Manipulated ssDNase-free CRISPR endonuclease

Abstract

The present disclosure provides compositions related to engineered CRISPR endonuclease proteins that have a reduced ability to non-specifically cleave single-stranded DNA (ssDNA) compared to their reference wild-type protein. The present disclosure also provides methods for using and producing engineered CRISPR endonuclease proteins that have a reduced ability to non-specifically cleave ssDNA compared to their reference wild-type protein.

Description

[Technical Field]

[0001] Cross-referencing of related applications and incorporation of sequence listings This application claims the interests of U.S. Provisional Patent Application No. 63 / 126,983, filed on 17 December 2020, which is incorporated herein by reference in its entirety. The sequence listing, which is 185,550 bytes (measured on MS-Windows®) and contained in the filename "P34731WO00_SL.txt" created on 29 November 2021, contains 23 sequences and was filed electronically with this specification, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to compositions and methods relating to the use of RNA-induced CRISPR endonucleases engineered to reduce nonspecific cleavage of single-stranded DNA (ssDNA). This disclosure also relates to compositions and methods relating to the refinement of magnesium concentration to reduce nonspecific cleavage of ssDNA by RNA-induced CRISPR endonucleases. [Background technology]

[0003] CRISPR (clustered, regularly spaced, short-interval palindromic repeating) nucleases (e.g., Cas9, CasX, Cas12a, CasY) are proteins that are guided by guide RNA to target nucleic acid molecules, and their endonucleases can then cleave one or two strands of the target nucleic acid molecule. Recent reports have shown that Cas12a (also known as Cpf1) can perform unregulated, non-targeted cleavage of single-stranded DNA (ssDNA).

[0004] This disclosure demonstrates that CRISPR endonucleases can be modified to cleave double-stranded DNA (dsDNA) while reducing or eliminating their ability to non-specifically cleave single-stranded DNA (ssDNA). This disclosure also demonstrates that the magnesium chloride concentration of a solution containing a CRISPR endonuclease can be manipulated such that the CRISPR endonuclease can cleave dsDNA while reducing or eliminating its ability to non-specifically cleave ssDNA.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

[0006] [Non-Patent Document 1] 「The American Heritage Science Dictionary」(Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York) [Non-Patent Document 2] 「McGraw-Hill Dictionary of Scientific and Technical Terms」(6th Edition, 2002, McGraw-Hill, New York) [Non-Patent Document 3] 「Oxford Dictionary of Biology」(6th Edition, 2008, Oxford University Press, Oxford and New York) [Non-Patent Document 4] Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012) [Non-Patent Document 5] Current Protocols in Molecular Biology (FM Ausubel et al. (eds.) (1987)) [Non-Patent Document 6] Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)) [Non-Patent Document 7] Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor (eds.) (1995)) [Non-Patent Document 8] Harlow and Lane (eds.) (1988) Antibodies, A Laboratory Manual [Non-Patent Document 9] Animal Cell Culture (RI Freshney (ed.) (1987)) [Non-Patent Document 10] Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences [Non-Patent Document 11] CN Stewart, A. Touraev, V. Citovsky, T. Tzfira (eds.) (2011) Plant Transformation Technologies (Wiley-Blackwell) [Non-Patent Document 12] RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.) [Non-licensed Document 13] www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR041383 [Non-licensed Document 14] www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR040852 [Non-licensed Document 15] www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR040882 [Non-licensed Document 16] www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR003615 [Non-licensed Document 17] PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler (eds.), Cold Spring Harbor Laboratory Press, 1995 [Non-licensed Document 18] Sambrook (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) [Non-licensed Document 19] Chenna R.ら, "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Research 31: 3497~3500 pages (2003) [Non-licensed Document 20] Thompson JD, "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Research 22: 4673~4680 pages (1994) [Non-licensed Document 21] Larkin MAら, "Clustal W and Clustal X version 2.0", Bioinformatics 23: pages 2947~48 (2007) [Non-licensed Document 22] Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool", J. Mol. Biol. 215:403~410 pages (1990) [Non-licensed Document 23] Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) [Non-licensed Document 24] Zhang and Madden, Genome Res., 1997, 7, pages 649~656 [Non-licensed Document 25] Smith and Waterman, Adv. Appl. Math., 1981, 2, pages 482~489 [Non-licensed Document 26] Compendium of Transgenic Crop Plants (2009) Blackwell Publishing [Non-licensed Document 27] www.herbicide.adjuvants.com [Non-Patent Document 28] Kam et al. (2004) / . Am. Chem. Soc, 126 (22): pp. 6850-6851 [Non-Patent Document 29] Liu et al. (2009) Nano Lett, 9(3): pp. 1007-1010 [Non-Patent Document 30] Khodakovskaya et al. (2009) ACS Nano, 3(10):3221-3227 [Non-Patent Document 31] Chen et al., Science, 360:436-439 (2018) Apr 27;360(6387):436-439 [Non-Patent Document 32] Zetsche et al., Cell, 163:759 (2015) [Non-Patent Document 33] Yamano et al., Cell 165, 4:949 (2016) [Non-Patent Document 34] Moreno-Mateos et al., 2017, DOI: 10.1038 / s41467-017-01836-2 [Non-Patent Document 35] Stella et al., Nature, 546: pp. 559-563 (2017) [Overview of the Initiative] [Means for solving the problem]

[0007] In one embodiment, the disclosure provides an engineered RNA-inducible CRISPR nuclease comprising at least one mutation in its DNA catalytic domain, which exhibits reduced nonspecific cleavage of single-stranded DNA (ssDNA) compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation.

[0008] In one embodiment, the disclosure provides a method for producing an engineered RNA-inducible CRISPR nuclease, comprising the step of editing a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease to generate at least one mutation in the DNA catalytic domain, wherein the engineered RNA-inducible CRISPR nuclease exhibits reduced nonspecific cleavage of single-stranded DNA compared to a wild-type RNA-inducible CRISPR nuclease lacking at least one mutation.

[0009] In one embodiment, the present disclosure provides a method for reducing nonspecific single-stranded DNA (ssDNA) breaks caused by an RNA-induced CRISPR nuclease, comprising the step of providing cells with an engineered RNA-induced CRISPR nuclease having at least one mutation in its DNA catalytic domain compared to a reference wild-type RNA-induced CRISPR nuclease, wherein the engineered RNA-induced CRISPR nuclease exhibits reduced nonspecific breaks of non-targeted ssDNA compared to a reference wild-type RNA-induced CRISPR nuclease lacking at least one mutation.

[0010] In one embodiment, the present disclosure provides a method for reducing nonspecific single-stranded DNA (ssDNA) breaks caused by RNA-induced CRISPR nucleases, comprising the step of contacting non-target ssDNA with RNA-induced CRISPR nucleases in a test solution, wherein the test solution contains MgCl2 at a concentration of less than 10 mM, and the nonspecific ssDNA breaks are reduced compared to nonspecific ssDNA breaks caused by RNA-induced CRISPR nucleases in a control solution containing MgCl2 at a concentration of 10 mM or more.

[0011] Some embodiments relate to engineered RNA-inducible CRISPR nucleases comprising at least one mutation in the DNA catalytic domain, exhibiting reduced nonspecific cleavage of single-stranded DNA (ssDNA) compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation. In some embodiments, the engineered RNA-inducible CRISPR nuclease is part of a ribonucleoprotein. In some embodiments, the ribonucleoprotein comprises at least one guide nucleic acid. In some embodiments, the at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the at least one guide nucleic acid does not contain tracr. In some embodiments, the engineered RNA-inducible CRISPR nuclease is a Cas12a nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease is selected from the group consisting of Cas9 nuclease, CasX nuclease, CasY nuclease, and C2c2 nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In some embodiments, the engineered RNA-inducible CRISPR nuclease exhibits the ability to cleave double-stranded DNA (dsDNA). In some embodiments, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of wild-type RNA-inducible CRISPR nuclease. In some embodiments, the DNA catalytic domain includes a RuvC domain, a Nuc domain, and / or an HNH domain. In some embodiments, at least one mutation in the DNA catalytic domain is selected from the group consisting of insertions, deletions, and substitutions. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position selected from the group consisting of positions 925 and 1138 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position corresponding to position 1138 compared to SEQ ID NO: 2.In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1146 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1148 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1218 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1225 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1227 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1226 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1234 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1235 of wtAsCas12a.

[0012] Some embodiments relate to a method for producing an engineered RNA-inducible CRISPR nuclease, comprising the step of editing a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease to generate at least one mutation in the DNA catalytic domain, wherein the engineered RNA-inducible CRISPR nuclease exhibits reduced nonspecific cleavage of single-stranded DNA compared to a wild-type RNA-inducible CRISPR nuclease lacking at least one mutation. In some embodiments, the ribonucleoprotein includes at least one guide nucleic acid. In some embodiments, the at least one guide nucleic acid includes at least one guide RNA. In some embodiments, the at least one guide nucleic acid does not include tracr. In some embodiments, the engineered RNA-inducible CRISPR nuclease is a Cas12a nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease is selected from the group consisting of Cas9 nuclease, CasX nuclease, CasY nuclease, and C2c2 nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In some embodiments, the engineered RNA-inducible CRISPR nuclease exhibits the ability to cleave double-stranded DNA (dsDNA). In some embodiments, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of wild-type RNA-inducible CRISPR nuclease. In some embodiments, the DNA catalytic domain includes a RuvC domain, a Nuc domain, and / or an HNH domain. In some embodiments, at least one mutation in the DNA catalytic domain is selected from the group consisting of insertions, deletions, and substitutions. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position selected from the group consisting of positions 925 and 1138 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position corresponding to position 1138 compared to SEQ ID NO: 2.In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1146 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1148 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1218 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1225 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1227 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1226 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1234 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1235 of wtAsCas12a.

[0013] Some embodiments relate to a method for reducing nonspecific single-stranded DNA (ssDNA) breaks caused by RNA-induced CRISPR nucleases, comprising the step of providing cells with an engineered RNA-induced CRISPR nuclease having at least one mutation in its DNA catalytic domain compared to a reference wild-type RNA-induced CRISPR nuclease, wherein the engineered RNA-induced CRISPR nuclease exhibits reduced nonspecific breaks of non-targeted ssDNA compared to a reference wild-type RNA-induced CRISPR nuclease lacking at least one mutation. In some embodiments, the ribonucleoprotein comprises at least one guide nucleic acid. In some embodiments, the at least one guide nucleic acid comprises at least one guide RNA. In some embodiments, the at least one guide nucleic acid does not contain tracr. In some embodiments, the engineered RNA-induced CRISPR nuclease is a Cas12a nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease is selected from the group consisting of Cas9 nuclease, CasX nuclease, CasY nuclease, and C2c2 nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In some embodiments, the engineered RNA-inducible CRISPR nuclease exhibits the ability to cleave double-stranded DNA (dsDNA). In some embodiments, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of wild-type RNA-inducible CRISPR nuclease. In some embodiments, the DNA catalytic domain includes a RuvC domain, a Nuc domain, and / or an HNH domain. In some embodiments, at least one mutation in the DNA catalytic domain is selected from the group consisting of insertions, deletions, and substitutions. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position selected from the group consisting of positions 925 and 1138 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at a position corresponding to position 1138 compared to SEQ ID NO: 2.In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1146 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1148 compared to SEQ ID NO: 2. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1218 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1225 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1227 of wtFnCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1226 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1234 of wtAsCas12a. In some embodiments, at least one mutation in the DNA catalytic domain corresponds to an amino acid substitution at the position corresponding to position 1235 of wtAsCas12a.

[0014] Some embodiments describe a method for reducing nonspecific single-stranded DNA (ssDNA) breaks induced by RNA-inducible CRISPR nucleases, comprising the step of contacting non-target ssDNA in a test solution with an RNA-inducible CRISPR nuclease, wherein the test solution contains MgCl2 at a concentration of less than 10 mM, and the reduction in nonspecific ssDNA breaks is compared to nonspecific ssDNA breaks induced by the RNA-inducible CRISPR nuclease in a control solution containing MgCl2 at a concentration of 10 mM or more. In some embodiments, the test solution contains MgCl2 at a concentration of 5 mM or less. In some embodiments, the test solution contains MgCl2 at a concentration of 0.02 mM or less. In some embodiments, the RNA-inducible CRISPR nuclease is an engineered RNA-inducible CRISPR nuclease containing at least one mutation in its DNA catalytic domain compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation. In some embodiments, the DNA catalytic domain is a RuvC domain or a Nuc domain. In some embodiments, the test solution is intracellular. In some embodiments, the cell is either a prokaryotic or eukaryotic cell. In some embodiments, the eukaryotic cell is a plant cell. In some embodiments, the RNA-inducible CRISPR nuclease is Cas12a nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease is engineered Cas12a nuclease, and the wild-type RNA-inducible CRISPR nuclease contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the engineered RNA-inducible CRISPR nuclease is selected from the group consisting of Cas9 nuclease, CasX nuclease, CasY nuclease, and C2c2 nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12. [Brief explanation of the drawing]

[0015] [Figure 1] This shows the predicted size of the template DNA cleaved by SpCas9 or LbCas12a. [Figure 2]Sequence alignments of a fragment of LbCas12a containing the conserved R1138 residue with its homologs FnCas12a and AsCas12a are shown. Conserved residues are shown in gray. Potential amino acid substitutions that can alter charge / donor ability are shown in italics, and amino acid residues that affect Mg2+-mediated ssDNAse activity are underlined. The positions of important amino acid residues are indicated above the Wt sequence. [Modes for carrying out the invention]

[0016] Unless otherwise defined, all technical and scientific terms used have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Where a term is provided in the singular form, the inventors also intend aspects of this disclosure described by that term in the plural form. Where there are differences in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their common meanings in the industry in which they are used, as exemplified by various industry-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to limit themselves to mechanisms or modes of operation. The references are provided for illustrative purposes only.

[0017] Unless otherwise noted, the implementation of this disclosure includes, but is not limited to, prior art in biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the scope of the art in this field. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (FM Ausubel et al. (eds.) (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)); Methods In Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor (eds.) (1995)); Harlow and Lane (eds.) (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney (ed.) (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; CN Stewart, A. Touraev, V. Citovsky, T. Tzfira (eds.) (2011) Plant Transformation Technologies See Wiley-Blackwell and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0018] For example, all references cited herein, including all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0019] When a set of options is presented, all possible combinations of the members constituting that set of options are specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each option individually (e.g., A only, B only, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc.

[0020] As used herein, singular terms and singular forms of “a,” “an,” and “the” include multiple referents, for example, unless otherwise explicitly indicated by the text.

[0021] Any composition, nucleic acid molecule, polypeptide, cell, plant, etc., provided herein is specifically intended for use in conjunction with any method provided herein.

[0022] In one embodiment, the disclosure provides an engineered RNA-inducible CRISPR nuclease comprising at least one mutation in the DNA catalytic domain, which exhibits reduced nonspecific cleavage of single-stranded DNA (ssDNA) compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation.

[0023] In another aspect, the disclosure provides a method for producing an engineered RNA-inducible CRISPR nuclease, comprising the step of editing a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease to generate at least one mutation in the DNA catalytic domain, wherein the engineered RNA-inducible CRISPR nuclease exhibits reduced nonspecific cleavage of ssDNA compared to a wild-type RNA-inducible CRISPR nuclease lacking at least one mutation.

[0024] In a further embodiment, the present disclosure provides a method for reducing nonspecific ssDNA cleavage induced by an RNA-inducible CRISPR nuclease, comprising the step of providing cells with an engineered RNA-inducible CRISPR nuclease having at least one mutation in its DNA catalytic domain, wherein the engineered RNA-inducible CRISPR nuclease exhibits reduced nonspecific ssDNA cleavage compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation.

[0025] As used herein, “cleavage” refers to the disruption of the phosphate diester bond between two nucleotides. When only one strand of a nucleic acid molecule is cleaved, such cleavage is referred to as a “single-strand break.” When both strands of a nucleic acid molecule are cleaved, such cleavage is referred to as a “double-strand break.” In some embodiments, a double-strand break produces a blunt-end break product. A blunt-end break product is produced when two nucleic acid molecular strands are cleaved at the same location within the nucleic acid molecule. In other embodiments, a double-strand break produces an overhang break product. An overhang break product is produced when two nucleic acid molecular strands are cleaved at locations one or more nucleotides apart within the nucleic acid molecule.

[0026] RNA-induced CRISPR nuclease As used herein, “RNA-inducible CRISPR nuclease” refers to any nuclease derived from the CRISPR (clustered, regularly spaced, short-repeated palindromic structure) family of nucleases found in bacterial and archaeal species. In some embodiments, the RNA-inducible CRISPR nucleases provided herein are engineered RNA-inducible CRISPR nucleases.

[0027] As used herein, “manipulated” RNA-inducible CRISPR nuclease means an RNA-inducible CRISPR nuclease that contains at least one non-naturally occurring mutation introduced into a wild-type RNA-inducible CRISPR nuclease. “Wild-type RNA-inducible CRISPR nuclease” means a naturally occurring, endogenous RNA-inducible CRISPR nuclease found in organisms.

[0028] Manipulated RNA-inducible CRISPR nucleases can be produced by modifying wild-type RNA-inducible CRISPR nucleases. Methods for editing protein-coding polynucleotides are well known in the art. For example, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease can be modified by subjecting it to a mutagenerator (e.g., ethyl methanesulfonate (EMS), ionizing radiation) or a nuclease (e.g., CRISPR nuclease, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease). A polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease can also be modified using other standard techniques in the art, such as PCR-mediated site-directed mutagenesis, but are not limited to these.

[0029] In one embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an engineered RNA-inducible CRISPR nuclease by subjecting the polynucleotide to a mutagenerator. As used herein, “mutagenerator” means any agent capable of altering or inducing mutations in nucleic acid sequences. In one embodiment, the mutagenerator is a chemical mutagenerator. In one embodiment, the mutagenerator is a physical mutagenerator. In another embodiment, the mutagenerator is ionizing radiation. In another embodiment, the mutagenerator is ultraviolet irradiation. In another embodiment, the mutagenerator is a reactive oxygen species. In another embodiment, the mutagenerator is a deamination agent. In another embodiment, the mutagenerator is an alkylating agent. In another embodiment, the mutagenerator is an aromatic amine. In another embodiment, the mutagenerator is an insertor such as ethidium bromide or proflavin. In another embodiment, the mutagenerator is X-rays.

[0030] In one embodiment, the chemical mutagenic agent is selected from the group consisting of ethylmethanesulfonate (EMS), methylmethanesulfonate, diethylsulfonate, dimethyl sulfate, dimethyl sulfoxide, diethylnitrosamine, N-nitroso-N-methylurea, N-methyl-N-nitrosourea, N-nitroso-N-diethylurea, arsenic, colchicine, ethyleneimine, nitrosomethylurea, nitrosoguanidine, nitrite, hydroxylamine, ethylene oxide, diepoxybutane, sodium azide, maleic acid hydrazide, cyclophosphamide, diazoacetylbutane, psoralen, benzene, datura extract, bromodeoxyuridine, and beryllium oxide.

[0031] In another embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an RNA-inducible CRISPR nuclease manipulated by feeding the polynucleotide to a ribonucleoprotein. In another embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an RNA-inducible CRISPR nuclease manipulated by feeding the polynucleotide to a CRISPR nuclease. In another embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an RNA-inducible CRISPR nuclease manipulated by feeding the polynucleotide to a zinc finger nuclease. In another embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an RNA-inducible CRISPR nuclease manipulated by feeding the polynucleotide to a TALEN. In another embodiment, a polynucleotide encoding a wild-type RNA-inducible CRISPR nuclease is edited to produce an engineered RNA-inducible CRISPR nuclease by subjecting the polynucleotide to a meganuclease.

[0032] In some embodiments, the RNA-inducible CRISPR nuclease is a class 1 RNA-inducible CRISPR nuclease. In some embodiments, the RNA-inducible CRISPR nuclease is a class 1 RNA-inducible CRISPR nuclease selected from the group consisting of types I, IA, IB, IC, ID, IE, IF, IU, III, IIIA, IIIB, IIIC, IIID, IV, IVA, and IVB. In some embodiments, the RNA-inducible CRISPR nuclease is a class 2 CRISPR-Cas. In some embodiments, the RNA-inducible CRISPR nuclease is a class 2 RNA-inducible CRISPR nuclease selected from the group consisting of types II, IIA, IIB, IIC, V, and VI.

[0033] In one embodiment, the RNA-inducible CRISPR nuclease is Cas12a nuclease (also called Cpf1 nuclease). In another embodiment, the RNA-inducible CRISPR nuclease is Cas9 nuclease. In another embodiment, the RNA-inducible CRISPR nuclease is CasX nuclease. In another embodiment, the RNA-inducible CRISPR nuclease is CasY nuclease. In another embodiment, the RNA-inducible CRISPR nuclease is C2c2 nuclease. In one embodiment, the RNA-inducible CRISPR nuclease is selected from the group consisting of Cas12a nuclease, Cas9 nuclease, CasX nuclease, CasY nuclease, and C2c2 nuclease.

[0034] In some embodiments, the RNA-induced CRISPR nuclease is the Cas12a nuclease (also known as the Cpf1 nuclease). In other embodiments, the RNA-induced CRISPR nuclease is the Cas12a (LbCas12a) nuclease of Lachnospiraceae bacteria.

[0035] In another embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated Cas9 nuclease. In another embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated CasX nuclease. In another embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated CasY nuclease. In another embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated C2c2 nuclease. In one embodiment, the manipulated RNA-inducible CRISPR nuclease is selected from the group consisting of a manipulated Cas12a nuclease, a manipulated Cas9 nuclease, a manipulated CasX nuclease, a manipulated CasY nuclease, and a manipulated C2c2 nuclease.

[0036] In one embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated Cas12a nuclease (also known as Cpf1 nuclease). In another embodiment, the manipulated RNA-inducible CRISPR nuclease is a manipulated Cas12a (LbCas12a) nuclease from a Lachnospiraceae bacterium.

[0037] In one embodiment, the Cas12a nuclease contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 2. In another embodiment, the Cas12a nuclease contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 2. In yet another embodiment, the Cas12a nuclease contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. In yet another embodiment, the Cas12a nuclease contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 2. In yet another embodiment, the Cas12a nuclease contains an amino acid sequence that is 100% identical to SEQ ID NO: 2.

[0038] In one embodiment, the manipulated RNA-inducible CRISPR nuclease contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

[0039] In another embodiment, the manipulated Cas9 nuclease was used against Streptococcus, Haloferax, Anabaena, Mycobacterium, Aeropyvrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, and Methanobacterium. Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacteriunum, Streptomyces, Aquifex, Porphybromonas, Chlorobium, Thermus (T Hermus), Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desperadoxes Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium,It is derived from a bacterial genus selected from the group consisting of Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0040] In another embodiment, the manipulated Cas12a nuclease is effective against Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Spirochete, Lactobacillus, and Eubacterium. terium), Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus Bacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus (B It is derived from bacterial genera selected from the group consisting of revibacilus, methylobacterium, acidaminococcus, peregrinibacteria, butyrivibrio, parcubacteria, Smithella, candidatus, moraxella, and leptospira.

[0041] In some embodiments, the present disclosure provides nucleic acid sequences encoding manipulated RNA-inducible CRISPR nucleases provided herein.

[0042] When an RNA-induced CRISPR nuclease forms a complex with a guide RNA, the entire system is called a "ribonucleoprotein." The guide RNA guides the ribonucleoprotein to a complementary target sequence where the CRISPR-related protein cleaves one or both strands of DNA. Depending on the protein, the cleavage can occur within a specific number of nucleotides from the PAM site (e.g., between 18 and 23 nucleotides for Cas12a). The PAM site is required only for type I and type II CRISPR-related proteins; type III CRISPR-related proteins do not require a PAM site for proper targeting or cleavage.

[0043] In one embodiment, the manipulated RNA-inducible CRISPR nuclease provided herein is part of a ribonucleoprotein. In another embodiment, the RNA-inducible CRISPR nuclease provided herein is part of a ribonucleoprotein.

[0044] mutation As used herein, “mutation” refers to a modification to a nucleic acid or amino acid sequence that does not exist in nature, compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. When identifying a mutation, it will be understood that the reference sequence should be derived from the same nucleic acid (e.g., gene, non-coding RNA) or amino acid (e.g., protein). As a non-limiting example, when an engineered LbCas12a nuclease containing a mutation is compared to a wild-type sequence, the wild-type sequence must be an endogenous LbCas12a sequence from the same species, rather than a homogeneous Cas12a sequence from a different bacterial species or a different RNA-induced CRISPR nuclease sequence (e.g., a Cas9 sequence). As used herein, “wild-type” sequence refers to a naturally occurring amino acid or nucleotide sequence.

[0045] In one embodiment, the mutation includes the insertion of at least one nucleotide or amino acid. In another embodiment, the mutation includes the deletion of at least one nucleotide or amino acid. In yet another embodiment, the mutation includes the substitution of at least one nucleotide or amino acid. In yet another embodiment, the mutation includes the inversion of at least two nucleotides or amino acids. In yet another embodiment, the mutation is selected from the group consisting of insertions, deletions, and substitutions.

[0046] In one embodiment, the manipulated Cas12a nuclease includes a substitution of the amino acid at position 925 compared to SEQ ID NO: 2. In another embodiment, the manipulated Cas12a nuclease includes a substitution of the amino acid at position 1138 compared to SEQ ID NO: 2. In one embodiment, the manipulated Cas12a nuclease includes a deletion of the amino acid at position 925 compared to SEQ ID NO: 2. In another embodiment, the manipulated Cas12a nuclease includes a deletion of the amino acid at position 1138 compared to SEQ ID NO: 2. In one embodiment, the manipulated Cas12a nuclease includes an insertion of at least one amino acid at position 925 compared to SEQ ID NO: 2. In another embodiment, the manipulated Cas12a nuclease includes an insertion of at least one amino acid at position 1138 compared to SEQ ID NO: 2.

[0047] DNA catalytic domain As used herein, “DNA catalytic domain” refers to a domain (or region) of an amino acid sequence that can influence the cleavage of nucleic acid molecules.

[0048] In one embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the DNA catalytic domain. In another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least two mutations in the DNA catalytic domain. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least three mutations in the DNA catalytic domain. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in each of at least two DNA catalytic domains.

[0049] In one embodiment, the DNA catalytic domain includes a RuvC domain.

[0050] In Cas9 and similar proteins, the RuvC domain may contain three discontinuous regions (RuvC-I, RuvC-II, and RuvC-III) including an HNH domain inserted between RuvC-II and RuvC-III. All three RuvC regions contribute to the nuclease activity of RuvC. In Cas9, RuvC cleaves the untargeted strand of double-stranded nucleic acids. The RuvC domain contains six beta sheets surrounded by four alpha helices. The RuvC domain is characterized by InterPro as belonging to Pfam number PF18541. See, for example, RuvC endonuclease subdomain 3 at www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR041383.

[0051] Alternatively, in Cas12a, the RuvC domain includes the Nuc domain and the arginine-rich bridge helix domain. See, for example, Cas12a, RuvC nuclease domain at www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR040852. In some embodiments, the Nuc domain is located between the RuvC-II domain and the RuvC-III domain.

[0052] In some embodiments, the DNA catalytic domain includes a Nuc domain. See, for example, the Cas12a nuclease domain at www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR040882.

[0053] In one embodiment, the RuvC domain includes a RuvC-I domain, a RuvC-II domain, a RuvC-III domain, or any combination thereof. In another embodiment, the RuvC domain further includes an HNH domain. In yet another embodiment, the RuvC domain includes an HNH domain between the RuvC-II domain and the RuvC-III domain. In yet another embodiment, the RuvC domain further includes a Nuc domain. In yet another embodiment, the RuvC domain further includes an arginine-rich bridge helix domain.

[0054] In some embodiments, the DNA catalytic domain includes an HNH domain. In Cas9, HNH cleaves a targeted strand of double-stranded nucleic acid. See, for example, the HNH nuclease at www(dot)ebi(dot)ac(dot)uk / interpro / entry / IPR003615.

[0055] In one embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the RuvC domain. In another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the RuvC-I domain. In another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the RuvC-II domain. In another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the RuvC-III domain. In another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the Nuc domain. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease contains at least one mutation in the HNH domain. In a further embodiment, the engineered RNA-inducible CRISPR nuclease contains any combination of mutations in the RuvC-I domain, RuvC-II domain, RuvC-III domain, Nuc domain, or HNH domain.

[0056] Decreased nonspecific cleavage of ssDNA In some embodiments, manipulated RNA-induced CRISPR nucleases exhibit the ability to cleave target double-stranded DNA (dsDNA).

[0057] In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at the same rate as its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 90% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 80% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 70% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 60% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 50% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 40% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 30% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 25% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease.In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 20% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 15% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 10% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of at least 5% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the manipulated RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate that is at least 1% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease.

[0058] In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 5% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 10% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 25% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 50% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 75% to 95% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 50% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 35% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 25% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 15% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease.In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 1% to 10% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 5% to 35% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves target dsDNA at a rate of 5% to 15% of the dsDNA cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease.

[0059] In some embodiments, wild-type RNA-inducible CRISPR nucleases nonspecifically cleave non-target ssDNA. As used herein, “nonspecific cleavage” or “nonspecific cleavage” means that an RNA-inducible CRISPR nuclease cleaves a nucleic acid sequence that is not complementary to the nuclease’s guide RNA. As used herein, “non-target ssDNA” means an ssDNA molecule that is not complementary to the guide nucleic acid.

[0060] In some embodiments, the engineered RNA-inducible CRISPR nuclease is unable to nonspecifically cleave nontarget ssDNA. In some embodiments, the engineered RNA-inducible CRISPR nuclease has a reduced ability to nonspecifically cleave nontarget ssDNA compared to its reference wild-type RNA-inducible CRISPR nuclease. In some embodiments, the engineered RNA-inducible CRISPR nuclease includes a DNA catalytic domain that is unable to nonspecifically cleave nontarget ssDNA. In other embodiments, the engineered RNA-inducible CRISPR nuclease includes a DNA catalytic domain that has a reduced ability to nonspecifically cleave nontarget ssDNA compared to its reference wild-type RNA-inducible CRISPR nuclease.

[0061] In some embodiments, the engineered RNA-inducible CRISPR nuclease does not exhibit detectable nonspecific cleavage of ssDNA. ssDNA cleavage can be detected, for example, by isolating ssDNA exposed to the engineered RNA-inducible CRISPR nuclease for at least 180 minutes at 37°C, and by electrophoresis of the isolated DNA on an agarose gel to detect the cleavage fragments. If no cleavage fragments are observed, those skilled in the art will determine that the engineered RNA-inducible CRISPR nuclease does not exhibit detectable cleavage of ssDNA.

[0062] In another embodiment, the manipulated RNA-inducible CRISPR nuclease exhibits a reduced nonspecific ssDNA cleavage rate compared to its reference wild-type RNA-inducible CRISPR nuclease.

[0063] In one embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 90% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 80% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 70% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 60% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 50% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 40% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 30% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 25% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA.In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 20% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 15% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 10% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the manipulated RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate less than 5% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA.

[0064] In one embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 5% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 10% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 25% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 50% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 75% to 95% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 50% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 35% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 25% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA.In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 15% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 1% to 10% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In yet another embodiment, the engineered RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 5% to 35% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA. In another embodiment, the manipulated RNA-inducible CRISPR nuclease nonspecifically cleaves nontarget ssDNA at a rate of 5% to 15% of the nonspecific cleavage rate of its reference wild-type RNA-inducible CRISPR nuclease at the same nontarget ssDNA.

[0065] The cleavage rate of ssDNA or dsDNA can be measured by supplying a known amount of ssDNA or dsDNA to a manipulated RNA-inducible CRISPR nuclease or its reference wild-type RNA-inducible CRISPR nuclease for a specific amount of time, and then determining how much of the original ssDNA or dsDNA remains intact and how much of the original ssDNA or dsDNA has been cleaved.

[0066] In one embodiment, the cleavage rate of ssDNA or dsDNA is measured within 180 minutes of introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 150 minutes of introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 120 minutes of introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 90 minutes of introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 60 minutes of introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 30 minutes of introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 15 minutes of introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 10 minutes of introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule.

[0067] In one embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 45°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 42°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 40°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 37°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 35°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 30°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature below 25°C.

[0068] In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 20°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 25°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 30°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 35°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 37°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 40°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of at least 42°C.

[0069] In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 20°C to 45°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 20°C to 40°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 20°C to 37°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 20°C to 35°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 20°C to 30°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 25°C to 45°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 25°C to 40°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 25°C to 37°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 25°C to 35°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 30°C to 45°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 30°C to 40°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 30°C to 37°C. In another embodiment, the cleavage rate of ssDNA or dsRNA is measured when the cleavage occurs at a temperature of 35°C to 42°C.

[0070] In one embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 300 minutes after introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 250 minutes after introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 200 minutes after introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 180 minutes after introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 150 minutes after introducing a modified RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 120 minutes after introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 90 minutes after introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 60 minutes after introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 30 minutes after introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 15 minutes after introducing a manipulated RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule.

[0071] In one embodiment, the cleavage rate of ssDNA or dsDNA is measured within 180 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 150 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 120 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 90 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 60 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 30 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 15 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured within 10 minutes of introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule.

[0072] In one embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 300 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 250 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 200 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 180 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In yet another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 150 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 120 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 90 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 60 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 30 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule. In another embodiment, the cleavage rate of ssDNA or dsDNA is measured between 5 and 15 minutes after introducing RNA-induced CRISPR nuclease into the ssDNA or dsDNA molecule.

[0073] In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules in vivo. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules in vivo. In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules in vitro. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules in vitro. In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules ex vivo. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules ex vivo.

[0074] In one embodiment, RNA-induced CRISPR nucleases cleave dsDNA molecules in vivo. In another embodiment, RNA-induced CRISPR nucleases cleave ssDNA molecules in vivo. In one embodiment, RNA-induced CRISPR nucleases cleave dsDNA molecules in vitro. In another embodiment, RNA-induced CRISPR nucleases cleave ssDNA molecules in vitro. In one embodiment, RNA-induced CRISPR nucleases cleave dsDNA molecules ex vivo. In another embodiment, RNA-induced CRISPR nucleases cleave ssDNA molecules ex vivo.

[0075] As used herein, “in vivo” means within living cells, tissues, or organisms. As used herein, “in vitro” means within laboratory equipment. Non-limiting examples of laboratory equipment include test tubes, flasks, beakers, graduated cylinders, pipettes, Petri dishes, and microtiter plates. As used herein, “ex vivo” means cells or tissues of biological origin in the external environment. Non-limiting examples include plant protoplasts in Petri dishes or test tubes, which would be considered ex vivo.

[0076] magnesium DNA catalytic domains often require magnesium for proper function. In one embodiment, the disclosure provides a method for reducing ssDNA cleavage induced by RNA-induced CRISPR nucleases, comprising the step of contacting an RNA-induced CRISPR nuclease with a target site in a solution, wherein the solution contains MgCl2 at a concentration of less than 10 mM, and the reduction in ssDNA cleavage is compared to cleavage induced by RNA-induced CRISPR nucleases in a control solution containing MgCl2 at a concentration of 10 mM or more.

[0077] In another embodiment, the disclosure provides a method for reducing ssDNA cleavage induced by RNA-induced CRISPR nucleases, comprising the step of contacting an RNA-induced CRISPR nuclease with a target site in a solution, wherein the solution contains Mg at a concentration of less than 10 mM. 2+ It contains Mg, and a decrease in ssDNA cleavage occurs at concentrations of 10 mM or higher. 2+ The present invention provides a method that compares cleavage induced by RNA-induced CRISPR nuclease in a control solution containing [the specified substance].

[0078] In one embodiment, the solution contains MgCl2 at a concentration of 10 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 9.5 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 9 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 8.5 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 8 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 7.5 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 7 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 6.5 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 6 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 5 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 4.5 mM or less. In one embodiment, the solution contains MgCl2 at a concentration of 4 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 3.5 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 3 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 2.5 mM or less. In one embodiment, the solution contains MgCl2 at a concentration of 2 mM or less. In one embodiment, the solution contains MgCl2 at a concentration of 1.5 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 1 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 0.5 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 0.2 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 0.1 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 0.05 mM or less. In yet another embodiment, the solution contains MgCl2 at a concentration of 0.02 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.01 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.005 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.001 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.0005 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.0001 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.00005 mM or less. In another embodiment, the solution contains MgCl2 at a concentration of 0.00001 mM or less. In another embodiment, the solution does not contain MgCl2.

[0079] In certain embodiments, the solution contains MgCl2 at a concentration of 0.00001 mM to 10 mM. In certain embodiments, the solution contains MgCl2 at a concentration of 0.00001 mM to 5 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.0001 mM to 10 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.0001 mM to 5 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.001 mM to 10 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.001 mM to 5 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.01 mM to 10 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.01 mM to 5 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.1 mM to 10 mM. In other embodiments, the solution contains MgCl2 at a concentration of 0.1 mM to 5 mM. In other embodiments, the solution contains MgCl2 at a concentration of 1 mM to 10 mM. In other embodiments, the solution contains MgCl2 at a concentration of 5 mM to 10 mM.

[0080] In certain embodiments, the control solution contains MgCl2 at a concentration of 5 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 7.5 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 10 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 12.5 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 15 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 17.5 mM or higher. In other embodiments, the control solution contains MgCl2 at a concentration of 20 mM or higher.

[0081] In certain embodiments, the solution contains Mg at a concentration of 10 mM or less. 2+ In certain embodiments, the solution contains Mg at a concentration of 7.5 mM or less. 2+ In other embodiments, the solution contains Mg at a concentration of 5 mM or less. 2+ In other embodiments, the solution contains Mg at a concentration of 5 mM or less. 2+ In other embodiments, the solution contains Mg at a concentration of 2.5 mM or less. 2+ In other embodiments, the solution contains Mg at a concentration of 1 mM or less. 2+ In other embodiments, the solution contains Mg at a concentration of 0.5 mM or less. 2+It contains. In another embodiment, the solution contains Mg at a concentration of 0.2 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.1 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.05 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.02 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.01 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.005 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.001 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.0005 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.0001 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.00005 mM or less. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.00001 mM or less. 2+ It contains. In another embodiment, the solution contains Mg 2+ It does not include.

[0082] In one embodiment, the solution contains Mg at a concentration of 0.00001 mM to 10 mM. 2+ It contains. In one embodiment, the solution contains Mg at a concentration of 0.00001 mM to 5 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.0001 mM to 10 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.0001 mM to 5 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.001 mM to 10 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.001 mM to 5 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.01 mM to 10 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.01 mM to 5 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.1 mM to 10 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 0.1 mM to 5 mM. 2+It contains. In another embodiment, the solution contains Mg at a concentration of 1 mM to 10 mM. 2+ It contains. In another embodiment, the solution contains Mg at a concentration of 5 mM to 10 mM. 2+ Includes.

[0083] In one embodiment, the control solution contains Mg at a concentration of 5 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 7.5 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 10 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 12.5 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 15 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 17.5 mM or higher. 2+ It includes. In another embodiment, the control solution contains Mg at a concentration of 20 mM or higher. 2+ Includes.

[0084] In one embodiment, the solution is provided in vivo. In another embodiment, the solution is provided in vitro. In yet another embodiment, the solution is provided ex vivo. In one embodiment, the solution is provided to cells. In another embodiment, the solution is intracellular.

[0085] In one embodiment, the control solution is provided in vivo. In another embodiment, the control solution is provided in vitro. In yet another embodiment, the control solution is provided ex vivo. In one embodiment, the control solution is provided to cells. In another embodiment, the control solution is intracellular.

[0086] In one embodiment, the RNA-inducible CRISPR nuclease cleaves dsDNA in the solution provided herein. In another embodiment, the RNA-inducible CRISPR nuclease cleaves ssDNA in the solution provided herein. In one embodiment, the RNA-inducible CRISPR nuclease cleaves dsDNA but not ssDNA in the solution provided herein. In another embodiment, the RNA-inducible CRISPR nuclease cleaves ssDNA at a reduced rate in the solution provided herein compared to the ssDNA cleavage rate of the RNA-inducible CRISPR nuclease in a control solution.

[0087] EDTA EDTA (ethylenediaminetetraacetic acid) is a well-known chelating agent that captures divalent and trivalent metal ions such as calcium and magnesium. This ability prevents DNA and RNA degradation by inactivating metal-dependent enzymes that act as nucleases.

[0088] In another embodiment, the disclosure provides a method for reducing ssDNA cleavage induced by RNA-induced CRISPR nucleases, comprising the step of contacting an RNA-induced CRISPR nuclease with a target site in a solution, wherein the solution contains EDTA at a concentration of 0.1 mM or higher, and the reduction in ssDNA cleavage is compared to cleavage induced by RNA-induced CRISPR nucleases in a control solution containing EDTA at a concentration of less than 0.1 mM. In another embodiment, the solution contains EDTA at a concentration of 0.1 mM or higher and MgCl2 at a concentration of 10 mM or higher.

[0089] In some embodiments, the solution contains EDTA at a concentration of 0.1 mM or higher. In some embodiments, the solution contains EDTA at a concentration of 1 mM or higher. In some embodiments, the solution contains EDTA at a concentration of 5 mM or higher. In some embodiments, the solution contains EDTA at a concentration of 10 mM or higher. In some embodiments, the solution contains EDTA at a concentration of 15 mM or higher. In some embodiments, the solution contains EDTA at a concentration of 20 mM or higher.

[0090] cell In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules in cells. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules in cells. In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules in prokaryotic cells. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules in prokaryotic cells. In one embodiment, the engineered RNA-inducible CRISPR nuclease cleaves dsDNA molecules in eukaryotic cells. In another embodiment, the engineered RNA-inducible CRISPR nuclease cleaves ssDNA molecules in eukaryotic cells.

[0091] In one embodiment, the target nucleic acid is located within a cell. In another embodiment, the target nucleic acid is located within a prokaryotic cell. In yet another embodiment, the target nucleic acid is located within a eukaryotic cell.

[0092] In some aspects, prokaryotic cells are cells derived from phyla selected from a group consisting of prokaryotic cells, such as Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydie, Chlorobi, Chloroflexi, Chrysiogenetes, Coprothermobacterota, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, and Dicty These are cells derived from a phylum selected from the group consisting of oglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotoga, and Verrucomicrobia. In another embodiment, prokaryotic cells are Escherichia coli cells. In another embodiment, the prokaryotic cells are selected from genera chosen from the group consisting of Escherichia, Agrobacterium, Rhizobium, Sinorhizobium, and Staphylococcus.

[0093] In one aspect, eukaryotic cells are ex vivo cells. In another aspect, eukaryotic cells are plant cells. In another aspect, eukaryotic cells are plant cells in culture. In another aspect, eukaryotic cells are angiosperm cells. In another aspect, eukaryotic cells are gymnosperm cells. In another aspect, eukaryotic cells are monocotyledonous plant cells. In another aspect, eukaryotic cells are dicotyledonous plant cells. In another aspect, eukaryotic cells are maize cells. In another aspect, eukaryotic cells are rice cells. In another aspect, eukaryotic cells are sorghum cells. In another aspect, eukaryotic cells are wheat cells. In another aspect, eukaryotic cells are canola cells. In another aspect, eukaryotic cells are alfalfa cells. In another aspect, eukaryotic cells are soybean cells. In another aspect, eukaryotic cells are cotton cells. In another aspect, eukaryotic cells are tomato cells. In another aspect, eukaryotic cells are potato cells. In yet another aspect, eukaryotic cells are cucumber cells. In another aspect, the eukaryotic cell is a millet cell. In yet another aspect, the eukaryotic cell is a barley cell. In yet another aspect, the eukaryotic cell is a Brassica cell. In yet another aspect, the eukaryotic cell is a grass cell. In yet another aspect, the eukaryotic cell is a Setaria cell. In yet another aspect, the eukaryotic cell is an Arabidopsis cell. In yet another aspect, the eukaryotic cell is an algal cell.

[0094] In one aspect, plant cells are epithelial cells. In another aspect, plant cells are stomatal cells. In another aspect, plant cells are trichome cells. In another aspect, plant cells are root cells. In another aspect, plant cells are leaf cells. In another aspect, plant cells are callus cells. In another aspect, plant cells are protoplast cells. In another aspect, plant cells are pollen cells. In another aspect, plant cells are ovary cells. In another aspect, plant cells are flower cells. In another aspect, plant cells are meristem cells. In another aspect, plant cells are endosperm cells. In another aspect, plant cells do not contain reproductive material and do not mediate the natural reproduction of plants. In another aspect, plant cells are somatic plant cells.

[0095] In addition, the plant cells, tissues, and organs provided may be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristematic tissue, embryos, endosperm, roots, shoots, stems, sheaths, flowers, inflorescences, stalks, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarps, phloems, and vascular tissues.

[0096] In a further aspect, eukaryotic cells are animal cells. In another aspect, eukaryotic cells are animal cells in culture. In a further aspect, eukaryotic cells are human cells. In another aspect, eukaryotic cells are not human stem cells. In a further aspect, eukaryotic cells are human cells in culture. In a further aspect, eukaryotic cells are human somatic cells. In a further aspect, eukaryotic cells are cancer cells. In a further aspect, eukaryotic cells are mammalian cells. In a further aspect, eukaryotic cells are mouse cells. In a further aspect, eukaryotic cells are pig cells. In a further aspect, eukaryotic cells are bovine cells. In a further aspect, eukaryotic cells are chicken cells. In a further aspect, eukaryotic cells are reptile cells. In a further aspect, eukaryotic cells are amphibian cells. In a further aspect, eukaryotic cells are insect cells. In a further aspect, eukaryotic cells are arthropod cells. In a further aspect, eukaryotic cells are cephalopod cells. In a further embodiment, the eukaryotic cell is an arachnid cell. In a further embodiment, the eukaryotic cell is a mollusk cell. In a further embodiment, the eukaryotic cell is a nematode cell. In a further embodiment, the eukaryotic cell is a fish cell.

[0097] In another aspect, eukaryotic cells are protozoan cells. In another aspect, eukaryotic cells are fungal cells. In one aspect, fungal cells are yeast cells. In one aspect, yeast cells are Schizosaccharomyces pombe cells. In another aspect, yeast cells are Saccharomyces cerevisiae cells.

[0098] Guide nucleic acids In one embodiment, the method or composition provided herein comprises at least one guide nucleic acid or a nucleic acid encoding at least one guide nucleic acid, wherein the at least one guide nucleic acid forms a complex with an engineered RNA-inducible CRISPR nuclease, and the at least one guide nucleic acid hybridizes with a target nucleic acid molecule. In another embodiment, the ribonucleoprotein provided herein comprises an engineered RNA-inducible CRISPR nuclease and at least one guide nucleic acid. In yet another embodiment, the ribonucleoprotein provided herein comprises an RNA-inducible CRISPR nuclease and at least one guide nucleic acid.

[0099] As used herein, “guide nucleic acid” refers to a nucleic acid that forms a complex with a nuclease and subsequently guides the complex to a specific sequence in the target nucleic acid molecule, from which the guide nucleic acid and the target nucleic acid molecule share complementary sequences.

[0100] In one embodiment, the guide nucleic acid includes DNA. In another embodiment, the guide nucleic acid includes RNA. When the guide nucleic acid includes RNA, it may also be called “guide RNA.” In another embodiment, the guide nucleic acid includes both DNA and RNA. In another embodiment, the guide nucleic acid is single-stranded. In another embodiment, the guide nucleic acid is double-stranded. In yet another embodiment, the guide nucleic acid is partially double-stranded.

[0101] In another embodiment, the ribonucleoprotein provided herein comprises an engineered RNA-inducible CRISPR nuclease and at least one guide RNA.

[0102] In another embodiment, the guide nucleic acid contains at least 10 nucleotides. In another embodiment, the guide nucleic acid contains at least 11 nucleotides. In another embodiment, the guide nucleic acid contains at least 12 nucleotides. In another embodiment, the guide nucleic acid contains at least 13 nucleotides. In another embodiment, the guide nucleic acid contains at least 14 nucleotides. In another embodiment, the guide nucleic acid contains at least 15 nucleotides. In another embodiment, the guide nucleic acid contains at least 16 nucleotides. In another embodiment, the guide nucleic acid contains at least 17 nucleotides. In another embodiment, the guide nucleic acid contains at least 18 nucleotides. In another embodiment, the guide nucleic acid contains at least 19 nucleotides. In another embodiment, the guide nucleic acid contains at least 20 nucleotides. In another embodiment, the guide nucleic acid contains at least 21 nucleotides. In another embodiment, the guide nucleic acid contains at least 22 nucleotides. In another embodiment, the guide nucleic acid contains at least 23 nucleotides. In another embodiment, the guide nucleic acid contains at least 24 nucleotides. In another embodiment, the guide nucleic acid contains at least 25 nucleotides. In another embodiment, the guide nucleic acid contains at least 26 nucleotides. In another embodiment, the guide nucleic acid contains at least 27 nucleotides. In another embodiment, the guide nucleic acid contains at least 28 nucleotides. In another embodiment, the guide nucleic acid contains at least 30 nucleotides. In another embodiment, the guide nucleic acid contains at least 35 nucleotides. In another embodiment, the guide nucleic acid contains at least 40 nucleotides. In another embodiment, the guide nucleic acid contains at least 45 nucleotides. In another embodiment, the guide nucleic acid contains at least 50 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 50 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 40 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 30 nucleotides. In another embodiment, the guide nucleic acid contains 10 to 20 nucleotides. In another embodiment, the guide nucleic acid contains 16 to 28 nucleotides.In another embodiment, the guide nucleic acid comprises 16 to 25 nucleotides. In yet another embodiment, the guide nucleic acid comprises 16 to 20 nucleotides.

[0103] In some embodiments, the guide nucleic acid includes at least 70% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 75% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 80% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 85% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 90% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 91% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 92% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 93% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 94% sequence complementarity to the target nucleic acid sequence. In some embodiments, the guide nucleic acid includes at least 95% sequence complementarity to the target nucleic acid sequence. In one embodiment, the guide nucleic acid contains at least 96% sequence complementarity to the target nucleic acid sequence. In another embodiment, the guide nucleic acid contains at least 97% sequence complementarity to the target nucleic acid sequence. In another embodiment, the guide nucleic acid contains at least 98% sequence complementarity to the target nucleic acid sequence. In another embodiment, the guide nucleic acid contains at least 99% sequence complementarity to the target nucleic acid sequence. In another embodiment, the guide nucleic acid contains 100% sequence complementarity to the target nucleic acid sequence. In yet another embodiment, the guide nucleic acid contains 70% to 100% sequence complementarity to the target nucleic acid sequence. In yet another embodiment, the guide nucleic acid contains 80% to 100% sequence complementarity to the target nucleic acid sequence. In yet another embodiment, the guide nucleic acid contains 90% to 100% sequence complementarity to the target nucleic acid sequence.

[0104] Some RNA-induced CRISPR nucleases, such as CasX and Cas9, require another non-coding RNA component, called transactivating crRNA (tracrRNA), to be functionally active. The guide nucleic acid molecules provided herein can combine crRNA and tracrRNA into a single nucleic acid molecule, referred herein as “single guide RNA” (sgRNA). The gRNA guides the active CasX complex to a target site where CasX can cleave the target site.

[0105] In one embodiment, the guide nucleic acid includes crRNA. In another embodiment, the guide nucleic acid includes tracrRNA. In yet another embodiment, the guide nucleic acid includes sgRNA.

[0106] In some embodiments, the guide nucleic acids provided herein can be expressed from a recombinant vector in vivo. In some embodiments, the guide nucleic acids provided herein can be expressed from a recombinant vector in vitro. In some embodiments, the guide nucleic acids provided herein can be expressed from a recombinant vector ex vivo. In some embodiments, the guide nucleic acids provided herein can be expressed from a nucleic acid molecule in vivo. In some embodiments, the guide nucleic acids provided herein can be expressed from a nucleic acid molecule in vitro. In some embodiments, the guide nucleic acids provided herein can be expressed from a nucleic acid molecule ex vivo. In other embodiments, the guide nucleic acids provided herein can be synthesized synthetically.

[0107] target nucleic acid In one embodiment, the dsRNA molecule contains a target nucleic acid. In another embodiment, the dsRNA molecule contains a target region.

[0108] As used herein, “target nucleic acid,” “target nucleic acid molecule,” or “target nucleic acid sequence” refers to a selected nucleic acid molecule or a selected sequence or region of a nucleic acid molecule that is to be modified (e.g., cleaved). Similarly, “target dsRNA” refers to a selected double-stranded DNA molecule that is to be modified (e.g., cleaved).

[0109] As used herein, “target region” or “targeted region” refers to the portion of a target nucleic acid that is cleaved by an engineered RNA-induced CRISPR nuclease. In contrast to non-target nucleic acids (e.g., non-target ssDNA) or non-target regions, a target region contains significant complementarity to a guide nucleic acid or guide RNA. In one embodiment, the target region is 100% complementary to the guide nucleic acid. In another embodiment, the target region is 99% complementary to the guide nucleic acid. In another embodiment, the target region is 98% complementary to the guide nucleic acid. In another embodiment, the target region is 97% complementary to the guide nucleic acid. In another embodiment, the target region is 96% complementary to the guide nucleic acid. In another embodiment, the target region is 95% complementary to the guide nucleic acid. In another embodiment, the target region is 94% complementary to the guide nucleic acid. In another embodiment, the target region is 93% complementary to the guide nucleic acid. In another embodiment, the target region is 92% complementary to the guide nucleic acid. In another embodiment, the target region is 91% complementary to the guide nucleic acid. In another embodiment, the target region is 90% complementary to the guide nucleic acid. In another embodiment, the target region is 85% complementary to the guide nucleic acid. In another embodiment, the target region is 80% complementary to the guide nucleic acid. In one embodiment, the target region is adjacent to a nucleic acid sequence that is 100% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 99% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 98% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 97% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 96% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 95% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 94% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 93% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 92% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 91% complementary to the guide nucleic acid. In another embodiment, the target region is adjacent to a nucleic acid sequence that is 90% complementary to the guide nucleic acid.In another embodiment, the target region is adjacent to a nucleic acid sequence that is 85% complementary to the guide nucleic acid. In yet another embodiment, the target region is adjacent to a nucleic acid sequence that is 80% complementary to the guide nucleic acid.

[0110] In one embodiment, the target region includes at least one PAM site. In another embodiment, the target region is adjacent to a nucleic acid sequence containing at least one PAM site. In yet another embodiment, the target region is within 5 nucleotides of at least one PAM site. In yet another embodiment, the target region is within 10 nucleotides of at least one PAM site. In yet another embodiment, the target region is within 15 nucleotides of at least one PAM site. In yet another embodiment, the target region is within 20 nucleotides of at least one PAM site. In yet another embodiment, the target region is within 25 nucleotides of at least one PAM site. In yet another embodiment, the target region is within 30 nucleotides of at least one PAM site.

[0111] In some embodiments, the target nucleic acid includes RNA. In other embodiments, the target nucleic acid includes DNA. In some embodiments, the target nucleic acid is single-stranded. In other embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid includes single-stranded RNA. In some embodiments, the target nucleic acid includes ssDNA. In some embodiments, the target nucleic acid includes double-stranded RNA. In some embodiments, the target nucleic acid includes dsDNA. In some embodiments, the target nucleic acid includes genomic DNA. In some embodiments, the target nucleic acid is located within the nuclear genome. In some embodiments, the target nucleic acid includes chromosomal DNA. In some embodiments, the target nucleic acid includes plasmid DNA. In some embodiments, the target nucleic acid is located within a plasmid. In some embodiments, the target nucleic acid includes mitochondrial DNA. In some embodiments, the target nucleic acid is located within the mitochondrial genome. In some embodiments, the target nucleic acid includes plastid DNA. In some embodiments, the target nucleic acid is located within the plastid genome. In some embodiments, the target nucleic acid includes chloroplast DNA. In some embodiments, the target nucleic acid is located within the chloroplast genome. In one embodiment, the target nucleic acid is located within a genome selected from the group consisting of the nuclear genome, the mitochondrial genome, and the plastid genome.

[0112] In one embodiment, the target nucleic acid codes for a gene. As used herein, “gene” means a polynucleotide capable of producing a functional unit (e.g., a protein or a non-coding RNA molecule). A gene may include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. A “gene sequence” may include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination thereof. In one embodiment, the gene codes for a non-protein-coding RNA molecule or its precursor. In another embodiment, the gene codes for a protein. In some embodiments, the target nucleic acid is selected from the group consisting of a promoter, enhancer sequence, leader sequence, transcription start site, transcription stop site, polyadenylation site, exon, intron, splice site, 5'-UTR, 3'-UTR, protein-coding sequence, non-protein-coding sequence, miRNA, pre-miRNA, and miRNA-binding site.

[0113] Non-protein-coding RNA molecules include, but are not limited to, microRNAs (miRNAs), miRNA precursors (premiRNAs), small interfering RNAs (siRNAs), small RNAs (18-26 nt in length) and their encoding precursors, heterochromatin siRNAs (hc-siRNAs), Piwi-binding RNAs (piRNAs), hairpin double-stranded RNAs (hairpin dsRNAs), trans-acting siRNAs (ta-siRNAs), naturally occurring antisense siRNAs (nat-siRNAs), CRISPR RNAs (crRNAs), tracer RNAs (tracrRNAs), guide RNAs (gRNAs), and single guide RNAs (sgRNAs).

[0114] Nucleic acids and polypeptides The use of the terms “polynucleotide” or “nucleic acid molecule” is not intended to limit this disclosure to polynucleotides containing deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules may include deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of this disclosure also encompass, but are not limited to, all forms of sequences, including single-stranded, double-stranded, hairpin, stem-loop structures, etc. In some embodiments, the nucleic acid molecule provided herein is a DNA molecule. In other embodiments, the nucleic acid molecule provided herein is an RNA molecule. In some embodiments, the nucleic acid molecule provided herein is single-stranded. In other embodiments, the nucleic acid molecule provided herein is double-stranded.

[0115] In one embodiment, the methods and compositions provided herein include a vector. As used herein, the terms “vector” and “plasmid” are interchangeable and refer to a circular, double-stranded DNA molecule physically separated from chromosomal DNA. In one embodiment, the plasmid or vector used herein can be replicated in vivo. In another embodiment, a nucleic acid encoding a catalytically inactive inducible nuclease is provided in the vector. In yet another embodiment, a nucleic acid encoding a guide nucleic acid is provided in the vector. In yet yet another embodiment, the nucleic acid encoding the catalytically inactive inducible nuclease and the nucleic acid encoding the guide nucleic acid are provided in a single vector.

[0116] In one embodiment, the disclosure provides a polynucleotide encoding an engineered RNA-inducible CRISPR nuclease. In another embodiment, the vector comprises a polynucleotide encoding an engineered RNA-inducible CRISPR nuclease. In one embodiment, the disclosure provides a polynucleotide encoding an RNA-inducible CRISPR nuclease. In another embodiment, the vector comprises a polynucleotide encoding an RNA-inducible CRISPR nuclease. In one embodiment, the disclosure provides a polynucleotide encoding a guide nucleic acid. In another embodiment, the disclosure provides a vector encoding a guide nucleic acid.

[0117] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides may be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein may be encoded by nucleic acid molecules provided herein.

[0118] Nucleic acids can be isolated using techniques commonplace in the art. For example, nucleic acids can be isolated using any method, including, but not limited to, recombinant nucleic acid techniques and / or polymerase chain reaction (PCR). Common PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual*, edited by Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing nucleic acids in an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The purity of polypeptides can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0119] Nucleic acids can be detected using hybridization, though not exclusively. Hybridization between nucleic acids is discussed in detail by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0120] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity to the polypeptides provided herein can be produced using methods known in the art. The antibodies provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0121] As used herein in reference to two or more nucleotide or protein sequences, the term “percent identity” or “percent identical” is calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) on a comparison window; (ii) determining the number of positions in both sequences where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) exist, thereby obtaining the number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and then (iv) multiplying this quotient by 100% to obtain the percentage identity. When “percent identity” is calculated with respect to a reference sequence without specifying a particular comparison window, the percentage identity is determined by dividing the number of matching positions on the alignment region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and target) are optimally aligned (allowing for gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences obtained by dividing by the total number of positions in the query sequence over its entire length (or comparison window) and then multiplying by 100%. When the percentage of sequence identity is used in reference to a protein, it is recognized that non-identical residue positions are often distinguished by conservative amino acid substitutions, where the amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and therefore does not change the functional properties of the molecule. If sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to compensate for the conservative nature of the substitutions. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0122] The term “percent sequence complementarity” or “percent complementarity” as used herein in reference to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that optimally base-pair with or hybridize with the nucleotides of the target sequence, given that the query sequence and the target sequence are linearly arranged and optimally base-pair without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity may be between two DNA strands, between two RNA strands, or between a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folding or secondary structure) on a comparison window, (ii) determining the number of base-pairing positions between the two sequences on the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percentage complementarity of the two sequences. Optimal base-pairing of two sequences can be determined based on the pairing of known nucleotide bases such as GC, AT, and AU via hydrogen bonding. When "percent complementarity" is calculated with respect to a reference sequence without specifying a particular comparison window, percentage identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when the two sequences (query and target) optimally form base pairs (allowing for mismatches or nucleotides that do not form base pairs), the "percent complementarity" of the query sequence is equal to the number of base-paired positions between the two sequences, obtained by dividing the query sequence by the total number of positions in the query sequence over its entire length and then multiplying by 100%.

[0123] Various pairwise or multi-sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), are known in the art and can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences for their optimal alignment to calculate the percentage identity of sequences. Although other alignment and comparison methods are known in the art, the alignment and percentage identity (including the percentage identity range described above) between two sequences can be determined by the ClustalW algorithm. For example, Chenna R. et al., "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Research 31: 3497-3500 (2003); Thompson JD et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Research 22: pp. 4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal See 215:403–410 (1990) (the entire contents and disclosures of these are incorporated herein by reference).

[0124] As used herein, a first nucleic acid molecule can "hybridize" to a second nucleic acid molecule by non-covalent interactions (e.g., Watson-Crick base pairs) in a sequence-specific, antiparallel manner (i.e., nucleic acid specifically binds to complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairs include adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine [DNA, RNA]. Furthermore, for hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases pair with uracil. For example, G / U base pairs contribute to the degenerate (i.e., redundancy) of the genetic code in the context of tRNA anticodons that base-pair with codons in mRNA. In the context of this disclosure, guanine in the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule is considered complementary to uracil, and vice versa. Therefore, if a G / U base pair can be constructed at a given nucleotide position on the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule, that position is not considered non-complementary, but rather complementary.

[0125] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly in Chapter 11 and Table 11.1; and in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the "stringency" of hybridization.

[0126] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are acceptable. The appropriate conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are well-known variables in the art. The higher the degree of complementarity between two nucleotide sequences, the higher the melting point (Tm) value for the hybrid of nucleic acids having these sequences. For hybridization between nucleic acids with a short complementarity range (e.g., complementarity over 35 nucleotides or less), the location of the mismatch becomes important (see Sambrook et al.). Typically, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Examples of the minimum lengths of hybridizable nucleic acids are at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and the salt concentration of the washing solution can be adjusted as needed depending on factors such as the length and degree of complementarity of the complementary region.

[0127] It is understood in the art that the sequence of a polynucleotide does not need to be specifically hybridizable or 100% complementary to the sequence of its target nucleic acid that is hybridizable. Furthermore, a polynucleotide may hybridize across one or more segments (e.g., loop or hairpin structures) such that intervening or adjacent segments do not participate in the hybridization event. For example, if 18 of the 20 nucleotides in an antisense compound are complementary to the target region, and therefore the antisense nucleic acid that specifically hybridizes represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides may cluster with complementary nucleotides or be scattered, and do not need to be contiguous with each other or with complementary nucleotides. The percentage complementarity between specific ranges of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (basic local alignment search tools) and the PowerBLAST program (see Altschul et al., J. Mol. Biol., 1990, 215, pp. 403-410; Zhang and Madden, Genome Res., 1997, 7, pp. 649-656), which are known in the art, or by using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, pp. 482-489) and the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings.

[0128] Transformation / Transfection Any method provided herein can be used for transient translocation or stable transformation of target cells (e.g., eukaryotic cells, prokaryotic cells). In one embodiment, a nucleic acid molecule encoding an engineered RNA-induced CRISPR nuclease is stably transformed. In another embodiment, a nucleic acid molecule encoding an engineered RNA-induced CRISPR nuclease is transiently transformed. In one embodiment, a nucleic acid molecule encoding an RNA-induced CRISPR nuclease is stably transformed. In another embodiment, a nucleic acid molecule encoding an RNA-induced CRISPR nuclease is transiently transformed. In one embodiment, a nucleic acid molecule encoding a guide nucleic acid is stably transformed. In another embodiment, a nucleic acid molecule encoding a guide nucleic acid is transiently transformed.

[0129] Several methods for transforming cells with recombinant nucleic acid molecules or constructs are known in the art and can be used according to the method of this application. Any suitable method or technique for cell transformation known in the art can be used according to the method of the present invention. Effective methods for plant transformation include bacterial-mediated transformation such as Agrobacterium-mediated or Rhizobium-mediated transformation, and microprojectile bombardment-mediated transformation. Various methods for regenerating or developing transgenic plants by transforming explants using transformation vectors via bacterial-mediated transformation or microprojectile bombardment, and then culturing these explants, are known in the art.

[0130] In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in cells by Agrobacterium-mediated transformation, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in cells by polyethylene glycol-mediated transformation, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in cells by bioristic transformation, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in cells by liposome-mediated transfection, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in cells by viral transduction, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in a cell by the use of one or more delivery particles, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in a cell by microinjection, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease. In some embodiments, the method includes providing an RNA-inducible CRISPR nuclease engineered in a cell by electroporation, or a nucleic acid encoding an engineered RNA-inducible CRISPR nuclease.

[0131] In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by Agrobacterium-mediated transformation. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by polyethylene glycol-mediated transformation. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by bioristic transformation. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by liposome-mediated transfection. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by viral transduction. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by the use of one or more delivery particles. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by microinjection. In some embodiments, the method includes providing a guide nucleic acid or a nucleic acid encoding a guide nucleic acid to a cell by electroporation.

[0132] In some embodiments, ribonucleoproteins are delivered to cells by a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, bioristic transformation, liposome-mediated transfection, viral transduction, use of one or more delivery particles, microinjection, and electroporation.

[0133] Other methods for transformation, such as vacuum immersion, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and are intended for use in conjunction with any method provided herein.

[0134] Methods for transforming cells are well known to those skilled in the art. For example, specific instructions for transforming plant cells by microprojectile bombardment (e.g., bioristic transformation) using recombinant DNA-coated particles can be found in U.S. Patents 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,153,812, and Agrobacterium-mediated transformation can be found in U.S. Patents 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Plant cells can be transformed with any of the nucleic acid molecules provided herein using any suitable method known to those skilled in the art.

[0135] Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Suitable cationic and neutral lipids for efficient receptor recognition lipofection of polynucleotides include those described in Felgner's WO91 / 17424; WO91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration).

[0136] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for the expression of one or more elements of nucleic acid molecules or proteins are as used in WO2014 / 093622 (PCT / US2013 / 074667). In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by delivery particles. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by delivery vesicles. In one embodiment, the delivery vesicles are selected from the group consisting of exosomes and liposomes. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by viral vectors. In one embodiment, the viral vectors are selected from the group consisting of adenovirus vectors, lentiviral vectors, and adeno-associated virus vectors. In another embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by nanoparticles. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes microinjection. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes polycations. In one embodiment, a method for providing nucleic acid molecules or proteins to cells includes a cationic oligopeptide.

[0137] In some embodiments, the delivery particles are selected from the group consisting of exosomes, adenovirus vectors, lentiviral vectors, adeno-associated virus vectors, nanoparticles, polycations, and cationic oligopeptides. In some embodiments, the methods provided herein include the use of one or more delivery particles. In other embodiments, the methods provided herein include the use of two or more delivery particles. In yet another embodiment, the methods provided herein include the use of three or more delivery particles.

[0138] Suitable agents for facilitating the transfer of proteins, nucleic acids, mutagens, and ribonucleoproteins into plant cells include agents that increase the permeability to the outside of the plant, or agents that increase the permeability of plant cells to oligonucleotides, polynucleotides, proteins, or ribonucleoproteins. Such agents for facilitating the transfer of compositions into plant cells include chemical agents, physical agents, or combinations thereof. Chemical agents for conditioning include (a) surfactants, (b) organic solvents or aqueous solutions, or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0139] Organic solvents useful for conditioning plants to permeate with polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents that are miscible with water or dissolve phosphonucleotides in non-aqueous systems (such as those used in synthetic reactions). Natural or synthetic oils, with or without surfactants or emulsifiers, can be used, such as plant-derived oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, which is publicly available online at www.herbicide.adjuvants.com), and oils having short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0140] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as animal fats, animal fat amines, or phospholipids) and organosilicon surfactants. Other useful surfactants include nonionic organosilicon surfactants, such as organosilicon surfactants containing trisiloxane ethoxylate surfactants, or silicone polyether copolymers such as the copolymer of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxypolypropylene glycol methyl ether (commercially available as Silwet® L-77).

[0141] Useful physical agents may include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, and garnet; (b) nanoparticles such as carbon nanotubes; or (c) physical forces. Carbon nanotubes are disclosed by Kam et al. (2004) / . Am. Chem. Soc, 126 (22): pp. 6850-6851, Liu et al. (2009) Nano Lett, 9(3): pp. 1007-1010, and Khodakovskaya et al. (2009) ACS Nano, 3(10): pp. 3221-3227. Examples of physical force agents include heating, cooling, application of positive pressure, or sonication. Embodiments of the method may optionally include incubation steps, neutralization steps (e.g., to neutralize acids, bases, or oxidizing agents, or to inactivate enzymes), washing steps, or a combination thereof. The method of the present invention may further include the application of other agents that would have an enhanced effect due to the silencing of certain genes. For example, if a polynucleotide is designed to modulate a gene that provides herbicide resistance, subsequent application of the herbicide may have a dramatic effect on the efficacy of the herbicide.

[0142] Examples of agents used for laboratory conditioning of plant cells for polynucleotide permeability include the application of chemical agents, enzymatic treatment, heating or cooling, treatment using positive or negative pressure, or sonication. Examples of agents used for conditioning plants in the field include chemical agents such as surfactants and salts.

[0143] In one embodiment, ssDNA or dsDNA is contacted with an RNA-inducible CRISPR nuclease manipulated in vivo. In another embodiment, ssDNA or dsDNA is contacted with an RNA-inducible CRISPR nuclease manipulated ex vivo. In yet another embodiment, ssDNA or dsDNA is contacted with an RNA-inducible CRISPR nuclease manipulated in vitro.

[0144] In one embodiment, the target nucleic acid is contacted by a ribonucleoprotein in vivo. In another embodiment, the target nucleic acid is contacted by a ribonucleoprotein ex vivo. In yet another embodiment, the target nucleic acid is contacted by a ribonucleoprotein in vitro.

[0145] Examples of recipient plant cells or explant targets for transformation include, but are not limited to, seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, sheath cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, phloem cells, bud cells, or vascular tissue cells. In another embodiment, the disclosure provides plant chloroplasts. In a further embodiment, the disclosure provides epidermal cells, stomatal cells, trichome cells, root hair cells, storage root cells, or tuber cells. In another embodiment, the disclosure provides protoplasts. In another embodiment, the disclosure provides plant callus cells. Any cells capable of regenerating a fertile plant are intended as useful recipient cells for the implementation of the disclosure. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or embryonic portions, apical meristem of seedlings, and microspores. These cells, which can proliferate as callus, can serve as recipient cells for transformation. Practical transformation methods and materials for producing transgenic plants of this disclosure (e.g., various culture media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Transformed explants, cells, or tissues can be subjected to further culture steps, such as callus induction, selection, and regeneration, as known in the art. Transformed cells, tissues, or explants containing recombinant DNA insertions can be propagated, developed, or regenerated into transgenic plants in cultures, plugs, or soil according to methods known in the art. In one embodiment, the disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of plants. In another embodiment, the disclosure provides plant cells that are reproductive material and mediate the natural reproduction of plants. In yet another embodiment, the disclosure provides plant cells that cannot sustain themselves by photosynthesis. In yet another embodiment, the disclosure provides somatic plant cells.Somatic cells, in contrast to germline cells, do not mediate reproduction in plants. In one embodiment, this disclosure provides non-reproductive plant cells. [Examples]

[0146] (Example 1) in vitro DNase activity assay An in vitro deoxyribonuclease (DNase) activity assay was developed to investigate the single-stranded (ss) and double-stranded (ds) DNase activity of the RNA-inducible CRISPR nuclease LbCas12a (Lachnospiraceae bacterium ND2006 Cas12a). Two DNA substrates were used in this assay. The synthetic dsDNA substrate used in the assay was Zm7.1, a 1700 bp PCR product (SEQ ID NO: 1), containing two unique target sites. The Cas9 target site (Cas9_Zm7.1) is a 350 nucleotide sequenced and recognized by a Cas9-specific single guide RNA (Cas9_Zm7.1_sgRNA), the sequence of which was previously disclosed in U.S. Patent Application Publication No. 2017 / 0166912, which is incorporated herein by reference in its entirety. This 1700bp product also contains a 382-nucleotide LbCas1 target site (Cas12a_Zm7.1) located in a sequence recognized by the Cas-specific guide RNA (Cas12a-Zm7.1_gRNA) (SEQ ID NO: 21). Demonstration of dsDNA cleavage activity by SpCas9 (Streptococcus pyogenes Cas9) and its homolog Cas9-zm7.1_sgRNA will yield 350bp and 1350bp DNA fragments. See Figure 1. Demonstration of dsDNA cleavage activity by LbCas12a and its homolog Cas12a-Zm7.1 gRNA will yield 382bp and 1318bp DNA fragments. See Figure 1.

[0147] The ssDNA substrate used in the assay was the M13mp18 ssDNA phage sequence (New England Biolabs, #N4040s) previously described and used in Chen et al., Science, 360:436-439 (2018) Apr 27;360(6387):436-439. These substrates were evaluated in individual and combined reactions to assess whether they could separate LbCas12a dsDNA cleavage from ssDNase activity.

[0148] Wild-type LbCas12a protein (SEQ ID NO: 2) and variants were expressed and purified from E. coli. For this purpose, the LbCas12a coding region was codon-optimized for optimal expression in E. coli cells (SEQ ID NO: 3). A histidine tag sequence (SEQ ID NO: 4) was introduced into the 5' end of the gene. In addition, two nuclear localization signals (NLS) (SEQ ID NOs: 5 and 6) were introduced into the 5' and 3' ends of the LbCas12a coding region. Finally, a unique Sph1 site was introduced into the 3' end of the DNA to produce an alanine residue at the C-terminus of the protein. The LbCas12a fusion protein used in the in vitro DNAse assay had the following configuration: HIStag:NLS:LbCas12a:NLS.

[0149] The reaction was carried out in a cleavage buffer consisting of 20 mM HEPES, 10 mM MgCl2, and 0.5 mM DTT, containing 26.7 nM dsDNA substrate and / or 12.54 nM M13 ssDNA substrate. Purified LbCas12a or LbCas12a variant protein was incubated with or without assembled with congeneral gRNA (100 μM) with dsDNA, ssDNA, or a combination of dsDNA and ssDNA. Protein amounts were adjusted to match the specific protein-DNA ratio being investigated for each reaction. Unless otherwise specified, the reaction was carried out at 37°C for 45 minutes and quenched at 65°C for 15 minutes using proteinase K treatment. Samples were separated and analyzed on 1.8% TBE agarose gels.

[0150] (Example 2) LbCas12a ssDNase activity It has been previously reported that Cas12a, when paired with its guide RNA and in the presence of target DNA, exhibits non-specific single-stranded (ss)DNAse activity, leading to the degradation of non-targeted ssDNA (see, for example, Chen et al., Science, 360:436-439 (2018) Apr 27;360(6387):436-439). The in vitro DNAse assay described in Example 1 was used to investigate the DNAse activity of LbCas12a. Specifically, the guide RNA-directed DNA cleavage activity of LbCas12a was tested for dsDNA, ssDNA, and combinations of dsDNA and ssDNA templates. The experimental setup is shown in Table 1. The gRNA-directed and substrate-specific targeted dsDNA cleavage activity of LbCas12a was tested in Assay 4 (see Table 1). The reaction mixture was substantially as described in Experiment 1, containing purified LbCas12a protein mixed with Zm7.1 dsDNA in a 60:1 ratio, along with Cas12a-zm7.1 gRNA. Three controls were performed in parallel (see Assays 1-3, Table 1). Assay 1 contained Zm7.1 template dsDNA but lacked Cas12a nuclease and gRNA. Assay 2 contained the template and nuclease but lacked the congeneral Cas12a gRNA. Assay 3 contained the template, Cas12a nuclease, and Cas9 guide RNA, which is not expected to be recognized by Cas12a.

[0151] The non-target-specific ssDNase activity of LbCas12a was tested in assay 8, Table 1. The reaction mixture contained purified LbCas12a mixed with M13mp18 ssDNA in a 60:1 ratio, along with Cas12a-zm7.1 gRNA. Three controls were performed in parallel (see assays 5-7, Table 1), detailed in Table 1.

[0152] The cleavage activity of LbCas12a in the presence of a mixture of dsDNA and ssDNA templates was tested in assay 12. See Table 1. The reaction mixture contained purified LbCas12a mixed with Zm7.1 dsDNA and ssDNA M13mp18 at a DNA-to-protein ratio of 60:1, along with Cas12a-zm7.1 gRNA. Three controls were performed in parallel (assays 9, 10, and 11), and are listed in Table 1.

[0153] The reaction was carried out at 37°C for 45 minutes and quenched with proteinase K. The samples were then separated and analyzed on a 1.8% TBE agarose gel. As shown in Table 1, in reactions (Assays 4 and 12) containing Zm7.1 template DNA together with LbCas12a and its congener gRNA, approximately 382 bp and 1318 bp DNA fragment bands were observed. This suggests that, in the presence of congener Cas12a guide RNA, LbCas12a performed sequence-specific cleavage of both strands of approximately 1700 bp Zm7.1 dsDNA, releasing approximately 382 bp and 1318 bp fragments with near completion. In reactions containing M13mp18 ssDNA along with LbCas12a and its gRNA (Table 1, Assays 8 and 12), the M13mp18 ssDNA band was either absent or its band intensity was significantly lower than that observed in the control. This suggests that, in the presence of its homologous guide RNA, LbCas12a degrades M13mp18 ssDNA, thereby confirming its nonspecific ssDNAse activity.

[0154] It has been previously reported that mutations at key residues within the DNA targeting domain of the Cas12a protein can completely eliminate its DNA cleavage activity (see, for example, Zetsche et al., Cell, 163:759 (2015)). We mutated the D832 and E925 residues in LbCas12a to alanine residues and named the resulting Cas12a variant dLbCas12a (Dead LbCas12a) (SEQ ID NO: 7). dLbCas12a was tested for its in vitro DNAse activity using the assay described in Example 1. Details of the experiment are shown in Table 2. As shown in Table 2 (Assays 4 and 12), in reactions containing Zm7.1 template DNA along with dLbCas12a and its related gRNA, full-length Zm7.1 DNA of approximately 1700 bp was observed, while fragments of approximately 382 bp and 1318 bp were not observed. This suggests that dLbCas12a did not cleave dsDNA in the presence of congenital Cas12a guide RNA at a 60:1 protein-to-DNA ratio. As shown in Table 2 (Assays 8 and 12), M13 ssDNA was observed in reactions containing M13mp18 ssDNA together with dLbCas12a and its gRNA, and the band intensity was comparable to that of the control. This suggests that dLbCas12a did not degrade M13mp18 ssDNA in the presence of congenital Cas12a guide RNA at a 60:1 protein-to-DNA ratio.

[0155] [Table 1]

[0156] [Table 2A]

[0157] [Table 2B]

[0158] (Example 3) Identification of LbCas12a variants with reduced ssDNase activity. DNA nuclease activity arises at the RuvC-Nuc domain boundary of the Cas12a protein (see, e.g., Yamano et al., Cell 165, 4:949 (2016)). Two candidate residues within this region, R1138 and E925, were mutated to alanine, and the ssDNase activity of the variants was tested. The amino acid sequence of LbCas12aR1138A is described as SEQ ID NO: 8, and the amino acid sequence of LbCas12aE925A is described as SEQ ID NO: 9. Since the R1138A mutation occurs within the predicted DNA catalytic domain of Cas12a, this LbCas12a variant is predicted to be a nickase and to cleave only single strands of target DNA (see, e.g., U.S. Patent Application Publication No. 2018 / 0030425). The variants were investigated for their in vitro DNase activity using the assay described in Example 1. The test assay involved mixing purified LbCas12a protein variant with Zm7.1 dsDNA in a 60:1 ratio, along with Cas12a-zm7.1 gRNA. Three controls were performed in parallel. The assays and results for LbCas12aE925A and LbCas12aR1138A are described in Tables 3 and 4. All reactions were carried out at 37°C for 45 minutes, quenched with proteinase K, and samples were separated and analyzed on 1.8% TBE agarose gels.

[0159] [Table 3]

[0160] [Table 4A]

[0161] [Table 4B]

[0162] As shown in Table 3 (Assays 4 and 12), in the reaction containing Zm7.1 template DNA along with LbCas12aE925A and its congener gRNA, only approximately 1700 nucleotides of full-length Zm7.1 DNA were observed. This data suggests that LbCas12aE925A did not cleave both strands of the target dsDNA in the presence of a 60:1 protein-to-DNA ratio and its congener gRNA. As shown in Table 3 (Assays 8 and 12), in the reaction containing M13mp18 ssDNA along with LbCas12aE925A and its gRNA, the full-length M13mp18 ssDNA band intensity was comparable to that observed in the control. This suggests that LbCas12aE925A did not degrade M13mp18 ssDNA in the presence of congener Cas12a guide RNA in the presence of a 60:1 protein-to-DNA ratio.

[0163] As shown in Table 4 (Assays 4 and 12), three bands were observed in the reaction containing Zm7.1 template DNA, LbCas12aR1138A, and its homologous gRNA: a full-length Zm7.1 DNA band of approximately 1700 nucleotides, a band of approximately 383 nucleotides, and a band of approximately 1318 nucleotides. This data suggests that LbCas12aR1138A still possesses dsDNA cleavage activity, despite being predicted as a nickase, and that it produces site-specific cleavage of both strands of Zm7.1 dsDNA. The dsDNA processing activity of LbCas12aR1138A is thought to be less than that of wtLbCas12a, as evidenced by the presence of some uncleaved Zm7.1 dsDNA. As shown in Table 4 (Assays 8 and 12), in reactions containing M13mp18ssDNA together with LbCas12aR1138A and its gRNA, the intensity of the ssDNA band was comparable to that observed in the control. This suggests that the substitution of arginine at position 1138 with alanine results in a significant decrease in LbCas12a ssDNase activity. These results were consistent with the increase in the DNA-to-protein ratio from 60:1 to 100:1 (see Table 4).

[0164] (Example 4) Effects of time and temperature on LbCas12a and LbCas12aR1138A dsDNA processing and ssDNase activity It is well known that temperature modulates Cas12a activity (see, e.g., Moreno-Mateos et al., 2017, DOI: 10.1038 / s41467-017-01836-2). To compare the DNase activity of LbCas12a and LbCas12aR1138A, time-course assays were performed using the two proteins, and processing activity was assayed at 25°C and 37°C. Each test reaction mixture contained purified LbCas12a protein or LbCas12aR1138A variant mixed with Zm7.1 dsDNA or M13mp18 ssDNA in a 60:1 ratio, along with Cas12a-zm7.1 gRNA. Three controls were performed in parallel. The first control lacked Cas12a nuclease and gRNA; the second control contained the template and nuclease but lacked the congeneral Cas12a gRNA; and the third control contained the nuclease and template along with Cas9 guide RNA, which is not known in the literature to be recognized by Cas12a. The test and control reaction mixtures were incubated at either 25°C or 37°C and quenched with proteinase K at 10, 20, 40, 90, or 180 minutes. Samples were separated and visually analyzed on 1.8% TBE agarose gels. The test assay results are shown in Table 5.

[0165] [Table 5]

[0166] As shown in Table 5, for LbCas12a, targeted dsDNA processing of Zm7.1 was completed in 40 minutes when the reaction mixture was incubated at 37°C. For reaction mixtures incubated at 25°C, complete processing was achieved by 180 minutes, suggesting a moderate decrease in activity at 25°C. The ssDNase activity of LbCas12a was comparable at both temperatures tested and was completed by 20 minutes.

[0167] Targeted dsDNA processing activity of LbCas12aR1138A was slower than that of the wild type at both temperatures, reaching completion by 180 minutes at 37°C. No evidence of ssDNase activity was noted for the LbCas12aR1138A variant at any time point and temperature tested. For all control assays, neither ssDNase nor targeted dsDNase activity was observed at any time point and temperature tested.

[0168] (Example 5) Further testing of DNase activity of LbCas12a variants Analysis of the crystal structure and point mutations of FnCas12a revealed that DNA nuclease activity arises in the pocket at the boundary between the RuvC and Nuc domains (see Stella et al., Nature, 546: pp. 559-563 (2017)). The R1138 residue in LbCas12a is located within this boundary. With the goal of altering ssDNase activity, a series of substitutions were designed at the R1138 position to change the charge, alter donor ability, and change the potential catalytic residue length (Figure 2). Further mutants were also created at residues D1146 and D1148, both located at the RuvC-Nuc boundary. The dsDNase and ssDNase activity of all mutants was investigated using the in vitro DNase assay described in Experiment 1. Each test reaction mixture contained a purified LbCas12a variant mixed with Zm7.1 dsDNA or M13mp18 ssDNA in a 60:1 ratio, along with Cas12a-zm7.1 gRNA. Three negative controls were run in parallel. The first control contained only the template and lacked Cas12a nuclease and gRNA; the second control contained the template and nuclease but lacked the congeneral Cas12a gRNA; and the third control contained the nuclease and template along with Cas9 guide RNA, which is not known in the literature to be recognized by Cas12a. The test and control reaction mixtures were incubated at 37°C and quenched with proteinase K after 45 minutes. Samples were separated and visually analyzed on 1.8% TBE agarose gels. The tested variants and results are disclosed in Table 6.

[0169] [Table 6A]

[0170] [Table 6B]

[0171] [Table 6C]

[0172] Among the protein variants tested, LbCas12a-R1138A and LbCas12a-R1138H maintained dsDNA cleavage activity, while ssDNase activity was not observed (Table 6). A time-course assay was performed using LbCas12a-R1138H as described in Example 4, and it was noted that targeted dsDNA processing of LbCpf-1R1138H was completed by 180 minutes. No ssDNase activity was observed at 180 minutes.

[0173] (Example 6) DNase activity of LbCas12a and LbCas12a variants in the presence of various guide RNAs and congeneral target sites. The experiments described in Examples 1-5 test the DNase activity of Cas12a in the presence of Cas12a-Zm7.1 gRNA. To investigate whether this activation is independent of the guide RNA sequence, the in vitro cleavage activity of LbCas12a, LbCas12aR1138A, and LbCas12aR1138H was tested in the presence of six further individual gRNAs. Three synthetic dsDNA substrates were created for this purpose. E_1088 was a 1716-nucleotide PCR product containing three unique target sites: ZmTS1; ZmTS2 and ZmTS3. Each TS site was first identified in the maize genome, was 23 nucleotides long, and contained the Cas12a PAM sequence TTTN at 5' of the target sequence. gRNAs were designed to recognize each target site, and these are listed in Table 7. Completely targeted cleavage of E_1088 by Cas12a and each gRNA will yield digest products 1 and 2, as shown in Table 7.

[0174] [Table 7]

[0175] E_1090 is a 1702-nucleotide PCR product containing three unique target sites: GmTS1, GmTS2, and GmTS3. The 23-nucleotide GmTS1, 2, and 3 sites were first identified in the soybean genome and each contained the Cas12a PAM sequence TTTN at the 5' of the target site. gRNAs were designed to recognize each target site and are listed in Table 8. Fully targeted cleavage of E_1090 dsDNA by LbCas12a and each gRNA would yield the digest products listed in Table 8.

[0176] [Table 8]

[0177] E_1089 was a 1747-nucleotide PCR product containing seven unique target sites: Cas12a-Zm7.1; GmTS1; ZmTS1; GmTS2 site, ZmTS2, ZmTS3, and GmTS3 site. Fully targeted cleavage of E_1090 dsDNA by LbCas12a and each gRNA would yield the digest products listed in Table 9.

[0178] [Table 9]

[0179] The in vitro DNase assay described in Example 1 was used to investigate the guide RNA-directed DNA cleavage activity of LbCas12a, LbCas12a R1138A, and LbCas12a R1138H with the three dsDNA templates and M13 ssDNA. The experimental setup is shown in Table 10. For the test assays, purified LbCas12a protein or LbCas12a variants were mixed with gRNA and either one of the three dsDNA templates or one of the M13mp18 ssDNA templates. The DNA-to-protein ratio was maintained at 60:1. Two control reactions were performed in parallel for each test assay. The first control lacked both the nuclease and gRNA, while the second control lacked only the gRNA. All reaction products were incubated at 37°C for 45 minutes and quenched with proteinase K. The samples were separated and analyzed using 1.8% TBE agarose gel. The results are shown in Table 10.

[0180] [Table 10A]

[0181] [Table 10B]

[0182] [Table 10C]

[0183] [Table 10D]

[0184] Results from in vitro DNase assays indicate that when LbCas12a complexes with any of the six gRNAs tested, it completes targeted dsDNA cleavage of all tested dsDNA templates within 45 minutes and degrades ssDNA. Under the same conditions, LbCas12aR1138A and LbCas12aR1138H showed partial dsDNA cleavage and no visual evidence of ssDNA degradation.

[0185] (Example 7) Cleavage activity of LbCas12a, LbCas12aR1138A, and LbCas12aR1138H in soybean protoplasts To test whether LbCas12aR1138A and LbCas12aR1138H can recognize, cleave, and induce mutagenesis in soybean chromosome DNA in the presence of gRNA, three different genomic target sites were targeted for cleavage, and the presence of mutations indicative of cleavage was examined by deep sequencing. Wild-type LbCas12a was used as a positive control, and dead LbCas12a (dLbCas12a) was used as a negative control. Since LbCas12a shows a preference for TTTN PAM sequences, target sites GmTS1, GmTS2, and GmTS3 were selected based on the presence of appropriate PAM sequences at the 5' end. GmTS1 gRNA, GmTS2 gRNA, and GmTS3 gRNA were designed to target these three sites.

[0186] [Table 11]

[0187] LbCas12a variants listed in Table 11 were expressed from E. coli and purified. The nuclease was mixed with the corresponding gRNA in a 1:2 ratio (gRNA:nuclease) to form a ribonucleoprotein complex, and soy protoplasts were transformed using standard polyethylene glycol (PEG)-mediated transformation. To quantify the transformation frequency, a vector containing a GFP expression cassette was co-delivered. As a control, protoplasts were transformed using only the nuclease without guide RNA. Multiple technical replicates were performed for each assay. Following transformation, protoplasts were incubated in incubation buffer in the dark and collected after 48 hours. Genomic DNA was isolated, the region around the target site of interest was amplified, and deep sequencing was performed by Illumina sequencing using standard methods known in the art. The obtained reads were evaluated for the presence of mutations, specifically insertions or deletions (indels), at predicted cleavage sites. Table 12 and Figure 3 summarize the average indel rates observed for each test treatment using nucleases and guide RNA at each site.

[0188] [Table 12]

[0189] For R1138, the Wilcoxon rank-sum test was performed for each variant within each site to assess the statistical significance of the difference in indel rates between the treated and gRNA-free controls. Both uncorrected and Holmes-Bonferroni corrected p-values ​​are provided. The results are summarized in Table 13, showing that the R1138A and R1138H variants have significantly higher indel rates compared to their respective gRNA-free controls.

[0190] [Table 13]

[0191] Within each target site, the difference in indel rates between the two R1138 variants was evaluated using the Wilcoxon rank-sum test (Table 14). The indel rate for R1137H was significantly higher than that of R1138A across all sites.

[0192] [Table 14]

[0193] (Example 8) The role of magnesium in non-targeted ssDNA cleavage. The role of magnesium in non-targeted ssDNA cleavage by wild-type RNA-inducible CRISPR nucleases was investigated. The activity of wild-type RNA-inducible CRISPR nucleases SpCas9 (SEQ ID NO: 22) and LbCas12a (SEQ ID NO: 2) was investigated using the in vitro assays described in Examples 1 and 2. The coding region of the SpCas9 sequence was codon-optimized for optimal expression in E. coli cells (SEQ ID NO: 23). A histidine tag sequence (SEQ ID NO: 4) was introduced into the 5' end of the gene. In addition, two nuclear localization signals (NLS) (SEQ ID NOs: 5 and 6) were introduced into the 5' and 3' ends of the SpCas9 coding region to create HIStag:NLS:SpCas9:NLS. The design of the HIStag:NLS:LbCas12a:NLS fusion protein is described in Example 1. Next, purified E. coli LbCas12a or SpCas9 fusion proteins were incubated with or without nuclease-appropriate gRNA (100 μM) in a cleavage buffer containing 20 mM HEPES, 0.5 mM DTT, and either 0.02 mM MgCl2 or 10 mM MgCl2, along with dsDNA (26.7 nM), M13 ssDNA (12.54 nM), or a combination of dsDNA and ssDNA. The protein amount was adjusted for each reaction to suit the specific protein-to-DNA ratio of 60:1. The reaction was carried out at 37°C for 45 minutes and quenched with proteinase K treatment at 65°C for 15 minutes. Samples were separated and analyzed on 1.8% TBE agarose gels. Observations regarding LbCas12a are summarized in Table 15, and observations regarding SpCas9 are summarized in Table 16.

[0194] Nonspecific ssDNA cleavage was not observed for SpCas9 or LbCas12a in the presence of 0.02 mM MgCl2. In contrast, nonspecific ssDNA cleavage was observed for both SpCas9 and LbCas12a in the presence of 10 mM MgCl2 when paired with a nuclease-appropriate gRNA.

[0195] [Table 15A]

[0196]

Table 15B

[0197]

Table 16A

[0198]

Table 16B

[0199] (Example 9) MgCl2 Titration Assay MgCl2 titrations were performed to establish a buffer formulation that decreases the ssDNase activity of CRISPR nuclease while maintaining desirable dsDNA cleavage activity. Purified LbCas12a or SpCas9 at 1604 nM was incubated with dsDNA (26.7 nM), M13 ssDNA (12.54 nM), or a combination of dsDNA and ssDNA in cleavage buffer containing 20 mM HEPES and 0.5 mM DTT, with or without assembly with nuclease-appropriate gRNA (100 μM). For the titration assay, cleavage buffer was supplemented with increasing concentrations of MgCl2 as shown in Tables 17 - 22. Since the protein concentration was kept constant, the specific DNA-to-protein ratios were 60:1 for nuclease:dsDNA; 128:1 for nuclease:ssDNA, and 40:1 for nuclease:dsDNA + ssDNA. Reactions were run at 37 °C for 45 minutes and quenched using proteinase K treatment at 65 °C for 15 minutes. Samples were separated and analyzed on a 1.8% TBE agarose gel. Observations for LbCas12a are summarized in Tables 17 - 19, and observations for SpCas9 are summarized in Tables 20 - 22.

[0200] [Table 17]

[0201] [Table 18]

[0202] [Table 19]

[0203] [Table 20]

[0204] [Table 21]

[0205] [Table 22]

[0206] Furthermore, the data from Tables 17-22 suggest that reducing the Mg concentration in the cleavage buffer can reduce the nonspecific ssDNase activity of CRISPR nucleases.

[0207] (Example 10) EDTA Chelation Assay To establish a buffer formulation that reduces the ssDNase activity of CRISPR nuclease while maintaining desirable dsDNA cleavage activity, an EDTA titration was performed. Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that can capture metal ions such as Mg2+. Purified LbCas12a or SpCas9 was incubated with or without a nuclease-appropriate gRNA (100 μM) in cleavage buffer containing 20 mM HEPES, 0.5 mM DTT, and 10 mM MgCl2 with dsDNA (26.7 nM), M13 ssDNA (12.54 nM), or a combination of dsDNA and ssDNA. For the titration assay, the cleavage buffer was supplemented with increasing concentrations of EDTA as shown in Tables 22-28 (Tables 23-28). The amount of protein was adjusted to accommodate a DNA-to-specific protein ratio of 60:1 for each reaction. Reactions were run at 37 °C for 45 minutes and quenched using proteinase K treatment at 65 °C for 15 minutes. Samples were separated and analyzed on a 1.8% TBE agarose gel. Observations for LbCas12a are summarized in Tables 23-25 (Tables 23-25), and observations for SpCas9 are summarized in Tables 26-28 (Tables 26-28).

[0208] Taken together, the data from Tables 23-28 (Tables 23-28) suggest that the addition of EDTA to the cleavage buffer containing Mg2+ can reduce the non-specific ssDNase activity of CRISPR nuclease.

[0209] [Table 23]

[0210] [Table 24]

[0211] [Table 25]

[0212] Table 26

[0213] Table 27

[0214] Table 28

Claims

1. An engineered RNA-inducible CRISPRCas12a nuclease comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, wherein the engineered RNA-inducible CRISPRCas12a nuclease exhibits reduced nonspecific cleavage of single-stranded DNA (ssDNA) compared to a reference wild-type RNA-inducible CRISPR nuclease comprising the amino acid sequence of SEQ ID NO:

2.

2. A method for producing an engineered RNA-inducible CRISPR Cas12a nuclease, comprising the step of editing a polynucleotide encoding a wild-type RNA-inducible CRISPR Cas12a nuclease to generate at least one mutation in the DNA catalytic domain, wherein the engineered RNA-inducible CRISPR Cas12a nuclease exhibits reduced nonspecific cleavage of single-stranded DNA compared to a wild-type RNA-inducible CRISPR nuclease containing the amino acid sequence of SEQ ID NO: 2, and the engineered RNA-inducible CRISPR Cas12a nuclease contains the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

12.

3. A method for reducing nonspecific single-stranded DNA (ssDNA) breaks caused by RNA-inducible CRISPR Cas12a nuclease, comprising the step of providing cells with an engineered RNA-inducible CRISPR Cas12a nuclease having at least one mutation in its DNA catalytic domain compared to a reference wild-type RNA-inducible CRISPR nuclease having the amino acid sequence of SEQ ID NO: 2, wherein the engineered RNA-inducible CRISPR Cas12a nuclease exhibits a reduction in nonspecific breaks of non-targeted ssDNA compared to a reference wild-type RNA-inducible CRISPR nuclease lacking at least one mutation, and the engineered RNA-inducible CRISPR Cas12a nuclease having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

12.

4. The manipulated RNA-inducible CRISPRCas12a nuclease according to claim 1, wherein the manipulated RNA-inducible CRISPRCas12a nuclease is part of a ribonucleoprotein.

5. The manipulated RNA-inducible CRISPRCas12a nuclease according to claim 4, wherein the ribonucleoprotein comprises at least one guide nucleic acid.

6. The modified RNA-inducible CRISPRCas12a nuclease according to claim 1, wherein the modified RNA-inducible CRISPRCas12a nuclease exhibits the ability to cleave double-stranded DNA (dsDNA).

7. The manipulated RNA-inducible CRISPRCas12a nuclease according to claim 6, wherein the manipulated RNA-inducible CRISPRCas12a nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of the wild-type RNA-inducible CRISPR nuclease.

8. The modified RNA-inducible CRISPR nuclease according to claim 1, wherein the reduction in ssDNA cleavage indicates a reduced cleavage rate compared to the wild-type RNA-inducible CRISPR nuclease.

9. The modified RNA-inducible CRISPR nuclease according to claim 1, comprising an ssDNA cleavage rate in which the reduction in ssDNA cleavage is less than 50% of the ssDNA cleavage rate of the wild-type RNA-inducible CRISPR nuclease.

10. The modified RNA-inducible CRISPR nuclease according to claim 1, wherein the reduction in ssDNA cleavage is measured within 180 minutes of introducing the modified RNA-inducible CRISPR nuclease into the ssDNA.

11. The modified RNA-inducible CRISPRCas12a nuclease according to claim 1, wherein the modified RNA-inducible CRISPRCas12a nuclease cleaves dsDNA in eukaryotic cells.

12. The manipulated RNA-inducible CRISPRCas12a nuclease according to claim 11, wherein the eukaryotic cell is selected from the group consisting of plant cells, animal cells, protozoan cells and fungal cells.

13. A method for reducing nonspecific single-stranded DNA (ssDNA) breaks caused by RNA-inducible CRISPRCas12a nuclease, comprising the step of contacting a modified RNA-inducible CRISPRCas12a nuclease containing the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12 with non-target ssDNA in a test solution, wherein the test solution contains MgCl at a concentration of less than 10 mM. 2 It contains MgCl at a concentration of 10 mM or higher. 2 A method in which nonspecific ssDNA cleavage is reduced compared to nonspecific ssDNA cleavage caused by RNA-induced CRISPRCas12a nuclease manipulated in a control solution containing [the specified substance].

14. The method according to claim 13, wherein the test solution is located inside the cell.

15. The method according to claim 14, wherein the cells are plant cells.

16. The method according to claim 13, wherein the manipulated RNA-induced CRISPRCas12a nuclease is part of a ribonucleoprotein.

17. The method according to claim 16, wherein the ribonucleoprotein comprises at least one guide nucleic acid.

18. The method according to claim 13, wherein the manipulated RNA-inducible CRISPRCas12a nuclease has the ability to cleave double-stranded DNA.

19. The method according to claim 2 or 3, wherein the manipulated RNA-induced CRISPRCas12a nuclease is part of a ribonucleoprotein.

20. The method according to claim 19, wherein the ribonucleoprotein comprises at least one guide nucleic acid.

21. The method according to claim 2 or 3, wherein the manipulated RNA-inducible CRISPRCas12a nuclease exhibits the ability to cleave double-stranded DNA (dsDNA).

22. The method according to claim 21, wherein the manipulated RNA-inducible CRISPR nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of the wild-type RNA-inducible CRISPR nuclease.

23. The method according to claim 2 or 3, wherein the reduction in ssDNA cleavage is shown as a reduction in the cleavage rate compared to wild-type RNA-induced CRISPR nuclease.

24. The method according to claim 2 or 3, wherein the reduction in ssDNA cleavage is less than 50% of the ssDNA cleavage rate of wild-type RNA-induced CRISPR nuclease.

25. The method according to claim 2 or 3, wherein the reduction in ssDNA cleavage is measured within 180 minutes of introducing a modified RNA-induced CRISPR nuclease into the ssDNA.

26. The method according to claim 2 or 3, wherein the manipulated RNA-induced CRISPRCas12a nuclease cleaves dsDNA in eukaryotic cells.

27. The method according to claim 26, wherein the eukaryotic cells are selected from the group consisting of plant cells, animal cells, protozoan cells and fungal cells.

Images