include RNA-inducible nuclease polypeptides and one or more guide RNAs. Therefore, the methods disclosed herein relate to the binding of target sequences of interest, and in some embodiments, to the cleaving or modification of target sequences of interest. Modification of target sequences of interest can be achieved, for example, as a result of non-homologous end joining or homologous recombination repair using a donor sequence to be introduced, or as a result of base editing. [Means for solving the problem]

[0006] Those skilled in the art will be able to conceive of numerous modifications and other embodiments of the invention described herein that have the same advantages as those shown in the above description and the accompanying drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments fall within the scope of the appended embodiments. Specialized terms are used herein, but they are used in a general and descriptive sense and are not intended to be limiting. [Effects of the Invention]

[0007] I. Overview

[0008] RNA-induced nucleases (RGNs) are useful in the context of gene targeting for therapeutic and research applications because they allow for the targeting and manipulation of specific sites within the genome. In a diverse range of organisms, including mammals, RNA-induced nucleases have been used for genomic manipulation, for example, by stimulating non-homologous end joining and homologous recombination. The compositions and methods described herein are useful for inducing single-strand or double-strand breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or altering the expression of specific genes.

[0009] The RNA-inducible nucleases disclosed herein can alter gene expression by modifying target sequences. In particular embodiments, the RNA-inducible nuclease is directed to the target sequence by a guide RNA (gRNA) as part of a clustered, regularly spaced, short palindromic repeat (CRISPR) RNA-inducible nuclease system. RGNs are considered "RNA-inducible" because the guide RNA forms a complex with the RNA-inducible nuclease, causing it to bind to the target sequence, and in some embodiments, introducing a single-strand or double-strand break at the location of the target sequence. After the target sequence is cleaved, this cleavage can be repaired, thus modifying the DNA sequence of the target sequence during the repair process. Therefore, this specification provides a method for modifying a target sequence in the DNA of a host cell using an RNA-inducible nuclease. For example, an RNA-inducible nuclease can be used to modify a target sequence located at a single genomic locus in a eukaryotic or prokaryotic cell.

[0010] II. RNA-inducible nucleases

[0011] This specification presents RNA-inducible nucleases. The term RNA-inducible nuclease (RGN) refers to a polypeptide that binds to a specific target nucleotide sequence in a sequence-specific manner, is directed to the target nucleotide sequence by a guide RNA molecule that forms a complex with the polypeptide, and hybridizes with the target sequence. While RNA-inducible nucleases can cleave the target sequence upon binding, the term RNA-inducible nuclease also includes nuclease-dead RNA-inducible nucleases that can bind to the target sequence but cannot cleave it. As a result of cleaving the target sequence by an RNA-inducible nuclease, single-stranded or double-stranded nucleic acid molecules can be cleaved. RNA-inducible nucleases that can only cleave a single strand of a double-stranded nucleic acid molecule are referred to herein as niccasses.

[0012] The RNA-inducible nucleases disclosed herein include APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, and APG01560. This includes RNA-inducible nucleases such as APG02777, APG05761, APG02479, APG08385, APG09217, and APG06657 (whose amino acid sequences are represented by SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579, respectively), and those in which active fragments or variants retain the ability to bind to target nucleotide sequences in a manner specific to RNA-inducible sequences. In some of these embodiments, APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 Active fragments or variants of RGN can cleave single-stranded or double-stranded target sequences.In some embodiments, APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 Active variants of RGN include amino acid sequences that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%, 96%, 103, 110, 117, 123, or 570–579 matching sequences.In one embodiment, these APG06622, APG02787, APG06248, APG06007, APG02874, APG03850, APG07553, APG03031, APG09208, APG05586, APG08770, APG08167, APG01604, APG03021, APG06015, APG09344, APG07991, APG01868, APG02998, APG09298, APG06251, APG03066, APG01560, APG02777, APG05761, APG02479, APG08385, APG09217, or APG06657 The active fragment of RGN contains at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1050, or more consecutive amino acid residues of the amino acid sequence indicated as SEQ ID NOs. 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. The RNA-inducible nucleases presented herein may include at least one nuclease domain (e.g., a DNase domain, an RNase domain) and at least one RNA recognition domain and / or RNA binding domain that interacts with a guide RNA. Non-limiting examples of other domains that may be found in the RNA-inducible nucleases presented herein include DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In particular embodiments, the RNA-inducible nucleases presented herein may include sequences that match at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, one or more of the DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains.

[0013] An RNA-inducible nuclease presented herein binds to a target nucleotide sequence, and the RNA-inducible nuclease hybridizes with a guide RNA associated with it. If the RNA-inducible nuclease has nuclease activity, the target sequence can then be cleaved by it. The terms "cleaved" or "cleaved" mean that at least one phosphodiester bond in the target sequence's backbone is hydrolyzed, resulting in the cleavage of a single or double strand within the target nucleic acid. The RGNs of this disclosure can function as endonucleases to cleave nucleotides within a polynucleotide, or as exonucleases to successively remove nucleotides from the ends (5' and / or 3' ends) of a polynucleotide. In another embodiment, the disclosed RGNs can function as both endonucleases and exonucleases, since they can cleave nucleotides of the target sequence at any position within the polynucleotide. The target polynucleotide may have sticky or blunt ends as a result of being cleaved by the RGNs of this disclosure.

[0014] The RNA-inducible nucleases disclosed herein may be wild-type sequences derived from bacterial or archaeal species. Alternatively, the RNA-inducible nucleases may be variants or fragments of wild-type polypeptides. Wild-type RGNs can be modified to alter, for example, nuclease activity or PAM specificity. In some embodiments, the RNA-inducible nucleases are not native.

[0015] In some embodiments, the RNA-induced nuclease functions as a nickase, cleaving only a single strand of the target nucleotide sequence. Such RNA-induced nucleases have a single-function nuclease domain. In specific embodiments, the nickase can cleave either the positive or negative strand. In some of these embodiments, mutations in additional nuclease domains resulted in reduced or absent nuclease activity.

[0016] In another embodiment, the RNA-induced nuclease has completely lost its nuclease activity and is therefore referred to herein as nuclease-dead or nuclease-inactive. Nicasse or nuclease-dead RGNs can be generated using any method known in the art for introducing mutations into the amino acid sequence (e.g., PCR-mediated mutagenesis and site-directed mutagenesis). See, for example, U.S. Patent Application Publication 2014 / 0068797 and U.S. Patent No. 9,790,490 (each incorporated herein by reference in its entirety).

[0017] RNA-induced nucleases lacking nuclease activity can be used to deliver fused polypeptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN polypeptide or guide RNA is fused to a detectable label to enable detection of specific sequences. As a non-limiting example, nuclease-dead RGNs can be fused to a detectable label (e.g., a fluorescent protein) to target specific disease-related sequences, thereby enabling detection of these disease-associated sequences.

[0018] Alternatively, nuclease-dead RGNs can be directed to specific genomic locations to alter the expression of desired sequences. In some embodiments, when a nuclease-dead RGN-inducible nuclease binds to a target sequence, it interferes with the binding of RNA polymerases or transcription factors within the targeted genomic region, thereby repressing the expression of the target sequence or a gene under transcriptional regulation by the target sequence. In another embodiment, the RGN (e.g., nuclease-dead RGN), or a guide RNA complexed with the RGN, further includes an expression modulator that, upon binding to the target sequence, represses or activates the expression of the target sequence or a gene under transcriptional regulation by the target sequence. In some of these embodiments, the expression modulator alters the expression of the target sequence or a gene regulated through an epigenetic mechanism.

[0019] In another embodiment, the sequence of a target polynucleotide can be modified by directing a nuclease-dead RGN, or an RGN having only nickase activity, to a specific genomic location and fusing it with a base-editing polypeptide (e.g., a deaminase polypeptide, or one that directly chemically modifies (e.g., deaminates) a nucleotide base with an active variant or fragment thereof, resulting in the conversion of one nucleotide base to another. The base-editing polypeptide can be fused to the RGN at its N-terminus or C-terminus. In addition, the base-editing polypeptide can be fused to the RGN via a peptide linker. A non-limiting example of a deaminase polypeptide useful in such compositions and methods includes cytidine deaminase or adenosine deaminase (adenosine deaminase base editor described in Gaudelli et al. (2017) Nature 551:464-471, United States Patent Application Publications 2017 / 0121693 and 2018 / 0073012, and International Patent Application Publication WO / 2018 / 027078, or International Patent Application Publication WO This includes any of the deaminases disclosed in U.S. Provisional Application No. 63 / 077,089 filed on 11 September 2020, No. 63 / 146,840 filed on 8 February 2021, and No. 63 / 164,273 filed on 22 March 2021 (each of these documents in their entirety is incorporated herein by reference). Furthermore, it is known in this art that certain fusion proteins between RGNs and base editing enzymes may also include at least one uracil-stabilizing polypeptide that increases the mutation rate from cytidine, deoxycytidine, or cytosine to thymidine, deoxythymidine, or thymine in nucleic acid molecules by the deaminase. Non-limiting examples of uracil-stabilized polypeptides include those disclosed in U.S. Provisional Application No. 63 / 052,175 filed on July 15, 2020 (which includes USP2 (SEQ ID NO: 1089)) and uracil glycosylase inhibitor (UGI) domains (SEQ ID NO: 212) that may increase base editing efficiency.Therefore, the fusion protein may include the RGN or a variant thereof as described herein, a deaminase, and optionally at least one uracil-stabilized polypeptide (such as UGI or USP2). In one embodiment, the RGN fused to the base-editing polypeptide is a nickasase that cleaves DNA strands that are not affected by the base-editing polypeptide (e.g., deaminase).

[0020] RNA-inducible nucleases fused to polypeptides or domains can be separated or joined by linkers. As used herein, the term “linker” means a chemical group or molecule that links two molecules or parts (e.g., the binding domain and cleavage domain of a nuclease). In some embodiments, the linker joins the gRNA-binding domain of an RNA-inducible nuclease to a base-editing polypeptide (e.g., a deaminase). In some embodiments, the linker links the nuclease-dead RGN to the deaminase. Typically, the linker is located between or adjacent to two groups, molecules, or other parts, and connects them by covalent bonds. In some embodiments, the linker is one amino acid or more amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical part. In some embodiments, the linker is 5 to 100 amino acids in length, for example, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids. Longer or shorter linkers are also conceivable.

[0021] The RNA-guided nuclease of the present disclosure can comprise at least one nuclear localization signal (NLS) to enhance transport of the RGN to the nucleus of a cell. Nuclear localization signals are known in the art and generally comprise a series of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In particular embodiments, the RGN comprises 2, 3, 4, 5, 6, or more nuclear localization signals. A heterologous NLS is permissible as the nuclear localization signal. Non-limiting examples of nuclear localization signals useful for the RGNs of the present disclosure are the nuclear localization signals of SV40 large T antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6):1004-7). In particular embodiments, the RGN comprises the NLS sequence set forth as SEQ ID NO: 251 or 253. The RGN can comprise one or more NLS sequences at its N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, the RGN can comprise two NLS sequences located in the N-terminal region and four NLS sequences located in the C-terminal region.

[0022] To target RGNs, other localization signals known in this field that localize polypeptides to specific locations within cells can also be used, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual-target signal sequences that target both plastids and mitochondria (e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (See also: (2009) FEBS J 276:1187-1195; Silva-Filho (2003) Curr Opin Plant Biol 6:589-595; Peeters and Small (2001) Biochim Biophys Acta 1541:54-63; Murcha et al. (2014) J Exp Bot 65:6301-6335; Mackenzie (2005) Trends Cell Biol 15:548-554; Glaser et al. (1998) Plant Mol Biol 38:311-338).

[0023] In certain embodiments, the RNA-guided nuclease of the present disclosure comprises at least one cell penetration domain that facilitates uptake of the RGN into a cell. Cell penetration domains are known in the art, and generally comprise a series of positively charged amino acid residues (i.e., polycationic cell penetration domains), alternating polar and non-polar amino acid residues (i.e., amphipathic cell penetration domains), or hydrophobic amino acid residues (i.e., hydrophobic cell penetration domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860). One non-limiting example of a cell penetration domain is the trans-activating transcriptional activator (TAT) from Human Immunodeficiency Virus 1.

[0024] The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dual targeting signal, and / or cell penetration domain can be located at the amino terminus (N-terminus), carboxyl terminus (C-terminus), or an internal position of the RNA-guided nuclease.

[0025] The RGN of the present disclosure can be fused directly to an effector domain (such as a cleavage domain, a deaminase domain, or an expression modulator domain), or indirectly fused via a linker peptide. Such a domain can be located at the N-terminus, C-terminus, or an internal position of the RNA-guided nuclease. In some of these embodiments, the RGN element of the fusion protein is a nuclease-dead RGN.

[0026] In some embodiments, the RGN fusion protein includes a cleavage domain. The cleavage domain is any domain capable of cleaving polynucleotides (i.e., RNA, DNA, or RNA / DNA hybrids), and non-limiting examples include restriction endonucleases and homing endonucleases (such as IIS-type endonucleases (e.g., FokI)) (see, e.g., Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0027] In another embodiment, the RGN fusion protein comprises a deaminase domain that deaminates nucleotide bases to convert one nucleotide base to another, and non-limiting examples thereof include a cytidine deaminase base editor or an adenosine deaminase base editor (see, for example, Gaudelli et al. (2017) Nature 551:464-471, United States Patent Application Publications 2017 / 0121693 and 2018 / 0073012, United States Patent No. 9,840,699, and International Patent Application Publication WO / 2018 / 027078).

[0028] In some embodiments, the effector domain of the RGN fusion protein can be an expression modulator domain, which is a domain that has the effect of upregulating or downregulating transcription. The expression modulator domain can be an epigenetic modification domain, a transcriptional repression domain, or a transcriptional activation domain.

[0029] In some of these embodiments, the RGN fusion protein expression modulator includes an epigenetic modification domain that modifies the covalent bonds of DNA or histone proteins to alter histone and / or chromosome structures without altering the DNA sequence, leading to changes in gene expression (i.e., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues in DNA, methylation of arginine, phosphorylation of serine and threonine, ubiquitination of lysine, sumoation of histone proteins, and methylation and hydroxymethylation of cytosine residues. Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.

[0030] In another embodiment, the expression modulator of the fusion protein includes a transcriptional repression domain. The transcriptional repression domain interacts with transcriptional regulatory elements and / or transcriptional regulatory proteins (such as RNA polymerases and transcription factors) to reduce or halt the transcription of at least one gene. Transcriptional repression domains known in this art include, but not limited to, Sp1-like repressor domains, IκB domains, and Krupper-associated box (KRAB) domains.

[0031] In yet another embodiment, the expression modulator of the fusion protein includes a transcriptional activation domain. The transcriptional activation domain interacts with transcriptional regulatory elements and / or transcriptional regulatory proteins (such as RNA polymerases and transcription factors) to increase or activate the transcription of at least one gene. Transcriptional activation domains are known in the art, and non-limiting examples include the herpes simplex virus VP16 activation domain and the NFAT activation domain.

[0032] The RGN polypeptides of this disclosure may include detectable labels or purified tags. The detectable labels or purified tags may be directly positioned at the N-terminus, C-terminus, or internal position of the RNA-induced nuclease, or indirectly via a linker peptide. In some of these embodiments, the RGN element of the fusion protein is a nuclease-dead RGN. In another embodiment, the RGN element of the fusion protein is an RGN having nickase activity.

[0033] A detectable label is a molecule that can be visualized or otherwise observed. A detectable label can be fused to an RGN as a fusion protein (e.g., a fluorescent protein). Alternatively, a small molecule complexed with an RGN polypeptide can be a detectable label, which can be detected visually or by other means. Detectable labels that can be fused to an RGN as a fusion protein in this disclosure include any detectable protein domain, and non-limiting examples include fluorescent proteins or protein domains detectable using specific antibodies. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of detectable small molecule labels include radioactive labels. 3 H and 35 Examples include S.

[0034] RGN polypeptides can also include purification tags. A purification tag is any molecule that can be used to isolate a protein or fusion protein from a mixture (e.g., a biological sample, culture medium). Non-limiting examples of purification tags include myc, maltose-binding protein (MBP), glutathione-S-transferase (GST), and the 3×FLAG tag.

[0035] II. Guide RNA

[0036] This disclosure provides guide RNAs and polynucleotides encoding them. The term “guide RNA” means a nucleotide sequence that hybridizes with the target nucleotide sequence to have sufficient complementarity with the target nucleotide sequence, thereby causing the associated RNA-induced nuclease to sequence-specifically bind to the target nucleotide sequence. Thus, each guide RNA in an RGN is one or more RNA molecules (generally one or two) that can bind to the RGN and guide it to bind to a specific target nucleotide sequence, and can also cleave the target nucleotide sequence in embodiments where the RGN has nickase activity or nuclease activity. Generally, guide RNAs include CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA). Natural guide RNAs containing both crRNA and tracrRNA generally consist of two separate RNA molecules that hybridize with each other through the repetitive sequence of crRNA and the anti-repetitive sequence of tracrRNA.

[0037] The native serial repeat sequences in a CRISPR array are generally in the range of 28–37 base pairs in length, but this length can vary between approximately 23 bp and 55 bp. The spacer sequences in a CRISPR array are generally in the range of approximately 32–38 bp in length, but this length can vary between approximately 21 bp and 72 bp. Each CRISPR array generally contains fewer than 50 units of CRISPR repeat spacer sequences. CRISPR is transcribed as part of a longer transcript called the primary CRISPR transcript. The primary CRISPR transcript contains much of the CRISPR array. The primary CRISPR transcript is cleaved by Cas proteins to produce crRNA, but in some cases, it produces precrRNA. The precrRNA is further processed by additional Cas proteins to become mature crRNA. Mature crRNA contains spacer sequences and CRISPR repeat sequences. In some embodiments, the processing of precrRNA to become mature (or processed) crRNA involves removing approximately 1 to approximately 6 or more 5' nucleotides, 3' nucleotides, or 5' and 3' nucleotides. For the purpose of genome editing or genome targeting of a specific target nucleotide sequence of interest, nucleotides removed during the maturation of the precrRNA molecule are not required for the generation or design of the guide RNA.

[0038] CRISPR RNA (crRNA) contains a spacer sequence and a CRISPR repeat sequence. The "spacer sequence" is a nucleotide sequence that directly hybridizes with the target nucleotide sequence of interest. The spacer sequence is manipulated to be fully or partially complementary to the target nucleotide sequence of interest. In various embodiments, the spacer sequence can contain approximately 8 to 30 or more nucleotides. For example, a spacer sequence can have a length of approximately 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. In some embodiments, the spacer sequence consists of 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. In some embodiments, the spacer sequence consists of approximately 10 to 26 nucleotides, or approximately 12 to 30 nucleotides. In a special embodiment, the spacer sequence consists of approximately 30 nucleotides. In some embodiments, the spacer sequence consists of 30 nucleotides. In some embodiments, the degree of complementarity between the spacer sequence and its corresponding target sequence is between 50% and 99% or more when optimally aligned using an appropriate alignment algorithm, and non-limiting examples include approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more.In a particular embodiment, the degree of complementarity between a spacer sequence and its corresponding target sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% when optimally aligned using a suitable alignment algorithm. In a particular embodiment, the spacer sequence is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art. Non-restrictive examples of algorithms include mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(1):23-24).

[0039] A CRISPR RNA repeat sequence contains a nucleotide sequence that forms a structure, either by itself or in cooperation with a hybridized tracrRNA, which is recognized by an RGN molecule. In various embodiments, a CRISPR RNA repeat sequence can contain approximately 8 to 30 or more nucleotides. For example, a CRISPR repeat sequence can have a length of approximately 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. In a particular embodiment, the CRISPR repeat sequence has a length of 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. In some embodiments, the degree of complementarity between the CRISPR repeat sequence and the corresponding tracrRNA sequence is approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more when optimal alignment is achieved using an appropriate alignment algorithm. In a particular embodiment, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% when optimally aligned using an appropriate alignment algorithm.

[0040] In a particular embodiment, the CRISPR repeat sequence comprises the nucleotide sequence of SEQ ID NOs. 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof, and when it is included in the guide RNA, the relevant RNA-inducible nucleases presented herein can be sequence-specifically bound to the target sequence of interest. In one embodiment, the active CRISPR repeat sequence variant of the wild-type sequence includes a nucleotide sequence that matches at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 97%, 104, 111, 118, or 124 of the nucleotide sequence shown as SEQ ID NOs. In one embodiment, the active CRISPR repeat sequence fragment of the wild-type sequence contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of the nucleotide sequence shown as SEQ ID NOs: 2, 9, 16, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124.

[0041] In some embodiments, the crRNA does not exist in nature. In some of these embodiments, the specific CRISPR repeat sequence does not naturally ligate to the manipulated spacer sequence, and this CRISPR repeat sequence is considered heterogeneous to the spacer sequence. In some embodiments, the spacer sequence is a manipulated sequence that does not exist in nature.

[0042] A transactivated CRISPR RNA molecule, i.e., a tracrRNA molecule, contains a nucleotide sequence that includes a region (referred to herein as an anti-repetitive region) that is sufficiently complementary to the CRISPR repeat sequence of the crRNA. In some embodiments, the tracrRNA molecule further contains a region with a secondary structure (e.g., a stem-loop) or forms a secondary structure when hybridized to the corresponding crRNA. In a particular embodiment, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is located at the 5' end of the molecule, and the 3' end of the tracrRNA contains a secondary structure. This region of the secondary structure generally contains several hairpin structures (including ligating hairpins found adjacent to the anti-repetitive sequence). These ligating hairpins form the core of the interaction between the guide RNA and the RGN, and are located at the intersection of the guide RNA, RGN, and target DNA. The ligating hairpins often have conserved nucleotide sequences in the bases of the hairpin stem, and among the ligating hairpins in tracrRNA, the motif UNANNC (SEQ ID NO: 132) is found. Interestingly, some of the RGNs of the present invention utilize tracrRNAs containing non-canonical sequences (including UNANNA, UNANNU, UNANNG, and CNANNC (sequences 129, 130, 131, and 133, respectively)) within the base of the hairpin stem of their ligating hairpins. A terminal hairpin is often present at the 3' end of the tracrRNA, and while its structure and number can vary, it frequently contains a CG-rich, Rho-independent transcription termination hairpin followed by a string of U located at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and United States Patent Application Publication 2017 / 0275648 (each in its entirety incorporated herein by reference).

[0043] In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence contains approximately 8 to 30 or more nucleotides. For example, the region that forms base pairs between the tracrRNA anti-repeat region and the CRISPR repeat sequence can have a length of approximately 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. In a particular embodiment, the region that forms a base pair between the tracrRNA anti-repeat region and the CRISPR repeat sequence has a length of 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. In some embodiments, the degree of complementarity between the CRISPR repeat sequence and the corresponding tracrRNA anti-repeat sequence is approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 81% or more, approximately 82% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more when optimally aligned using an appropriate alignment algorithm. In a particular embodiment, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% when optimally aligned using an appropriate alignment algorithm.

[0044] In various embodiments, the entire tracrRNA can contain approximately 60 to over 210 nucleotides. For example, a tracrRNA can have a length of approximately 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210, or more nucleotides. In a particular embodiment, the tracrRNA is a number of nucleotides of 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210, or more. In a particular embodiment, the tracrRNA is a number of nucleotides of approximately 80 to 90 in length, including approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 in length. In a particular embodiment, tracrRNA is 80 to 90 nucleotides in length, including nucleotides of lengths of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90.

[0045] In a particular embodiment, the tracrRNA contains the nucleotide sequence of SEQ ID NOs. 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof, and when it is included in the guide RNA, the relevant RNA-inducible nucleases presented herein can be sequence-specifically bound to the target sequence of interest. In one embodiment, the active tracrRNA sequence variant of the wild-type sequence includes a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 72, 78, 85, 91, 98, 105, 112, 119, or 125 matching sequence to sequence 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98%, 98%, 99%, or more matching sequence to sequence 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more matching sequence. In one embodiment, the wild-type active tracrRNA sequence fragment contains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125 consecutive nucleotides of the nucleotide sequence shown as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125.

[0046] Two polynucleotide sequences can be considered substantially complementary when they hybridize with each other under harsh conditions. Similarly, a RGN is considered to bind to a specific target sequence in a sequence-specific manner when the guide RNA bound to that RGN binds to that target sequence under harsh conditions. "Harsh conditions" or "harsh hybridization conditions" refer to conditions under which two polynucleotide sequences hybridize with each other to a greater degree than other sequences (e.g., at least twice the detection level of the background). Harsh conditions are sequence-dependent and will vary depending on the environment. Typically, harsh conditions are salt concentrations of less than about 1.5 M Na ions at pH 7.0–8.3, typically about 0.01–1.0 M Na ions (or other salts), and temperatures of at least about 30°C for short sequences (e.g., 10–50 nucleotides) and at least about 60°C for long sequences (e.g., more than 50 nucleotides). Harsh conditions can also be achieved by adding destabilizers (e.g., formamide). Typical low-severity conditions include hybridization at 37°C using a buffer solution of 30-35% formamide, 1M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing in 1×~2×SSC (20×SSC = 3.0M NaCl / 0.3M trisodium citrate) at 50-55°C. Typical moderate-severity conditions include hybridization at 37°C using 40-45% formamide, 1.0M NaCl, and 1% SDS, followed by washing in 0.5×~1×SSC at 55-60°C. Typical high-severity conditions include hybridization at 37°C using 50% formamide, 1M NaCl, and 1% SDS, followed by washing in 0.1×SSC at 60-65°C. Depending on the case, the washing buffer may contain approximately 0.1% to 1% SDS. Hybridization time is generally less than 24 hours, typically around 4 to 12 hours. Washing time should be long enough to reach equilibrium.

[0047] Tm is the temperature at which 50% of the complementary target sequence hybridizes to a perfectly matched sequence (under specified ionic strength and pH). For DNA-DNA hybrids, Tm can be roughly determined from the formula in Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (logM) + 0.41 (%GC) - 0.61 (%form) - 500 / L (where M is the number of moles of monovalent cations, %GC is the ratio of guanosine nucleotides to cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in the base pair). Generally, harsh conditions are chosen so that they are about 5°C lower than the melting point (Tm) of the specific sequence and its complementary sequence at the specified ionic strength and pH. However, extremely severe conditions can utilize hybridization and / or washing at temperatures 1, 2, 3, or 4°C below the melting point (Tm); moderately severe conditions can utilize hybridization and / or washing at temperatures 6, 7, 8, 9, or 10°C below the melting point (Tm); and less severe conditions can utilize hybridization and / or washing at temperatures 11, 12, 13, 14, 15, or 20°C below the melting point (Tm). Those skilled in the art will understand that variations in the severity of the hybridization and / or washing solution are essentially described using the above formula, the hybridization and washing compositions, and the desired Tm.Comprehensive guides on nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0048] The term "sequence-specific" can also mean binding to the target sequence at a greater frequency than binding to a randomized background sequence.

[0049] As guide RNA, either a single guide RNA or a dual guide RNA system is possible. A single guide RNA system contains crRNA and tracrRNA on a single RNA molecule, while a dual guide RNA system contains crRNA and tracrRNA on two different RNA molecules, hybridized with each other through at least a portion of the CRISPR repeat sequence of crRNA and at least a portion of the tracrRNA (which can be completely or partially complementary to the CRISPR repeat sequence of crRNA). In some embodiments where the guide RNA is a single guide RNA, crRNA and tracrRNA are separated by a linker nucleotide sequence. Generally, the linker nucleotide sequence is a sequence that does not contain complementary bases in order to avoid the formation of secondary structures within the nucleotides of the linker nucleotide sequence, or to avoid the formation of secondary structures containing the nucleotides of the linker nucleotide sequence. In some embodiments, the linker nucleotide sequence between crRNA and tracrRNA is a number of nucleotides of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more. In a particular embodiment, the linker nucleotide sequence of a single guide RNA is a number of nucleotides of at least 4. In one embodiment, the linker nucleotide sequence is the nucleotide sequence shown as SEQ ID NO: 249.

[0050] Single or dual guide RNAs can be synthesized chemically or through in vitro transcription. Assays for elucidating sequence-specific binding between RGNs and guide RNAs are known in this field, and non-limiting examples include in vitro binding assays between expressed RGNs and guide RNAs. Guide RNAs can be tagged with a detectable label (e.g., biotin) and used in pull-down detection assays, in which the guide RNA:RGN complex is captured through the detectable label (e.g., using streptavidin beads). A control guide RNA with a sequence or structure unrelated to the guide RNA can be used as a negative control for nonspecific binding of RGNs to RNA. In some embodiments, the guide RNA is SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 59, 66, 73, 79, 86, 92, 99, 106, 113, 120, or 126, where the spacer sequence can be any sequence and is shown as a poly-N sequence.

[0051] In some embodiments, the guide RNA can be introduced as an RNA molecule into a target cell, organelle, or embryo. The guide RNA can be transcribed in vitro or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into the target cell, organelle, or embryo. In some of these embodiments, the nucleotide sequence encoding the guide RNA is functionally ligated to a promoter (e.g., an RNA polymerase III promoter). The promoter can be a native promoter or a heterogeneous promoter for the nucleotide sequence encoding the guide RNA.

[0052] In various embodiments, the guide RNA can be introduced into target cells, organelles, or embryos as a ribonucleoprotein complex, as described herein, and the guide RNA binds to an RNA-inducible nuclease polypeptide.

[0053] Guide RNA directs the relevant RNA-induced nuclease to a specific target nucleotide sequence of interest by hybridizing to that target nucleotide sequence. The target nucleotide sequence can include DNA, RNA, or a combination of both, and can be single-stranded or double-stranded. Possible target nucleotide sequences include genomic DNA (i.e., chromosomal DNA), plasmid DNA, or RNA molecules (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). The target nucleotide sequence can bind to (and in some cases cleave by) an RNA-induced nuclease in vitro or intracellularly. Possible chromosomal sequences targeted by RGN include nuclear, plastid, or mitochondrial chromosome sequences. In some embodiments, there is only one target nucleotide sequence within the target genome.

[0054] The target nucleotide sequence is adjacent to a protospacer motif (PAM). The protospacer motif is generally located within approximately 1 to 10 nucleotides of the target nucleotide sequence and contains approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the target nucleotide sequence. In a particular embodiment, the PAM is located within 1 to 10 nucleotides of the target nucleotide sequence and contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the target nucleotide sequence. The PAM can be the 5' or 3' of the target sequence. In some embodiments, the PAM is the 3' of the target sequence for the RGN of this disclosure. Generally, PAMs are consensus sequences consisting of approximately 3 to 4 nucleotides, but in special embodiments, they can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length. In various embodiments, the PAM sequences recognized by the RGN of this disclosure include consensus sequences designated as SEQ ID NOs. 7, 14, 21, 28, 35, 42, 49, 62, 69, 79, 82, 95, 102, 109, or 116.

[0055] In a particular embodiment, an RNA-inducible nuclease having SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence, respectively, indicated as SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 62, 69, 79, 82, 95, 102, 109, or 116. In some of these embodiments, the RGN binds to a CRISPR repeat sequence, or an active variant or fragment thereof, shown as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, respectively, and to a guide sequence containing a tracrRNA sequence, or an active variant or fragment thereof, shown as SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, respectively. The RGN systems are described in more detail in Examples 1-3 and Tables 1 and 2 of this specification.

[0056] We constructed the RGN variant APG05586 (SEQ ID NO: 63). These variants have the amino acid sequences of SEQ ID NOs. 570–579. RGNs having either SEQ ID NOs. 63 or either SEQ ID NOs. 570–579 can bind to a target nucleotide sequence adjacent to the PAM sequence shown as SEQ ID NO: 79. In some embodiments, variants of RGN APG05586 bind to a guide sequence containing the CRISPR repeat sequence shown as SEQ ID NO: 64, and may also include the tracrRNA sequence shown as SEQ ID NO: 65. These RGN systems are further described in Example 5 of this specification.

[0057] It is well known that the PAM sequence specificity for a given nuclease enzyme is affected by the enzyme concentration (see, for example, Karvelis et al. (2015) Genome Biol 16:253), and this can be altered by changing the promoter used for RGN expression or by changing the amount of ribonucleoprotein complex delivered to cells, organelles, or embryos.

[0058] Upon recognizing a corresponding PAM sequence, the RGN can cleave a target nucleotide sequence at a specific cleavage site. In this specification, a cleavage site consists of two specific nucleotides within the target nucleotide sequence, between which the nucleotide sequence is cleaved by the RGN. Cleavage sites can include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides in the 5' or 3' direction from the PAM. In some embodiments, cleavage sites can be located beyond 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in the 5' or 3' direction from the PAM. Because the RGN can cleave the target nucleotide sequence into sticky ends, in some embodiments, cleavage sites are defined based on the distance of two nucleotides from the PAM on the positive (+) strand of the polynucleotide and the distance of two nucleotides from the PAM on the negative (-) strand of the polynucleotide.

[0059] III. Nucleotides encoding RNA-induced nucleases, CRISPR RNA, and / or tracrRNA

[0060] This disclosure provides polynucleotides comprising CRISPR RNA, tracrRNA, and / or sgRNA as disclosed, and polynucleotides comprising nucleotide sequences encoding RNA-inducible nucleases, CRISPR RNA, tracrRNA, and / or sgRNA as disclosed. The polynucleotides of this disclosure include polynucleotides comprising or encoding CRISPR repeat sequences comprising the nucleotide sequences of SEQ ID NOs. 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or active variants or fragments thereof, which, when contained in a guide RNA, can sequence-specifically bind the associated RNA-inducible nuclease to a target sequence of interest. Also disclosed are polynucleotides containing or encoding tracrRNAs containing the nucleotide sequences of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or active variants or fragments thereof, which, when contained in a guide RNA, can sequence-specifically bind the associated RNA-inducible nuclease to a target sequence of interest. Also provided are RNA-inducible nucleases containing amino acid sequences shown as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579, and polynucleotides encoding those active fragments or variants thereof that retain the ability to bind to target nuclease sequences in an RNA-inducible sequence-specific manner.

[0061] The use of the terms “polynucleotide” or “nucleic acid molecule” is not limited in this disclosure to polynucleotides containing DNA. Those skilled in the art will recognize that polynucleotides can include ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. These include peptide nucleic acids (PNA), PNA-DNA chimeras, cross-linked nucleic acids (LNA), and phosphothiolate cross-linked sequences. Polynucleotides disclosed herein also encompass all forms of sequences, including, but not limited to, single-stranded forms, double-stranded forms, DNA-RNA hybrids, triple-stranded structures, stem-loop structures, and the like.

[0062] Nucleic acid molecules encoding RGNs can be codon-optimized for expression in organisms of interest. A “codon-optimized” coding sequence is a polynucleotide that encodes a sequence with codon usage frequencies designed to mimic the preferred codon usage frequencies or transcription conditions of a particular host cell. Increased expression in that particular host cell or organism results from changes in one or more codons at the nucleic acid level, but without changes in the translated amino acid sequence. Codon optimization can be performed on the whole or a portion of a nucleic acid molecule. Codon tables and other references providing preferred information for a wide range of organisms are available in this field (for example, regarding the use of plant-preferred codons, see Campbell and Gowri (1990) Plant Physiol. 92:1-11). Methods for synthesizing plant-preferred genes are available in this field. See, for example, United States Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498 (which are incorporated herein by reference).

[0063] Polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA as presented herein are provided in expression cassettes that can be expressed in vitro or in cells, organelles, embryos, or organisms of interest. The cassette will enable the expression of the polynucleotide by including 5' and 3' regulatory sequences functionally ligated to the polynucleotide encoding RGN, crRNA, tracrRNA, and / or sgRNA as presented herein. The cassette may include at least one additional gene or gene element for co-transformation in the organism. If an additional gene or element is included, these elements are functionally ligated. The expression "functionally ligated" means a functional link between two or more elements. For example, a functional link between a promoter and a coding region of interest (e.g., a region encoding RGN, crRNA, tracrRNA, and / or sgRNA) is a functional link that enables the expression of this coding region of interest. Functionally ligated elements may be contiguous or discontinuous. When functionally linked is used to refer to the junction of two protein-coding regions, it means that those coding regions are in the same reading frame. Alternatively, additional genes or elements may be provided on multiple expression cassettes. For example, the nucleotide sequence encoding the RGN of this disclosure may be on one expression cassette, while the nucleotide sequence encoding crRNA, trancrRNA, or complete guide RNA may be on another expression cassette. Such an expression cassette is provided with multiple restriction and / or recombination sites for inserting polynucleotides under transcriptional regulation of the regulatory region. The expression cassette may additionally include a selection marker gene.

[0064] The expression cassette would include, in the 5'→3' direction of transcription, a transcription (and, in some cases, translation) initiation region (i.e., promoter) that functions in the organism of interest, a polynucleotide encoding the RGN, crRNA, tracrRNA, and / or sgRNA of the present invention, and a transcription (and, in some embodiments, translation) termination region (i.e., termination region). The promoter of the present invention can direct or command the expression of the coding sequence in the host cell. These regulatory regions (e.g., promoter, transcription regulatory region, and translation termination region) can be endogenous or heterologous to or from the host cell. As used herein with respect to sequences, “heterologous” means a sequence derived from an exotic species, or, if derived from the same species, a sequence whose composition and / or which is substantially modified from its native form within a genomic locus by artificial intervention. Hereinafter, a chimeric gene includes, as a coding sequence, a coding sequence functionally ligated to a heterologous transcription initiation region for this coding sequence.

[0065] Convenient termination regions (e.g., octopine synthase and nopalin synthase termination regions) can be obtained from the Ti plasmid of A. tumefaciens. Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Proudfoot (1991) Cell 64:671-674;Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272;Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0066] Non-exclusive examples of additional regulatory signals include transcription start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, and stop signals. See, for example, U.S. Patents No. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) (hereafter referred to as "Sambrook 11"); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, New York, and the references cited therein.

[0067] When preparing expression cassettes, various DNA fragments are manipulated to provide DNA sequences with the appropriate orientation and, if necessary, in the appropriate reading frame. To this end, this may include joining DNA fragments using adapters or linkers, or other operations such as providing convenient restriction sites, removing excess DNA, or removing restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and resubstitution (e.g., transitions and transversions) may be included.

[0068] Numerous promoters can be used to carry out the present invention. Promoters can be selected based on the desired results. Nucleic acids can be expressed in organisms of interest by combining them with constitutive promoters, inductive promoters, growth stage-specific promoters, cell type-specific promoters, tissue-selective promoters, tissue-specific promoters, or other promoters. See, for example, WO 99 / 43838 and the promoters listed in United States Patents No. 8,575,425; No. 7,790,846; No. 8,147,856; No. 8,586,832; No. 7,772,369; No. 7,534,939; No. 6,072,050; No. 5,659,026; No. 5,608,149; No. 5,608,144; No. 5,604,121; No. 5,569,597; No. 5,466,785; No. 5,399,680; No. 5,268,463; No. 5,608,142; and No. 6,177,611 (these are incorporated herein by reference).

[0069] To express these compounds in plants, constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten et al. (1984) EMBO J. 3:2723-2730).

[0070] Examples of inducible promoters include the Adh1 promoter, which can be induced by hypoxic or cold stress; the Hsp70 promoter, which can be induced by thermal stress; and the PPDK and pepcarboxylase promoters, which can be induced by light. Chemically inducible promoters are also useful, such as the In2-2 promoter induced by drug-induced mitigating agents (United States Patent No. 5,364,780), the Axig1 promoter induced by auxin and specific to tapetum tissue but active in callus (PCT US01 / 22169), steroid-responsive promoters (e.g., the ERE promoter induced by estrogen and the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257), and tetracycline-inducible and tetracycline-inhibiting promoters (e.g., Gatz et al. (1991) Mol. Gen. Genet. See 227:229-237 and United States Patents No. 5,814,618 and No. 5,789,156 (these documents are incorporated herein by reference).

[0071] Tissue-specific or tissue-selective promoters can be used to express an expression construct within a specific tissue. In some embodiments, the tissue-specific or tissue-selective promoter is active within plant tissue. Examples of promoters under developmental control within plants include promoters that selectively initiate transcription within a given tissue (e.g., leaves, roots, fruits, seeds, or flowers). A “tissue-specific” promoter is one that initiates transcription only within a given tissue. Tissue-specific expression, unlike constitutive expression of a gene, is a result of several levels of gene regulatory interaction. Therefore, promoters from homologous or closely related plant species can be selectively used to achieve effective and reliable expression of a transgene within a specific tissue. In some embodiments, expression includes a tissue-selective promoter. A “tissue-selective” promoter is one that selectively initiates transcription, but does not necessarily initiate transcription entirely within or only within a given tissue.

[0072] In some embodiments, nucleic acid molecules encoding RGN, crRNA, and / or tracrRNA include cell type-specific promoters. A “cell type-specific” promoter is a promoter that primarily drives expression in several types of cells within one or more organs. Some examples of plant cells in which cell type-specific promoters functioning in plants can be primarily active include, for example, BELT cells, vascular cells in roots and leaves, stem cells, and stem cells. Nucleic acid molecules can also include cell type-selective promoters. A “cell type-selective” promoter is a promoter that primarily drives expression in several types of cells within one or more organs, but does not necessarily drive expression entirely in or only in a given tissue. Some examples of plant cells in which cell type-selective promoters functioning in plants can be selectively active include, for example, BELT cells, vascular cells in roots and leaves, stem cells, and stem cells.

[0073] Nucleic acid molecules encoding RGN, crRNA, tracrRNA, and / or sgRNA can be functionally ligated to a promoter sequence recognized by phage RNA polymerase, for example, for in vitro mRNA synthesis. In such embodiments, the RNA transcribed in vitro can be purified and used in the methods described herein. For example, the promoter sequence can be a T7 promoter sequence, a T3 promoter sequence, or an SP6 promoter sequence, or a variation thereof. In such embodiments, the expressed protein and / or RNA can be purified and used in the genome modification methods described herein.

[0074] In one embodiment, polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA may also be ligated to a polyadenylation signal (e.g., an SV40 polyA signal and other signals that function in plants) and / or at least one transcription termination sequence. In addition, as described elsewhere herein, the sequence encoding RGN may also be ligated to a sequence encoding at least one nuclear localization signal, at least one cell entry domain, and / or at least one signal peptide capable of moving the protein to a specific intracellular location.

[0075] Polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA can be present in one or more vectors. “Vector” refers to a polynucleotide composition for transferring, delivering, or introducing nucleic acids into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated virus vectors, baculovirus vectors). Vectors may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), selective marker sequences (e.g., antibiotic resistance genes), origins of replication, etc. Additional information can be found in “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003, or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, New York, 3rd edition, 2001.

[0076] The vector may also contain selection marker genes for selecting transformed cells. These selection marker genes are used to select transformed cells or tissues. Examples of marker genes include those encoding antibiotic resistance (such as neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) genes), as well as those conferring resistance to herbicides (e.g., glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D)).

[0077] In some embodiments, an expression cassette or expression vector containing a sequence encoding an RGN polypeptide may further include a sequence encoding crRNA and / or tracrRNA, or a combination thereof, to generate a guide RNA. The sequence encoding crRNA and / or tracrRNA can be functionally ligated to at least one transcriptional regulatory sequence to express crRNA and / or tracrRNA in the organism or host cell of interest. For example, a polynucleotide encoding crRNA and / or tracrRNA can be functionally ligated to a promoter sequence recognized by RNA polymerase III (Pol III). Non-limiting examples of suitable Pol III promoters include the mammalian U6 promoter, U3 promoter, H1 promoter, and 7SL RNA promoter, and the rice U6 promoter and U3 promoter.

[0078] As shown, organisms of interest can be transformed using expression constructs encoding RGN, crRNA, tracrRNA, and / or sgRNA. The transformation method involves introducing a nucleotide construct into the organism of interest. "Introducing" means introducing the nucleotide construct into a host cell so that the construct can access the interior of the host cell. The method of the present invention does not require a special method for introducing the nucleotide construct into the host organism; it is sufficient that the nucleotide construct can access the interior of at least one cell of the host organism. The host cell can be a eukaryotic cell or a prokaryotic cell. In particular embodiments, the eukaryotic cell is a plant cell, a mammalian cell, or an insect cell. In some embodiments, the eukaryotic cell containing or expressing the RGN of the present disclosure, or a eukaryotic cell modified by the RGN of the present disclosure, is a human cell. In some embodiments, eukaryotic cells containing or expressing the RGNs of the Disclosure, or eukaryotic cells modified by the RGNs of the Disclosure, are hematopoietic-derived cells, such as immune cells (i.e., cells of the innate or adaptive immune system), and non-limiting examples include B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

[0079] Methods for introducing nucleotide constructs into plants and other host cells are known in this field, and non-exclusive examples include methods for stable transformation, transient transformation, and virus-mediated transformation.

[0080] These methods yield transformed organisms (e.g., plants, including the entire plant body, as well as plant organs (e.g., leaves, stems, roots), seeds, plant cells, bulbils, and their embryos and offspring). Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0081] A “transgenic organism,” “transformed organism,” or “stable transformed” organism, cell, or tissue means an organism in which polynucleotides encoding RGN, crRNA, and / or tracrRNA of the present invention are incorporated or integrated. Other exogenous or endogenous nucleic acid sequences or DNA fragments are also known to be incorporated into host cells. Agrobacterium-mediated transformation and bioristic transformation remain the two main approaches used to transform plant cells. However, host cell transformation can also be achieved by infection, transfection, microinjection, electroporation, microprojection, bioristic or particle impact, electroporation, silica / carbon fiber, sonication, PEG-mediated, calcium phosphate coprecipitation, polycationic DMSO technology, DEAE dextran procedure, and virus-mediated, liposome-mediated, and others. Viral introduction of polynucleotides encoding RGN, crRNA, and / or tracrRNA includes introduction and expression via retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, as well as the use of karimoviruses, geminiviruses, and RNA plant viruses.

[0082] In addition to transformation protocols, protocols for introducing polypeptide or polynucleotide sequences into plants may vary depending on the type of host cell targeted for transformation (e.g., monocotyledonous or dicotyledonous cells). Transformation methods are known in this art, including those described in U.S. Patents No. 8,575,425; No. 7,692,068; No. 8,802,934; and No. 7,541,517 (each of which is incorporated herein by reference). Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858;Jones et al. (2005) Plant Methods 1:5;Rivera et al. (2012) Physics of Life Reviews 9:308-345;Bartlett et al. (2008) Plant Methods 4:1-12;Bates, GW (1999) Methods in Molecular Biology 111:359-366;Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606;Christou, P. (1992) The Plant Journal 2:275-281;Christou, P. (1995) Euphytica 85:13-27;Tzfira et al. (2004) TRENDS in Genetics See also 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1:5;

[0083] Cell transformation allows for the stable or transient integration of nucleic acids into cells. "Stable transformation" means that the nucleotide construct introduced into the host cell is integrated into the host cell's genome and can be inherited by its offspring. "Transient transformation" means that the polynucleotide is introduced into the host cell but is not integrated into the host cell's genome.

[0084] Methods for transforming chloroplasts are known in this field. See, for example, Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. This method relies on delivering DNA containing a selection marker using a particle gun, directing that DNA to the plastid genome through homologous recombination. In addition, plastid transformation can be achieved by cross-activating the silent transgene carried by the plastid through tissue-selective expression of plastid-inducible RNA polymerase encoded by the nucleus. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0085] Transformed cells can be introduced into transgenic organisms (such as plants) and propagated according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants are then propagated and pollinated using the same or different transformed strains, and from the resulting hybrids, those constitutively expressing the desired phenotypic characteristics are identified. After propagating for two or more generations to confirm that the expression of the desired phenotypic characteristics is stably maintained and inherited, the seeds are collected to confirm that the desired phenotypic characteristics have been realized. In this way, the present invention provides transformed seeds (also called "transgenic seeds") in which the nucleotide construct of the present invention (e.g., the expression cassette of the present invention) is stably incorporated into the genome.

[0086] Alternatively, transformed cells can be introduced into an organism. These cells may originate from that organism, in which case the cells are transformed using an in vitro approach.

[0087] The sequences presented herein can be used for the transformation of any plant species (including, but not limited to, monocots and dicots). Examples of plants of interest include corn, maize, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and rapeseed, Brassica species, alfalfa, rye, grains, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus fruits, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia nut, almond, oat, vegetables, ornamental plants, and conifers.

[0088] Non-limiting examples of vegetables include tomatoes, lettuce, green peas, lima beans, peas, and members of the genus Cucumis (such as cucumbers, cantaloupes, and muskmelons). Non-limiting examples of ornamental plants include azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. The plants of the present invention are preferably cultivated crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, pepper, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0089] In this specification, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant populations, and plant cells that are complete or part of a plant (e.g., embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, spike, rachis, husk, stem, root, root cap, anther). "Grain" means a mature seed produced by a crop grower for purposes other than propagation or reproduction. Progeny, variants, and mutants of regenerated plants are also within the scope of this invention, provided that these parts contain the introduced polynucleotides. Furthermore, processed plant products or by-products (e.g., soybean meal) retaining the sequences disclosed herein are also provided.

[0090] Polynucleotides encoding RGN, crRNA, and / or tracrRNA can also be used to transform any prokaryotic species. Non-exclusive examples of prokaryotic species include archaea and bacteria (e.g., species of Bacillus, Klebsiella, Streptomyces, Rhizobium, Escherichia coli, Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycoplasma, Agrobacterium, Lactobacillus).

[0091] Polynucleotides encoding RGN, crRNA, and / or tracrRNA can also be used to transform any eukaryotic species. Non-limiting examples of eukaryotic species include animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebas, algae, and yeast.

[0092] Nucleic acids can be introduced into mammalian, insect, or avian cells or target tissues using conventional gene transfer methods based on viruses and nonvirals. Such methods can be used to administer nucleic acids encoding components of the CRISPR system to cultured cells or host organisms. Nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles (such as liposomes). Viral vector delivery systems include DNA viruses and RNA viruses, which possess an episomal genome or an integrated genome after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Current Topics in Microbiology and Immunology, Doerfler and Bohm See Haddada et al. in (eds) (1995) and Yu et al., Gene Therapy 1:13-26 (1994).

[0093] Nonviral methods of nucleic acid delivery include lipofection, nucleofection, microinjection, bioristic, virosome, liposome, immunoliposome, polycational or lipid:nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patents No. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include the lipids of Felgner WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or in vitro) or target tissues (e.g., intra vivo). The preparation of lipid:nucleic acid complexes, including the preparation of targeted liposomes (such as immunolipid complexes), is well known to those skilled in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); see United States Patents Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0094] Using RNA or DNA virus-based systems for nucleic acid delivery involves utilizing highly evolved methods to direct viruses towards specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro, and these modified cells can, in some cases, be administered to patients (extra vivo). Conventional virus-based systems can include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transport. Integration into the host genome is made possible by gene transport methods using retroviruses, lentiviruses, and adeno-associated viruses, and the resulting inserted transgenes are often expressed over long periods. In addition, high gene transfer efficiencies have been observed in many different cell types and target tissues.

[0095] The targeting of retroviruses can be altered by incorporating foreign envelope proteins and amplifying a potential target population within target cells. Lentiviral vectors are retroviral vectors capable of introducing or infecting non-dividing cells, typically producing high viral titers. Therefore, the selection of retroviral gene transport systems is considered to depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and have the ability to package foreign sequences up to 6–10 kb. Minimal cis-acting LTRs are sufficient for vector replication and packaging, and these vectors are used to introduce therapeutic genes into target cells and permanently express the introduced gene. Widely used retroviral vectors include vectors based on mouse leukemia virus (MuLV), Gibbon's leukemia virus (GaLV), simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV), as well as combinations thereof (see, for example, Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., 1. Viral. 65: 2220-2224 (1991); PCT / US94 / 05700).

[0096] For applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors enable very high gene transfer efficiency in many cell types and do not require cell division. High titers and high expression levels have been obtained using such vectors. These vectors can be produced in large quantities in relatively simple systems. For example, adeno-associated virus ("AAV") vectors can be used to introduce target nucleic acids into cells for in vitro production of nucleic acids and peptides, and for in vivo and in vitro gene therapy (see, e.g., West et al., Virology 160:38-47 (1987); United States Patent No. 4,797,368; WO 93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, 1. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in numerous publications, including United States Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., 1. Viral. 63:03822-3828 (1989). Typically, packaging cells are used to form viral particles that can infect host cells. These cells include 293 cells (for packaging adenoviruses) and ψJ2 cells or PA317 cells (for packaging retroviruses).

[0097] Viral vectors used in gene therapy are typically constructed by generating a cell line that packages the nucleic acid vector within a viral particle. The vector typically contains the minimal viral sequence necessary for packaging and subsequent integration into the host, with other viral sequences replaced by expression cassettes for the polynucleotides that will be expressed. The lost viral function is typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the ITR sequence from the AAV genome necessary for packaging and integration into the host genome. The viral DNA is packaged within a cell line containing helper plasmids that encode other AAV genes (i.e., rep and cap) but lack the ITR sequence.

[0098] Cell lines can also be infected with an adenovirus as a helper virus. The helper virus promotes replication of the AAV vector and expression of the AAV gene from the helper plasmid. The helper plasmid is not packaged in large quantities due to the lack of an ITR sequence. Adenovirus contamination can be reduced by heat treatment, for example, to which adenovirus is more susceptible than AAV. Additional methods for delivering nucleic acids to cells are known to those skilled in the art. See, for example, United States Patent Application Publication 2003 / 0087817 (incorporated herein by reference).

[0099] In some embodiments, one or more vectors described herein are transfected transiently or non-transiently into host cells. In some embodiments, the cells are transfected in a manner similar to what occurs naturally in the subject. In some embodiments, the transfected cells are harvested from the subject. In some embodiments, the cells are derived from cells harvested from the subject (e.g., a cell line). In some embodiments, the cell line can be mammalian cells, insect cells, or avian cells. A wide variety of cell lines for tissue culture are known in this field. Non-limiting examples of cell lines include: C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TFl, CTLL-2, CIR, Rat6, CVI, RPTE, AlO, T24, 182 , A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, T IB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2 780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10Tl / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-l0A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC These include cell lines 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THPl cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and their transgenic variants. Cell lines from a variety of sources known to those skilled in the art can be utilized (see, for example, the American Type Culture Collection (ATCC) (Manassas, Virginia)).

[0100] In some embodiments, a novel cell line containing one or more vector-derived sequences is established using cells transfected with one or more vectors described herein. In some embodiments, a novel cell line containing the modification but completely lacking other exogenous sequences is established using cells that have been transiently transfected with components of the RGN system described herein (e.g., by transient transfection of one or more vectors or by RNA transfection) and modified through the activity of the RGN system. In some embodiments, one or more test compounds are evaluated using cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells.

[0101] In some embodiments, non-human transgenic animals or transgenic plants are produced using one or more vectors described herein. In some embodiments, the transgenic animal is a mammal (e.g., mouse, rat, hamster, rabbit, cow, or pig). In some embodiments, the transgenic animal is a bird (e.g., chicken or duck). In some embodiments, the transgenic animal is an insect (mosquito or tick).

[0102] IV. Variants and Fragments of Polypeptides and Polynucleotides

[0103] This disclosure provides active variants and fragments of natural (i.e., wild-type) RNA-induced nucleases (whose amino acid sequences are indicated as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579), as well as natural CRISPR repeats (e.g., SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71) Active variants and fragments of sequences (represented as 77, 84, 90, 97, 104, 111, 118, or 124), and active variants and fragments of native tracrRNA (represented as sequences 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125), and polynucleotides encoding these are provided.

[0104] The activity of a variant or fragment may be altered compared to the polynucleotide or polypeptide of interest, but the variant or fragment must retain the functionality of the polynucleotide or polypeptide of interest. For example, the variant or fragment may have increased activity, decreased activity, a different activity spectrum, or some other alteration of activity compared to the polynucleotide or polypeptide of interest.

[0105] Fragments and variants of natural RGN polypeptides (such as those disclosed herein) would retain sequence-specific RNA-induced DNA-binding activity. In specific embodiments, fragments and variants of natural RGN polypeptides (such as those disclosed herein) would retain nuclease activity (single-stranded or double-stranded).

[0106] Natural CRISPR repeat fragments and variants (such as those disclosed herein), when part of a guide RNA (including tracrRNA), would retain the ability to bind to an RNA-inducible nuclease (forming a complex with the guide RNA) and guide it to target a target nucleotide sequence in a sequence-specific manner.

[0107] Natural tracrRNA fragments and variants (such as those disclosed herein), when part of a guide RNA (including CRISPR RNA), would retain the ability to bind to an RNA-inducible nuclease (forming a complex with the guide RNA) and guide it to target a target nucleotide sequence in a sequence-specific manner.

[0108] The term "fragment" means a portion of a polynucleotide sequence or polypeptide sequence according to the present invention. A "fragment" or "bioactive portion" includes a polynucleotide containing a sufficient number of consecutive nucleotides to maintain bioactivity (i.e., when contained within a guide RNA, it binds to an RGN and directs this RGN sequence-specifically to a target nucleotide sequence). A "fragment" or "bioactive portion" includes a polypeptide containing a sufficient number of consecutive amino acid residues to maintain bioactivity (i.e., when complexed with a guide RNA, it sequence-specifically binds to a target nucleotide sequence). Fragments of RGN proteins may be shorter than the full-length sequence due to the use of an alternative downstream start site. The biologically active portion of the RGN protein is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 copies of SEQ ID NOs. 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579. Polypeptides containing 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or more consecutive amino acid residues are possible. Such bioactive moieties can be prepared by recombinant technology and then evaluated for sequence-specific RNA-induced DNA binding activity. A bioactive fragment of a CRISPR repeat sequence may contain at least eight consecutive nucleic acids of sequence numbers 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124.The bioactive portion of the CRISPR repeat sequence can be a polynucleotide containing, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides, such as sequence numbers 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124. The biologically active portion of tracrRNA can be a polynucleotide containing a number of consecutive nucleotides, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or more, from sequence numbers 3, 10, 17, 24, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or more.

[0109] Generally, a “variant” refers to a substantially similar sequence. With respect to polynucleotides, a variant includes the deletion and / or addition of one or more nucleotides to one or more internal sites within a native polynucleotide, and / or the substitution of one or more nucleotides to one or more internal sites within a native polynucleotide. In this specification, “native” or “wild-type” polynucleotides or polypeptides include the native nucleotide sequence or amino acid sequence, respectively. With respect to polynucleotides, conserved variants include sequences that, due to the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Such native allele variants can be identified using well-known molecular biology techniques (e.g., polymerase chain reaction (PCR) and hybridization techniques outlined below). Variant polynucleotides also include polynucleotides of synthetic origin that still encode the polypeptide or polynucleotide of interest (e.g., polynucleotides produced by site-directed mutagenesis). Generally, any special polynucleotide variant disclosed herein, when examined by the sequence alignment programs and parameters described elsewhere herein, will have sequences that match at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of that special polynucleotide.

[0110] A variant of a particular polynucleotide (i.e., a reference polynucleotide) disclosed herein can also be evaluated by comparing the degree of sequence agreement between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The degree of sequence agreement between any two polypeptides can be calculated using the sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides disclosed herein is evaluated by comparing the degree of sequence agreement between the two polypeptides encoded by these polynucleotides, the degree of sequence agreement between these two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater.

[0111] In a particular embodiment, the polynucleotides of the present disclosure encode RNA-inducible nuclease polypeptides comprising amino acid sequences that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579, or more, identical to the amino acid sequences of SEQ ID NOs. 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579. In some embodiments, variants of SEQ ID NO: 63 retain isoleucine at the amino acid position corresponding to 305, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405 of SEQ ID NO: 63. An amino acid position in the first amino acid sequence that "corresponds" to a specific position in the second amino acid sequence means a position in the first amino acid sequence that coincides with the position of that particular amino acid residue in the second sequence when the first and second amino acid sequences are optimally aligned. In a particular embodiment, the variant of SEQ ID NO: 63 is identical to SEQ ID NO: 63 by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, except for the retained amino acid residues from SEQ ID NO: 63 (i.e., I305, V328, L366, T368, and V405).

[0112] The biologically active variants of the RGN polypeptide of the present invention may differ by a small number of amino acid residues, such as about 1 to 15, about 1 to 10, for example, about 6 to 10, 5, 4, 3, 2, or 1. In particular embodiments, these polypeptides may include N-terminal or C-terminal truncations, where 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 45 from the N-terminus or C-terminus of the polypeptide. It may contain at least 0, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or more amino acid deletions.

[0113] In one embodiment, the polynucleotides of the present disclosure include or encode CRISPR repeats that contain nucleotide sequences that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 87%, 88%, 89%, 90%, 97%, 104, 111, 118, or 124, or more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the nucleotide sequences shown as SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 84%, 84%, 84%, 95%, 96%, 97%, 98%, 99%.

[0114] The polynucleotides of this disclosure may include or encode tracrRNAs that contain nucleotide sequences that are at least 40%, 45%, 50%, 55%, 60%, 65%, 72, 78, 85, 91, 98, 105, 112, 119, or 125 matched by a larger proportion of nucleotide sequences.

[0115] The biologically active variants of the CRISPR repeat or tracrRNA of the present invention may differ by a small number of nucleotides, approximately 1 to 15, approximately 1 to 10, approximately 6 to 10, approximately 5, 4, 3, 2, or 1. In particular embodiments, these polynucleotides may include 5' or 3' truncations, which may include at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, or more nucleotide deletions from either the 5' or 3' end of the polynucleotide.

[0116] It is recognized that the RGN polypeptides, CRISPR repeats, and tracrRNAs presented herein can be modified to produce variant proteins and variant polynucleotides. Human-designed alterations can be introduced using site-directed mutagenesis techniques. Alternatively, it may be possible to identify natural, but unknown or unidentified, polynucleotides and / or polypeptides related to the sequences, structures, and / or functions disclosed herein that fall within the scope of the present invention. Conserved amino acid substitutions that do not alter the function of RGN proteins can be implemented in non-conserved regions. Alternatively, modifications can be made that improve or alter the activity of RGNs.

[0117] Variant polynucleotides and variant proteins also include sequences and proteins obtained from procedures that induce mutation and recombination (such as DNA shuffling). Using such procedures, one or more different RGN proteins disclosed herein (e.g., SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579) are manipulated to create new RGN proteins with desired properties. In this way, a library of recombinant polynucleotides is generated from a population of polynucleotides with related sequences, i.e., a population of polynucleotides with substantially identical sequences and containing sequence regions capable of homologous recombination in vitro or in vivo. For example, this approach can be used to shuffle sequence motifs encoding domains of interest between RGN sequences presented herein and other known RGN genes, resulting in improved properties of interest (e.g., K in the case of enzymes). mThis allows us to obtain new genes encoding proteins (with increased density). Strategies for such DNA shuffling are known in this field. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and United States Patents Nos. 5,605,793 and 5,837,458. A “shuffled” nucleic acid is a nucleic acid produced by a shuffling procedure (e.g., any shuffling procedure described herein). A shuffled nucleic acid is produced by rearranging two or more nucleic acids (or strings) (physically or virtually) in an artificial, and possibly recurrent, manner. Generally, the shuffling process utilizes one or more screening steps to identify nucleic acids of interest. This screening step can be performed before or after any recombination step. In some (but not all) embodiments of shuffling, it is desirable to perform multiple recombinations before selection to increase the diversity of the pool being screened. The entire process of recombination and selection is sometimes repeated recursively. Depending on the context, shuffling may mean the entire process of recombination and selection, or only the recombination portion of the whole process.

[0118] In this specification, “sequence matching” or “match” in the context of two polynucleotide or polypeptide sequences means that the residues in the two sequences are the same when aligned to maximize correspondence on a given comparison window. When the degree of sequence matching is used in relation to proteins, the residue positions that are not the same are often differences in conserved amino acid substitutions, and it is recognized that the functional properties of the molecule do not change because the amino acid residue at that position is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity). When sequences differ with respect to conserved substitutions, the degree of sequence matching can be adjusted upward to correct for the conserved nature of those substitutions. Sequences that differ in such conserved substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those skilled in the art. Typically, this means involves increasing the degree of sequence matching by scoring conserved substitutions as partial mismatches rather than complete mismatches. Thus, for example, if matching amino acids are given 1 point and non-conserved substitutions are given 0 points, conserved substitutions are given a score between 0 and 1. The saved substitution scores are calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0119] In this specification, “sequence matching percentage” refers to a value obtained by comparing two sequences that have been optimally aligned within a comparison window. In this case, portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where nucleic acid bases or amino acid residues are the same in both sequences, obtaining the number of matching positions, dividing this number of matching positions by the total number of positions within the comparison window, and multiplying by 100 to obtain the sequence matching percentage.

[0120] Unless otherwise specified, the sequence match / similarity values ​​presented herein refer to values ​​obtained using GAP Version 10 (using the following parameters: for nucleotide sequences, GAP Weight 50, Length Weight 3, and % match and % similarity obtained using the nwsgapdna.cmp scoring matrix; for amino acid sequences, GAP Weight 8, Length Weight 2, and % match and % similarity obtained using the BLOSUM62 scoring matrix), or values ​​obtained using any equivalent program. "Equivalent program" means any sequence comparison program that, when compared to the corresponding alignment generated by GAP Version 10 for any two sequences in question, produces alignments with the same nucleotide or amino acid residue match and the same percentage of sequence match.

[0121] Two sequences are considered "optimally aligned" when, for the purpose of determining a similarity score, they are aligned using a predetermined amino acid substitution matrix (e.g., BLOSUM62), a gap presence penalty, and a gap extension penalty, and the highest possible score for the pair of sequences is achieved. Amino acid substitution matrices and their use in quantifying similarity between two sequences are well-known in this field, and are described, for example, in Dayhoff et al. (1978), "Models of Evolutionary Change in Proteins," pp. 345-352, in "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. MO Dayhoff), and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is ​​often used as the default scoring substitution matrix in sequence alignment protocols. A gap presence penalty is imposed for introducing a single amino acid gap into one of the aligned sequences, and a gap extension penalty is imposed for introducing additional empty amino acid positions into already existing gaps. Alignment is defined within each sequence by the amino acid positions where the alignment begins and ends, and in some cases, one or more gaps are inserted into one or both sequences to obtain the highest possible score. Optimal alignment and scoring can be achieved manually, but this process is facilitated by computer-implemented alignment algorithms (e.g., Gap-Included BLAST 2.0 described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, which is available from the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov)).The optimal alignment can be prepared using a variety of methods, such as PSI-BLAST (described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and available from www.ncbi.nlm.nih.gov).

[0122] For an amino acid sequence optimally aligned with a reference sequence, each amino acid residue "corresponds" to a position in the reference sequence that pairs with that residue within the alignment. This "position" is represented by the number of each amino acid, sequentially identified based on its relative position to the N-terminus in the reference sequence. Because deletions, insertions, truncations, and fusions must be considered when determining optimal alignment, the numbers of amino acid residues in a test sequence, obtained by simply counting from the N-terminus, are generally not the same as the numbers of their corresponding positions in the reference sequence. For example, if there is a deletion in the aligned test sequence, there is no amino acid in the reference sequence that corresponds to the location of that deletion. If there is an insertion in the aligned test sequence, that insertion does not correspond to any amino acid position in the reference sequence. If there is a truncation or fusion, there may be a series of amino acids in the reference sequence or the aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0123] V. Antibodies

[0124] The present invention also includes antibodies against the RGN polypeptide, or ribonucleoproteins containing this RGN polypeptide (including those having amino acid sequences represented as SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579), or against active variants or fragments thereof. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; U.S. Patent No. 4,196,265). These antibodies can be used in kits for detecting and isolating RGN polypeptides or ribonucleoproteins. Accordingly, this disclosure provides a kit comprising antibodies that specifically bind to polypeptides or ribonucleoproteins described herein (including, for example, polypeptides having sequences SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570-579).

[0125] VI. Systems and ribonucleoprotein complexes for binding to target sequences of interest, and methods for producing them.

[0126] This disclosure provides a system for binding to a target sequence of interest. The system comprises at least one guide RNA, or a nucleotide sequence encoding it, and at least one RNA-inducible nuclease, or a nucleotide sequence encoding it. The guide RNA hybridizes to the target sequence of interest and also forms a complex with the RGN polypeptide, thereby binding the RGN polypeptide to the target sequence. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence containing the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In a special embodiment, the guide RNA comprises a tracrRNA containing the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. A single guide RNA or a dual guide RNA is possible as the guide RNA in this system. In a special embodiment, the system includes a heterologous RNA-inducible nuclease for the guide RNA, so that the RGN and guide RNA do not spontaneously form a complex (i.e., bind to each other).

[0127] A ribonucleoprotein complex (RGN complex) is a possible system presented herein for binding to a target sequence of interest, and this RGN complex is at least one RNA molecule bound to at least one protein. The RGN complex presented herein comprises at least one guide RNA as an RNA component and one RNA-inducible nuclease as a protein component. Such a nuclear protein complex can be purified from cells or organisms that spontaneously express an RGN polypeptide and have been engineered to express a specific guide RNA specific to a target sequence of interest. Alternatively, the RGN complex can be purified from cells or organisms that have been transformed with polynucleotides encoding the RGN polypeptide and the guide RNA, and then cultured under conditions that allow for the expression of the RGN polypeptide and the guide RNA. Thus, a method for producing an RGN polypeptide or RGN ribonucleoprotein complex is provided. Such a method involves culturing cells containing a nucleotide sequence encoding the RGN polypeptide and, in some embodiments, a nucleotide sequence encoding the guide RNA, under conditions that allow for the expression of the RGN polypeptide (and, in some embodiments, its guide RNA). The RGN polypeptide, or RGN ribonucleoprotein complex, can then be purified from the lysate of the cultured cells.

[0128] Methods for purifying RGN polypeptides or RGN ribonucleoprotein complexes from lysates of biological samples are known in this field (e.g., size exclusion chromatography and / or affinity chromatography, 2D-PAGE, HPLC, reverse-phase chromatography, immunoprecipitation). In special methods, the RGN polypeptides are recombinantly produced and contain purification tags to aid in their purification. Non-limiting examples of purification tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His, 10×His, biotin carboxyl carrier protein (BCCP), and calmodulin. Generally, the tagged polypeptides or ribonucleoprotein complexes of the present invention are purified using immobilized metal affinity chromatography. It will be found that other similar methods known in the art (including other forms of chromatography, or, for example, immunoprecipitation) can be used alone or in combination.

[0129] "Isolated" or "purified" polypeptides, or their bioactive portions, substantially or essentially contain no components that normally accompany polypeptides found in their natural environment, or components that interact with such polypeptides. Therefore, isolated or purified polypeptides, when prepared by recombinant techniques, substantially contain no other cellular material or cell medium, and when chemically synthesized, substantially contain no chemical precursors or other compounds. Proteins substantially free of cellular material include protein preparations containing less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the proteins of the present invention, or their bioactive portions, are prepared by recombinant techniques, the culture medium optimally contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals without the protein of interest.

[0130] The special methods presented herein for binding to and / or cleaving target sequences of interest include the use of RGN ribonucleoprotein complexes assembled in vitro. In vitro assembly of RGN ribonucleoprotein complexes can be carried out using any method known in the art, in which the RGN polypeptide is brought into contact with the guide RNA under conditions that allow the RGN polypeptide to bind to the guide RNA. Herein, “in contact,” “in contact,” and “contacted” mean bringing together the components of a desired reaction and placing them under conditions suitable for the performance of that desired reaction. RGN polypeptides can be purified from biological samples, cell lysates, or culture media, transcribed in vitro, or chemically synthesized. Guide RNA can be produced through in vitro transcription or chemical synthesis, and then purified from biological samples, cell lysates, or culture media. RGN polypeptides and guide RNA can be brought into contact in solution (e.g., buffered saline solution) to assemble RGN ribonucleoprotein complexes in vitro.

[0131] VII. Methods for binding to a target sequence, cleaving a target sequence, or modifying a target sequence

[0132] This disclosure provides methods for binding to a target nucleotide of interest, cleaving a target nucleotide of interest, and / or modifying a target nucleotide of interest. These methods include delivering a system comprising at least one guide RNA or polynucleotide encoding it and at least one RGN polypeptide or polynucleotide encoding it to a target sequence or to a cell, organelle, or embryo containing the target sequence. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence containing the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In a special embodiment, the guide RNA comprises a tracrRNA containing the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a double guide RNA. The RGN of the system can be a nuclease-dead RGN, have nickasase activity, or be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base-editing polypeptide (e.g., cytidine deaminase or adenosine deaminase). In other embodiments, the RGN fusion protein comprises a reverse transcriptase.In another embodiment, the RGN fusion protein comprises a polypeptide that recruits members of a functional nucleic acid repair complex (such as members of the nucleotide excision repair (NER) pathway or the transcription-coupled nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA 112(27):E3495-504; Troelstra et al., 1992, Cell 71:939-953; Marnef et al., 2017, J Mol Biol 429(9):1277-1288)), as described in U.S. Provisional Application No. 62 / 966,203 (filed on 27 January 2020, which is incorporated in its entirety by reference). In some embodiments, the RGN fusion protein includes a CSB (corner subsequence) that is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members (van den Boom et al., 2004, J Cell Biol 166(1):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; one example is shown as SEQ ID NO: 608). In further embodiments, the RGN fusion protein includes the active domain of the CSB (such as the acidic domain of the CSB containing amino acid residues 356-394 of SEQ ID NO: 608 (Teng et al., 2018, Nat Commun 9(1):4115)).

[0133] In a particular embodiment, the RGN and / or guide RNA are heterogeneous to the cell, organelle, or embryo into which the RGN and / or guide RNA (or the polynucleotide encoding at least one of the RGN and / or guide RNA) is introduced.

[0134] In embodiments where the method includes delivering a polynucleotide encoding a guide RNA and / or RGN polypeptide, cells or embryos can then be cultured under conditions in which the guide RNA and / or RGN polypeptide is expressed. In various embodiments, the method includes contacting a target sequence with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex may include nuclease-dead or nickas-active RGNs. In some embodiments, the RGNs in the ribonucleoprotein complex are fusion polypeptides containing base-editing polypeptides. In some embodiments, the method includes introducing the RGN ribonucleoprotein complex into cells, organelles, or embryos containing the target sequence. The RGN ribonucleoprotein complex can be purified from a biological sample, recombinantly produced and then purified, or assembled in vitro as described herein. In embodiments where the RGN ribonucleoprotein complex to be contacted with the target sequence, or cell organelle, or embryo, is assembled in vitro, the method may further include assembling the complex in vitro before contacting the target sequence, cell, organelle, or embryo.

[0135] Purified or in vitro assembled RGN ribonucleoprotein complexes can be introduced into cells, organelles, or embryos using any method known in this art (including, but not limited to, electroporation). Alternatively, RGN polypeptides and / or polynucleotides encoding or containing guide RNA can be introduced into cells, organelles, or embryos using any method known in this art (e.g., electroporation).

[0136] When the guide RNA is delivered to or comes into contact with a target sequence, or a cell, organelle, or embryo containing this target sequence, it causes the RGN polypeptide or fusion protein to bind to the target sequence in a sequence-specific manner. In embodiments where the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence upon binding to it. The target sequence is then modified using a provided donor polynucleotide through an endogenous repair mechanism (e.g., non-homologous end join repair or homologous recombination repair).

[0137] Methods for measuring the binding of RNA-induced DNA-binding polypeptides to target sequences are known in this field and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, and microplate capture and detection assays. Similarly, methods for measuring cleavage or modification of target sequences are known in this field and include in vitro or in vivo cleavage assays. These cleavage assays confirm cleavage using PCR sequencing or gel electrophoresis, with or without appropriate labeling (e.g., radioisotopes, fluorescent substances) attached to the target sequence to facilitate the detection of degradation products. Alternatively, cleavage-triggered exponential amplification (NTEXPAR) assays can be used (e.g., Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo cleavage can be evaluated using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

[0138] In some embodiments, the method involves using a single type of RGN complexed with two or more guide RNAs. These two or more guide RNAs can target different regions of a single gene or multiple genes.

[0139] In embodiments where a donor polynucleotide is not provided, double-strand breaks introduced by the RGN polypeptide can be repaired by a non-homologous end-joining (NHEJ) repair process. Because NHEJs are error-prone, repair of double-strand breaks may modify the target sequence. As used herein with respect to nucleic acid molecules, “modification” means a change in the nucleotide sequence of the nucleic acid molecule, which may include the deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of the target sequence may result in the expression of an altered protein product or inactivation of the coding sequence.

[0140] In embodiments where a donor polynucleotide is present, during the repair of introduced double-strand breaks, the donor sequence within the donor polypeptide can be incorporated into or replaced with the target nucleotide sequence, resulting in the introduction of an exogenous donor sequence. Therefore, the donor polynucleotide contains a donor sequence that is desirable to be introduced into the target sequence of interest. In some embodiments, the donor sequence alters the original target nucleotide sequence so that this newly incorporated donor sequence is not recognized by RGNs and is not cleaved by RGNs. Donor sequence incorporation can be enhanced by including a flanking sequence (referred to herein as a “homologous arm”) within the donor polynucleotide that substantially matches a sequence adjacent to the target nucleotide sequence, enabling the homologous recombination repair process. In some embodiments, the homologous arm has a length of at least 50 base pairs, at least 100 base pairs, and up to 2000 base pairs, or longer, and has at least 90%, at least 95%, or greater sequence homology with the corresponding sequence in the target nucleotide sequence.

[0141] In embodiments in which the RGN polypeptide introduces double-stranded sticky ends, the donor polynucleotide may contain a donor sequence adjacent to a fitted overhang, allowing the non-homologous recombination repair process to directly ligate the donor sequence to the cleaved target nucleotide sequence containing the overhang while the double-strand break is being repaired.

[0142] In embodiments of the present invention in which the method is a nickase (i.e., capable of cleaving only a single strand of a double-stranded polynucleotide), the method may include introducing two RGN nickases that target multiple identical or overlapping target sequences and cleave different strands of the polynucleotide. For example, a second RGN nickase that cleaves only the minus (-) strand of the double-stranded polynucleotide may be introduced along with an RGN nickase that cleaves only the plus (+) strand of the double-stranded polynucleotide.

[0143] In various embodiments, a method is provided for detecting a target nucleotide sequence by binding to that target sequence, the method comprising introducing at least one guide RNA or polynucleotide encoding it and at least one RGN polypeptide or polynucleotide encoding it into a cell, organelle, or embryo, expressing the guide RNA and / or RGN polypeptide (if a coding sequence is introduced; the RGN polypeptide is a nuclease-dead RGN and further comprises a detectable label), the method further comprising detecting the detectable label. The detectable label can be fused to the RGN as a fusion protein (e.g., a fluorescent protein), or as a detectable label, it can be a small molecule that is complexed with the RGN polypeptide or incorporated into the RGN polypeptide and can be detected visually or by other means.

[0144] This specification also provides methods for altering the expression of a target sequence or the expression of a gene of interest that is regulated by the target sequence. These methods involve introducing at least one guide RNA or polynucleotide encoding it and at least one RGN polypeptide or polynucleotide encoding it into a cell, organelle, or embryo and expressing this guide RNA and / or RGN polypeptide (if a coding sequence is introduced; this RGN polypeptide is a nuclease-dead RGN). In some of these embodiments, the nuclease-dead RGN is a fusion protein comprising an expression modulator domain (i.e., an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain) as described herein.

[0145] This disclosure also provides methods for conjugating to and / or modifying a target nucleotide sequence of interest. These methods involve delivering a system comprising at least one guide RNA or a polynucleotide encoding it, wherein at least one fusion polypeptide comprises the RGN and base-editing polypeptide of the present invention (e.g., cytidine deaminase or adenosine deaminase) or a polynucleotide encoding the fusion polypeptide, to the target sequence or to a cell, organelle, or embryo containing the target sequence.

[0146] Those skilled in the art will see that any one of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. For example, the method involves using a single RGN polypeptide in combination with a number of different guide RNAs, which can target a number of different sequences in a single gene and / or multiple genes. This specification also encompasses methods for introducing a number of different guide RNAs in combination with a number of different RGN polypeptides. These guide RNA and guide RNA / RGN polypeptide systems can target a number of different sequences in a single gene and / or multiple genes.

[0147] In one aspect, the present invention provides a kit comprising any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a DNA sequence encoding a crRNA sequence and a first regulatory element functionally ligated to one or more insertion sites for inserting a guide sequence upstream of the encoded crRNA sequence (the guide sequence, when expressed, causes the RGN complex (containing the RGN enzyme as a complex with guide RNA polynucleotides) to sequence-specifically bind to a target sequence in a eukaryotic cell); and / or (b) a second regulatory element functionally ligated to an enzyme-coding sequence encoding the RGN enzyme, which includes a nuclear localization sequence. The elements may be provided individually or in combination and may be provided in any suitable container (such as a vial, bottle, or tube).

[0148] In some embodiments, the kit includes instructions in one or more languages. In some embodiments, the kit includes one or more reagents for use in a manner utilizing one or more elements described herein. Reagents can be provided in any suitable container. For example, the kit can provide one or more reaction buffers or storage buffers. Reagents can be provided in a form usable in a particular assay, or in a form that requires the addition of one or more other components before use (e.g., in a concentrated or lyophilized form). Any buffer is possible as a buffer, and non-limiting examples include sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10.

[0149] In some embodiments, the kit includes one or more oligonucleotides corresponding to guide sequences for insertion into a vector, which is to functionally link the guide sequence with the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide. In one aspect, the present invention provides a method for using one or more elements of an RGN system. The RGN system of the present invention provides an effective means for modifying a target polynucleotide. The RGN system of the present invention has diverse utility, including modification of a target polynucleotide in many types of cells (e.g., deletion, insertion, transposition, inactivation, activation, base editing). Thus, the RGN system of the present invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis prediction. A typical RGN system or RGN complex includes an RGN enzyme complexed with a guide sequence hybridized to a target sequence in the target polynucleotide.

[0150] VIII. Target Polynucleotides

[0151] In one aspect, the present invention provides a method for modifying a target polynucleotide in eukaryotic cells present in vivo, in vitro, or in vitro. In some embodiments, the method involves sampling one cell or population of cells from a human, non-human animal, or plant (including microalgae), and modifying one or more of those cells. Culturing can be carried out in vitro at any stage. The one or more of those cells can even be reintroduced into a non-human animal or plant (including microalgae).

[0152] Plant growers utilize natural variability to combine highly useful genes in search of desirable qualities (e.g., yield, quality, uniformity, hardiness, and resistance to pathogens). These desirable qualities also include growth, sunlight preferences, temperature conditions, flowering or reproductive development start date, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, and tolerance to various environmental factors (drought, heat, humidity, cold, wind, and unfavorable soil conditions, including high salinity). Sources of these useful genes include native or introduced species, heirloom varieties, relatives of wild plants, and induced mutations (e.g., by treating plant material with mutagenic agents). By utilizing the present invention, plant growers are provided with a new tool for inducing mutations. Thus, those skilled in the art can analyze genomes to search for useful genes and utilize the present invention to induce an increase in useful genes in varieties with desired features or traits more accurately than with previous mutagenic agents, thereby accelerating and improving plant cultivation programs.

[0153] Any polynucleotide, endogenous or exogenous to eukaryotic cells, can be used as a target polynucleotide in the RGN system. For example, a polynucleotide present in the nucleus of a eukaryotic cell can be a target polynucleotide. The target polynucleotide can be a sequence that codes for a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). While we do not want to be constrained by theory, it is thought that the target sequence should be related to a PAM (protospacer adjacent motif), i.e., a short sequence recognized by the RGN system. The exact sequence and length requirements of the PAM vary depending on the RGN used, but typically, a PAM is a sequence of 2 to 5 base pairs adjacent to the protospacer (i.e., the target sequence).

[0154] RGN target polynucleotides can include a wide range of genes and polynucleotides related to biochemical signaling pathways, as well as those related to disease-related genes and polynucleotides. Examples of target polynucleotides include sequences related to biochemical signaling pathways, such as genes or polynucleotides related to biochemical signaling pathways. Examples of target polynucleotides include genes or polynucleotides related to disease. A “disease-related” gene or polynucleotide means any gene or polynucleotide that produces transcripts or translated products at abnormal levels or in abnormal forms in cells derived from diseased tissue compared to control tissue or cells without the disease. It may be a gene that becomes expressed at abnormally high levels, or a gene that becomes expressed at abnormally low levels, and the altered expression correlates with the onset and / or progression of the disease. Disease-related genes also mean genes with mutations or genetic variations directly responsible for the origin of the disease (e.g., causative mutations), or genes with mutations or genetic variations in linkage equilibrium with genes responsible for the origin of the disease. The transcribed or translated products may be known or unknown, and may be at normal or abnormal levels. In some embodiments, animal diseases can be considered diseases. In some embodiments, the disease can be a avian disease. In other embodiments, the disease can be a mammalian disease. In further embodiments, the disease can be a human disease. Examples of genes and polynucleotides associated with human diseases are available on the World Wide Web from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Maryland) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Maryland).

[0155] While RGN systems are particularly useful because they allow for relatively easy targeting of genomic sequences of interest, the question remains as to what RGNs can do to address causative mutations. One approach is to construct a fusion protein between an RGN (preferably an inactive variant or nickase variant of the RGN) and a base-editing enzyme (e.g., cytidine deaminase base editor or adenosine deaminase base editor) or the active domain of a base-editing enzyme (United States Patent No. 9,840,699; incorporated herein by reference). In some embodiments, the method comprises (a) contacting a DNA molecule with a fusion protein comprising the RGN of the present invention and a base-editing polypeptide (such as a deaminase); and (b) contacting a gRNA that directs the fusion protein of (a) to a target nucleotide sequence on the DNA strand; the DNA molecule then contacts the fusion protein and gRNA in an amount effective for deamination of the nucleotide base and under suitable conditions. In some embodiments, the target DNA sequence contains a sequence related to a disease or disorder, where the nucleotide base in question is deaminated to become a sequence not related to the disease or disorder. In some embodiments, the target DNA sequence is present in one allele of a crop plant, and this allele is special for the trait of interest, resulting in a plant with lower agricultural value. Deamination of the nucleotide base in question results in an allele that improves that trait, increasing the agricultural value of the plant.

[0156] In some embodiments, the DNA sequence contains a T→C or A→G point mutation related to a disease or disorder, and deamination of the mutated C or G base results in a sequence that is not related to the disease or disorder. In some embodiments, deamination corrects the point mutations within the sequence that are related to the disease or disorder.

[0157] In some embodiments, the disease-or-disorder-related sequence codes for a protein in which a stop codon is introduced into the disease-or-disorder-related sequence by deamination, resulting in truncation of the encoded protein. In some embodiments, the contact operation is performed in vivo in subjects likely to have the disease or disorder, subjects with the disease or disorder, or subjects diagnosed with the disease or disorder. In some embodiments, the disease or disorder is a disease associated with a point mutation or single nucleotide mutation in the genome. In some embodiments, the disease is a genetic disorder, cancer, metabolic disorder, or lysosomal storage disorder.

[0158] IX. Pharmaceutical Compositions and Methods of Treatment

[0159] A pharmaceutical composition is provided comprising the RGN polypeptide and its active variants and fragments thereof, as well as polynucleotides encoding them; the gRNA or polynucleotide encoding it; the system of the disclosure; or a cell containing any of the RGN polypeptide or polynucleotide encoding RGN, gRNA or polynucleotide encoding gRNA, or RGN system; and a pharmaceutically acceptable base.

[0160] A pharmaceutical composition is a composition used for the prevention, reduction, cure, or other treatment of a target condition or target disease, and comprises an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding an RGN, a gRNA, a polynucleotide encoding a gRNA, an RGN system, or a cell containing any one of these) and a pharmaceutically acceptable base.

[0161] In this specification, “pharmaceutically acceptable base” means a material that does not significantly irritate living organisms and does not impair the activity and properties of the active ingredient (i.e., RGN polypeptide, polynucleotide encoding RGN, gRNA, polynucleotide encoding gRNA, RGN system, or a cell containing any one of these). The base must be sufficiently pure and sufficiently low in toxicity to be suitable for administration to a subject under treatment. The base can be inactive or have pharmaceutically beneficial properties. In some embodiments, the pharmaceutically acceptable base comprises one or more suitable solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans or other vertebrates. In some embodiments, the pharmaceutically acceptable base is not natural. In some embodiments, the pharmaceutically acceptable base and the active ingredient are not found together in nature.

[0162] The pharmaceutical compositions used in the methods of this disclosure can be formulated with a suitable base, excipients, and other agents that provide suitable mobility, delivery, tolerance, etc. Many suitable formulations are known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st ted. 2005). Suitable formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA complexes, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. Pharmaceutical compositions for oral or parenteral use can be prepared into unit dose dosage forms suitable for one dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0163] In some embodiments in which cells containing RGNs, gRNAs, RGN systems, or polynucleotides encoding them, or cells modified therein, are administered to a subject, the cells are administered as a suspension with a pharmaceutically acceptable base. Those skilled in the art will recognize that the pharmaceutically acceptable base used in the cell composition does not contain buffers, compounds, cryopreservants, preservatives, or other agents in amounts that substantially interfere with the viability of the cells delivered to the subject. The cell-containing formulation may include, for example, an osmotic buffer that allows for the maintenance of cell membrane integrity and may optionally contain nutrients to maintain cell viability or enhance colonization after administration. Such formulations and suspensions can be adapted for use with the cells described herein using experiments known to those skilled in the art and / or standardized experiments.

[0164] The cell composition may be provided as an emulsified or liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients in amounts suitable for use in the therapeutics described herein.

[0165] Other agents contained in the cell composition may include pharmaceutically acceptable salts of its components. Pharmaceutically acceptable salts include acid-added salts (formed with the amino group of the polypeptide) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, tartaric acid, mandelic acid). Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine).

[0166] Physiologically acceptable and pharmaceutically acceptable bases are well known in this field. Typical liquid bases are sterile aqueous solutions containing no materials other than the active ingredient and water, or contain a buffer (sodium phosphate, physiological saline, or both at a physiological pH value, e.g., phosphate-buffered physiological saline). Furthermore, aqueous bases may contain two or more buffer salts, as well as salts (e.g., sodium chloride and potassium chloride), dextrose, polyethylene glycol, and other solutes. Liquid compositions may contain a liquid phase in addition to water, or a liquid phase excluding water. Examples of such additional liquid phases are glycerin, vegetable oils (e.g., cottonseed oil), and water-oil emulsions. The amount of active ingredient used in cell compositions to be effective in treating a particular disorder or condition may depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0167] The RGN polypeptides, guide RNAs, RGN systems, or polynucleotides encoding them herein can be formulated with pharmaceutically acceptable excipients (such as bases, solvents, stabilizers, adjuvants, and diluents) depending on the specific mode of administration and dosage form. In some embodiments, these pharmaceutical compositions achieve a physiologically suitable pH through formulation according to the formulation and route of administration, in the range of about 3 to about 11 or about 3 to about 7. In some embodiments, the pH can be adjusted to a range of about 5.0 to about 8. In some embodiments, the composition may contain at least one compound described herein in a therapeutically effective amount, together with one or more pharmaceutically acceptable excipients. In some embodiments, the composition may contain a combination of compounds described herein, a second active ingredient that is useful in treating or preventing bacterial growth (e.g., an antimicrobial agent or antimicrobial agent, in non-limiting examples), or a combination of reagents herein.

[0168] Suitable excipients include base molecules containing large, slowly metabolized macromolecules (such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles). Other typical excipients may include antioxidants (e.g., ascorbic acid as an unspecified example), chelating agents (e.g., EDTA as an unspecified example), carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose as unspecified examples), stearic acid, liquids (e.g., oils, water, saline, glycerol, and ethanol as unspecified examples), wetting agents or emulsifiers, and pH buffering agents.

[0169] In some embodiments, the formulations are provided in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in a lyophilized (freeze-dried) state (a sterile liquid base, e.g., saline, water for injection, semi-liquid foam, or gel, must be added immediately before use). Instantaneous injectable solutions and suspensions can be prepared from the types of sterile powders, granules, and tablets previously described. In some embodiments, the active ingredient is dissolved in a buffered solution and frozen in single-dose or multi-dose containers, and subsequently thawed for injection or kept / stabilized under refrigeration until use.

[0170] Therapeutic agents can be incorporated into controlled-release systems. To prolong the effects of a drug, it is often desirable to delay its absorption from subcutaneous, intrathecal, or intramuscular injections. This can be achieved by using suspensions of crystalline or amorphous materials with low water solubility. The absorption rate of the drug then depends on its dissolution rate, which may depend on the size and morphology of the crystals. Alternatively, delayed absorption of parenterally administered forms of drugs can be achieved by dissolving or suspending the drug in an oil carrier. In some embodiments, the use of long-acting sustained-release implants may be particularly suitable for the treatment of chronic conditions. Long-acting sustained-release implants are well known to those skilled in the art.

[0171] Methods for treating diseases of subjects requiring treatment are provided herein. These methods involve administering to subjects requiring treatment in an effective amount of the RGN polypeptide or its active variant or fragment thereof, or polynucleotides encoding them, the gRNA or polynucleotides encoding it, the RGN system of the Disclosure, cells modified with any one of these compositions, or cells containing any one of these compositions.

[0172] In some embodiments, the treatment includes in vivo gene editing by administering RGN polypeptides, gRNAs, or RGN systems of the Disclosure, or polynucleotides encoding them. In some embodiments, the treatment includes in vitro gene editing in which cells are genetically modified in vitro using RGN polypeptides, gRNAs, or RGN systems of the Disclosure, or polynucleotides encoding them, and then the modified cells are administered to a subject. In some embodiments, the genetically modified cells are derived from a subject, and the modified cells are then administered to that subject. These transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells are derived from a different subject (i.e., a donor) of the same species as the subject (i.e., recipient) to which these modified cells are administered, and the transplanted cells are referred to herein as allogeneic. In some examples described herein, the cells may be grown in a culture medium and then administered to a subject that requires them.

[0173] In some embodiments, the diseases treated with the compositions of this disclosure are diseases that can be treated with immunotherapy (such as chimeric antigen receptor (CAR) T cells). Non-limiting examples of such diseases include cancer. In some embodiments, the diseases treated with the compositions of this disclosure are related to causative mutations. Herein, “causative mutation” means a specific nucleotide, a group of nucleotides, or a nucleotide sequence in the genome that contributes to the severity or presence of a disease or disorder in a subject. Correction of the causative mutation leads to improvement of at least one symptom resulting from the disease or disorder. In some embodiments, the causative mutation is adjacent to a PAM site recognized by the RGN disclosed herein. The causative mutation can be corrected using the RGN of this disclosure, or a fusion polypeptide containing the RGN of this disclosure, and a base-editing polypeptide (i.e., a base editor). Non-limiting examples of diseases related to causative mutations include cystic fibrosis, Hurler syndrome, Friedreich’s ataxia, Huntington’s disease, and sickle cell anemia. In some embodiments, the diseases treated with the RGN of this disclosure are those listed in Table 11. Additional, non-exclusive examples of disease-related genes and mutations are available on the World Wide Web from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Maryland) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Maryland).

[0174] In this specification, “treatment” or “to treat,” or “alleviate” or “improve,” are used interchangeably. These terms mean an approach to obtain a beneficial or desired outcome (including, in non-limiting examples, therapeutic benefits and / or preventive benefits). Therapeutic benefits mean any therapeutically significant improvement in one or more diseases, conditions, or symptoms being treated, or any therapeutically significant effect on one or more diseases, conditions, or symptoms being treated. To obtain preventive benefits, a composition may be administered to a subject at risk of progression of a particular disease, condition, or symptom, or to a subject who has reported one or more physiological symptoms of a disease, condition, or symptom, even if that disease, condition, or symptom has not yet manifested.

[0175] The expression "effective dose" or "therapeutic dose" means the amount of drug sufficient to obtain a beneficial or desired outcome. The therapeutic dose may vary depending on one or more of the following: the subject and the disease condition being treated, the subject's weight and age, the severity of the disease condition, and the mode of administration, and can be easily determined by a person skilled in the art. The specific dose may vary depending on one or more of the specific drug selected, the administration plan followed, whether it is administered in combination with other compounds, the timing of administration, and the delivery system carrying the drug.

[0176] The term "administration" means placing an active ingredient within a subject by a certain method or route, resulting in at least partial localization of the introduced active ingredient to a desired site (such as a site of injury or repair) and producing the desired effect. In embodiments in which cells are administered, cells can be administered via any appropriate route to deliver them to a desired location within the subject, so that at least some of the transplanted cells or components of these cells remain viable at that location. The survival period of cells after administration to a subject can range from a short period of a few hours (e.g., 24 hours) to several days, several years, or even the patient's lifetime, i.e., long-term engraftment. For example, in some aspects described herein, an effective dose of photoreceptor cells or retinal progenitor cells is administered via a systemic administration route (such as an intraperitoneal or intravenous route).

[0177] In some embodiments, administration includes administration by viral delivery. In some embodiments, administration includes administration by electroporation. In some embodiments, administration includes administration by nanoparticle delivery. In some embodiments, administration includes administration by liposome delivery. An effective amount of the pharmaceutical composition described herein can be administered using any effective route of administration. In some embodiments, administration includes administration by intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, ear, nasal, and topical administration, or by a method selected from the group consisting of any combination thereof. In some embodiments, administration by injection or infusion is utilized to deliver cells.

[0178] In this specification, the term “subject” means any individual for which diagnosis, treatment, or therapy is desired. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0179] The effectiveness of treatment can be determined by a skilled clinician. However, a treatment is considered "effective" if any one or all of the signs or symptoms of the disease or disorder change in a beneficial way (e.g., a reduction of at least 10%), or if other clinically accepted symptoms or markers of the disease improve or improve. Effectiveness can also be measured by not showing a worsening that would be assessed by the need for hospitalization or medical intervention (e.g., the progression of the disease stops or at least slows). Methods for measuring these indicators are known to those skilled in the art. Treatment includes (1) suppression of the disease (e.g., cessation or delay of symptom progression); or (2) alleviation of the disease (e.g., induction of symptom regression); and (3) prevention or reduction of the likelihood of symptom progression.

[0180] A. Modify the causative mutation using base editing.

[0181] One example of a genetic disorder that can be corrected using the RGN-base editor fusion protein approach of the present invention is Hurler syndrome. Hurler syndrome (also known as MPS-1) is a lysosomal storage disorder resulting from a deficiency of α-L-iduronidase (IDUA), characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes. This disorder is generally a hereditary genetic disorder caused by mutations in the IDUA gene that encodes α-L-iduronidase. Common IDUA mutations are W402X and Q70X, both of which are nonsense mutations that cause translation to stop midway. Such mutations can be successfully addressed by precise genome editing (PGE) approaches because, for example, by reversing a single nucleotide using a base editing approach, the wild-type coding sequence is restored, and protein expression is regulated by the endogenous regulatory mechanisms of the gene locus. In addition, since heterozygotes are known to be asymptomatic, PGE therapy targeting one of these mutations is considered useful for many patients with this disease because only one of the mutated alleles needs to be corrected (Bunge et al. (1994) Hum. Mol. Genet. 3(6): 861-866; incorporated herein by reference).

[0182] Current treatments for Hurler syndrome include enzyme replacement therapy and bone marrow transplantation (Vellodi et al. (1997) Arch. Dis. Child. 76(2): 92-99; Peters et al. (1998) Blood 91(7): 2601-2608; incorporated herein by reference). Enzyme replacement therapy has had a dramatic effect on the survival and quality of life of patients with Hurler syndrome, but this approach requires costly and time-consuming weekly fluid infusions. Additional approaches include delivery of the IDUA gene via an expression vector, or insertion of this gene into a highly expressed locus (such as the serum albumin locus) (United States Patent No. 9,956,247; incorporated herein by reference). However, these approaches do not restore the original IDUA locus to the correct coding sequence. Genome editing strategies are thought to have numerous advantages, most notably that gene expression regulation is believed to be controlled by natural mechanisms present in healthy individuals. In addition, using base editing eliminates the need to cause double-strand DNA breaks that could lead to cancer development through large-scale chromosomal rearrangements, cell death, or disruption of tumor suppressor mechanisms. One common strategy is to use the RGN-base editor fusion proteins of the present invention to target and correct specific disease-causing mutations in the human genome. It will be further found that similar approaches targeting disease-causing mutations in other species (particularly common pets or livestock) can be developed using the RGNs of the present invention. Common pets and livestock include dogs, cats, horses, pigs, cattle, sheep, chickens, donkeys, snakes, ferrets, and fish (including salmon and shrimp).

[0183] B. Modify the causative mutation through targeted deletion.

[0184] The RGN of the present invention may also be useful in therapies for human conditions with more complex causative mutations. For example, several diseases, such as Friedreich's ataxia and Huntington's disease, result from a significant increase in the number of 3-nucleotide motif repeats in specific regions of genes, which affects the ability of expressed proteins to function or to be expressed. Friedreich's ataxia (FRDA) is an autosomal recessive disorder in which nerve tissue in the spine gradually degenerates. Reduced levels of frataxin (FXN) protein in mitochondria cause oxidative damage and iron deficiency at the cellular level. Reduced FXN expression has been linked to GAA triplet elongation in intron 1 of the FXN gene in somatic cells and germline cells. In FRDA patients, GAA repeats often consist of more than 70 triplets, and sometimes even more than 1000 triplets (most commonly 600-900), compared to approximately 40 repeats or fewer in individuals without the disease (Pandolfo et al. (2012) Handbook of Clinical Neurology 103: 275-294; Campuzano et al. (1996) Science 271: 1423-1427; Pandolfo (2002) Adv. Exp. Med. Biol. 516: 99-118; all of these are incorporated herein by reference).

[0185] The elongation of a 3-nucleotide repeat sequence that causes Friedreich's ataxia (FRDA) occurs within a specific locus in the FXN gene, known as the FRDA instability region. RNA-induced nucleases (RGNs) can be used to excise this instability region within the cells of FRDA patients. This approach requires 1) a sequence of RGN and guide RNA that can be programmed to target an allele in the human genome, and 2) a method for delivering this RGN and guide sequence. Many nucleases used in genome editing (such as the commonly used Cas9 nuclease from Streptococcus pyogenes (SpCas9)) are too large to be packaged within adeno-associated virus (AAV) vectors. This is particularly true when considering the length of the SpCas9 gene and guide RNA, in addition to the lengths of other genetic elements required for a functional expression cassette. Therefore, approaches utilizing SpCas9 are more challenging.

[0186] A certain RNA-induced nuclease of the present invention is well-suited for packaging with guide RNA in an AAV vector. While a second vector is likely required to package two guide RNAs, this approach is still more advantageous than the vectors that would be required for larger nucleases (e.g., SpCas9, which may require splitting the protein sequence into two vectors). The present invention encompasses strategies for eliminating regions of genomic instability using the RGN of the present invention. Such strategies can be applied to other diseases and disorders with similar genetic basis (e.g., Huntington's disease). Similar strategies using the RGN of the present invention can also be applied to diseases and disorders in agriculturally or economically important non-human animals. Such non-human animals include dogs, cats, horses, pigs, cattle, sheep, chickens, donkeys, snakes, ferrets, and fish (including salmon and shrimp).

[0187] C. Modify the causative mutation through targeted mutagenesis.

[0188] The RGN of the present invention may also introduce disruptive mutations that could yield beneficial effects. Genetic defects in genes encoding hemoglobin, particularly the beta-globin chain (HBB gene), may be the cause of numerous diseases known as hemoglobin disorders (including sickle cell anemia and thalassemia).

[0189] In adults, hemoglobin is a heterotetramer containing two alpha (α)-like globin chains, two beta (β)-like globin chains, and four heme groups. In adults, this α2β2 tetramer is called hemoglobin A (HbA), or adult hemoglobin. Typically, alpha globin chains and beta globin chains are synthesized in a ratio of approximately 1:1, and this ratio appears to be crucial for the stability of hemoglobin and red blood cells (RBCs). In developing fetuses, a different form of hemoglobin (fetal hemoglobin (HbF)) is produced that has a greater affinity for oxygen than hemoglobin A, allowing oxygen to be delivered to the fetal system through the maternal bloodstream. Fetal hemoglobin also contains two α-globin chains, but instead of adult β-globin chains, it has two fetal gamma (γ)-globin chains (i.e., fetal hemoglobin is α2γ2). The regulation of the switch from gamma-globin production to beta-globin production is extremely complex, mainly involving the downregulation of gamma-globin transcription and the simultaneous upregulation of beta-globin transcription. Around 30 weeks of gestation, gamma-globin synthesis in the fetus begins to decrease, while beta-globin production increases. Neonatal hemoglobin is almost entirely α2β2 until around 10 months of age, but some HbF remains into adulthood (about 1-3% of total hemoglobin). As explained above, the majority of patients with hemoglobin disorders still have the gene encoding gamma-globin, but its expression is relatively low due to the suppression of normal genes that occurs around the time of delivery, as described above.

[0190] Sickle cell anemia is caused by a V6E mutation in the β-globin gene (HBB) (from GAG to GTG at the DNA level), and the resulting hemoglobin is called "hemoglobin S" or "HbS." Under conditions of lower oxygen levels, HbS molecules aggregate to form fibrous precipitates. These aggregates cause abnormalities in red blood cell clusters (RBCs), or "sickling," resulting in a loss of cellular flexibility. Because these sickle RBCs can no longer enter the capillary bed, patients with sickle cell anemia may face a risk of vascular occlusion. In addition, sickle RBCs are more fragile and prone to hemolysis than normal RBCs, ultimately leading to anemia in patients.

[0191] The treatment and management of sickle cell disease patients is a lifelong challenge, involving antibiotic therapy, pain management, and acute blood transfusions. One approach involves the use of hydroxyureas, which exert some of their effect by increasing gammaglobin production. While the long-term side effects of chronic hydroxyurea therapy are not yet known, the effectiveness may vary from patient to patient due to the possibility of unwanted side effects. Despite the increasing effectiveness of sickle cell therapy, the average life expectancy of patients is still only in their mid-to-late 50s, and the pathological conditions associated with this disease have a significant impact on their quality of life.

[0192] Thalassemia (alpha-thalassemia and beta-thalassemia) is also a hemoglobin-related disorder, typically involving reduced globin chain expression. This can occur through mutations in the regulatory regions of genes or through mutations in the globin coding sequence, resulting in reduced expression or levels of functional globin protein. Treatment for thalassemia usually includes blood transfusions and iron chelation therapy. Bone marrow transplantation is also used to treat severely ill patients when a suitable donor can be identified, although this method can carry significant risks.

[0193] One approach proposed to treat both SCD and beta-thalassemia is to increase gamma-globin expression and functionally replace abnormal adult hemoglobin A with HbF. As mentioned above, treatment of SCD patients with hydroxyurea is considered successful, partly because hydroxyurea is effective in increasing gamma-globin expression (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31; Ley, et al., (1982) N. Engl. J. Medicine, 307: 1469-1475; Ley, et al., (1983) Blood 62: 370-380; Constantoulakis et al., (1988) Blood 72(6):1961-1967; all of these are incorporated herein by reference). Increased HbF expression is associated with the identification of genes that produce products that play a role in regulating gammaglobin expression. One such gene is BCL11A. BCL11A encodes a zinc finger protein expressed in adult erythrocyte progenitor cells, and downregulation of its expression leads to increased gammaglobin expression (Sankaran et al. at (2008) Science 322: 1839; incorporated herein by reference). The use of repressive RNA targeting the BCL11A gene has been proposed (e.g., U.S. Patent Application Publication 2011 / 0182867; incorporated herein by reference), but this technique has several potential drawbacks. These include the possibility of not achieving complete knockdown, potential problems with the delivery of such RNA, and the need for multiple lifetime treatments because the RNA must be continuously present.

[0194] Gammaglobin expression can be increased by targeting the BCL11A enhancer region and interfering with BCL11A expression using the RGN of the present invention. This targeted interference can be achieved by non-homologous end joining (NHEJ). Through NHEJ, the RGN of the present invention cleaves a double-stranded molecule targeting a specific sequence within the BCL11A enhancer region, and the cellular mechanism repairs the cleavage, typically simultaneously introducing a harmful mutation. As described for other disease targets, the RGN of the present invention has advantages over other known RGNs because its relatively small size allows the expression cassette for the RGN and its corresponding guide RNA to be packaged within a single AAV vector for in vivo delivery. Similar strategies using the RGN of the present invention can be applied to similar diseases and disorders in both humans and agriculturally or economically important non-human animals.

[0195] Cells containing X polynucleotide gene modifications

[0196] This specification provides cells and organisms containing target sequences of interest modified using methods mediated by RGNs, crRNAs, and / or tracrRNAs as described herein. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, or 570–579, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence containing the nucleotide sequence of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, or 124, or an active variant or fragment thereof. In a particular embodiment, the guide RNA includes a tracrRNA containing the nucleotide sequence of SEQ ID NOs: 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, or an active variant or fragment thereof. The guide RNA in the system can be a single guide RNA or a double guide RNA.

[0197] Modified cells can be eukaryotic cells (e.g., mammalian cells, plant cells, insect cells) or prokaryotic cells. Organelles and embryos containing at least one nucleotide sequence modified by the methods using RGN, crRNA, and / or tracrRNA described herein are also provided. Genetically modified cells, organisms, organelles, and embryos can be heterozygous or homozygous for the modified nucleotide sequence.

[0198] Modification of chromosomes in cells, organisms, organelles, or embryos can result in altered (upward or downward) expression, inactivation, or expression of altered protein products or incorporated sequences. When chromosomal modification results in gene inactivation or expression of non-functional protein products, the genetically modified cells, organisms, organelles, or embryos are called “knockout.” Knockout phenotypes can result from deletion mutations (i.e., deletion of at least one nucleotide), insertion mutations (i.e., insertion of at least one nucleotide), or nonsense mutations (i.e., substitution of at least one nucleotide and introduction of a stop codon).

[0199] Alternatively, "knock-ins" may be generated as a result of modifications to the chromosomes of cells, organisms, organelles, or embryos. This is a result of the integration of a nucleotide sequence encoding a certain protein into the chromosome. In some of these embodiments, the integration of the coding sequence into the chromosome inactivates the chromosomal sequence encoding the wild-type protein, while the externally introduced protein is expressed.

[0200] In another embodiment, a variant protein product is generated as a result of chromosome modification. The expressed variant protein product may have at least one amino acid substitution and / or at least one amino acid addition or deletion. The variant protein product encoded by the altered chromosomal sequence may exhibit modified characteristics or activities compared to the wild-type protein (non-limiting examples include altered enzyme activity or substrate specificity).

[0201] In yet another embodiment, alterations to chromosomes may result in changes to the protein expression pattern. As a non-limiting example, alterations to chromosomes within regulatory regions that control the expression of a protein product may result in overexpression or downregulation of that protein product, or altered tissue expression patterns or altered temporal expression patterns.

[0202] Modified cells can be propagated according to conventional methods to form a single organism (such as a plant). See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be propagated and pollinated with the same modified strain or a different strain to obtain a hybrid with genetic modification. The present invention provides genetically modified seeds. Offspring, variants, and mutants of the regenerated plants are also within the scope of the present invention, provided that these parts include genetic modification. Furthermore, processed plant products or by-products (e.g., soybean meal) that retain genetic modification are provided.

[0203] The methods presented herein can be used to modify any plant species (including, but not limited to, monocots and dicots). Examples of plants of interest include corn, maize, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and rapeseed, Brassica species, alfalfa, rye, grains, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus fruits, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew nut, macadamia nut, almond, oat, vegetables, ornamental plants, and conifers.

[0204] Non-limiting examples of vegetables include tomatoes, lettuce, green peas, lima beans, peas, and members of the genus Cucumis (such as cucumbers, cantaloupes, and muskmelons). Non-limiting examples of ornamental plants include azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. The plants of the present invention are preferably cultivated crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, pepper, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0205] The methods presented herein can also be used to transform any prokaryotic species. Non-exclusive examples of prokaryotic species include archaea and bacteria (e.g., species of Bacillus, Klebsiella, Streptomyces, Rhizobium, Escherichia coli, Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycoplasma, Agrobacterium, Lactobacillus).

[0206] Any eukaryotic species or cells derived therefrom can be genetically modified using the methods presented herein. Non-limiting examples of eukaryotic species include animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebas, algae, and yeasts. In some embodiments, the cells modified by the methods of the present disclosure include hematopoietic cells, such as immune cells (i.e., cells of the innate or adaptive immune system), non-limiting examples of which include B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

[0207] Modified cells can be introduced into living organisms. These cells are modified using in vitro approaches, and in the case of autologous cell transplantation, it is thought that they could be derived from the same organism (e.g., a human). Alternatively, in the case of allogeneic cell transplantation, the cells could be derived from a different organism of the same species (e.g., another human).

[0208] The articles "one" and "one" are used in this specification to refer to one or more (i.e., at least one) objects that are grammatically relevant. For example, "one polypeptide" means one or more polypeptides.

[0209] All publications and patent applications referenced herein represent the level of skill of those skilled in the art relating to this disclosure. All publications and patent applications are incorporated herein by reference in such a manner as it is indicated that individual publications or patent applications are incorporated by reference specifically and individually.

[0210] While the above invention has been described in some detail with explanations and examples for the purpose of clear understanding, it will be clear that several changes and modifications can be made within the scope of the attached embodiments.

[0211] Non-limiting embodiments include the following:

[0212] 1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-induced nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide having an amino acid sequence that is at least 90% identical to one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; When the RGN polypeptide binds to a guide RNA (gRNA) that can hybridize to a target DNA sequence, it can bind to the target DNA sequence in a manner specific to the RNA-inducible sequence. The polynucleotide encoding the RGN polypeptide is a nucleic acid molecule in which a heterogeneous promoter is functionally linked to this polynucleotide.

[0213] 2. The nucleic acid molecule of Embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is at least 95% identical to one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0214] 3. The nucleic acid molecule of Embodiment 1, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0215] 4. The nucleic acid molecule of Embodiment 1, wherein the RGN polypeptide has a sequence that is at least 90% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405 of SEQ ID NO: 63.

[0216] 5. Any one nucleic acid molecule from Embodiments 1 to 4, wherein the RGN polypeptide can cleave the target DNA sequence upon binding.

[0217] 6. The nucleic acid molecule of Embodiment 5, wherein the RGN polypeptide can cause double-strand breaks.

[0218] 7. The nucleic acid molecule of Embodiment 5, wherein the RGN polypeptide can undergo single-strand breaks.

[0219] 8. Any one of Embodiments 1 to 4, wherein the RGN polypeptide is either nuclease-inactive or nicasse.

[0220] 9. A nucleic acid molecule according to any one of embodiments 1 to 8, wherein the RGN polypeptide is functionally fused to a base-editing polypeptide.

[0221] 10. The nucleic acid molecule of Embodiment 9, wherein the base-editing polypeptide is a deaminase.

[0222] 11. A nucleic acid molecule of Embodiment 10, wherein the deaminase is cytidine deaminase or adenine deaminase.

[0223] 12. A nucleic acid molecule according to any one of Embodiments 1 to 11, wherein the RGN polypeptide contains one or more nuclear localization signals.

[0224] 13. A nucleic acid molecule according to any one of Embodiments 1 to 12, wherein the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0225] 14. A nucleic acid molecule according to any one of Embodiments 1 to 13, wherein the target DNA sequence is located adjacent to a protospacer adjacency motif (PAM).

[0226] 15. A vector containing one nucleic acid molecule from any one of embodiments 1 to 14.

[0227] 16. The vector of Embodiment 15, further comprising at least one nucleotide sequence encoding the gRNA that can be hybridized to the target DNA sequence.

[0228] 17. The selection of the guide RNA is a)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 2; ii) A tracrRN having a sequence that is at least 90% identical to sequence number 3. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% agreement with SEQ ID NO: 1); b)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 9; ii) tracrRN having a sequence that is at least 90% identical to sequence number 10 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% agreement with SEQ ID NO: 8); c)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 16; ii) tracrRN with a sequence that is at least 90% identical to sequence number 17 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 15); d)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 23; ii) tracrRN with a sequence that is at least 90% identical to sequence number 24 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% matching sequence number 22); e)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 30; ii) A tracrRN having a sequence that is at least 90% identical to sequence number 31. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% agreement with SEQ ID NO: 29); f)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 37; ii) A tracrRN having a sequence that is at least 90% identical to sequence number 38. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 36); g)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 44; ii) tracrRN with a sequence that is at least 90% identical to sequence number 45 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% matching sequence number 43); h)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 51; ii) tracrRN with a sequence that is at least 90% identical to sequence number 52 A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 50); i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 57; ii) A tracrRN sequence that is at least 90% identical to sequence number 58. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 56); j)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 90% identical to sequence number 64; ii) tracrRN with a sequence that is at least 90% identical to sequence number 65 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to one of sequence numbers 63 and 570-579); k)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% identical to sequence number 71; ii) tracrRN having a sequence that is at least 90% identical to sequence number 72 A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 70); l)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% identical to sequence number 77; ii) A tracrRN sequence that is at least 90% identical to sequence number 78. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 76); m)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 84; ii) A tracrRN sequence that is at least 90% identical to sequence number 85. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 83); n)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 90; ii) tracrRN having a sequence that is at least 90% identical to sequence number 91 A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 89); o)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 97; ii) A tracrRN with a sequence that is at least 90% identical to sequence number 98. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 96); p)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% matching sequence number 104; ii) tracrRN with a sequence that is at least 90% identical to sequence number 105 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% agreement with sequence number 103); q)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 111; ii) A tracrRN with a sequence that is at least 90% identical to sequence number 112. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 110); r)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 118; ii) tracrRN with a sequence that is at least 90% identical to sequence number 119 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% matching sequence number 117); s)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 124; ii) A tracrRN with a sequence that is at least 90% identical to sequence number 125. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 123); t)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 90% agreement with sequence number 84; ii) A tracrRN sequence that is at least 90% identical to sequence number 78. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 90% matching sequence number 83) A vector of embodiment 16, consisting of a group of the following.

[0229] 18. The selection of the guide RNA is a)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 2; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 3. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 1); b)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 9; ii) A tracrRN sequence that is at least 95% identical to sequence number 10. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 8); c)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 16; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 17. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 15); d)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 23; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 24. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 95% agreement with SEQ ID NO: 22); e)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% matching sequence number 30; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 31. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 29); f)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 37; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 38. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 36); g)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 44; ii) A tracrRN sequence that is at least 95% identical to sequence number 45. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 43); h)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 51; ii) tracrRN with a sequence that is at least 95% identical to sequence number 52 A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 50); i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 57; ii) A tracrRN sequence that is at least 95% identical to sequence number 58. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 56); j)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 64; ii) A tracrRN sequence that is at least 95% identical to sequence number 65. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to one of sequence numbers 63 and 570-579); k)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 71; ii) A tracrRN sequence that is at least 95% identical to sequence number 72. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 70); l)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 77; ii) A tracrRN sequence that is at least 95% identical to sequence number 78. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 76); m)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 84; ii) A tracrRN sequence that is at least 95% identical to sequence number 85. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 83); n)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 95% matching sequence number 95; ii) A tracrRN having a sequence that is at least 95% identical to sequence number 91. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 89); o)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 97; ii) A tracrRN sequence that is at least 95% identical to sequence number 98. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 96); p)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% identical to sequence number 104; ii) A tracrRN with a sequence that is at least 95% identical to sequence number 105. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 103); q)i) A CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 111; ii) A tracrRN with a sequence that is at least 95% identical to sequence number 112. A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 110); r)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 118; ii) tracrRN with a sequence that is at least 95% identical to sequence number 119 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 117); s)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 124; ii) tracrRN with a sequence that is at least 95% identical to sequence number 125 A guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 123); t)i) CRISPR RNA containing a CRISPR repeat sequence that has at least 95% agreement with sequence number 84; ii) A tracrRN sequence that is at least 95% identical to sequence number 78. Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that has at least 95% agreement with sequence number 83) A vector of embodiment 16, consisting of a group of the following.

[0230] 19. The selection of the guide RNA is a)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 2; ii) tracrRN with a sequence that is 100% identical to sequence number 3 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 1); b)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 9; ii) tracrRN with a sequence that is 100% identical to sequence number 10 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 8); c)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 16; ii) tracrRN with a sequence that is 100% identical to sequence number 17 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 15); d)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 23; ii) tracrRN with a sequence that is 100% identical to sequence number 24 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 22); e)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 30; ii) tracrRN with a sequence that is 100% identical to sequence number 31 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 29); f)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 37; ii) tracrRN with a sequence that is 100% identical to sequence number 38 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 36); g)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 44; ii) tracrRN with a sequence that is 100% identical to sequence number 45 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 43); h)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 51; ii) tracrRN with a sequence that is 100% identical to sequence number 52 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 50); i) A CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 57; ii) tracrRN with a sequence that is 100% identical to sequence number 58 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 56); j)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 64; ii) tracrRN with a sequence that is 100% identical to sequence number 65 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to one of sequence numbers 63 and 570-579); k)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence 100% identical to sequence number 71; ii) tracrRN with a sequence that is 100% identical to sequence number 72 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 70); l)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence 100% identical to sequence number 77; ii) tracrRN with a sequence that is 100% identical to sequence number 78 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 76); m)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 84; ii) tracrRN with a sequence that is 100% identical to sequence number 85 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 83); n)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 95; ii) tracrRN with a sequence that is 100% identical to sequence number 91 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 89); o)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence that is 100% identical to sequence number 97; ii) tracrRN with a sequence that is 100% identical to sequence number 98 Guide RNA containing (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 96); p)i) CRISPR RNA containing a CRISPR repeat sequence that has a sequence 100% identical to sequence number 104; ii) tracrRN with a sequence that is 100% identical to sequence number 105 a guide RNA (wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 103); q) i) a CRISPR RNA comprising a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 111; ii) a tracrRN having a sequence that is 100% identical to SEQ ID NO: 112 a guide RNA (wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 110); r) i) a CRISPR RNA comprising a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 118; ii) a tracrRN having a sequence that is 100% identical to SEQ ID NO: 119 a guide RNA (wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 117); s) i) a CRISPR RNA comprising a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 124; ii) a tracrRN having a sequence that is 100% identical to SEQ ID NO: 125 a guide RNA (wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 123); t) i) a CRISPR RNA comprising a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 84; ii) a tracrRN having a sequence that is 100% identical to SEQ ID NO: 78 a guide RNA (wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 83) The vector of embodiment 16, which is selected from the group consisting of.

[0231] 20. The vector of any one of embodiments 16 to 19, wherein the gRNA is a single guide RNA.

[0232] 21. The vector of any one of embodiments 16 to 19, wherein the gRNA is a dual guide RNA.

[0233] 22. A cell containing any one nucleic acid molecule from Embodiments 1 to 14, or any one vector from Embodiments 15 to 21.

[0234] 23. A method for producing an RGN polypeptide, comprising culturing the cells of Embodiment 22 under conditions in which the RGN polypeptide is expressed.

[0235] 24. A method for producing an RGN polypeptide, comprising introducing a heterogeneous nucleic acid molecule into a cell, the nucleotide sequence encoding an RNA-inducible nuclease (RGN) polypeptide containing an amino acid sequence having at least 90% agreement with one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579 (provided that the RGN polypeptide, when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence, can bind to the target DNA sequence in a manner specific to the RNA-inducible sequence); A method comprising culturing the cells under conditions in which the RGN polypeptide is expressed.

[0236] 25. The method of Embodiment 24, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0237] 26. The method of Embodiment 24, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0238] 27. The method of Embodiment 24, wherein the RGN polypeptide has a sequence that is at least 90% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

[0239] 28. Any one of embodiments 23 to 27, further comprising purifying the RGN polypeptide.

[0240] 29. Any one of embodiments 23 to 27, wherein the cells further express one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex.

[0241] 30. The method of Embodiment 29, further comprising purifying the RGN ribonucleoprotein complex.

[0242] 31. Contains an amino acid sequence that is at least 90% identical to one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; An isolated RNA-inducible nuclease (RGN) polypeptide that, when bound to a guide RNA (gRNA) capable of hybridizing to a target DNA sequence, can bind to the target DNA sequence in a manner specific to the RNA-inducible sequence.

[0243] 32. An isolated RGN polypeptide of Embodiment 31, comprising an amino acid sequence having a sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0244] 33. An isolated RGN polypeptide of Embodiment 31, comprising an amino acid sequence having a sequence that is 100% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579.

[0245] 34. An isolated RGN polypeptide of Embodiment 31 having a sequence that is at least 90% identical to SEQ ID NO: 63, and having isoleucine at the amino acid position corresponding to 305, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405 of SEQ ID NO: 63.

[0246] 35. An isolated RGN polypeptide from any one of embodiments 31 to 34, which can cleave the target DNA sequence upon binding.

[0247] 36. An isolated RGN polypeptide of Embodiment 35, wherein cleavage by the RGN polypeptide results in a double-strand break.

[0248] 37. An isolated RGN polypeptide of Embodiment 35, wherein cleavage by the RGN polypeptide results in a single-strand break.

[0249] 38. An isolated RGN polypeptide from any one of embodiments 31-34, which is either nuclease-inactive or nicasse.

[0250] 39. An isolated RGN polypeptide from any one of embodiments 31-38, functionally fused to a base-editing polypeptide.

[0251] 40. An isolated RGN polypeptide of Embodiment 39, wherein the base-editing polypeptide is a deaminase.

[0252] 41. The isolated RGN polypeptide of any one of embodiments 31 to 40, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0253] 42. The isolated RGN polypeptide of any one of embodiments 31 to 41, comprising one or more nuclear localization signals.

[0254] 43. A nucleic acid molecule comprising a polynucleotide encoding a CRISPR RNA (crRNA), wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124, a) said crRNA; b) a trans-activating CRISPR RNA (tracrRNA) hybridized to said CRISPR repeat sequence of said crRNA wherein when a guide RNA comprising the same binds to an RNA-guided nuclease (RGN) polypeptide, it is capable of hybridizing to a target DNA sequence in a sequence-specific manner through the spacer sequence of said crRNA, wherein said polynucleotide encoding the crRNA is operably linked to a promoter heterologous to said polynucleotide.

[0255] 44. The nucleic acid molecule of embodiment 43, wherein said CRISPR repeat sequence comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124.

[0256] 45. A nucleic acid molecule of Embodiment 43, wherein the CRISPR repeat sequence comprises a nucleotide sequence having a sequence that is 100% identical to any one of SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 57, 64, 71, 77, 84, 90, 97, 104, 111, 118, and 124.

[0257] 46. ​​A vector comprising any one nucleic acid molecule from Embodiments 43 to 45.

[0258] 47. A vector of embodiment 46, further comprising a polynucleotide encoding the tracrRNA.

[0259] 48. The selection of the tracrRNA is a) tracrRNA having a sequence that is at least 90% identical to sequence number 3 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 2); b) tracrRNA having a sequence that is at least 90% identical to sequence number 10 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 9); c) tracrRNA having a sequence that is at least 90% identical to sequence number 17 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 16); d) tracrRNA having a sequence that is at least 90% identical to sequence number 24 (in this case, the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 23); e) tracrRNA having a sequence that is at least 90% identical to sequence number 31 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 30); f) tracrRNA having a sequence that is at least 90% identical to sequence number 38 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 37); g) tracrRNA having a sequence that is at least 90% identical to sequence number 45 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 44); h) tracrRNA having a sequence that is at least 90% identical to sequence number 52 (in this case, the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 51); i) tracrRNA having a sequence that is at least 90% identical to sequence number 58 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 57); j) tracrRNA having a sequence that is at least 90% identical to sequence number 65 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 64); k) tracrRNA having a sequence that is at least 90% identical to sequence number 72 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 71); l) tracrRNA having a sequence that is at least 90% identical to sequence number 78 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 77); m) tracrRNA having a sequence that is at least 90% identical to sequence number 85 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 84); n) A tracrRNA having a sequence that is at least 90% identical to sequence number 91 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 90); o) tracrRNA having a sequence that is at least 90% identical to sequence number 98 (in this case, the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 97); p) tracrRNA having a sequence that is at least 90% identical to sequence number 105 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 104); q) tracrRNA having a sequence that is at least 90% identical to sequence number 112 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 111); r) tracrRNA having a sequence that is at least 90% identical to sequence number 119 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 118); and s) tracrRNA having a sequence that is at least 90% identical to sequence number 125 (in this case, the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 124). A vector of embodiment 47, consisting of a group comprising the following.

[0260] 49. The selection of the tracrRNA is a) tracrRNA having a sequence that is at least 95% identical to sequence number 3 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 2); b) tracrRNA having a sequence that is at least 95% identical to sequence number 10 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 9); c) tracrRNA having a sequence that is at least 95% identical to sequence number 17 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 16); d) tracrRNA having a sequence that is at least 95% identical to sequence number 24 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 23); e) tracrRNA having a sequence that is at least 95% identical to sequence number 31 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 30); f) tracrRNA having a sequence that is at least 95% identical to sequence number 38 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 37); g) tracrRNA having a sequence that is at least 95% identical to sequence number 45 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 44); h) tracrRNA having a sequence that is at least 95% identical to sequence number 52 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 51); i) tracrRNA having a sequence that is at least 95% identical to sequence number 58 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 57); j) tracrRNA having a sequence that is at least 95% identical to sequence number 65 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 64); k) tracrRNA having a sequence that is at least 95% identical to sequence number 72 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 71); l) tracrRNA having a sequence that is at least 95% identical to sequence number 78 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 77); m) tracrRNA having a sequence that is at least 95% identical to sequence number 85 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 84); n) tracrRNA having a sequence that is at least 95% identical to sequence number 91 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 90); o) tracrRNA having a sequence that is at least 95% identical to sequence number 98 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 97); p) tracrRNA having a sequence that is at least 95% identical to sequence number 105 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 104); q) A tracrRNA having a sequence that is at least 95% identical to sequence number 112 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 111); r) tracrRNA having a sequence that is at least 95% identical to sequence number 119 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 118); and s) tracrRNA having a sequence that is at least 95% identical to sequence number 125 (in this case, the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 124). A vector of embodiment 47, consisting of a group comprising the following.

[0261] 50. The selection of the tracrRNA is a) tracrRNA having a sequence that is 100% identical to sequence number 3 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 2); b) tracrRNA having a sequence that is 100% identical to sequence number 10 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 9); c) tracrRNA having a sequence that is 100% identical to sequence number 17 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 16); d) tracrRNA having a sequence that is 100% identical to sequence number 24 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 23); e) tracrRNA having a sequence that is 100% identical to sequence number 31 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 30); f) tracrRNA having a sequence that is 100% identical to sequence number 38 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 37); g) tracrRNA having a sequence that is 100% identical to sequence number 45 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 44); h) tracrRNA having a sequence that is 100% identical to sequence number 52 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 51); i) tracrRNA having a sequence that is 100% identical to sequence number 58 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 57); j) tracrRNA having a sequence that is 100% identical to sequence number 65 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 64); k) tracrRNA having a sequence that is 100% identical to sequence number 72 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 71); l) tracrRNA having a sequence that is 100% identical to sequence number 78 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 77); m) tracrRNA having a sequence that is 100% identical to sequence number 85 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 84); n) tracrRNA having a sequence that is 100% identical to sequence number 91 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 90); o) tracrRNA having a sequence that is 100% identical to sequence number 98 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 97); p) tracrRNA with a sequence that is 100% identical to sequence number 105 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 104); q) tracrRNA having a sequence that is 100% identical to sequence number 112 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 111); r) tracrRNA having a sequence that is 100% identical to sequence number 119 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 118); and s) tracrRNA with a sequence that is 100% identical to sequence number 125 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 124) A vector of embodiment 47, consisting of a group comprising the following.

[0262] 51. Any one of embodiments 47 to 50, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are functionally linked to the same promoter and encoded as a single guide RNA.

[0263] 52. Any one of embodiments 47 to 50, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are functionally linked to separate promoters.

[0264] 53. Any one of embodiments 46 to 52, further comprising a polynucleotide encoding the RGN polypeptide.

[0265] 54. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 1 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 8 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 10); c) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 15 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 17); d) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 22 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 23, and the tracrRNA has a sequence that is at least 90% identical to sequence number 24); e) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 29 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 30, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 31); f) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 36 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 38); g) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 43 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 45); h) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 50 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 51, and the tracrRNA has a sequence that is at least 90% identical to sequence number 52); i) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 56 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is at least 90% identical to one of sequence numbers 63 and 570-579 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 64, and the tracrRNA has a sequence that is at least 90% identical to sequence number 65); k) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 70 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to SEQ ID NO: 71, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 72); l) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 76 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 77, and the tracrRNA has a sequence that is at least 90% identical to sequence number 78); m) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 83 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 84, and the tracrRNA has a sequence that is at least 90% identical to sequence number 85); n) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 89 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 90, and the tracrRNA has a sequence that is at least 90% identical to sequence number 91); o) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 96 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 97, and the tracrRNA has a sequence that is at least 90% identical to sequence number 98); p) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 103 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 104, and the tracrRNA has a sequence that is at least 90% identical to sequence number 105); q) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 110 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 111, and the tracrRNA has a sequence that is at least 90% identical to sequence number 112); r) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 117 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 118, and the tracrRNA has a sequence that is at least 90% identical to sequence number 119); s) RGN polypeptide having a sequence that is at least 90% identical to sequence number 123 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 124, and the tracrRNA has a sequence that is at least 90% identical to sequence number 125); and t) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 83 (where the CRISPR repeat sequence has a sequence that is at least 90% identical to sequence number 84, and the tracrRNA has a sequence that is at least 90% identical to sequence number 78). A vector of embodiment 53, consisting of a group of the following.

[0266] 55. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 1 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 8 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 10); c) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 15 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 17); d) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 22 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 23, and the tracrRNA has a sequence that is at least 95% identical to sequence number 24); e) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 29 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 30, and the tracrRNA has a sequence that is at least 95% identical to sequence number 31); f) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 36 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 38); g) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 43 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 45); h) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 50 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 51, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 52); i) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 56 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is at least 95% identical to one of sequence numbers 63 and 570-579 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 64, and the tracrRNA has a sequence that is at least 95% identical to sequence number 65); k) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 70 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 71, and the tracrRNA has a sequence that is at least 95% identical to sequence number 72); l) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 76 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 77, and the tracrRNA has a sequence that is at least 95% identical to sequence number 78); m) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 83 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 84, and the tracrRNA has a sequence that is at least 95% identical to sequence number 85); n) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 89 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 90, and the tracrRNA has a sequence that is at least 95% identical to sequence number 91); o) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 96 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 97, and the tracrRNA has a sequence that is at least 95% identical to sequence number 98); p) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 103 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 104, and the tracrRNA has a sequence that is at least 95% identical to sequence number 105); q) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 110 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 111, and the tracrRNA has a sequence that is at least 95% identical to sequence number 112); r) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 117 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 118, and the tracrRNA has a sequence that is at least 95% identical to sequence number 119); s) RGN polypeptide having a sequence that is at least 95% identical to sequence number 123 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 124, and the tracrRNA has a sequence that is at least 95% identical to sequence number 125); and t) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 83 (where the CRISPR repeat sequence has a sequence that is at least 95% identical to sequence number 84, and the tracrRNA has a sequence that is at least 95% identical to sequence number 78). A vector of embodiment 53, consisting of a group of the following.

[0267] 56. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 1 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 8 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 10); c) An RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 15 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 17); d) An RGN polypeptide having a sequence that is 100% identical to sequence number 22 (wherein the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 23, and the tracrRNA has a sequence that is 100% identical to sequence number 24); e) An RGN polypeptide having a sequence that is 100% identical to sequence number 29 (wherein the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 30, and the tracrRNA has a sequence that is 100% identical to sequence number 31); f) An RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 36 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 38); g) An RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 43 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 45); h) An RGN polypeptide having a sequence that is 100% identical to sequence number 50 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 51, and the tracrRNA has a sequence that is 100% identical to sequence number 52); i) An RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 56 (where the CRISPR repeat sequence has a sequence that is 100% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is 100% identical to one of sequence numbers 63 and 570-579 (here, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 64, and the tracrRNA has a sequence that is 100% identical to sequence number 65); k) An RGN polypeptide having a sequence that is 100% identical to sequence number 70 (wherein the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 71, and the tracrRNA has a sequence that is 100% identical to sequence number 72); l) An RGN polypeptide having a sequence that is 100% identical to sequence number 76 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 77, and the tracrRNA has a sequence that is 100% identical to sequence number 78); m) An RGN polypeptide having a sequence that is 100% identical to sequence number 83 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 84, and the tracrRNA has a sequence that is 100% identical to sequence number 85); n) An RGN polypeptide having a sequence that is 100% identical to sequence number 89 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 90, and the tracrRNA has a sequence that is 100% identical to sequence number 91); o) An RGN polypeptide having a sequence that is 100% identical to sequence number 96 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 97, and the tracrRNA has a sequence that is 100% identical to sequence number 98); p) An RGN polypeptide having a sequence that is 100% identical to sequence number 103 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 104, and the tracrRNA has a sequence that is 100% identical to sequence number 105); q) An RGN polypeptide having a sequence that is 100% identical to sequence number 110 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 111, and the tracrRNA has a sequence that is 100% identical to sequence number 112); r) An RGN polypeptide having a sequence that is 100% identical to sequence number 117 (wherein the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 118, and the tracrRNA has a sequence that is 100% identical to sequence number 119); s) RGN polypeptide having a sequence that is 100% identical to sequence number 123 (where the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 124, and the tracrRNA has a sequence that is 100% identical to sequence number 125); and t) RGN polypeptide having a sequence that is 100% identical to sequence number 83 (in this case, the CRISPR repeat sequence has a sequence that is 100% identical to sequence number 84, and the tracrRNA has a sequence that is 100% identical to sequence number 78). A vector of embodiment 53, consisting of a group of the following.

[0268] 57. A nucleic acid molecule comprising a polynucleotide encoding a transactivated CRISPR RNA (tracrRNA) having a nucleotide sequence that is at least 90% identical to sequence numbers 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125, a) The tracrRNA and; b) crRNA containing spacer sequences and CRISPR repeat sequences When a guide RNA containing (wherein the tracrRNA hybridizes to the CRISPR repeat sequence of the crRNA) binds to an RNA-inducible nuclease (RGN) polypeptide, it can hybridize to the target DNA sequence in a sequence-specific manner through the spacer sequence of the crRNA. A nucleic acid molecule in which the polynucleotide encoding tracrRNA is functionally linked to a heterogeneous promoter for the polynucleotide.

[0269] 58. A nucleic acid molecule of Embodiment 57, wherein the tracrRNA comprises a nucleotide sequence having a sequence that is at least 95% identical to sequence numbers 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125.

[0270] 59. A nucleic acid molecule of Embodiment 57, wherein the tracrRNA contains a nucleotide sequence having a sequence that is 100% identical to sequence numbers 3, 10, 17, 24, 31, 38, 45, 52, 58, 65, 72, 78, 85, 91, 98, 105, 112, 119, or 125.

[0271] 60. A vector comprising any one nucleic acid molecule from Embodiments 57 to 59.

[0272] 61. A vector of embodiment 60, further comprising a polynucleotide encoding the crRNA.

[0273] 62. The crRNA mentioned above, a) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 2 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 3); b) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 9 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 10); c) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 16 (where the tracrRNA has a sequence that is at least 90% identical to sequence number 17); d) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 23 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 24); e) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 30 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 31); f) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 37 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 38); g) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 44 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 45); h) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 51 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 52); i) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 57 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 58); j) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 64 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 65); k) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 71 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 72); l) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 77 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 78); m) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 84 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 85); n) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 90 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 91); o) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 97 (in this case, the tracrRNA has a sequence that is at least 90% identical to sequence number 98); p) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 104 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 105); q) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 111 (where the tracrRNA has a sequence that is at least 90% identical to sequence number 112); r) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 118 (wherein the tracrRNA has a sequence that is at least 90% identical to sequence number 119); and s) A CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 124 (in this case, the tracrRNA has a sequence that is at least 90% identical to sequence number 125). A vector of embodiment 61 comprising a CRISPR repeat sequence selected from the group consisting of the following.

[0274] 63. The aforementioned crRNA, a) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 2 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 3); b) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 9 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 10); c) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 16 (where the tracrRNA has a sequence that is at least 95% identical to sequence number 17); d) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 23 (where the tracrRNA has a sequence that is at least 95% identical to sequence number 24); e) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 30 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 31); f) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 37 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 38); g) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 44 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 45); h) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 51 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 52); i) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 57 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 58); j) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 64 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 65); k) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 71 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 72); l) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 77 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 78); m) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 84 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 85); n) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 90 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 91); o) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 97 (in this case, the tracrRNA has a sequence that is at least 95% identical to sequence number 98); p) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 104 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 105); q) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 111 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 112); r) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 118 (wherein the tracrRNA has a sequence that is at least 95% identical to sequence number 119); and s) A CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 124 (in this case, the tracrRNA has a sequence that is at least 95% identical to sequence number 125). A vector of embodiment 61 comprising a CRISPR repeat sequence selected from the group consisting of the following.

[0275] 64. The aforementioned crRNA, a) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 2 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 3); b) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 9 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 10); c) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 16 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 17); d) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 23 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 24); e) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 30 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 31); f) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 37 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 38); g) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 44 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 45); h) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 51 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 52); i) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 57 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 58); j) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 64 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 65); k) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 71 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 72); l) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 77 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 78); m) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 84 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 85); n) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 90 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 91); o) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 97 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 98); p) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 104 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 105); q) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 111 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 112); r) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 118 (where the tracrRNA has a sequence that is 100% identical to sequence number 119); and s) A CRISPR repeat sequence having a sequence that is 100% identical to sequence number 124 (in this case, the tracrRNA has a sequence that is 100% identical to sequence number 125). A vector of embodiment 61 comprising a CRISPR repeat sequence selected from the group consisting of the following.

[0276] 65. Any one of embodiments 61 to 64, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are functionally linked to the same promoter and encoded as a single guide RNA.

[0277] 66. Any one of embodiments 61 to 64, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are functionally linked to separate promoters.

[0278] 67. Any one of embodiments 60 to 66, further comprising a polynucleotide encoding the RGN polypeptide.

[0279] 68. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 1 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 8 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 10); c) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 15 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 17); d) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 22 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 23, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 24); e) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 29 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 30, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 31); f) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 36 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 38); g) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 43 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 45); h) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 50 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 51, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 52); i) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 56 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is at least 90% identical to one of sequence numbers 63 and 570-579 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to sequence number 64, and the tracrRNA has a sequence that is at least 90% identical to sequence number 65); k) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 70 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 71, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 72); l) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 76 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 77, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 78); m) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 85); n) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 89 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 90, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 91); o) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 96 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 97, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 98); p) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 103 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 104, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 105); q) An RGN polypeptide having a sequence that is at least 90% identical to sequence number 110 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to sequence number 111, and the tracrRNA has a sequence that is at least 90% identical to sequence number 112); r) An RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 117 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 118, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 119); s) RGN polypeptide having a sequence that is at least 90% identical to sequence number 123 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to sequence number 124, and the tracrRNA has a sequence that is at least 90% identical to sequence number 125); t) RGN polypeptide having a sequence that is at least 90% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is at least 90% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is at least 90% identical to SEQ ID NO: 78) A vector of embodiment 67, consisting of a group of the following.

[0280] 69. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 1 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 8 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 10); c) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 15 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 17); d) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 22 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 23, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 24); e) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 29 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 30, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 31); f) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 36 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 38); g) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 43 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 45); h) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 50 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 51, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 52); i) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 56 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is at least 95% identical to one of sequence numbers 63 and 570-579 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to sequence number 64, and the tracrRNA has a sequence that is at least 95% identical to sequence number 65); k) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 70 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 71, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 72); l) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 76 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 77, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 78); m) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 85); n) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 89 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 90, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 91); o) RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 96 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 97, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 98); p) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 103 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 104, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 105); q) An RGN polypeptide having a sequence that is at least 95% identical to sequence number 110 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to sequence number 111, and the tracrRNA has a sequence that is at least 95% identical to sequence number 112); r) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 117 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 118, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 119); s) RGN polypeptide having a sequence that is at least 95% identical to sequence number 123 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to sequence number 124, and the tracrRNA has a sequence that is at least 95% identical to sequence number 125); t) An RGN polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is at least 95% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is at least 95% identical to SEQ ID NO: 78). A vector of embodiment 67, consisting of a group of the following.

[0281] 70. The selection of the RGN polypeptide is a) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 1 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 2, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 3); b) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 8 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 9, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 10); c) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 15 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 16, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 17); d) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 22 (wherein the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 23, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 24); e) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 29 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 30, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 31); f) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 36 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 37, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 38); g) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 43 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 44, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 45); h) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 50 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 51, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 52); i) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 56 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 57, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 58); j) An RGN polypeptide having a sequence that is 100% identical to one of sequence numbers 63 and 570-579 (wherein the crRNA contains a CRISPR repeat sequence that is 100% identical to sequence number 64, and the tracrRNA has a sequence that is 100% identical to sequence number 65); k) An RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 70 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 71, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 72); l) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 76 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 77, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 78); m) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 85); n) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 89 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 90, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 91); o) An RGN polypeptide having a sequence that is 100% identical to sequence number 96 (wherein the crRNA contains a CRISPR repeat sequence that is 100% identical to sequence number 97, and the tracrRNA has a sequence that is 100% identical to sequence number 98); p) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 103 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 104, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 105); q) An RGN polypeptide having a sequence that is 100% identical to sequence number 110 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to sequence number 111, and the tracrRNA has a sequence that is 100% identical to sequence number 112); r) An RGN polypeptide having a sequence that is 100% identical to sequence number 117 (wherein the crRNA contains a CRISPR repeat sequence that is 100% identical to sequence number 118, and the tracrRNA has a sequence that is 100% identical to sequence number 119); s) RGN polypeptide having a sequence that is 100% identical to sequence number 123 (wherein the crRNA contains a CRISPR repeat sequence that is 100% identical to sequence number 124, and the tracrRNA has a sequence that is 100% identical to sequence number 125); t) RGN polypeptide having a sequence that is 100% identical to SEQ ID NO: 83 (where the crRNA contains a CRISPR repeat sequence that is 100% identical to SEQ ID NO: 84, and the tracrRNA has a sequence that is 100% identical to SEQ ID NO: 78) A vector of embodiment 67, consisting of a group of the following.

[0282] 71. A system for binding a DNA molecule to a target DNA sequence, a) One or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); b) comprising an RNA-inducible nuclease (RGN) polypeptide having an amino acid sequence that is at least 90% identical to one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; At least one of the nucleotide sequences encoding one or more guide RNAs and the nucleotide sequences encoding the RGN polypeptide are functionally linked to heterologous promoters for this nucleotide sequence; The one or more guide RNAs can hybridize to the target DNA sequence. A system in which one or more guide RNAs form a complex with the RGN polypeptide, thereby enabling the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

[0283] 72. A system for binding a DNA molecule to a target DNA sequence, a) One or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); b) comprising an RNA-inducible nuclease (RGN) polypeptide having an amino acid sequence that is at least 90% identical to one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; The one or more guide RNAs can hybridize to the target DNA sequence. A system in which one or more guide RNAs form a complex with the RGN polypeptide, thereby enabling the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

[0284] 73. The system of Embodiment 71 or 72, wherein at least one of the nucleotide sequences encoding one or more guide RNAs is functionally linked to a heterogeneous promoter for the nucleotide sequence.

[0285] 74. Any one of the systems of Embodiments 71 to 73, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570 to 579.

[0286] 75. Any one of the systems of Embodiments 71 to 73, wherein the RGN polypeptide comprises an amino acid sequence having a sequence that is 100% identical to any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570 to 579.

[0287] 76. Any one of the systems from Embodiments 71 to 73, wherein the RGN polypeptide has a sequence that is at least 90% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

[0288] 77. Any one of the systems of Embodiments 71 to 76, wherein the RGN polypeptide and the one or more guide RNAs are not found to be complexed with each other in nature.

[0289] 78. Any one of the systems from Embodiments 71 to 77, wherein the target DNA sequence is a eukaryotic target DNA sequence.

[0290] 79. Any one of the systems from Embodiments 71 to 78, wherein the gRNA is a single guide RNA (sgRNA).

[0291] 80. Any one of the systems from Embodiments 71 to 78, wherein the gRNA is a double guide RNA.

[0292] 81. The selection of the gRNA is a) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 2, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 3 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1); b) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 9, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 10 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 8); c) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 16, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 17 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 15); d) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 23, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 24 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 22); e) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 30, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 31 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 29); f) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 37, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 38 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 36); g) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 44, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 45 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 43); h) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 51, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 52 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 50); i) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 57, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 58 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 56); j) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 64, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 65 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 63 and 570-579); k) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 71, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 72 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 70); l) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 77, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 78 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 76); m) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 85 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 83); n) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 90, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 91 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 89); o) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 97, and a tracrRNA having a sequence that is at least 90% identical to sequence number 98 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 96); p) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 104, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 105 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 103); q) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 111, and a tracrRNA having a sequence that is at least 90% identical to sequence number 112 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 110); r) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to sequence number 118, and a tracrRNA having a sequence that is at least 90% identical to sequence number 119 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to sequence number 117); s) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 124, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 125 (wherein the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 123); and t) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 78 (here, the RGN polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 83). A system consisting of any one of embodiments 71 to 80, comprising the group.

[0293] 82. The selection of the gRNA is a) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 2, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 3 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 1); b) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 9, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 10 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 8); c) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 16, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 17 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 15); d) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 23, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 24 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 22); e) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 30, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 31 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 29); f) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 37, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 38 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 36); g) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 44, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 45 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 43); h) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 51, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 52 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 50); i) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 57, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 58 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 56); j) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 64, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 65 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 63 and 570-579); k) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 71, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 72 (wherein the RGN polypeptide, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 70); l) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 77, and a tracrRNA having a sequence that is at least 95% identical to sequence number 78 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 76); m) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 85 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 83); n) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 90, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 91 (wherein the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 89); o) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 97, and a tracrRNA having a sequence that is at least 95% identical to sequence number 98 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 96); p) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 104, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 105 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 103); q) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 111, and a tracrRNA having a sequence that is at least 95% identical to sequence number 112 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 110); r) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to sequence number 118, and a tracrRNA having a sequence that is at least 95% identical to sequence number 119 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to sequence number 117); s) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 124, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 125 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 123); and t) A gRNA containing a CRISPR repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 78 (here, the RGN polypeptide contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 83). A system consisting of any one of embodiments 71 to 80, comprising the group.

[0294] 83. The selection of the gRNA is a) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 2, and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 3 (here, the RGN polypeptide contains an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 1); b) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 9 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 10 (here, the RGN polypeptide contains an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 8); c) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 16, and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 17 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 15); d) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 23 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 24 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 22); e) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 30 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 31 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 29); f) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 37 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 38 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 36); g) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 44 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 45 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 43); h) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 51 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 52 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 50); i) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 57 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 58 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 56); j) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 64 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 65 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 63 and one of 570-579); k) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 71 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 72 (here, the RGN polypeptide contains an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 70); l) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 77 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 78 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 76); m) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 85 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 83); n) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 90 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 91 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 89); o) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 97 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 98 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 96); p) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 104, and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 105 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 103); q) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to sequence number 111, and a tracrRNA having a sequence that is 100% identical to sequence number 112 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to sequence number 110); r) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to sequence number 118, and a tracrRNA having a sequence that is 100% identical to sequence number 119 (here, the RGN polypeptide contains an amino acid sequence having a sequence that is 100% identical to sequence number 117); s) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 124 and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 125 (here, the RGN polypeptide contains an amino acid sequence having a sequence that is 100% identical to SEQ ID NO: 123); and t) A gRNA containing a CRISPR repeat sequence having a sequence that is 100% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is 100% identical to SEQ ID NO: 78 (here, the RGN polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 83). A system consisting of any one of embodiments 71 to 80, comprising the group.

[0295] 84. Any one of the systems from Embodiments 71 to 83, wherein the target DNA sequence is located adjacent to a protospacer adjacency motif (PAM).

[0296] 85. Any one of the systems from Embodiments 71 to 84, wherein the target DNA sequence is located within a cell.

[0297] 86. The system of Embodiment 85, wherein the cells are eukaryotic cells.

[0298] 87. The system of Embodiment 86, wherein the eukaryotic cells are plant cells.

[0299] 88. The system of Embodiment 86, wherein the eukaryotic cells are mammalian cells.

[0300] 89. The system of Embodiment 88, wherein the mammalian cells are human cells.

[0301] 90. The system of Embodiment 89, wherein the human cells are immune cells.

[0302] 91. The system of Embodiment 90, wherein the immune cells are stem cells.

[0303] 92. The system of Embodiment 91, wherein the stem cells are induced pluripotent stem cells.

[0304] 93. The system of Embodiment 86, wherein the eukaryotic cells are insect cells.

[0305] 94. The system of Embodiment 85, wherein the cells are prokaryotic cells.

[0306] 95. Any one of the systems from Embodiments 71 to 94, wherein, when transcribed, one or more guide RNAs can hybridize to the target DNA sequence, and the guide RNAs can form a complex with the RGN polypeptide to cleave the target DNA sequence.

[0307] 96. The system of embodiment 95, wherein the break results in a double-strand break.

[0308] 97. The system of embodiment 95, wherein the break results in a single-strand break.

[0309] 98. Any one of the systems from Embodiments 71 to 94, wherein the RGN polypeptide is either nuclease-inactive or nicasse.

[0310] 99. Any one of the systems from Embodiments 71 to 98, wherein the RGN polypeptide is functionally linked to a base-editing polypeptide.

[0311] 100. The system of Embodiment 99, wherein the base-editing polypeptide is a deaminase.

[0312] 101. The system of Embodiment 100, wherein the deaminase is cytidine deaminase or adenine deaminase.

[0313] 102. Any one of the systems from Embodiments 71 to 101, wherein the RGN polypeptide comprises one or more nuclear localization signals.

[0314] 103. Any one of the systems from Embodiments 71 to 102, wherein the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0315] 104. Any one of the systems from Embodiments 71 to 103, wherein the nucleotide sequences encoding one or more guide RNAs and the nucleotide sequences encoding the RGN polypeptide are located on a single vector.

[0316] 105. Any one of the systems from Embodiments 71 to 104, further comprising one or more donor polynucleotides, or one or more nucleotide sequences encoding the one or more donor polynucleotides.

[0317] 106. A pharmaceutical composition comprising any one nucleic acid molecule from Embodiments 1-14, 43-45, and 57-59, any one vector from Embodiments 15-21, 46-56, and 60-70, a cell from Embodiment 22, any one isolated RGN polypeptide from Embodiments 31-42, or any one system from Embodiments 71-105, and a pharmaceutically acceptable base.

[0318] 107. A method for binding a DNA molecule to a target DNA sequence, comprising delivering a system according to any one of embodiments 71 to 105 to the target DNA sequence or to a cell containing the target DNA sequence.

[0319] 108. The method of Embodiment 107, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby enabling detection of the target DNA sequence.

[0320] 109. The method of Embodiment 107, wherein the guide RNA or the RGN polypeptide further comprises an expression modulator, thereby altering the expression of the target DNA sequence or the expression of a gene under transcriptional control by the target DNA sequence.

[0321] 110. A method for cleaving or modifying a target DNA sequence of a DNA molecule, comprising delivering a system according to any one of embodiments 71 to 105 to the target DNA sequence or to a cell containing the DNA sequence, wherein the cleavage or modification of the target DNA sequence occurs.

[0322] 111. The method of Embodiment 110, wherein the modified target DNA sequence includes the insertion of heterologous DNA into the target DNA sequence.

[0323] 112. The method of Embodiment 110, wherein the modified target DNA sequence comprises the deletion of at least one nucleotide from the target DNA sequence.

[0324] 113. The method of Embodiment 110, wherein the modified target DNA sequence comprises a mutation in at least one nucleotide within the target DNA sequence.

[0325] 114. In order to bind the DNA molecule to the target DNA sequence, a)i) one or more guide RNAs capable of hybridizing to the target DNA sequence; ii) RGN polypeptides containing an amino acid sequence that has at least 90% matching with one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579. By combining these, the RNA-induced nuclease (RGN) ribonucleotide complex can be assembled in vitro under conditions suitable for the formation of this RGN ribonucleotide complex; b) A method comprising contacting the target DNA sequence, or a cell containing the target DNA sequence, with the RGN ribonucleotide complex assembled in vitro, A method in which one or more guide RNAs hybridize to the target DNA sequence, thereby causing the RGN polypeptide to bind to the target DNA sequence.

[0326] 115. The method of Embodiment 114, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby enabling detection of the target DNA sequence.

[0327] 116. The method of Embodiment 114, wherein the guide RNA or the RGN polypeptide further comprises an expression modulator, thereby enabling alteration of the expression of the target DNA sequence.

[0328] 117. To cleave and / or modify the target DNA sequence of a DNA molecule, the DNA molecule is used a) RNA-inducible nuclease (RGN) polypeptides containing amino acid sequences that are at least 90% identical to any one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570-579; and b) A method comprising contacting the RGN of (a) with one or more guide RNAs capable of directing it to the target DNA sequence, A method in which one or more guide RNAs hybridize to the target DNA sequence, thereby causing the RGN polypeptide to bind to the target DNA sequence, and resulting in cleavage and / or modification of the target DNA sequence.

[0329] 118. The method of Embodiment 117, wherein cleavage by the RGN polypeptide causes double-strand breaks.

[0330] 119. The method of Embodiment 117, wherein cleavage by the RGN polypeptide causes single-strand breaks.

[0331] 120. The method of Embodiment 117, wherein the RGN polypeptide is nuclease-inactive or nickasase and functionally linked to a base-editing polypeptide.

[0332] 121. The method of Embodiment 120, wherein the base-editing polypeptide is a deaminase.

[0333] 122. The method of Embodiment 121, wherein the deaminase is cytidine deaminase or adenine deaminase.

[0334] 123. The method of Embodiment 117, wherein the modified target DNA sequence includes the insertion of heterologous DNA into the target DNA sequence.

[0335] 124. The method of Embodiment 117, wherein the modified target DNA sequence comprises the deletion of at least one nucleotide from the target DNA sequence.

[0336] 125. The method of Embodiment 117, wherein the modified target DNA sequence comprises a mutation in at least one nucleotide within the target DNA sequence.

[0337] 126. Any one of embodiments 114 to 125, wherein the target DNA sequence is located adjacent to a protospacer adjacency motif (PAM).

[0338] 127. Any one of embodiments 114 to 126, wherein the target DNA sequence is a eukaryotic target DNA sequence.

[0339] 128. Any one of embodiments 114 to 127, wherein the gRNA is a single guide RNA (sgRNA).

[0340] 129. Any one of embodiments 114 to 127, wherein the sgRNA is a double guide RNA.

[0341] 130. Any one of the embodiments 114 to 129, wherein the RGN comprises an amino acid sequence having a sequence that is at least 95% identical to any one of sequence numbers 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570 to 579.

[0342] 131. Any one of Embodiments 114 to 129, wherein the RGN comprises an amino acid sequence having a sequence that is 100% identical to any one of Sequence IDs 1, 8, 15, 22, 29, 36, 43, 50, 56, 63, 70, 76, 83, 89, 96, 103, 110, 117, 123, and 570 to 579.

[0343] 132. Any one of Embodiments 114 to 129, wherein the RGN polypeptide has a sequence that is at least 90% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

[0344] 133.a) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 1, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 2, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 3; b) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 8, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 9, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 10; c) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 15, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 16, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 17; d) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 22, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 23, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 24; e) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 29, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 30, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 31; f) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 36, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 37, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 38; g) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 43, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 44, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 45; h) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 50, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 51, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 52; i) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 56, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 57, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 58; j) The RGN has a sequence that is at least 90% identical to one of sequence numbers 63 and 570-579, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to sequence number 64, and a tracrRNA having a sequence that is at least 90% identical to sequence number 65; k) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 70, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 71, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 72; l) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 76, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 77, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 78; m) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 83, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 85; n) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 89, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 90, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 91; o) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 96, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 97, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 98; p) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 103, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 104, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 105; q) The RGN has a sequence that is at least 90% identical to sequence number 110, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to sequence number 111, and a tracrRNA having a sequence that is at least 90% identical to sequence number 112; r) The RGN has a sequence that is at least 90% identical to sequence number 117, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to sequence number 118, and a tracrRNA having a sequence that is at least 90% identical to sequence number 119; s) The RGN has a sequence that is at least 90% identical to SEQ ID NO: 123, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 124, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 125; or t) Any one of Embodiments 114 to 129, wherein the RGN has a sequence that is at least 90% identical to SEQ ID NO: 83, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 90% identical to SEQ ID NO: 84, and a tracrRNA having a sequence that is at least 90% identical to SEQ ID NO: 78.

[0345] 134.a) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 1, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 2, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 3; b) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 8, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 9, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 10; c) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 15, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 16, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 17; d) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 22, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 23, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 24; e) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 29, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 30, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 31; f) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 36, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 37, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 38; g) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 43, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 44, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 45; h) The RGN has a sequence that is at least 95% identical to SEQ ID NO: 50, and the guide RNA comprises a crRNA repeat sequence having a sequence that is at least 95% identical to SEQ ID NO: 51, and a tracrRNA having a sequence that is at least 95% identical to SEQ ID NO: 52; i) The RGN ha...

Claims

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-inducible nuclease (RGN) polypeptide, wherein the polynucleotide encoding the RGN polypeptide has an amino acid sequence that is at least 92% identical to SEQ ID NO:

63.

2. below: (a) The polynucleotide encoding the RGN polypeptide is functionally linked to a different promoter for this polynucleotide, (b) The polynucleotide encoding the RGN polypeptide is codon-optimized for expression in eukaryotic cells, and / or (c) The RGN polypeptide comprises one or more nuclear localization signals, A nucleic acid molecule according to claim 1, characterized in that...

3. The nucleic acid molecule according to claim 1 or 2, wherein the RGN polypeptide has a sequence that is at least 92% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

4. The RGN polypeptide is subject to the following conditions: (a) Nuclease-inactive or nicasse; (b) functionally fused to a base-editing polypeptide; and / or (c) Functionally integrated into the effects domain; A nucleic acid molecule according to any one of claims 1 to 3, wherein the base-editing polypeptide and / or effector domain is functionally fused to the N-terminus or C-terminus of the RGN polypeptide.

5. The nucleic acid molecule according to claim 4, wherein the base-editing polypeptide is a deaminase.

6. The nucleic acid molecule according to claim 4, wherein the effector domain comprises a cleavage domain, a deaminase domain, or an expression modulator domain.

7. The nucleic acid molecule according to claim 6, wherein the expression modulator domain is an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain.

8. A vector comprising a nucleic acid molecule according to any one of claims 1 to 7.

9. The vector according to claim 8, further comprising at least one nucleotide sequence encoding a guide RNA capable of hybridizing to a target DNA sequence, wherein the RGN polypeptide can bind to the target DNA sequence in an RNA-inducible manner when it binds to the guide RNA.

10. The aforementioned guide RNA i) CRISPR RNA containing a CRISPR repeat sequence that is at least 90%, 95%, or 100% identical to SEQ ID NO: 64; ii) TracrRNA that is at least 90%, 95%, or 100% identical to Sequence ID No. 65; The vector according to claim 9, wherein the RGN polypeptide comprises an amino acid sequence that is at least 92%, 95%, or 100% identical to SEQ ID NO:

63.

11. A cell that is not a human embryonic cell, comprising a nucleic acid molecule according to any one of claims 1 to 9, or a vector according to any one of claims 8 to 10.

12. A method for producing RGN polypeptide, comprising the following steps: (a) A step of culturing the cells according to claim 11 under conditions in which the RGN polypeptide is expressed; or (b) A step of introducing a heterogeneous nucleic acid molecule into a cell that contains a nucleotide sequence encoding an RNA-induced nuclease (RGN) polypeptide having an amino acid sequence that is at least 92% identical to SEQ ID NO: 63; A method comprising culturing the cells under conditions in which the RGN polypeptide is expressed.

13. The above method further includes the following steps: (i) a step of purifying the RGN polypeptide; and / or (ii) The cells of (b) here further express one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex; The method according to claim 12, further comprising the step of purifying the RGN ribonucleoprotein complex.

14. RNA-induced nuclease (RGN) polypeptides comprising an amino acid sequence that is at least 92%, 95%, or 100% identical to SEQ ID NO:

63.

15. The RGN polypeptide according to claim 14, having a sequence that is at least 92% identical to SEQ ID NO: 63, and having isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

16. The RGN polypeptide is subject to the following conditions: (a) Nuclease-inactive or nicasse; (b) containing one or more nuclear localization signals; (c) functionally fused to a base-editing polypeptide; and / or (d) Functionally integrated into the effects domain; The RGN polypeptide according to claim 14 or 15, wherein the condition is met, and thereafter, one or more nuclear localization signals, base editing polypeptides, and / or effector domains are functionally fused to the N-terminus or C-terminus of the RGN polypeptide.

17. The RGN polypeptide according to claim 16, wherein the base-editing polypeptide is a deaminase.

18. The RGN polypeptide according to claim 16, wherein the effector domain comprises a cleavage domain, a deaminase domain, or an expression modulator domain.

19. The RGN polypeptide according to claim 18, wherein the expression modulator domain is an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain.

20. A system for binding a DNA molecule to a target DNA sequence, a) One or more guide RNAs (gRNAs) capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more gRNAs; b) RNA-induced nuclease (RGN) polypeptides comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 63, or polynucleotides comprising a nucleotide sequence encoding the RGN polypeptide; A system that includes this.

21. The system according to claim 20, wherein at least one of the nucleotide sequences encoding one or more gRNAs and / or the nucleotide sequences encoding the RGN polypeptide is functionally linked to a heterogeneous promoter for the nucleotide sequence.

22. The system according to claim 20 or 21, wherein the RGN polypeptide has a sequence that is at least 92% identical to SEQ ID NO: 63, and has isoleucine at the amino acid position corresponding to 305 of SEQ ID NO: 63, valine at the amino acid position corresponding to 328, leucine at the amino acid position corresponding to 366, threonine at the amino acid position corresponding to 368, and valine at the amino acid position corresponding to 405.

23. The system according to any one of claims 20 to 22, wherein the target DNA sequence is a eukaryotic target DNA sequence.

24. The system according to any one of claims 20 to 23, wherein the one or more gRNAs are single guide RNAs (sgRNAs) or dual guide RNAs.

25. The one or more gRNAs mentioned above, A CRISPR repeat sequence having a sequence that is at least 90%, 95%, or 100% identical to sequence number 64, and a tracrRNA having a sequence that is at least 90%, 95%, or 100% identical to sequence number 65; The system according to any one of claims 20 to 24, wherein the RGN polypeptide comprises an amino acid sequence that is at least 92%, 95%, or 100% identical to SEQ ID NO:

63.

26. The system according to any one of claims 20 to 25, wherein the target DNA sequence is located within a cell and the cell is not a human embryonic cell.

27. The RGN polypeptide is subject to the following conditions: (a) Nuclease-inactive or nicasse; (b) containing one or more nuclear localization signals; (c) functionally fused to a base-editing polypeptide; and / or (d) Functionally integrated into the effects domain; The system according to any one of claims 20 to 26, wherein the one or more nuclear localization signals, base-editing polypeptides, and / or effector domains are functionally fused to the N-terminus or C-terminus of the RGN polypeptide.

28. The system according to claim 27, wherein the base-editing polypeptide is a deaminase.

29. The system according to claim 27, wherein the effector domain includes a cleavage domain, a deaminase domain, or an expression modulator domain.

30. The system according to claim 29, wherein the expression modulator domain is an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain.

31. The system according to any one of claims 20 to 30, wherein the nucleotide sequences encoding one or more gRNAs and the nucleotide sequences encoding an RGN polypeptide are located on a single vector, and / or the system further comprises one or more donor polynucleotides, or one or more nucleotide sequences encoding the one or more donor polynucleotides.

32. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 7, a vector according to any one of claims 8 to 10, a cell according to claim 13, an RGN polypeptide according to any one of claims 14 to 19, or a system according to any one of claims 20 to 31, and a pharmaceutically acceptable base.

33. An in vitro or ex vivo method for binding, cleaving, and / or modifying a target DNA sequence of a DNA molecule, comprising delivering a system according to any one of claims 20 to 31 to the target DNA sequence or to a cell containing the target DNA sequence, wherein the cell is not a human embryonic cell.

34. To cleave and / or modify the target DNA sequence of a DNA molecule, the DNA molecule is used (a) RNA-induced nuclease (RGN) polypeptides having an amino acid sequence that is at least 92% identical to SEQ ID NO: 63; and (b) One or more guide RNAs capable of directing the RGN polypeptide of (a) to the target DNA sequence; An in vitro or ex vivo method, which includes contacting with A method wherein the target DNA sequence is not located in a human embryonic cell, and one or more guide RNAs hybridize to the target DNA sequence, thereby causing the RGN polypeptide to bind to the target DNA sequence, and cleavage and / or modification of the target DNA sequence occurs.

35. The in vitro or ex vivo method according to claim 34, wherein the cleavage by the RGN polypeptide causes a double-strand break or a single-strand break.

36. The in vitro or ex vivo method according to claim 34, wherein the RGN polypeptide is nuclease-inactive or nicasse and functionally linked to a base-editing polypeptide, wherein the base-editing polypeptide is functionally fused to the N-terminus or C-terminus of the RGN polypeptide.

37. The in vitro or ex vivo method according to claim 36, wherein the base-editing polypeptide comprises a deaminase.

38. The in vitro or ex vivo method according to claim 34, wherein the target DNA sequence cut and / or modified by the in vitro or ex vivo method according to claim 34 comprises: insertion of heterologous DNA into the target DNA sequence; deletion of at least one nucleotide from the target DNA sequence; and / or mutation of at least one nucleotide in the target DNA sequence.

39. The in vitro or ex vivo method according to any one of claims 33 to 38, wherein the target DNA sequence is a eukaryotic target DNA sequence.

40. The in vitro or ex vivo method according to any one of claims 33 to 39, wherein the target DNA sequence is located within a cell, and the cell is a eukaryotic cell or a prokaryotic cell.

41. The in vitro or ex vivo method according to claim 40, further comprising: culturing the cells under conditions in which the RGN polypeptide is expressed and cleaves the target DNA sequence to produce a DNA molecule containing the modified DNA sequence; and selecting the cells containing the modified target DNA sequence.

42. A pharmaceutical composition for the treatment of a disease, comprising a nucleic acid molecule according to any one of claims 1 to 7, a vector according to any one of claims 8 to 10, a cell according to claim 11, an RGN polypeptide according to any one of claims 14 to 19, or a system according to any one of claims 20 to 31.

43. The pharmaceutical composition according to claim 42, wherein the disease is related to a causative mutation, and the pharmaceutical composition modifies the causative mutation.

44. A system for binding to a target DNA sequence of a DNA molecule, as follows: (a) an RNA-induced nuclease (RGN) polypeptide comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 63, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide; and (b) One or more guide RNAs comprising (i) a spacer complementary to the target DNA sequence, and (ii) a CRISPR repeat recognized by the RGN polypeptide; or one or more polynucleotides comprising one or more nucleotide sequences encoding one or more guide RNAs; A system that includes this.

45. It is a complex, and the following: (i) an isolated RNA-inducible nuclease (RGN) polypeptide containing an amino acid sequence that is at least 92% identical to SEQ ID NO: 63; and (ii) Non-natural guide RNA (gRNA); A complex that includes this.

46. The aforementioned non-natural guide RNAs are as follows: (a) gRNA containing one or more synthetic ribonucleotide analogs; (b) Single guide RNA; (c) gRNA comprising one or more peptide nucleic acids (PNAs), PNA-DNA chimeras, cross-linked nucleic acids (LNAs), and / or phosphorothioate crosslinks; A composite according to claim 45, selected from the group consisting of the following.

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