Switchable Cas9 nuclease and its use

Abstract

Some aspects of the present disclosure provide compositions, methods, systems, and kits for modulating the activity and / or improving the specificity of RNA-programmable endonucleases such as Cas9. [Solution] For example, provided herein are guide RNAs (gRNAs) that can be engineered to exist in an "on" or "off" state and regulate the binding and thus cleavage activity of an RNA-programmable endonuclease. Some aspects of the present disclosure provide gRNAs that sense mRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of a target mRNA. Some aspects of the present disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of extended DNA (xDNA).

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. 365(c) to U.S. patent application Ser. No. 14 / 326,329, filed July 8, 2014, U.S. patent application Ser. No. 14 / 326,340, filed July 8, 2014, and U.S. patent application Ser. No. 14 / 326,361, filed July 8, 2014, and also claims the benefit of priority under 35 U.S.C. 365(e) to U.S. provisional patent application Ser. No. 61 / 874,682, filed September 6, 2013, the entire disclosures of which are incorporated herein by reference. [Background technology]

[0002] Site-specific endonucleases theoretically allow for targeted manipulation of a single site within the genome, making them useful for targeted gene recombination and for therapeutic applications. In various organisms, including mammals, site-specific endonucleases have been used for genome engineering by stimulating either nonhomologous end-joining or homologous recombination. In addition to providing powerful research tools, site-specific nucleases also hold promise as gene therapy agents, and two site-specific endonucleases have recently entered clinical trials: CCR5-2246, which targets the human CCR-5 allele as part of an anti-HIV treatment (NCT00842634, NCT01044654, NCT01252641), and VF24684, which targets the human VEGF-A promoter as part of an anti-cancer treatment (NCT01082926).

[0003] Specific cleavage at the intended nuclease target site with minimal or no off-target activity is a prerequisite for the clinical application of site-specific endonucleases and for highly efficient genome engineering in basic research applications. For example, imperfect specificity of engineered site-specific binding domains is thought to be associated with cytotoxicity and unwanted alterations at genomic loci other than the intended target. However, the majority of currently available nucleases exhibit significant off-target activity and therefore would be unsuitable for clinical applications. A nuclease platform gaining attention for use in clinical and research settings is the RNA-guided nuclease, such as Cas9. These nucleases can bind guide RNAs (gRNAs) that direct cleavage at specific target sites, but off-target activity is still observed in certain Cas9:gRNA complexes (Pattanayak et al., "High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nat Biotechnol. 2013; doi: 10.1038 / nbt.2673). Therefore, there is a need for technologies to engineer nucleases with improved specificity. Summary of the Invention

[0004] Some aspects of the present disclosure are based on the recognition that the reported toxicity of engineered site-specific endonucleases is due to off-target DNA cleavage. Furthermore, the activity of existing RNA-guided nucleases generally cannot be regulated at the molecular level, e.g., by switching the nuclease from an "off" to an "on" state. Modulating the activity of the nuclease may reduce the potential for off-target effects. Some aspects of the present disclosure provide strategies, compositions, systems, and methods for modulating the binding and / or cleavage activity of RNA-programmable endonucleases, such as Cas9 endonuclease.

[0005] Thus, one embodiment of the present disclosure provides an RNA-guided nuclease complex comprising a "switchable" guide RNA (gRNA). For example, in some embodiments, the present invention provides a complex comprising: (i) a gRNA comprising an aptamer that does not hybridize to a target nucleic acid in the absence of a specific ligand that binds to the aptamer; and (ii) a Cas9 protein. In some embodiments, the aptamer is bound to a ligand. In some aspects, the ligand is any molecule. In some aspects, the ligand is a small molecule, metabolite, carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the gRNA:ligand:Cas9 complex binds to and mediates cleavage of the target nucleic acid. See, e.g., Figure 1.

[0006] In other embodiments, a gRNA comprising an aptamer is provided. In some embodiments, the gRNA does not hybridize to a target nucleic acid in the absence of a ligand bound to the aptamer. Such a gRNA can be referred to as a "switchable gRNA." For example, in some aspects, the gRNA does not bind Cas9 in the absence of a ligand bound to the aptamer. See, e.g., Figures 1A and 1B. In some embodiments, the gRNA binds to Cas9 when its specific ligand binds to the aptamer. In some embodiments, the gRNA binds to Cas9 in the presence or absence of a ligand bound to the aptamer, but only in the presence of a ligand bound to the aptamer. In some aspects, the ligand is any molecule. In some aspects, the ligand is a small molecule, metabolite, carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the aptamer is an RNA aptamer, such as an RNA aptamer derived from a riboswitch. In some embodiments, the riboswitch from which the aptamer is derived is selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCbl) riboswitch, an S-adenosylmethionine (SAM) riboswitch, a SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a prekeosin 1 (PreQ1) riboswitch. In some embodiments, the aptamer is derived from the theophylline riboswitch and comprises SEQ ID NO: 3. In other embodiments, the aptamer is not naturally occurring and, in some aspects, has been engineered to bind a specific ligand using a SELEX platform.In some embodiments, the non-aptamer portion of the gRNA comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 nucleotides, and the aptamer comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, or at least 300 nucleotides.

[0007] In another embodiment, a method for site-specific DNA cleavage using the Cas9 mutant of the present invention is provided. For example, in some aspects, the method comprises contacting DNA with a complex comprising (i) a gRNA comprising an aptamer having a sequence that binds to a portion of DNA, (ii) a specific ligand bound to the aptamer of the gRNA, and (iii) a Cas9 protein under conditions in which the Cas9 protein cleaves the DNA.

[0008] According to other embodiments, methods for inducing site-specific DNA cleavage in cells are provided. For example, in some embodiments, the methods include: (a) contacting a cell with or expressing in the cell a gRNA comprising an aptamer, the gRNA comprising a sequence capable of binding to a DNA target sequence; (b) contacting the cell with or expressing in the cell a Cas9 protein; and (c) contacting the cell with a ligand that binds the aptamer of the gRNA, thereby forming a gRNA:ligand:Cas9 complex that cleaves the DNA target. In some embodiments, the cell produces the ligand intracellularly, e.g., as part of a physiological or pathophysiological process. In some embodiments, the methods include: (a) contacting the cell with a complex comprising a gRNA comprising an aptamer, the gRNA comprising a sequence capable of binding to a DNA target sequence, and a Cas9 protein; and (b) contacting the cell with a ligand that binds to the aptamer of the gRNA, thereby forming a gRNA:ligand:Cas9 complex that cleaves the DNA target. In some aspects, steps (a) and (b) are performed simultaneously or sequentially in any order. In some embodiments, the method is performed in vitro, while in other embodiments, the method is performed in vivo.

[0009] In other embodiments, an RNA-guided nuclease complex is provided, which comprises a gRNA that senses an mRNA. For example, in some embodiments, the complex comprises a gRNA that comprises (i) a region that hybridizes with a region of a target nucleic acid; (ii) another region that partially or completely hybridizes with the sequence of region (i); and (iii) a region that hybridizes with a region of a transcript (mRNA), and a Cas9 protein.

[0010] In other embodiments, gRNAs are provided that sense mRNAs, including, for example, (i) a region that hybridizes to a region of the target nucleic acid; (ii) another region that partially or completely hybridizes to the sequence of region (i); and (iii) a region that hybridizes to a region of the transcript (mRNA). See, e.g., Figure 2. In some embodiments, each of the sequences of region (i), region (ii), and region (iii) comprises at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides. In some aspects, the gRNA forms a stem-loop structure in which the stem comprises the sequence of region (i) hybridized to part or all of the sequence of region (ii), and the loop is formed from part or all of the sequence of region (iii). In some embodiments, both region (ii) and region (iii) are located either 5' or 3' to region (i). See, e.g., Figure 2A versus Figure 2C. In some embodiments, a stem-loop structure forms in the absence of a transcript that hybridizes to the sequence in region (iii). In this instance, the gRNA is said to be in the "off" state. See, e.g., Figures 2A and 2C. In some embodiments, binding of a transcript to the sequence in region (iii) unfolds or prevents the formation of the stem-loop structure such that the sequence in region (ii) does not hybridize to the sequence in region (i). In this instance, the gRNA is said to be in the "on" state. See, e.g., Figures 2B and 2D. In some embodiments, the gRNA binds a Cas9 protein, and when the sequence in region (iii) binds (e.g., "senses") the transcript, the sequence in region (i) hybridizes to the target nucleic acid.

[0011] In other embodiments, methods for site-specific DNA cleavage are provided, including contacting DNA with a complex comprising a Cas9 protein associated with a gRNA that senses an mRNA, to which the mRNA binds such that the complex can bind and cleave the DNA.

[0012] In other embodiments, extended DNA-recognition (xDNA-sensing) gRNAs are provided. See, e.g., Figure 3. In some embodiments, the xDNA-sensing gRNA comprises (i) a region that hybridizes to a region of the target nucleic acid; (ii) another region that partially or completely hybridizes with the sequence of region (i); and (iii) a region that hybridizes to another region of the target nucleic acid. In some embodiments, the sequences of region (i) and region (ii) each comprise at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides, and the sequence of region (iii) comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides. In some embodiments, the gRNA forms a stem-loop structure in which the stem comprises the sequence of region (i) hybridized with part or all of the sequence of region (ii), and the loop is formed by part or all of the sequence of region (iii). In some embodiments, both region (ii) and region (iii) are either 5' or 3' to region (i). See, e.g., Figure 3A versus Figure 3C. In some embodiments, the stem-loop structure forms in the absence of a region of the target nucleic acid that complements or binds to a sequence in region (iii). See, e.g., Figures 3A and 3C. In some embodiments, hybridization of a region of the target nucleic acid to a sequence in region (iii) unfolds or prevents the formation of the stem-loop structure such that the sequence in region (ii) does not hybridize to the sequence in region (i). See, e.g., Figures 3B and 3D. In some embodiments, the gRNA binds the Cas9 protein, and the sequence in (i) binds the target nucleic acid when the sequence in region (iii) binds the target nucleic acid.

[0013] In other embodiments, a complex is provided comprising a gRNA that senses xDNA and a Cas9 protein, optionally including a target nucleic acid, and in some embodiments, formation of the complex results in cleavage of the target nucleic acid.

[0014] According to another embodiment, there is provided a method for site-specific DNA cleavage, comprising contacting DNA with a complex comprising a gRNA that senses xDNA and a Cas9 protein.

[0015] Any of the methods provided herein can be performed on DNA in a cell, e.g., a cell in vitro or in vivo. In some embodiments, any of the methods provided herein is performed on DNA in a eukaryotic cell. In some embodiments, the eukaryotic cell is in an individual, e.g., a human.

[0016] In other embodiments, polynucleotides are provided that encode, for example, any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein. In some embodiments, vectors are provided that include the polynucleotides described herein. In some embodiments, vectors are provided for the recombinant expression of any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein. In some embodiments, cells are provided that include a genetic construct that expresses any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein.

[0017] In some embodiments, kits are provided. For example, kits are provided that include any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein. In some embodiments, kits are provided that include any of the polynucleotides described herein. In some embodiments, kits are provided that include a recombinant expression vector that includes a polynucleotide encoding any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein. In some embodiments, kits are provided that include cells that include a genetic construct that expresses any of the gRNAs, complexes, or proteins (e.g., Cas9 proteins) described herein.

[0018] Other advantages, features and uses of the present invention will become apparent from the detailed description of several embodiments of the invention, the schematic and not-to-scale drawings, and the claims. [Brief explanation of the drawings]

[0019] [Figure 1] Figures 1A-1D illustrate several embodiments of the invention relating to aptamer-linked gRNAs. (A) This figure shows a schematic of a switchable gRNA containing an aptamer. In the absence of a specific ligand (in this figure, a metabolite) that binds the aptamer, the sequence that binds to the target nucleic acid hybridizes to a portion of the aptamer (see the leftmost portion labeled "switching sequence"). Upon metabolite binding, the aptamer undergoes a conformational change such that the sequence that binds to the target nucleic acid no longer hybridizes to the aptamer sequence, allowing it to hybridize to the target. (B) Upon switching to the "on" state, upon binding to Cas9, the gRNA directs a nuclease to the hybridized target site, allowing Cas9 to cleave each strand of the target nucleic acid. (C and D) In ​​this figure, the aptamer linked to the gRNA is derived from a theophylline riboswitch. In the absence of theophylline (C), the portion of the aptamer (denoted "theophylline riboswitch") binds the portion of the sequence (denoted "target cleavage guide") that should bind to the target nucleic acid (denoted by the double-stranded sequence at the top of the panel), preventing the gRNA from hybridizing to the target nucleic acid. In Figure 1C, the sequences correspond, from top to bottom, to SEQ ID NOS: 4-6. Upon binding of theophylline (denoted as a solid small molecule bound to the aptamer sequence) to the aptamer (D), the aptamer undergoes a conformational change, allowing the "guide" sequence to freely hybridize to the target nucleic acid. In Figure 1D, the sequences correspond, from top to bottom, to SEQ ID NOS: 4, 5, and 11.

[0020] [Figure 2]Figures 2A-2D illustrate several embodiments of the present invention related to gRNAs that sense mRNA. (A and B) The figures show gRNAs containing a 5' transcript sensor / guide block motif. In the absence of a specific mRNA (A), the transcript sensor portion remains unbound, forming a stem-loop structure that blocks the specific portion of the sequence (denoted "target cleavage guide") that should bind the target nucleic acid (denoted by the double-stranded sequence at the top of the panel), thereby preventing the gRNA from hybridizing to the target nucleic acid. In Figure 2A, the sequences correspond, from top to bottom, to SEQ ID NOS: 4, 5, and 7. In the presence of an mRNA to which the transcript sensor hybridizes (B), the gRNA undergoes a conformational change, allowing the "guide" sequence to freely hybridize to the target nucleic acid. The sequences correspond, from top to bottom, to SEQ ID NOS: 4, 5, 7, and 12. (C and D) Similarly, this strategy can be applied to gRNAs containing a 3' transcript sensor / guide block such that in the absence of mRNA (C), the gRNA is in the "off" state (sequences, from top to bottom, correspond to SEQ ID NOS: 4, 5, and 8), and in the presence of mRNA (D), the gRNA is in the "on" state (sequences, from top to bottom, correspond to SEQ ID NOS: 4, 5, and 12).

[0021] [Figure 3]Figures 3A-3D illustrate several embodiments of the present invention relating to extended DNA (xDNA) recognition strategies. (A and B) In this embodiment, a gRNA is shown containing a 5' xDNA sensor / guide block motif. The xDNA sensor motif is complementary to and hybridizes to another portion of the target nucleic acid (e.g., in addition to a "target cleavage guide" sequence). (A) In the absence of the correct target sequence (e.g., including both the "guide" sequence target and the xDNA sensor sequence target), the xDNA sensor portion remains unbound, forming a stem-loop structure that blocks the specific portion of the sequence (shown as the "target cleavage guide") that should bind to the target nucleic acid (shown as the double-stranded sequence at the top of the panel), preventing the gRNA from hybridizing to the target nucleic acid. The sequences, from top to bottom, correspond to SEQ ID NOS: 4, 5, and 9. In the presence of the correct target nucleic acid to which the xDNA sensor portion hybridizes (B), the gRNA undergoes a conformational change that frees the "guide" sequence to hybridize to the target nucleic acid. The sequences, from top to bottom, correspond to SEQ ID NOS: 13, 14, and 9. Thus, binding of the gRNA (and associated Cas9 protein) occurs only in the presence of the correct target nucleic acid. This effectively increases (i.e., lengthens) the number of target nucleotides recognized by, for example, the Cas9:gRNA complex, increasing specificity. (C and D) Similarly, this strategy can be applied to gRNAs containing 3'x DNA sensor / guide blocks, such that in the absence of target nucleic acid (C), the gRNA is in the "off" state (sequences, from top to bottom, are SEQ ID NOS: 4, 5, and 10), and in the presence of target nucleic acid (D), the gRNA is in the "on" state. The sequences, from top to bottom and left to right, correspond to SEQ ID NOS: 4, 15, 5, 16, and 10.

[0022] definition As used in this specification and claims, the singular forms "a," "an," and "the" include both the singular and the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes a single drug and multiple drugs.

[0023] The term "aptamer" refers to a nucleic acid or peptide molecule that binds to a specific target molecule, e.g., a specific ligand. In some embodiments, binding of the ligand to the aptamer induces a conformational change in the aptamer and another molecule, e.g., conjugated or linked to the aptamer. In some embodiments, nucleic acid (e.g., DNA or RNA) aptamers have been engineered by iterative in vitro selection or equivalently, SELEX (enrichment of exogenous sequences by enrichment in vitro) to bind to a variety of molecular targets, e.g., small molecules, large molecules, metabolites, proteins, carbohydrates, metals, nucleic acids, cells, tissues, and organisms.Methods for engineering aptamers to bind small molecules are known in the art, see, for example, U.S. Pat. Nos. 5,580,737 and 8,492,082; Ellington and Szostak, "In vitro selection of RNA molecules that bind specific ligands." Nature. 1990; 346:818-822; Tuerk and Gold, "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science. 1990; 249:505-510; Burke and Gold, "RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX." Nucleic Acids Res. 1997; 25(10):2020-4; Ulrich et al., "DNA and RNA aptamers: from tools for basic research towards therapeutic applications." Comb Chem High Throughput Screen. 2006; 9(8):619-32; Svobodova et al., "Comparison of different methods for generation of single-stranded DNA for SELEX processes. Anal Bioanal Chem. 2012; 404:835-842. Nucleic acid aptamers also exist in nature, for example, they form part of riboswitches.A "riboswitch" is a regulatory segment of an mRNA molecule that binds a small molecule, e.g., a metabolite, resulting in a change in the production of a protein encoded by the mRNA (e.g., a protein involved in producing the metabolite that binds to the riboswitch). Riboswitches are often conceptually divided into two parts: an aptamer and an expression platform (e.g., mRNA). The aptamer directly binds the small molecule (e.g., a metabolite), and the mRNA undergoes a conformational change in response to the aptamer. The change in mRNA conformation typically results in a reduction or inhibition of protein expression. Aptamers can be cloned (e.g., isolated) from riboswitches using methods known in the art and used to regulate the activity of other molecules (e.g., RNA, DNA) linked to them. Furthermore, naturally occurring aptamers can be reengineered to bind synthetic, non-natural small molecule ligands using known methods to regulate the activity of other molecules linked to them. See, e.g., Dixon et al., "Reengineering orthogonally selective riboswitches." PNAS 2010; 107(7):2830-2835, the entire disclosure of which is incorporated herein by reference. The following is a non-limiting list of riboswitches, including aptamers:

[0024] Cobalamin riboswitches (also called B12 elements) bind adenosylcobalamin (the coenzyme form of vitamin B12) to control cobalamin biosynthesis and transport of cobalamin and similar metabolites, as well as other genes. See, e.g., Nahvi et al., "Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes." Nucleic Acids Res. 2004; 32: 143-150; Vitreschak et al., "Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element." RNA. 2003; 9:1084-1097, the entire contents of which are incorporated herein by reference.

[0025] Cyclic di-GMP riboswitches bind the second messenger cyclic di-GMP to control various genes regulated by this signaling molecule. At least two classes of cyclic di-GMP riboswitches are known: cyclic di-GMP-I riboswitches and cyclic di-GMP-II riboswitches. See, e.g., Sudarsan et al., "Riboswitches in eubacteria sense the second messenger cyclic di-GMP." Science. 2008; 321 (5887): 411-3; Lee et al., "An allosteric self-splicing ribozyme triggered by a bacterial second messenger." Science. 2010; 329 (5993): 845-8, the entire contents of which are incorporated herein by reference.

[0026] The FMN riboswitch (also called an RFN element) binds flavin mononucleotide (FMN) to control riboflavin biosynthesis and transport. See, e.g., Winkler et al., "An mRNA structure that controls gene expression by binding FMN." Proc Natl Acad Sci USA. 2002; 99 (25): 15908-15913; Serganov et al., "Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch." Nature. 2009; 458 (7235): 233-7, the entire contents of which are incorporated herein by reference.

[0027] The GlmS riboswitch is a ribozyme that cleaves itself when glucosamine-6-phosphate is bound. See, e.g., Winkler et al., "Control of gene expression by a natural metabolite-responsive ribozyme," Nature. 2004; 428: 281-286; Jansen et al., "Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme," Nat Struct Mol Biol. 2006; 13: 517-523; Hampel and Tinsley, "Evidence for preorganization of the glmS ribozyme ligand binding pocket," Biochemistry. 2006; 45: 7861-7871, the entire contents of which are incorporated herein by reference.

[0028] Glycine riboswitches bind glycine and regulate glycine metabolic genes, such as the use of glycine as an energy source. See, e.g., "A glycine-dependent riboswitch that uses cooperative binding to control gene expression." Science. 2004; 306 (5694): 275-279; Kwon and Strobel, "Chemical basis of glycine riboswitch cooperativity." RNA. 2008; 14 (1): 25-34, the entire contents of which are incorporated herein by reference.

[0029] Lysine riboswitches (also called L-boxes) bind lysine and control its biosynthesis, catabolism, and transport. See, e.g., Sudarsan et al., "An mRNA structure in bacteria that controls gene expression by binding lysine." Genes Dev. 2003;17:2688-2697; Grundy et al., "The L box regulon: Lysine sensing by leader RNAs of bacterial lysine biosynthesis genes." Proc. Natl. Acad. Sci. USA. 2003;100:12057-12062, the entire contents of which are incorporated herein by reference.

[0030] PreQ1 riboswitches bind prechaeosin 1 and control genes involved in the synthesis or transport of this precursor to chaeosin. At least two different classes of PreQ1 riboswitches are known: PreQ1-I riboswitches and PreQ1-II riboswitches. For example, Roth et al., "A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain," Nat. Struct Mol Biol. 2007; 14 (4): 308-317; Klein et al., "Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase," Nat. Struct. Mol. Biol. 2009; 16 (3): 343-344; Kang et al., "Structural Insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA." Mol. Cell 33 2009; (6): 784-90; Meyer et al., "Confirmation of a second natural preQ1 aptamer class in Streptococccaceae bacteria." See RNA 2008; 14 (4): 685, the entire contents of which are incorporated herein by reference.

[0031] Purine riboswitches bind purines to control purine metabolism and transport. Different forms of purine riboswitches bind either guanine (a form originally known as the G-box) or adenine. Specificity for either guanine or adenine depends entirely on Watson-Crick interactions with a single pyrimidine in the riboswitch at a specific position, such as Y74. In guanine riboswitches, this residue is usually cytosine (e.g., C74), and in adenine riboswitches, it is usually uracil (e.g., U74). Homologous forms of purine riboswitches bind deoxyguanosine but differ significantly by more than a single base mutation. See, e.g., Serganov et al., "Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs." Chem Biol. 2004; 11 (12): 1729-41; Batey et al., "Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine." Nature. 2004; 432 (7015): 411-415; Mandal and Breaker, "Adenine riboswitches and gene activation by disruption of a transcription terminator." Nat Struct Mol Biol. 2004; 11 (1): 29-35, the entire contents of which are incorporated herein by reference.

[0032] SAH riboswitches bind S-adenosylhomocysteine ​​and control genes involved in recycling this metabolite, which is produced when S-adenosylmethionine is used in methylation reactions. See, e.g., Wang et al., "Riboswitches that Sense S-adenosylhomocysteine ​​and Activate Genes Involved in Coenzyme Recycling," Mol. Cell 2008; 29 (6): 691-702; Edwards et al., "Structural basis for recognition of S-adenosylhomocysteine ​​by riboswitches," RNA 2010; 16 (11): 2144-2155, the entire contents of which are incorporated herein by reference.

[0033] SAM riboswitches bind S-adenosylmethionine (SAM) and regulate the biosynthesis and transport of methionine and SAM. At least four SAM riboswitches are known: SAM-I (originally called S-box), SAM-II, SMK box riboswitch, and SAM-IV. SAM-I is common in bacteria, whereas SAM-II is present only in Proteobacteria α, Proteobacteria β, and some Proteobacteria γ. SMK box riboswitches are thought to be present only in the Lactobacillales order. SAM-IV riboswitches have a similar ligand-binding core to SAM-I riboswitches but are associated with a different scaffold. See, e.g., Montange et al., "Structure of the S-adenosylmethionine riboswitch regulatory mRNA element." Nature. 2006; 441:1172-1175; Winkler et al., "An mRNA structure that controls gene expression by binding Sadenosylmethionine." Nat Struct Biol. 2003; 10: 701-707; Zasha et al., "The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches." RNA. 2008; 14(5): 822-828, the entire contents of which are incorporated herein by reference.

[0034] Tetrahydrofolate riboswitches bind tetrahydrofolate to regulate synthesis and transport genes. See, e.g., Ames et al., "A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate." Chem. Biol. 2010; 17 (7): 681-5; Huang et al., "Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch." Proc. Natl. Acad. Sci. USA 2011; 108 (36): 14801-6; Trausch et al., "The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand binding sites in a single aptamer." Structure. 2011; 19 (10): 1413-23, the entire contents of which are incorporated herein by reference.

[0035] The theophylline riboswitch was identified by SELEX and selectively binds the small molecule theophylline. The aptamer contains a 15-nucleotide core motif required for theophylline binding. See, e.g., Jenison et al., "High-resolution molecular discrimination by RNA." Science. 1994; 263:1425-1429; Zimmerman et al., "Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer." RNA. 2000; 6(5):659-67; Suess et al., "A theophylline-responsive riboswitch based on helix slipping controls gene expression in vivo." Nucleic Acids Res. 2004; 32(4): 1610-1614, the entire contents of which are incorporated herein by reference. See, e.g., Figures 1C and 1D.

[0036] The TPP riboswitch (also called the THI-box) binds thiamine pyrophosphate (TPP) to control the transport of metabolites as well as thiamine biosynthesis and transport. It is believed to be the only riboswitch yet found in eukaryotes. See, e.g., Edwards et al., "Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition." Structure 2006; 14 (9): 1459-68; Winkler et al., "Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression." Nature. 2002; 419 (6910): 952-956; Serganov et al., "Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch." Nature. 2006; 441 (7097): 1167-1171, the entire contents of which are incorporated herein by reference.

[0037] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease, including the Cas9 protein or a fragment thereof. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (e.g., viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPRRNA (crRNA). In type II CRISPR systems, proper processing of the crRNA precursor requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of the crRNA precursor. The Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to the crRNA are first cleaved intranucleolytically, followed by 3'-5' cleavage from the end. In nature, DNA binding and cleavage typically require proteins and both types of RNA. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA portions into a single RNA molecule. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire disclosure of which is incorporated herein by reference. Cas9 recognizes a short motif within the CRISPR repeat sequence (PAM, or protospacer adjacent motif) to help distinguish self from non-self.The sequence and structure of Cas9 nuclease are known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L. expand / collapse author list McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012), the entire disclosures of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire disclosure of which is incorporated herein by reference. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants are homologous to Cas9 or fragments thereof. For example, Cas9 variants are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO: 1 (nucleotide); SEQ ID NO: 2 (amino acid)).

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[0038] The terms "conjugated," "conjugated," and "conjugation" refer to the association of two entities, e.g., two molecules, such as two proteins, two domains (e.g., a binding domain and a cleavage domain), or a protein and an agent, e.g., a protein binding domain and a small molecule. In some aspects, the association occurs between a protein (e.g., an RNA programmable nuclease) and a nucleic acid (e.g., a guide RNA). The association can be via a covalent bond, e.g., a direct or indirect (e.g., via a linker). In some embodiments, the association is by a covalent bond. In some embodiments, the two molecules are conjugated via a linker that connects the two molecules. For example, in embodiments where two portions of an RNA, such as an aptamer (i.e., a nucleic acid sensing domain) and a gRNA, are conjugated to each other, the two RNAs can be conjugated via a polynucleotide linker, e.g., a nucleotide sequence that connects the 3' end of one RNA to the 5' end of the other RNA. In some embodiments, the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides.

[0039] The term "consensus sequence," as used herein in connection with nucleic acid sequences, refers to a calculated sequence representing the most frequently occurring nucleotide residue at each position in multiple similar sequences. Typically, a consensus sequence is determined by sequence alignment, in which similar sequences are compared to one another and similar sequence motifs are calculated.

[0040] As used herein, the term "effective amount" refers to an amount of a bioactive agent sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nuclease can refer to an amount of nuclease sufficient to induce cleavage at a desired target site to which the nuclease specifically binds and cleaves, preferably with minimal or no off-target cleavage. As will be understood by those skilled in the art, the effective amount of an agent, such as a nuclease, hybrid protein, fusion protein, protein dimer, protein (or protein dimer)-polynucleotide complex, or polynucleotide, will vary depending on various factors, including, for example, the desired biological response, the specific allele, genome, target site, cell, or tissue being targeted, and the agent being used.

[0041] As used herein, the term "engineered" refers to a nucleic acid molecule, protein molecule, complex, substance, or entity that is artificially designed, produced, prepared, synthesized, and / or manufactured. Thus, an engineered product is a product that does not exist in nature.

[0042] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties, such as, for example, an aptamer (i.e., a nucleic acid sense domain) and a gRNA. Typically, a linker is located between or adjacent to two groups, molecules, or other moieties and connects them by covalently binding each other. In some embodiments, the linker is a nucleotide linker. In some embodiments, the nucleotide linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety.

[0043] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, such as a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. As used herein, a mutation is typically represented by identifying the original residue, then placing the residue in the sequence, and identifying the newly substituted residue. Amino acid substitution (mutation) methods provided herein are known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0044] As used herein, the term "nuclease" refers to an agent, e.g., a protein or small molecule, capable of cleaving the phosphodiester bond linking nucleotide residues in a nucleic acid molecule. In some embodiments, a nuclease is a protein, e.g., an enzyme, capable of binding a nucleic acid molecule and cleaving the phosphodiester bond linking nucleotide residues within a nucleic acid molecule. A nuclease may be an endonuclease, which cleaves a phosphodiester bond within a polynucleotide chain, or an exonuclease, which cleaves the terminal phosphodiester bond of a polynucleotide chain. In some embodiments, a nuclease is a site-specific nuclease that ligates and / or cleaves a specific phosphodiester bond within a specific nucleotide sequence, also referred to herein as a "recognition sequence," "nuclease target site," or "target site." In some embodiments, a nuclease is an RNA-guided (i.e., RNA-programmable) nuclease that provides sequence specificity for the nuclease by complexing with (e.g., binding to) an RNA (e.g., guide RNA, "gRNA") having a sequence complementary to the target site. In some embodiments, the nuclease recognizes a single-stranded target site. In other embodiments, the nuclease recognizes a double-stranded target site, such as a double-stranded DNA target site. Many naturally occurring nuclease target sites, such as many naturally occurring DNA restriction nucleases, are known to those of skill in the art. In many cases, DNA nucleases, such as EcoRI, HindIII, or BamHI, recognize double-stranded DNA target sites that are palindromic, 4-10 base pairs in length, and cleave each strand of the two DNA strands at a specific location within the target site. Some endonucleases cleave double-stranded nucleic acid target sites symmetrically, i.e., at the same position on both strands so that the ends consist of base-paired nucleotides, also referred to herein as blunt ends. Other endonucleases cleave double-stranded nucleic acid target sites asymmetrically, i.e., at different positions on both strands so that the ends consist of unpaired nucleotides.Unpaired nucleotides at the ends of double-stranded DNA molecules are also referred to as "overhangs," e.g., "5' overhangs" or "3' overhangs," depending on whether the unpaired nucleotides form the 5' or 5' ends of each DNA strand. Ends of double-stranded DNA molecules that terminate in unpaired nucleotides are also called sticky ends because they can "stick" to the ends of other double-stranded DNA molecules containing complementary unpaired nucleotides. Nuclease proteins typically mediate the interaction of the protein with nucleic acid substrates and, in some cases, contain a "binding domain" that specifically binds to a target site and a "cleavage domain" that catalyzes cleavage of phosphodiester bonds within the nucleic acid backbone. In some embodiments, nuclease proteins are capable of binding and cleaving nucleic acid molecules in a monomeric form, while in other embodiments, nuclease proteins must dimerize or multimerize to cleave target nucleic acid molecules. Naturally occurring nuclease binding and cleavage domains, as well as modular binding and cleavage domains that can be fused to create nucleases that bind to specific target sites, are known to those of skill in the art. For example, the binding domain of an RNA-programmable nuclease (e.g., Cas9), i.e., a Cas9 protein having an inactive DNA cleavage domain, can be used as a binding domain that specifically binds a desired target site (e.g., binds to a gRNA to direct binding to the target site), and fused or joined to a cleavage domain, such as the cleavage domain of FokI, to create an engineered nuclease that cleaves the target site.

[0045] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds containing a nucleobase and an acidic moiety, such as, for example, a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, a nucleic acid polymer, such as, for example, a nucleic acid molecule containing three or more nucleotides, is a linear molecule in which adjacent nucleotides are linked to each other by phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a polymer of nucleotides (e.g., at least three nucleotides linked together). In some embodiments, "nucleic acid" includes RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids may occur naturally, for example, as genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. Alternatively, a nucleic acid molecule can be a non-naturally occurring molecule, such as, for example, recombinant DNA or RNA, an artificial chromosome, an engineered genome or fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or molecules containing non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, or chemically synthesized. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can contain nucleoside analogs, such as analogs with chemically modified bases or sugars and backbone modifications. Nucleic acid sequences are written in the 5' to 3' direction unless otherwise specified.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-isopropylur ... is, or contains, 5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0046] As used herein, the term "pharmaceutical composition" refers to a composition that can be administered to a subject in connection with the treatment of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, such as a nuclease or a nucleic acid encoding a nuclease, and a pharmaceutically acceptable excipient.

[0047] As used herein, the term "proliferative disorder" refers to a disorder in which cell or tissue homeostasis is disturbed, such that a cell or cell population exhibits an abnormally high rate of proliferation. Proliferative disorders include hyperproliferative disorders such as pre-neoplastic hyperplastic conditions and neoplastic disorders. Neoplastic disorders are characterized by abnormal cell proliferation and include both benign and malignant neoplasms. Malignant neoplasms are also called cancers.

[0048] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by conjugation, functionalization, or the addition of chemical moieties such as linkers for other modifications, carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, or fatty acid groups. A protein, peptide, or polypeptide can be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be natural, recombinant, synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. A protein may be placed at the amino-terminal (N-terminal) portion or the carboxy-terminal (C-terminal) portion of the fusion protein to form an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. The protein may comprise distinct domains, such as a nucleic acid-binding domain (e.g., a gRNA-binding domain of Cas9 that directs binding of the protein to a target site) and a nucleic acid-cleavage domain. In some embodiments, the protein comprises a proteinaceous portion, such as an amino acid sequence that constitutes the nucleic acid-binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed with or associated with a nucleic acid, such as RNA. Any of the proteins provided herein can be produced by any method known in the art.For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire disclosure of which is incorporated herein by reference.

[0049] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used interchangeably herein to refer to a nuclease that forms a complex (e.g., binds or associates) with one or more RNAs that are not targets for cleavage. In some embodiments, when complexed with an RNA, the RNA-programmable nuclease is referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). A gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. While "gRNA" is used interchangeably to refer to a guide RNA that exists either as a single molecule or as a complex of two or more molecules, a gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA). Typically, a gRNA that exists as a single RNA species has at least two domains: (1) a domain that shares homology with the target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) is the "sgRNA backbone" shown in any of Figures 1-4. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is homologous to the tracrRNA shown in Figure 1E of Jinek et al., Science 337:816-821 (2012), the entire disclosure of which is incorporated herein by reference. In some embodiments, domain 2 is at least 90%, at least 95%, at least 98%, or at least 99% identical to the "sgRNA backbone" of any of Figures 1-4 or to the tracrRNA described in Jinek et al., Science 337:816-821 (2012). In some embodiments, the gRNA comprises two or more domains (1) and (2) and can be referred to as an "extended gRNA." For example, an extended gRNA can bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions. The gRNA contains a nucleotide sequence complementary to the target site that mediates binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.The sequence of the gRNA that binds the target nucleic acid can be complementary to a region of the target and include a sequence suitable for binding of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L. expand / collapse author list McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012), the entire disclosures of which are incorporated herein by reference.

[0050] RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to determine target DNA cleavage sites, and these proteins can in principle cleave any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify genomes) are known in the art (e.g., Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013), the entire disclosure of which is incorporated herein by reference).

[0051] The terms "small molecule" and "organic compound" are used interchangeably herein and refer to naturally occurring or artificially created (e.g., by chemical synthesis) molecules with relatively small molecular weights. Organic compounds typically contain carbon. Organic compounds can contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxy, carbonyls, or heterocyclic rings). In some embodiments, the organic compound is monomeric and has a molecular weight of less than about 1500 g / mol. In some embodiments, the molecular weight of a small molecule is less than about 1000 g / mol or less than about 500 g / mol. In some embodiments, the small molecule is a drug, e.g., a drug already deemed safe and effective by a governmental or regulatory body for use in humans or animals. In some embodiments, the small molecule is known to bind an aptamer. In some embodiments, the organic compound is an antibiotic, e.g., an anticancer antibiotic, e.g., dynemycin, neocarzinostatin, calicheamicin, esperamicin, bleomycin, or derivatives thereof.

[0052] As used herein, the term "subject" refers to an individual organism, such as, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either gender and at any stage of development.

[0053] The terms "target nucleic acid" and "target genome" as used herein in connection with nucleases refer to a nucleic acid molecule or genome, respectively, that contains at least one target site targeted by a given nuclease.

[0054] The term "target site," used interchangeably herein with the term "nuclease target site," refers to a sequence within a nucleic acid molecule to which a nuclease binds and cleaves. A target site can be single-stranded or double-stranded. In the context of RNA-guided (e.g., RNA-programmable) nucleases (e.g., protein dimers comprising a Cas9 gRNA-binding domain and an active Cas9 DNA-cleavage domain), the target site typically comprises a nucleotide sequence complementary to the gRNA of the RNA-programmable nuclease and a protospacer adjacent motif (PAM) at the 3' end adjacent to the gRNA-complementary sequence. For the RNA-guided nuclease Cas9, in some embodiments, the target site can be 20 base pairs plus a 3 base pair PAM (e.g., NNN, where N represents any nucleotide). Typically, the first nucleotide of the PAM can be any nucleotide, while the downstream two nucleotides are specific depending on the particular RNA-guided nuclease. Examples of target sites for RNA-guided nucleases such as Cas9 are known to those skilled in the art and include, but are not limited to, NNG, NGN, NAG, and NGG, where N is any nucleotide. Furthermore, Cas9 nucleases from different species (e.g., S. thermophilus instead of S. pyogenes) recognize PAMs containing the sequence: NGGNG. Other PAM sequences are known and include, but are not limited to, NNAGAAW and NAAR (see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013), the entire disclosure of which is incorporated herein by reference). For example, target sites for RNA-guided nucleases such as Cas9 include [N Z)-[PAM] structure, where each N is independently any nucleotide, and Z is an integer between 1 and 50. In some embodiments, Z is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. In some embodiments, Z is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, Z is 20. In some embodiments, "target site" can also refer to a sequence within a nucleic acid molecule to which a nuclease binds but does not cleave.

[0055] The terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or preventing the progression of a disease or disorder as described herein, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or preventing the progression of a disease or disorder as described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have occurred and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or to prevent the onset or progression of a disease. For example, treatment can be administered to a susceptible individual before the onset of symptoms (e.g., in light of a history of symptoms and / or genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, e.g., to prevent or delay recurrence.

[0056] The term "vector" refers to a polynucleotide comprising one or more recombinant polynucleotides of the invention, such as those encoding the gRNA and / or Cas9 proteins provided herein. Vectors include, but are not limited to, plasmids, viral vectors, cosmids, artificial chromosomes, and phagemids. A vector is replicable in a host cell and is further characterized by one or more endonuclease restriction sites at which the vector can be cleaved and into which a desired nucleic acid sequence can be inserted. A vector can contain one or more marker sequences suitable for use in identifying and / or selecting cells transformed or genomically modified with the vector. Examples of markers include genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics (e.g., kanamycin, ampicillin) or other compounds, genes encoding enzymes (e.g., β-galactosidase, alkaline phosphatase, or luciferase) whose activity can be detected by routine assays known in the art, and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. Examples of any vector suitable for transforming host cells (e.g., E. coli, mammalian cells such as CHO cells, insect cells, etc.) encompassed by the present invention include vectors belonging to the pUC, pGEM, pET, pBAD, pTET, or pGEX series. In some embodiments, the vector is suitable for transforming host cells for recombinant protein production. Methods for selecting and engineering vectors and host cells that express gRNAs and / or proteins (e.g., those provided herein), transforming cells, and expressing and purifying recombinant proteins are known in the art, and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0057] Detailed Description of the Embodiments of the Invention Site-specific nucleases are powerful tools for targeted genome modification in vitro and in vivo. Some site-specific nucleases can theoretically achieve a level of target cleavage site specificity that allows cleavage to be targeted to a single, unique site in the genome without affecting other genomic sites. Nuclease cleavage in living cells has been reported to trigger DNA repair mechanisms that frequently result in modification of the cleaved and repaired genomic sequence, for example, by homologous recombination or nonhomologous end joining. Thus, targeted cleavage of specific sequences within the genome opens new avenues for targeted recombination and gene modification in living cells, including many human somatic or embryonic stem cells, which are difficult to manipulate using traditional targeted recombination methods. Nuclease-mediated modification of disease-associated sequences, such as CCR-5 alleles in HIV / AIDS patients, or genes required for tumor angiogenesis, can also be used in clinical settings, with two site-specific nucleases currently undergoing clinical trials (Perez, EE et al., "Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases." Nature Biotechnology. 26, 808-816 (2008); ClinicalTrials.gov identifiers: NCT00842634, NCT01044654, NCT01252641, NCT01082926). Other diseases treatable using site-specific nucleases include, for example, triplet disease and related diseases (e.g., Huntington's disease, myotonic dystrophy, spinocerebellar ataxia, etc.), cystic fibrosis (by targeting the CFTR gene), cancer, autoimmune diseases, and viral infections.

[0058] One of the key issues with site-specific nuclease-mediated modification is off-target nuclease effects, such as cleavage of genomic sequences that differ by one or more nucleotides from the intended target sequence. Undesirable side effects of off-target cleavage range from insertion into undesired loci during targeted gene recombination to severe complications in clinical settings. Off-target cleavage of tumor suppressor genes or sequences encoding critical gene functions by endonucleases administered to a subject can lead to disease or death in the subject. Therefore, it is desirable to employ new strategies to design nucleases that are most likely to minimize off-target effects.

[0059] The methods and compositions of the present disclosure, in some aspects, improve upon existing methods and compositions by providing a means for modulating the temporal activity and / or increasing the specificity of RNA-guided nucleases. For example, both naturally occurring and engineered RNA-guided nucleases known in the art typically bind to and cleave DNA by forming a complex with an RNA (e.g., gRNA) complementary to the target. Aspects of the present invention relate to the recognition that temporally modulating the timing of binding of the RNA-guided nuclease:RNA complex to its target reduces the potential for off-target effects by minimizing or modulating the length of time the complex is able to bind and cleave the target. Furthermore, engineering a gRNA to bind only to the target site to be cleaved, e.g., using a gRNA with an extended target recognition domain that blocks binding in the absence of the target, improves the specificity of the RNA-guided nuclease and reduces the potential for off-target effects.

[0060] The strategies, methods, compositions, kits, and systems provided herein can be used to modulate the activity and / or improve the specificity of any RNA-guided nuclease (e.g., Cas9). Suitable nucleases for use with the modified gRNAs described herein will be apparent to those of skill in the art based on this disclosure.

[0061] In certain embodiments, the strategies, methods, compositions, kits, and systems provided herein are used to control the timing of RNA-guided (e.g., RNA-programmable) nuclease activity. While typical RNA-guided nucleases recognize and cleave target sequences upon formation of a nuclease:RNA complex, the modified gRNAs provided herein allow for control of target binding and cleavage. Another aspect provides gRNAs that have been engineered to bind the intended target site only when it is present, thereby improving the specificity of the RNA-guided nuclease. Although Cas9:gRNA complexes have been effectively used to modify both cells (Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science. 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science. 339, 823-826 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013)) and organisms (Hwang, WY et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology. 31, 227-229 (2013)), studies using Cas9:guide RNA complexes to modify zebrafish embryos have not been performed using ZFNs and TALENs (Hwang, WY et al. Nature Biotechnology. 31, 227-229 (2013)). (2013)), observed toxicity (e.g., off-target effects) at rates similar to those observed in Cas9. Accordingly, aspects of the present disclosure aim to reduce the potential for Cas9 off-target effects using novel gRNA platforms that modulate the timing of target binding and cleavage and / or improve the specificity of RNA-guided nucleases.

[0062] With particular reference to DNA and DNA-cleaving nucleases such as Cas9, the inventive concepts, methods, compositions, strategies, kits, and systems provided herein are not limited in this respect, but are applicable to any nucleic acid:nuclease system that utilizes a nucleic acid template, such as RNA, to direct binding to a target nucleic acid.

[0063] Modified guide RNA (gRNA) Some aspects of the present disclosure provide gRNAs that are engineered to have both an "on" and an "off" state. In some aspects, these gRNAs can be collectively referred to as "switchable gRNAs." For example, a switchable gRNA is said to be in the "off" state when it is in a structural state that prevents the gRNA from binding to a target nucleic acid. In some aspects, a gRNA in the "off" state can bind to its cognate RNA-guided nuclease (e.g., Cas9), but the nuclease:gRNA complex (when the gRNA is in the "off" state) cannot bind to and mediate cleavage of the target nucleic acid. In other aspects, a gRNA in the "off" state cannot bind to a target sequence or an RNA-guided nuclease such as Cas9. Conversely, a switchable gRNA is said to be in the "on" state when the gRNA is in a structural state that allows the gRNA to bind to a target nucleic acid (e.g., in a complex with an RNA-guided nuclease such as Cas9). Some embodiments of the present disclosure provide complexes comprising the gRNAs of the present invention in association with an RNA-guided nuclease, such as Cas9, and methods for their use. Some embodiments of the present disclosure provide nucleic acids encoding such gRNAs and / or RNA-guided nucleases (e.g., Cas9). Some embodiments of the present disclosure provide expression constructs comprising such encoding nucleic acids.

[0064] Aptamer-based gRNA In one embodiment, a gRNA comprising an aptamer is provided. See, e.g., FIG. 1. For example, in some embodiments, the gRNA is linked to an aptamer via a nucleotide linker, as described herein. Aptamers are typically RNA- or peptide-based molecules that bind a specific ligand with an affinity that competes with, for example, an antibody-antigen interaction. In some embodiments, the aptamer has a K of about 1 nM to 10 μM, about 1 nM to 1 μM, about 1 nM to 500 nM, or about 1 nM to 100 nM. d 5'-GGUGAUACCAG CAUCGUCUUGAUG CCCUUGGCAGCACC-3' (SEQ ID NO: 3)

[0065] In some embodiments, the aptamer is not naturally occurring (e.g., not found in nature). For example, in some embodiments, the aptamer is engineered or selected from a library using SELEX. In some embodiments, the aptamer comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, or at least 300 nucleotides. In some embodiments, the aptamer comprises 20-200, 20-150, 20-100, or 20-80 nucleotides. In some embodiments, the gRNA portion of a provided RNA (e.g., an RNA comprising a gRNA linked to an aptamer) comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, or at least 200 nucleotides. In some embodiments, the gRNA portion comprises between 60 and 150, between 60 and 100, or between 60 and 80 nucleotides.

[0066] gRNA that senses mRNA In other embodiments, gRNAs are provided that bind to a target nucleic acid under certain conditions (e.g., in the presence of a metabolite, small molecule, nucleic acid, etc.). In some embodiments, the gRNA is structurally prevented from binding (e.g., hybridizing) to the target (e.g., in the "off" state) unless another molecule binds (e.g., hybridizes) to the gRNA, resulting in a structural rearrangement corresponding to the "on" state. In some embodiments, binding of a specific transcript (e.g., mRNA) to the gRNA transitions the gRNA from the "off" state to the "on" state. See, e.g., Figure 2. Such gRNAs are referred to as gRNAs that "sense mRNA." For example, in some aspects, gRNAs are provided that include: (i) a region that hybridizes to a region of the target nucleic acid (e.g., a "guide" sequence); (ii) another region that partially or fully hybridizes to the sequence of region (i) (e.g., a "guide block" sequence); and (iii) a region that hybridizes to a region of the transcript (mRNA) (e.g., a "transcript sensor"). In some embodiments, each region (e.g., i-iii) comprises at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides. In some embodiments, the gRNA forms a stem-loop structure. In some embodiments, the stem comprises the sequence of region (i) hybridized with part or all of the sequence of region (ii), and the loop consists of part or all of the sequence of region (iii). In some embodiments, both region (ii) and region (iii) are located either 5' or 3' to region (i). See, e.g., Figures 2A vs. 2C. The sequence of the gRNA that binds the target (e.g., the "guide" sequence) can be engineered to include any sequence that targets any desired nucleic acid target using methods known in the art and is therefore not limited to the sequences shown by way of example. Similarly, region (iii) (e.g., the transcript sensor) can be engineered to include any sequence that hybridizes to an mRNA of interest using methods known in the art. Similarly, region (ii) can be engineered, using methods routine in the art, to contain a sequence that hybridizes to part or all of a "guide" sequence.For example, in some aspects, mRNA, when expressed in a cell, is one for which genomic modification of a target nucleic acid (e.g., a gene) is desired. Thus, when delivered (or expressed) into a cell in the absence of mRNA, the gRNA remains in an "off" state. When mRNA is present (e.g., expressed), it binds the gRNA's transcript sensor, resulting in the unfolding of the stem-loop structure that prevented the "guide" sequence from hybridizing to the target nucleic acid, thereby turning the gRNA "on." See, e.g., Figures 2B and 2D. The provided "on" state gRNA can associate with an RNA-guided nuclease (e.g., Cas9 protein) to guide binding of the target nucleic acid.

[0067] gRNA that senses the extended DNA (xDNA) In other embodiments, modified gRNAs are provided that remain in an "off" state unless the gRNA hybridizes to a target nucleic acid in at least two distinct regions. See, e.g., Figure 3. Such gRNAs effectively extend the recognition sequence of a particular gRNA / target interaction, thereby improving specificity for RNA-guided nucleases (e.g., Cas9). Such gRNAs are referred to as "xDNA-sensing" gRNAs (the "x" stands for "extended" DNA recognition). For example, gRNAs are provided that include: (i) a region that hybridizes to a region of the target nucleic acid (e.g., a "guide" sequence); (ii) another region that partially or fully hybridizes to the sequence of region (i) (e.g., a "guide block"); and (iii) a region that hybridizes to another region of the target nucleic acid (e.g., an "xDNA sensor"). In some embodiments, the xDNA sensor must first bind the target nucleic acid before the guide sequence can bind to the target. In some embodiments, the xDNA sensor binds the same strand of the target that binds the guide sequence. In some embodiments, the xDNA sensor and guide sequences bind different strands of the target nucleic acid. In some embodiments, the sequences of region (i) and region (ii) comprise at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides. In some embodiments, the sequence of region (iii) comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides. In some embodiments, the gRNA forms a stem-loop structure. For example, in some embodiments, the stem comprises the sequence of region (i) hybridized with part or all of the sequence of region (ii), and the loop consists of part or all of the sequence of region (iii). In some embodiments, region (i) and region (iii) comprise adjacent sequences in the gRNA. In some embodiments, both region (ii) and region (iii) are either 5' or 3' to region (i). See, e.g., Figure 3A versus Figure 3C. In some embodiments, region (ii) is disposed between region (i) and region (iii).The sequence of the gRNA that binds the target (e.g., the "guide" sequence) can be engineered to include any sequence that targets any desired nucleic acid target using methods known in the art, and thus is not limited to the exemplary sequences shown in the figures. Similarly, region (iii) (e.g., the xDNA sensor) can be engineered using methods known in the art to include any sequence that hybridizes to other regions of the target nucleic acid (e.g., regions different from the region targeted by the "guide" sequence). Similarly, region (ii) can be engineered using methods known in the art to include a sequence that hybridizes to part or all of the "guide" sequence. Thus, in the absence of the appropriate target nucleic acid (e.g., a target that includes both regions to which the gRNA is designed to hybridize), the gRNA will remain in an "off" state once delivered to (or expressed in) a cell. Without being bound by theory, it is believed that when the gRNA (e.g., in association with Cas9) contacts a target nucleic acid, the xDNA sensor hybridizes to the target, which then unwinds the stem-loop structure that blocks the "guide" sequence, turning the gRNA "on." If it is the correct target nucleic acid, the guide sequence will hybridize to the target, and the complex will cleave the target nucleic acid at will. See, e.g., Figures 3B and 3D.

[0068] Complex In some embodiments, complexes are provided that include any RNA / gRNA described herein (e.g., RNA comprising a gRNA linked to an aptamer, a gRNA that senses an mRNA, or a gRNA comprising an xDNA sensor). In some aspects, complexes are provided that include a provided RNA / gRNA associated with an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is Cas9, a Cas9 mutant, or a Cas9 fragment, e.g., as described herein. In some embodiments, the RNA-guided nuclease is any form of the Cas9 protein provided in U.S. Provisional Patent Application No. 61 / 874,609, filed September 6, 2013, entitled "Cas9 Mutants and Uses Thereof," and U.S. Provisional Patent Application No. 61 / 874,746, filed September 6, 2014, entitled "Delivery System for Functional Nucleases," the entire contents of which are incorporated herein by reference.

[0069] In some embodiments, the complex further comprises a ligand, such as, for example, a ligand that binds an aptamer for RNA associated with an RNA-guided nuclease described herein. In some embodiments, the complex (e.g., comprising a provided RNA (gRNA):ligand:Cas9 protein) binds to and optionally cleaves a target nucleic acid. In some aspects, a complex comprising a "sensing" gRNA (e.g., mRNA or xDNA) and Cas9 binds to and optionally cleaves a target nucleic acid.

[0070] Pharmaceutical Composition In some embodiments, any gRNA described herein is provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises an RNA-guided nuclease (e.g., Cas9) that forms a complex with the gRNA of the invention. For example, some embodiments provide a pharmaceutical composition comprising a gRNA and an RNA-guided nuclease provided herein, or a nucleic acid encoding such a gRNA and / or nuclease, and a pharmaceutically acceptable excipient. The pharmaceutical composition can optionally further comprise one or more therapeutically active substances.

[0071] In some embodiments, the compositions provided herein are administered to a subject, e.g., a human subject, to effect targeted genome modification in the subject. In some embodiments, cells are obtained from the subject and contacted in vitro with the provided gRNA or a nucleic acid encoding the same, which is associated with an RNA-guided nuclease. In some embodiments, cells removed from the subject and contacted in vitro with the gRNA:nuclease complex of the present invention are reintroduced into the subject, optionally after the desired genome modification has been effected or detected in the cells. Methods for delivering pharmaceutical compositions containing nucleases are known and are described, for example, in U.S. Patent Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the entire disclosures of which are incorporated herein by reference. While the description of pharmaceutical compositions provided herein is primarily in terms of pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals or organisms. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and / or implement such modifications, if necessary, with no more than routine experimentation. Subjects to which the pharmaceutical compositions are intended for administration include, but are not limited to, humans and / or other primates; mammals, livestock, pets, and commercially important mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, such as commercially important birds such as chicken, duck, goose, and / or turkey.

[0072] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0073] As used herein, pharmaceutical formulations can further include pharmaceutically acceptable excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, the entire disclosure of which is incorporated herein by reference) describes various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. International Patent No. PCT / US2010 / 055131 (Publication No. WO2011053982A8, filed November 2, 2010), the entire disclosure of which is incorporated herein by reference, may also be referenced for other suitable methods, reagents, excipients, and solvents for the preparation of pharmaceutical compositions containing nucleases. Except insofar as any conventional excipient vehicle is incompatible with a substance or its derivatives, either by producing undesirable biological effects or by interacting in a deleterious manner with other components of the pharmaceutical composition, its use is considered to be within the scope of this disclosure.

[0074] In some embodiments, the compositions according to the present invention can be used to treat any of a variety of diseases, disorders and / or conditions, including, but not limited to, one or more of the following: autoimmune diseases (e.g., diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory diseases (e.g., arthritis, pelvic inflammatory disease); infectious diseases (e.g., viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological diseases (e.g., Alzheimer's disease, Huntington's disease; autism; Duchenne myopathy); dystrophies); cardiovascular disorders (e.g., atherosclerosis, hypercholesterolemia, thrombosis, coagulation disorders, angiogenesis disorders such as macular degeneration); proliferative disorders (e.g., cancer, benign tumors); respiratory diseases (e.g., chronic obstructive pulmonary disease); digestive disorders (e.g., inflammatory bowel disease, ulcers); musculoskeletal diseases (e.g., fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g., diabetes, osteoporosis); urinary disorders (e.g., renal disease); psychological disorders (e.g., depression, schizophrenia); skin disorders (e.g., wounds, eczema); blood and lymphatic disorders (e.g., anemia, hemophilia); etc.

[0075] Site-specific nucleic acid cleavage method In other embodiments of the present disclosure, methods for site-specific nucleic acid (e.g., DNA) cleavage are provided. In some embodiments, the methods involve contacting DNA with any of the Cas9:RNA complexes described herein. For example, in some embodiments, the methods involve contacting DNA with a complex comprising: (i) a gRNA that includes a sequence that binds to a portion of the DNA and is linked to an aptamer described herein; (ii) a ligand that is bound to the aptamer of the gRNA; and (iii) an RNA-guided nuclease (e.g., a Cas9 protein) under conditions suitable for the Cas9 nuclease to cleave the DNA.

[0076] In some embodiments, a method for inducing site-specific DNA cleavage in a cell is provided. In some embodiments, the method comprises: (a) contacting a cell with or expressing in the cell a gRNA comprising a sequence capable of binding to a DNA target sequence and comprising an aptamer described herein; (b) contacting the cell with or expressing in the cell an RNA-guided nuclease (e.g., a Cas9 protein); and (c) contacting the cell with a specific ligand that binds the aptamer of the gRNA to form a gRNA:ligand:Cas9 complex that cleaves the DNA target. In some embodiments, the method comprises: (a) contacting the cell with a complex comprising a gRNA comprising a sequence capable of binding to a DNA target sequence and comprising an aptamer described herein and a Cas9 protein; and (b) contacting the cell with a specific ligand that binds the aptamer of the gRNA to form a gRNA:ligand:Cas9 complex that cleaves the DNA target. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, steps (a) and (b) are performed sequentially. Thus, in some embodiments where the cell is contacted with the complex followed by the ligand, modulation of cleavage is achieved because cleavage occurs only after the ligand is delivered to the cell. In some embodiments of these methods, the ligand is not delivered to the cell but is produced internally by the cell, e.g., as part of a physiological or pathophysiological process.

[0077] In some embodiments, a site-specific DNA cleavage method using a gRNA that senses the mRNA described herein is provided. For example, in some embodiments, the method comprises contacting DNA with a complex comprising an RNA-guided nuclease (e.g., Cas9 protein) and a gRNA that senses the mRNA, the complex comprising: (i) a region that hybridizes with a region of the target nucleic acid; (ii) another region that partially or completely hybridizes with the sequence of region (i); and (iii) a region that hybridizes with a region of the transcript (mRNA). In some embodiments, cleavage occurs after the sequence in region (iii) hybridizes with the mRNA.

[0078] In other embodiments, methods for site-specific DNA cleavage using a gRNA that senses xDNA described herein are provided. For example, in some embodiments, the methods involve contacting DNA with a complex comprising an RNA-guided nuclease (e.g., a Cas9 protein) and a gRNA that senses xDNA, the complex comprising: (i) a region that hybridizes to a region of the target nucleic acid; (ii) another region that partially or completely hybridizes to the sequence in region (i); and (iii) a region that hybridizes to another region of the target nucleic acid. In some embodiments, cleavage occurs after the sequence in region (iii) hybridizes to a region of the target nucleic acid that is not targeted by the "guide" sequence.

[0079] In some embodiments, any of the methods provided herein can be performed on DNA in a cell. For example, in some embodiments, the DNA contacted by any of the RNA / gRNA-containing complexes provided herein is in a eukaryotic cell. In some embodiments, the eukaryotic cell is in an individual. In some embodiments, the individual is a human. In some embodiments, any of the methods provided herein are performed in vitro. In some embodiments, any of the methods provided herein are performed in vivo.

[0080] Polynucleotides, vectors, cells, kits In other embodiments of the present disclosure, polynucleotides encoding any of the gRNAs (and optionally any of the Cas9 proteins) described herein are provided. For example, polynucleotides encoding any of the gRNAs and / or Cas9 proteins described herein are provided for recombinant expression and purification of the gRNAs of the invention, or complexes comprising same, such as complexes comprising the gRNAs of the invention and an RNA-guided nuclease (e.g., Cas9 protein). In some embodiments, the provided polynucleotides comprise one or more sequences encoding a gRNA, alone or in combination with a sequence encoding any of the Cas9 proteins described herein.

[0081] In some embodiments, vectors encoding any of the gRNAs described herein (and optionally, any Cas9 protein) are provided for recombinant expression and purification, e.g., of a gRNA of the invention, or a complex comprising a gRNA of the invention and an RNA-guided nuclease (e.g., a Cas9 protein). In some embodiments, the vector comprises, or is engineered to comprise, a polynucleotide, e.g., such as those described herein. In some embodiments, the vector comprises one or more sequences encoding a gRNA and / or any Cas9 protein (e.g., those described herein). Typically, the vector comprises a sequence encoding a gRNA of the invention operably linked to a promoter such that the gRNA is expressed in a host cell.

[0082] In some embodiments, cells are provided for recombinant expression and purification of any gRNA (and optionally any Cas9 protein) described herein. Cells include any cell suitable for recombinant RNA expression and, optionally, protein expression, such as cells containing a genetic construct expressing or capable of expressing a gRNA of the invention (e.g., a cell transformed with one or more vectors described herein, or a cell with a genomic modification that expresses a gRNA of the invention and, optionally, any Cas9 protein provided herein, from an allele integrated into the cell's genome). Methods for transforming cells, genetically modifying cells, and expressing genes and proteins in such cells are known in the art, including, for example, those provided by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)) and Friedman and Rossi, Gene Transfer: Delivery and Expression of DNA and RNA, A Laboratory Manual (1st ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2006)).

[0083] Some aspects of the present disclosure provide kits comprising any of the gRNAs or complexes of the present invention provided herein and, optionally, any of the Cas9 proteins described herein. In some embodiments, the kits include any polynucleotide encoding a provided gRNA and, optionally, any of the Cas9 proteins. In some embodiments, the kits include a vector for recombinant expression of any of the gRNAs of the present invention and, optionally, any of the Cas9 proteins. In some embodiments, the kits include cells comprising a genetic construct expressing any of the gRNAs of the present invention, complexes, and, optionally, any of the Cas9 proteins provided herein. In some embodiments, the kits include an excipient and instructions for contacting the excipient with any of the compositions of the present invention to generate a composition suitable for contacting a nucleic acid, e.g., a complex of a gRNA and an RNA-guided nuclease, such as Cas9, with a nucleic acid. In some embodiments, the composition is suitable for contacting a nucleic acid within a genome. In some embodiments, the composition is suitable for delivering a composition of the present invention (e.g., a gRNA, a complex of gRNA and Cas9) to a cell. In some embodiments, the composition is suitable for delivering a composition of the invention (e.g., a gRNA, a complex of gRNA and Cas9) to a subject. In some embodiments, the excipient is a pharmaceutically acceptable excipient.

[0084] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0085] In the claims, articles such as "a," "an," and "the" are intended to mean one or more unless specifically stated otherwise or clear from context. Any claim or description containing "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the members of the group are present in, employed in, or otherwise involved in a given product or process, unless specifically stated otherwise or clear from context. The invention includes embodiments in which only one member of a group is present in, employed in, or otherwise involved in a given product or process. The invention also includes embodiments in which more than one, or all, are present in, employed in, or otherwise involved in a given product or process.

[0086] Furthermore, the present invention should be understood to include all variations, combinations, and substitutions of one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or relevant portions of the description incorporated into other claims. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same original claim. Furthermore, when a claim refers to a composition, it should be understood to include methods of using the composition for any purpose described herein, and to include methods of making the composition by any method described herein or by any other method known in the art, unless specifically stated otherwise or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art.

[0087] When elements are presented as a list, such as in Markush claim format, it should be understood that each subgroup of elements is also disclosed, and that any element may be removed from the group. It should also be noted that the term "comprising" is intended to be open and allow for the inclusion of additional elements or steps. Generally, when the invention, or aspects of the invention, are referred to as including particular elements, features, steps, etc., it should be understood that certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, those elements, features, steps, etc. For the sake of brevity, these embodiments are not specifically described in these terms herein. Thus, for each embodiment of the invention that includes one or more elements, features, steps, etc., the invention also provides embodiments that consist of, or consist essentially of, those elements, features, steps, etc.

[0088] When ranges are given, the endpoints are inclusive. Furthermore, unless otherwise specifically stated or apparent from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed in ranges can, in different embodiments of the invention, assume any specific value within the stated range, down to one-tenth of the unit of the minimum value of the range, unless the context clearly dictates otherwise. It should also be understood that unless otherwise specifically stated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed in ranges can assume any subranges within the given range, where the endpoints of the subranges are stated to the same degree of precision as one-tenth of the unit of the minimum value of the range.

[0089] Furthermore, it should be understood that any particular embodiment of the present invention can be explicitly excluded from any one or more claims. Where ranges are given, any value within the range can be explicitly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present invention can be excluded from any one or more claims. For the sake of brevity, not all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are explicitly set forth herein.

[0090] All publications, patents, and sequence database entries mentioned herein, including those listed above, are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including definitions herein, will control.

Claims

**Claim 1** A single guide RNA (sgRNA), comprising: (i) a region that hybridizes with a first portion of a target nucleic acid, the region comprising a sequence of at least 10 nucleotides; (ii) a region that hybridizes with the sequence of region (i), the region comprising a sequence of at least 10 nucleotides, wherein the sgRNA forms a stem-loop structure, and the stem comprises the sequence of region (i) that hybridizes with the sequence of region (ii); (iii) a region that hybridizes with a second portion of the target nucleic acid, the region comprising a sequence of at least 10 nucleotides and when the sequence of region (i) hybridizes with the sequence of region (ii), the loop of the stem-loop structure is formed by the sequence of region (iii); in the absence of a second portion of the target nucleic acid that hybridizes with the sequence of region (iii), a stem-loop structure is formed; when the second portion of the target nucleic acid hybridizes with the sequence of region (iii), the stem-loop structure is unfolded or the formation of the stem-loop structure is prevented so that the sequence of region (ii) does not hybridize with the sequence of region (i); and the sgRNA further comprises a domain that binds to the Cas9 protein the said sgRNA. **Claim 2** The sgRNA according to claim 1, wherein each of the sequences of region (i) and region (ii) comprises at least 15, at least 20, or at least 25 nucleotides. **Claim 3** The sgRNA according to claim 1 or 2, wherein the sequence of region (iii) comprises at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides. **Claim 4** Region (i) comprises a sequence of at least 10 or at least 15 consecutive nucleotides that is 100% complementary to a first portion of the target nucleic acid sequence, and region (ii) comprises a sequence of at least 10 or at least 15 consecutive nucleotides that is 100% complementary to the sequence of region (i). The sgRNA according to any one of claims 1 to 3. **Claim 5** The sgRNA according to any one of claims 1 to 4, wherein both region (ii) and region (iii) are either on the 5' or 3' side of region (i).

6. The sgRNA according to any one of claims 1 to 5, wherein when the sgRNA binds to the Cas9 protein and the sequence of region (iii) hybridizes to the second part of the target nucleic acid, the sequence of region (i) hybridizes to the first part of the target nucleic acid.

7. The sgRNA according to any one of claims 1 to 6, wherein the target nucleic acid is DNA.

8. A complex comprising the sgRNA according to any one of claims 1 to 7 and a Cas9 protein.

9. The complex according to claim 8, further comprising a target nucleic acid.

10. The complex according to claim 9, wherein the target nucleic acid is cleaved upon formation of the complex.

11. The complex according to claim 9 or 10, wherein the target nucleic acid is DNA.

12. A polynucleotide encoding the sgRNA according to any one of claims 1 to 7.

13. A vector comprising the polynucleotide according to claim 12.

14. The vector according to claim 13, further comprising a polynucleotide encoding a Cas9 protein.

15. A cell comprising a gene construct that expresses the sgRNA according to any one of claims 1 to 7.

16. The cell according to claim 15, further comprising a Cas9 protein.

17. A kit comprising the sgRNA according to any one of claims 1 to 7.

18. A kit comprising a polynucleotide encoding the sgRNA according to any one of claims 1 to 7.

19. A kit comprising a vector for recombinant expression, wherein the vector comprises a polynucleotide encoding the sgRNA according to any one of claims 1 to 7.

20. A kit comprising a cell comprising a gene construct that expresses the sgRNA according to any one of claims 1 to 7.

21. The kit according to any one of claims 17 to 20, further comprising one or more Cas9 proteins or a vector that expresses one or more Cas9 proteins.

22. An in vitro or ex vivo site-specific DNA cleavage method comprising contacting DNA with the complex according to any one of claims 8 to 11.

23. The method according to claim 22, wherein the DNA is in a cell.

24. The method according to claim 23, wherein the cell is in vitro.

25. The method according to claim 23, wherein the cell is ex vivo.

26. The method according to any one of claims 23 to 25, wherein the cell is a eukaryotic cell.

27. The method according to claim 23 or 26, wherein the cell is a eukaryotic cell from an individual.

28. The method according to claim 27, wherein the individual is a human.

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