RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional

Application, U.S. S.N. 62/505,175, filed May 12, 2017, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under ROl EB022376

(formerly ROl GM065400) and R35 GMl 18062 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Programmable sequence- specific genome editing agents such as CRISPR-

Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies.

SUMMARY

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is an RNA-programmed endonuclease that enables the efficient, sequence-specific modification of target loci in the human genome 1 ' 2. Catalytically inactive forms of Cas9 fused to transcriptional activator and repressor domains can serve as programmable transcriptional regulators ^3"6 . More recently, a fusion proteins containing a cytidine deaminase, a Cas9 nickase, and a base excision repair inhibitor were assembled to engineer "base editors" that introduce C:G to T:A mutations in the human genome without inducing double-stranded DNA breaks . Canonical forms of Cas9 are constitutively active and are not dependent on any metabolite, exogenous ligand, or cell state. The development of Cas9 systems that can be precisely controlled in living cells by exogenous, cell-permeable small molecules would enhance their ability to serve as research tools and as potential therapeutics to treat diseases with a genetic component by increasing their specificity and reducing the

8 9

likelihood of editing at off-target loci or in non-target tissues ' .

Substantial effort has been devoted to the development of controllable genome editing agents in mammalian cells ¹⁰ ' ¹¹ . Placing the expression of Cas9 under the control of inducible or tissue-specific promoters provides limited control over genome editing. Manipulation at the transcriptional level, however, offers poor temporal control compared with post-transcriptional and post-translational control, and is difficult to achieve in many cell types due to a lack of tissue- specific promoters. Post- translationally regulated Cas9 variants have been developed in which conditionally inactive Cas9 proteins are expressed and genome editing activity is restored only in

12-15

the presence of specific stimuli ^" . Controlling the activity of Cas9 to limit the temporal exposure of cells to genome editing activity has resulted in important

13

benefits such as reduced modification of off-target loci .

A complementary and much less-explored strategy to controlling Cas9- mediated genome editing or transcriptional regulation is to develop ligand-responsive guide RNAs. Both nature and researchers have evolved many RNAs that undergo secondary structure changes in the presence of small molecules ¹⁶ ' ¹⁷ , and these principles in theory could be used to engineer ligand-responsive guide RNAs.

Aptazymes are ligand-activate self-cleaving ribozymes that contain integrated

18

aptamer domains. Upon binding ligands of interest, aptazymes undergo structural changes that activate an associated ribozyme domain, triggering RNA cleavage.

In this study aptazyme-embedded guide RNAs (agRNAs) that enable small molecule control of guide RNA structure and genome engineering activity were developed. Different guide RNA architectures were tested and optimized both in vitro and in mammalian cells, resulting in the efficient liberation of active guide RNAs in the presence of the triggering molecules. The agRNAs were used to achieve theophylline-controlled nuclease-mediated genome editing and base editing, as well as guanine-controlled transcriptional activation in mammalian cells.

Thus, in some aspects the disclosure provides RNAs comprising an aptazyme that regulates the activity of (e.g., binding activity) of the RNA to direct a nucleic acid programmable DNA binding protein (napDNAbp) to a target sequence (e.g., target DNA sequence). In some embodiments, the RNA contains a blocking sequence that inhibits the RNA from binding to a target DNA or binding to a napDNAbp, thereby regulating its activity. In some embodiments, the blocking sequence binds to a portion of the RNA that is necessary for binding of the RNA to the napDNAbp, or its target sequence. In some embodiments, upon ligand binding of the aptazyme portion of the RNA, the blocking sequence is released and the RNA is capable of directing a napDNAbp (e.g., Cas9) to a target sequence.

In some embodiments, the disclosure provides an engineered ribonucleic acid

(RNA) comprising a single guide RNA (sgRNA) associated with an aptazyme, wherein the aptazyme hybridizes to a portion of the sgRNA, thereby making the sgRNA inactive. In some embodiments, the portion of the sgRNA hybridized to the aptazyme comprises a nucleotide sequence that is complementary to a target sequence of the sgRNA. In some embodiments, the sgRNA comprises a crRNA domain and a tracrRNA domain, and the portion of the sgRNA hybridized to the aptazyme comprises an annealing region between the crRNA domain and the tracrRNA domain. In some embodiments, the aptazyme comprises a ribozyme. In some embodiments, the ribozyme cleaves the aptazyme from the sgRNA. In some embodiments, cleavage of the aptazyme from the sgRNA activates the sgRNA. In some embodiments, the ribozyme is a hammerhead ribozyme. In some embodiments, the ribozyme is a ligand-responsive ribozyme. In some embodiments, the ligand-responsive ribozyme is inactive in the absence of a ligand. In some embodiments, the ligand-responsive ribozyme is activated via binding to a ligand. In some embodiments, the ligand- response ribozyme cleaves the aptazyme from the sgRNA upon binding to the ligand. In some embodiments, the ligand-responsive ribozyme is a ligand-response hammerhead ribozyme. In some embodiments, the ligand is a small molecule, a metabolite, a carbohydrate, a peptide, a protein, or a nucleic acid. In some

embodiments, the engineered RNA is encoded by a nucleic acid molecule comprising any one of the nucleotide sequences listed in Table 1.

Some aspects of the disclosure provide a complex comprising a Cas9 protein, a Cas9 variant, or a Cas9 fusion protein, and any of the engineered RNAs provided herein. In some embodiments, the sgRNA directs the Cas9 protein, the Cas9 variant, or the Cas9 fusion protein to a target sequence upon cleavage of the aptazyme from the sgRNA. In some embodiments, the Cas9 fusion protein comprises a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9) fused to a deaminase or a

transcriptional activator.

Some aspects of the disclosure provide nucleic acids encoding any of the RNAs provided herein. Other aspects of the disclosure provide vectors comprising any of the RNAs provided herein. Other aspects of the disclosure provide cells comprising any of the RNAs, nucleic acid molecules, or vectors provided herein. Other aspects of the disclosure provide kits comprising any of the RNAs, complexes, nucleic acids, vectors, or cells provided herein.

Some aspects of the disclosure provide methods that include contacting a cell with any of the RNAs, complexes, nucleic acid molecules, or vectors provided herein. In some embodiments, the method further comprises contacting the RNA with a ligand that activates the ligand-responsive ribozyme.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows strategies to abrogate the activity of guide RNAs by appending blocking sequences. Figure 1A is a Schematic representation of the regions of the guide RNA (SEQ ID NO: 36) that were targeted by candidate blocking sequences. Figure IB shows In vitro activity of unmodified guide RNAs and three blocked guide RNA variants are depicted in the lower right. The canonical and blocked sgRNAs were each incubated with the target DNA (the full-length GFP gene) in the presence of Cas9 for 1 h at 37 °C, while the crRNA and tracrRNA variants were incubated for an additional 4 h due to the lower overall activities of the corresponding Cas9:guide RNA complexes.

Figure 2 depicts restoring blocked guide RNA activity with an embedded hammerhead ribozyme in human cells. Figure 2A shows a schematic representation of the hammerhead ribozyme-embedded guide RNA and the expected activities in the presence of Cas9; Figure 2B shows genome-editing activities of hammerhead ribozyme-embedded guide RNAs in HEK293-GFP cells. The blocking sequence that is complementary to the spacer was fused to the guide RNA through a hammerhead ribozyme (HHR-bsgRNA). The length of the blocking sequence was varied from 10 to 17 nt. In HHR-bsgRNA variants, the hammerhead ribozyme is active and the blocking sequence is removed in situ once the guide RNA is transcribed, whereas in dHHR-bsgRNA variants, the hammerhead ribozyme is inactive and the blocking sequence remains appended to the guide RNA. The full activity of HHR-bsgRNA is shown by directly expressing the processed HHR-bsgRNA without the blocking sequence (pHHR-sgRNA). Canonical sgRNA (WT) served as the positive control and cells that were transfected with the Cas9 expression plasmid but no guide RNA plasmid were used as a negative control. The GFP fluorescence loss was quantified by comparing the mean cell fluorescence in transfected cells to that in cells treated with lipids only. Values and error bars reflect mean GFP fluorescence loss and the standard deviation of three biological replicates.

Figure 3 shows a small molecule-controlled genome editing and

transcriptional activation enabled by agRNAs in human cells. Figure 3A shows aschematic representation of the activation of theophylline- agRN A in the presence of theophylline. Figure 3B shows genome-editing activity of theophylline-agRNA in HEK293-GFP cells. Endogenous GFP sites were edited by the Cas9:guide RNA complex upon agRNA activation. Cells that were not transfected with any guide RNA plasmid served as the negative control, while cells transfected with Cas9 and a canonical sgRNA (+) served as a positive control. The GFP fluorescence loss was quantified by comparing the mean cell fluorescence in transfected cells to that in cells treated with lipids only. Figure 3C shows a schematic representation of the activation of guanine-agRNA in the presence of guanine. Figure 3D shows transcriptional activation activity of guanine-agRNA in the presence and absence of the ligand in HEK293T cells. GFP activation was achieved using dCas9-VPR:guide RNA complex upon agRNA activation. Values and error bars reflect mean GFP fluorescence and the standard deviation of three biological replicates.

Figure 4 shows theophylline-responsive base editing on the HEK-3 and FANCF loci in human cells. Figure 4A (SEQ ID NO: 64) and Figure 4B (SEQ ID NO: 65) show nuclease-mediated indel formation by HHR-bsgRNA and dHHR-bsgRNA on the HEK-3 (Figure 4A) and FANCF (Figure 4B) sites in the presence of Cas9. Figure 4C and Figure 4D show base editing by HHR-bsgRNA and dHHR-bsgRNA on the HEK-3 (Figure 4C) and FANCF (Figure 4D) sites in the presence of BE3. Figure 4E shows theophylline-dependent base editing on the FANCF site. Figure 4F, Figure 4G, and Figure 4H show theophylline-dependent base editing on the HEK-3 site with different doses of agRNA and BE3 plasmids. Values and error bars reflect mean editing percentage and the standard deviation of three biological replicates. Figure 5 shows matrix-assisted laser desorption ionization time-of-fight mass spectrometry analyses of cleaved HHR-bsgRNA (Figure 5A) and theophylline- agRNA (Figure 5B). Both the HHR-bsgRNA and theophylline- agRNA contain a 15 nt blocking sequence at the 5' end. HHRbsgRNA, calculated M: 22,429 (5' fragment) and 36,783 (3' fragment), average mass; observed M + H+: 22,439 and 36,808, average mass. Theophylline-agRNA, calculated M: 27,932 (5' fragment) and 36,783 (3' fragment), average mass; observed M + H+: 28,006 and 36,734, average mass.

Figure 6 shows genome-editing activity of theophylline- agRNA in the presence and absence of 4 mM (d)theophylline at endogenous GFP sites in HEK293- GFP cells.

Figure 7 shows a representative flow cytometry scatter plots showing GFP activation in cells that were transfected with different guide RNAs in the absence or presence of 50 μΜ guanine (Figures 7A-7G). APC signal was used to gate transfected cells using the iRFP fluorescence. Statistic results of the same set of experiments are shown in Fig. 3.

Figure 8 shows GFP activation in HEK293T cells by guanine-agRNA in the presence of different concentrations of (d)guanine.

Figure 9 shows GFP activation activities of two architectures of guanine- agRNA in HEK293T cells. Different blocking sequences were used in the two guanine-agRNAs.

Figure 10 shows RFP activation by guanine-agRNA with a different spacer in HEK293T cells. (d)Guanine-agRNA was included as a control.

Figure 11 shows Nuclease-mediated indel formation and base editing by HHRbsgRNA and dHHR-bsgRNA on the EMX-1 (SEQ ID NO: 37) and HEK-4 (SEQ ID NO: 38) sites.

Figure 12 shows base-editing activity of theophylline- agRNA in the presence and absence of theophylline and 3-methylxanthine on the FANCF site in HEK293T cells. DEFINITIONS

As used herein and in the claims, the singular forms "a," "an," and "the" include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to "an agent" includes a single agent and a plurality of such agents.

The term "aptamer" refers to nucleic acid or peptide molecules that bind to a specific target molecule, e.g., a specific ligand. In some embodiments, binding of the ligand to the aptamer induces conformational changes in the aptamer, and e.g., other molecules conjugated or linked to the aptamer. In some embodiments, nucleic acid (e.g., DNA or RNA) aptamers are engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, for example, small molecules, macromolecules, metabolites, proteins, proteins, carbohydrates, metals, nucleic acids, cells, tissues and organisms. Methods for engineering aptamers to bind small molecules are known in the art and include those described in 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; the entire contents of each are hereby incorporated by reference. Nucleic acid aptamers are also found in nature, for example, those that form part of a riboswitch. A "riboswitch", as used herein, is a regulatory segment of a mRNA molecule that binds a small molecule, for example, a metabolite, resulting in a change in production of the protein(s) encoded by the mRNA (e.g., proteins involved in the production of the metabolite binding 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., metabolite), and the mRNA undergoes structural changes in response to the changes in the aptamer. Typically, the structural changes in the mRNA result in a decrease or inhibition of protein expression. Aptamers can be cloned from (e.g., separated from) riboswitches and used to control the activity of other molecules (e.g., RNA, DNA) linked thereto using routine methods in the art. Additionally, aptamers found in nature can be re-engineered to bind to synthetic, non- natural small molecule ligands to control the activities of other molecules linked thereto using known methods. See, e.g., Dixon et al., "Reengineering orthogonally selective riboswitches." PNAS 2010; 107 (7): 2830-2835, the entire contents of which is hereby incorporated by reference. The following is a non-limiting list of riboswitches that include aptamers: cobalamin riboswitches (also B 12-element), cyclic di-GMP riboswitches, FMN riboswitches (also RFN-element), GlmS riboswitches, glycine riboswitches, lysine riboswitches (also L-box), PreQl riboswitches, purine riboswitches, SAH riboswitches, SAM riboswitches,

tetrahydrofolate riboswitches, theophylline riboswitches, and TPP riboswitches (also THI-box).

Cobalamin riboswitches (also B 12-element) refer to riboswitches that bind adenosylcobalamin (the coenzyme form of vitamin B 12) to regulate cobalamin biosynthesis and transport of cobalamin and similar metabolites, and other genes. See, e.g., Nahvi et al., "Coenzyme B 12 riboswitches are widespread genetic control elements in prokaryotes." Nucleic Acids Res. 2004; 32: 143-150; Vitreschak et al., "Regulation of the vitamin B 12 metabolism and transport in bacteria by a conserved RNA structural element." RNA. 2003; 9: 1084-1097; the entire contents of each are hereby incorporated by reference.

Cyclic di-GMP riboswitches refer to riboswitches that bind the signaling molecule cyclic di-GMP in order to regulate a variety of genes controlled by this second messenger. 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 each are hereby incorporated by reference.

FMN riboswitches (also RFN-element) refer to riboswitches that bind flavin mononucleotide (FMN) to regulate 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 each are hereby incorporated by reference.

GlmS riboswitches refer to a riboswitch that cleaves itself when bound by glucosamine-6-phosphate. 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 each are hereby incorporated by reference.

Glycine riboswitches refer to riboswitches that bind glycine to regulate glycine metabolism genes, including those that encode proteins that use of glycine as an energy source. See, e.g., Mandal et al., "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 each are hereby incorporated by reference.

Lysine riboswitches (also L-box) refer to riboswitches that bind lysine to regulate lysine 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 each are hereby incorporated by reference.

PreQl riboswitches refer to riboswitches that bind pre-queuosinel, to regulate genes involved in the synthesis or transport of this precursor to queuosine. At least two distinct classes of PreQl riboswitches are known: PreQl-I riboswitches and PreQl-II riboswitches. See, e.g., Roth et al., "A riboswitch selective for the queuosine precursor preQl contains an unusually small aptamer domain," Nat Struct Mol Biol. 2007; 14 (4): 308-317; Klein et al, "Cocrystal structure of a class I preQl 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 preQl aptamer class in Streptococcaceae bacteria." RNA 2008; 14 (4): 685; the entire contents of each are hereby incorporated by reference. Purine riboswitches refer to riboswitches that bind purines to regulate purine metabolism and transport. Different forms of the purine riboswitch bind guanine (a form originally known as the G-box) or adenine. The specificity for either guanine or adenine depends completely upon Watson-Crick interactions with a single pyrimidine in the riboswitch at a particular position, e.g., Y74. In the guanine riboswitch this residue is typically a cytosine (e.g., C74), in the adenine roboswitch it is typically a uracil (e.g., U74). Homologous types of purine riboswitches bind deoxyguanosine but have more significant differences than a single nucleotide 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 each are hereby incorporated by reference.

SAH riboswitches refer to riboswitches that bind S-adenosylhomocysteine to regulate genes involved in recycling this metabolite that 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 each are hereby incorporated by reference.

SAM riboswitches refer to riboswitches that bind S-adenosyl methionine (SAM) to regulate methionine and SAM biosynthesis and transport. At least four SAM riboswitches are known: SAM-I (originally called S-box), SAM-II, the SMK box riboswitch and Sam-IV. SAM-I is widespread in bacteria, but SAM-II is found only in alpha-, beta- and a few gamma-proteobacteria. The SMK box riboswitch is believed to be found only in the order Lactobacillales. SAM-IV riboswitches have a similar ligand-binding core to that of SAM-I riboswitches, but in the context of a distinct scaffold. See, e.g., Montange et al., "Structure of the S-adenosyl methionine 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 each are hereby incorporated by reference.

Tetrahydrofolate riboswitches refer to riboswitches that 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. U.S.A. 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 each are hereby incorporated by reference.

Theophylline riboswitches refer to riboswitches that selectively binds the small molecule theophylline. The theophylline riboswitch was identified by SELEX. The aptamer comprises a 15-nucleotide core motif that is 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 each are hereby incorporated by reference.

TPP riboswitches (also THI-box) refer to riboswitches that bind thiamin

pyrophosphate (TPP) to regulate thiamin biosynthesis and transport, as well as transport of similar metabolites. It is believed to be the only riboswitch found so far 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 each are hereby incorporated by reference.

The term "blocking sequence" refers to a nucleic acid {e.g., RNA) that is complementary to a portion of a nucleic acid sequence within a guide RNA. The blocking sequence may be designed to hybridize {e.g., via complementary base pairing) with a portion of a guide RNA in order to inhibit the guide RNA from binding to a Cas9 protein (e.g., by binding to a portion of the gRNA backbone), and/or to inhibit the guide RNA from binding to its target sequence (e.g., by binding to the spacer sequence of the gRNA). In some embodiments, the blocking sequence has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that are complementary to a nucleic acid sequence of a gRNA. The structure of gRNAs (e.g., sgRNAs) are known in the art and have several have been described, for example, in Briner et ah, "Guide RNA Functional Modules Direct Cas9 Activity and

Orthogonality." Molecular Cell. 56, 333-339, October 23, 2014; and Nowak et al, "Guide RNA engineering for versatile Cas9 functionality." Nucleic Acids Res. 2016 Nov 16; 44(20): 9555-9564; the contents of each of which are incorporated herein by reference. Typically, sgRNA sequences contain six modules, which include a "spacer" sequence that targets the sgRNA to a nucleic acid sequence (e.g., DNA sequence); the "upper stem", "bulge", and "lower stem" sequences that are formed by the CRISPR repeat (crRNA):tracrRNA duplex and the "nexus" and "hairpins" from the 3' end of the tracrRNA. The term "gRNA backbone" or "backbone sequence" refers to a portion of the gRNA sequence that does not include the spacer sequence. Targeting and cleavage by Cas9 systems rely on a RNA duplex consisting of CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). This native complex can be replaced by a synthetic single guide RNA (sgRNA) chimera that mimics the crRNA: tracrRNA duplex. sgRNAs in combination with Cas9 make convenient, compact, and portable sequence-specific targeting systems that are amenable to engineering and heterologous transfer into a variety of model systems of industrial and translational interest. See, e.g., Deltcheva, E., et ah, "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. (2011) 471, 602-607; and Jinek, M., et ah, "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (2012) 337, 816-821; the contents of each of which are hereby incorporated by reference. It should be appreciated that a skilled artisan would be able to identify the gRNA modules that are derived from any species (Streptococcus pyogenes) encoding CRISPR-Cas systems. Accordingly, the exemplary gRNA (e.g., sgRNA) sequences provided herein are not meant to be limiting and a blocking sequence may be designed to target a portion of any gRNA. The term "aptazyme" refers to ligand-activatable self-cleaving ribozymes that contain an integrated aptamer domain. In some embodiments, all, or at least a portion of the aptazyme is comprised of RNA. Without wishing to be bound by any particular theory, upon binding ligands of interest, aptazymes undergo structural changes that activate an associated ribozyme domain, which triggers RNA cleavage. In some embodiments, the aptazyme comprises at least one ribozyme and at least one aptamer. As these molecules are combinatorial in nature, they have been designed using a diverse series of known ribozymes and apatmers in different combinations.

Exemplary aptazymes known in the art include aptamers that bind theophylline, guanine, tetracycline, and thiamine pyrophosphate; and include ribozymes from the Pistol, Hammerhead, Hepatitis delta virus (HDV) and Twister families. See, e.g.

Wieland, M., and Hartig, J. S. "Improved aptazyme design and in vivo screening enable riboswitching in bacteria." Angew. Chem., Int. Ed. 2008 47, 2604-2607.;

Zhong et al. "Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells." eLife 2016 5, el8858.; Wieland et al. "Artificial ribozyme switches containing natural riboswitch aptamer domains." Angew. Chem., Int. Ed. 2009 48, 2715-2718.; Nomura et al. "Synthetic mammalian riboswitches based on guanine aptazyme." Chem. Commun. 2012, 48, 7215-7217.; Nomura et al. "Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes." ACS Synth. Biol, 2013 2, 684-689.; Felletti et al. "Twister ribozymes as highly versatile expression platforms for artificial riboswitches." Nat. Commun. 7, 12834.; Kobori et al. "Deep Sequencing Analysis of Aptazyme Variants Based on a Pistol Ribozyme" ACS Synth. Biol. 2017, 6, 1283-1288.; the entire contents of each are hereby incorporated by reference.

In some embodiments, the aptazyme of any of the RNAs herein will include a ribozyme, or a mutant thereof, from the Twister from O. sativa, Twister from env9, Twister from env22, Pistol, Hepatitis delta virus (HDV), Hammerhead, Hairpin, Neurospora Varkud satellite, glucosamine 6-phosphate synthase (glmS), Twister Sister, Pistol, or Hatchet families. In some embodiments, the aptazyme of any of the RNAs herein will include an aptamer from the following, non-limiting list of potential aptamers: cobalamin riboswitches (also B 12-element), cyclic di-GMP riboswitches, FMN riboswitches (also RFN-element), GlmS riboswitches, glycine riboswitches, lysine riboswitches (also L-box), PreQl riboswitches, purine riboswitches, SAH riboswitches, SAM riboswitches, tetrahydrofolate riboswitches, theophylline riboswitches, and TPP riboswitches (also THI-box).

The term "ribozyme" refers to an RNA molecule that is capable of catalyzing the cleavage of a nucleic acid (e.g., RNA). In some embodiments, a ribozyme is a site-specific and self-cleaving ribozyme. To date, several distinct classes of ribozymes that are site-specific and self-cleaving have been identified. These broad classes maintain unique active site architectures and can be differentially dependent upon a variety of divalent cations, pH, and mutations. These ribozyme classes include Twister from O. sativa, Twister from env9, Twister from env22, Pistol, Hepatitis delta virus (HDV), Hammerhead, Hairpin, Neurospora Varkud satellite, glucosamine 6- phosphate synthase (glmS), Twister Sister, Pistol, and Hatchet. See, e.g., Eiler et al., "Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme." Proc. Natl. Acad. Sci. USA 2014, 111, 13028-13033; Ren et al. "In-line alignment and Mg ²⁺ coordination at the cleavage site of the env22 twister ribozyme." Nat.

Commun. 2014, 5, 5534; Liu et al. "Crystal structure and mechanistic investigation of the twister ribozyme." Nat. Chem. Biol. 2014, 10, 739-744.; Ren et al. "Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage." Nat. Chem. Biol. 2016, 12, 702-708.; Chen, et al. "A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage." Biochemistry 2010, 49, 6508-6518.; Mir et al. "Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead

Ribozyme Bound to a Transition State Analogue." Biochemistry 2016, 55, 633-636.; Rupert et al. "Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis." Nature 2001, 410, 780-786.; Lilley, D.M. "The Varkud satellite ribozyme." RNA 2004, 10, 151-158.; Klein et al. "Structural basis of glmS ribozyme activation by glucosamine-6-phosphate." Science 2006, 313, 1752-1756.; Weinberg et al. "New classes of self-cleaving ribozymes revealed by comparative genomics analysis." Nat Chem Biol. 2015, 11:606-610.; the entire contents of each are hereby incorporated by reference.

Accordingly, in some embodiments, the ribozyme of any of the RNAs herein is a Twister ribozyme from O. sativa, or a related mutant thereof. In some

embodiments, the ribozyme of any of the RNAs herein is a Twister ribozyme from env9, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Twister ribozyme from env22, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Pistol ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Hepatitis delta virus (HDV) ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Neurospora Varkud satellite ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Hammerhead ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Hairpin ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a glucosamine 6-phosphate synthase (glmS) ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Twister Sister ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a Pistol ribozyme, or a related mutant thereof. In some embodiments, the ribozyme of any of the RNAs herein is a glucosamine Hatchet ribozyme, or a variant thereof.

The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g. , a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut

endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs ("sgRNA", or simply "gRNA") can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et ah, J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 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 Z.A., Eckert M.R., 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 J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences 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 contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive {e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

A nuclease-inactivated Cas9 protein may interchangeably be referred to as a "dCas9" protein (for nuclease-"dead" Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et ah, Science. 337:816-821(2012); Qi et ah, "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al, Science. 337:816-821(2012); Qi et al, Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% 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 may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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, 50 or more amino acid changes compared 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), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% 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 fragment is is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,

1250, or at least 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence:

NC_017053.1, SEQ ID NO: 39 (nucleotide); SEQ ID NO: 40 (amino acid)). ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG ATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGG TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT CTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGAC TTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCT ATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACT ATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTT GCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTT TTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTT ATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAAC GCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATC AAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATG GCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAA ATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATA CTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATG CTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGA TATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAA TGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTT TAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAAT CAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGA ATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGG AATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACC TTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCT ATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGA GAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATT GGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAA CAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTC AATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAA AAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAAC GAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATT TCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAA ATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATA GAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCT TCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTT TTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATT GACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATG CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATA CTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAG CAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGAT ATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGAT TGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGT AAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATA TCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAA AATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAG GAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAAT GAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGA CCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGT TCCACAAAGTTTCATTAAAGACGATTCAATAGACAATAAGGTACTAACGC GTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAAT CACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGA GTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCC AAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAA TACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAA ATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACG TGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGT TGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTA TGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCA AGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGA ACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGC CCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGG GCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATA TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATT TTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGA TCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCT AGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTT AAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAA TCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACT TAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTA AACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGC TCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAA GTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGC AGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTA AGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATA ACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCAT TTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATA CAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCC ACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTG AGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 39)

MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLF GSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILS ARLS KS RRLENLIAQLPGEKRNGLFGNLIALS LGLTPNFKS NFDLAED AKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLS GEQKKAIVDLLFKTNRKVT VKQLKED YFKKIECFDS VEIS G VED RFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDS LTFKEDIQKAQ VS GQGHS LHEQIANLAGSPAIKKGILQT VKIV DELVKVMGHKPENIVIEMARENOTTOKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR KRPLIETNGETGEIVWDKGRDFATVRKVLS MPQ VNIVKKTE VQTGGFS KES IL PKRNS DKLIARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 40)

(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO: 41 (nucleotide) and/or SEQ ID NO: 42 (amino acid):

ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGA TGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGT GTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCC TCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACC GCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGA AATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTT GGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCA TCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACG ATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCT GAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTT TCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGT TCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAA ATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAA TCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAA ATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATT TTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAG GACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCA GTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCT ATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCG CTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTC AAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTT TGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTC AAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGG ACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCA GCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAAT TGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACA ATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGG GACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCC GAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGC GTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTAC CGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACA GTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAA ACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTAT TCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTT AAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCG ATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGA TAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAG TGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTA AAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAG GCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGA TAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGAC GGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACC TTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATT GCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCA TACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGT CACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGA CTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGA GGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAA ATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGA AGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTA CGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGA CAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAAT GTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGC TCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAA GCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACG TCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAG ATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAA GTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGA TTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACG ACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCG AAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCG TAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAA TACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGG CAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGAC AGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAA GTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGAC CGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGC TCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGAT AGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGG AAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTA TGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAA GGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAG TCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAG AGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTC CTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAA CGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAA TCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCC AATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCAT ACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGG CGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGAT ACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATC ACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGG ATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGAC GGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGC TGCAGGA (SEQ ID NO: 41)

MDKKYS IGLAIGTNS VGW A VITDE YK VPS KKFKVLGNTDRHS IKKNLIG ALLF DSGETAEATRLKRTARRRYTRRKNRICYLOEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS

KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS

MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF

YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE

VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

GMRKPAFLS GEQKKAIVDLLFKTNRKVT VKQLKED YFKKIECFDS VEIS G VED

RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY

AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR

NFMQLIHDDS LTFKEDIQKAQ VS GQGDS LHEHIANLAGSPAIKKGILQT VKVV

DELVKVMGRHKPENIVIEMARENOTTOKGQKNSRERMKRIEEGIKELGSQILK

EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK

DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN

LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE

VKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL

ESEFVYGDYKVYDVRKMIAKSEOEIGKATAKYFFYSNIMNFFKTEITLANGEI

RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNrVKKTEVQTGGFSKESI

LPKRNS DKLIARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VK

ELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKYS LFELENGRKRMLA

SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL

DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP

AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID

NO: 42)

(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild type Cas9 corresponds to Cas9 from

Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, SEQ ID NO: 43 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 44 (amino acid).

ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG ATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGG TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT CTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGAC TTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCT ATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACT ATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTT GCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTT TTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTT ATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAA CGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAAT CAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAAT GGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTA AATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGAT ACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATAT GCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAG ATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCT TTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAA TCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAG AATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAG GAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGAC CTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGC TATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGA GAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATT GGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAA CAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTC AATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAA AAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAAC GAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATT TCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAA ATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATA GAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCT TCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTT TTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATT GACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATG CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATA CTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAG CAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGAC ATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATAT TGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGT AAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAA ATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAG AAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAAT TAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAA AATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGT GGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACAT TGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAAC GCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAG TAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTA ATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTC GCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACT AAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTT AAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGT ACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGT CGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGT CTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGA GCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCA TGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAA CGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAA AGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCA ATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCA ATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTG GGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAG TCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCC GTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAA AAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAG ACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTC GTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCT GGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGA

AAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGG

AGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTT

TCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCA

TATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTA

TTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTT

TGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAG

ATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTG

ATTTGAGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 43)

MDKKYS IGLDIGTNS VGW A VITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLF DSGETAEATRLKRTARRRYTRRKNRICYLOEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLS GEQKKAIVDLLFKTNRKVT VKQLKED YFKKIECFDS VEIS G VED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVV DELVKVMGRHKPENIVIEMARENOTTOKGQKNSRERMKRIEEGIKELGSQILK EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPOVNrVKKTEVOTGGFSKESI LPKRNS DKLIARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VK ELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKYS LFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 44) (single underline: HNH domain; double underline: RuvC domain)

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:

NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref:

NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref:

YP_002344900.1) or Neisseria, meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any naturally occurring organism..

In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and/or H840A mutation.

dCas9 (D10A and H840A):

MDKKYS IGLAIGTNS VGW A VITDE YK VPS KKFKVLGNTDRHS IKKNLIG ALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLS GEQKKAIVDLLFKTNRKVT VKQLKED YFKKIECFDS VEIS G VED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDS LTFKEDIQKAQVS GQGDS LHEHIANLAGSPAIKKGILQT VKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI LPKRNS DKLIARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VK ELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKYS LFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 63).

In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided in SEQ ID NO: 44, or at corresponding positions in any of the amino acid sequences provided in another Cas9 domain, such as any of the Cas9 proteins provided herein. In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g. , result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC l subdomain). In some

embodiments, variants or homologues of dCas9 are provided which 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 SEQ ID NO: 63. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 63) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 63, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:

NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref:

NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref:

YP_002344900.1); or Neisseria, meningitidis (NCBI Ref: YP_002342100.1).

The term "Cas9 nickase," as used herein, refers to a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule {e.g., a duplexed DNA molecule). In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position H840 of SEQ ID NO: 44, or a corresponding mutation in another Cas9 domain, such as any of the Cas9 proteins provided herein. In some embodiments, a Cas9 nickase comprises a H840A mutation and has an aspartic acid at position D10 of SEQ ID NO: 44, or a corresponding mutation in another Cas9 domain, such as any of the Cas9 proteins provided herein. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 63 Such a Cas9 nickase has an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA. Further, such a Cas9 nickase has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing is desired.

Exemplary Cas9 nickase (Cloning vector pPlatTET-gRNA2; Accession No.

BAV54124).

MDKKYS IGLAIGTNS VGW A VITDE YK VPS KKFKVLGNTDRHS IKKNLIG ALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA

HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS

ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS

KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS

MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF

YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE

VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

GMRKPAFLS GEQKKAIVDLLFKTNRKVT VKQLKED YFKKIECFDS VEIS G VED

RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY

AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR

NFMQLIHDDS LTFKEDIQKAQVS GQGDS LHEHIANLAGSPAIKKGILQT VKVV

DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK

EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK

DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN

LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE

VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL

ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI

RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNrVKKTEVQTGGFSKESI

LPKRNS DKLIARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VK

ELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKYS LFELENGRKRMLA

SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL

DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP

AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID

NO: 63)

The term "base editor (BE)," or "nucleobase editor (NBE)," as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating an cytosine (C) in DNA. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl , C2c2, C2c3, or Argonaute protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a Cas9 nickase (nCas9) fused to an cytidine deaminase. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a cytidine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain. In some embodiments, the base editor comprises a CasX protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a CasY protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a Cpfl protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2cl protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2c2 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2c3 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises an Argonaute protein fused to a cytidine deaminase.

Base editors are known in the art and have been described previously, for example, in PCT/US2014/070038 (Publication number WO 2015/089406 Al, filed December 12, 2014); PCT/US2016/058344 (Publication number WO 2017/070632 A2, filed October 22, 2016); and PCT/US2017/045381 (Publication number WO 2018/027078 Al, filed August 3, 2017); the entire contents of each of which are hereby incorporated by reference. A skilled artisan would appreciate that the gRNAs provided herein may be used to regulate the activity of base editors, which is in the scope of this disclosure. The terms "conjugating," "conjugated," and "conjugation" refer to an association of two entities, for example, of 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 is between a protein (e.g. , RNA-programmable nuclease) and a nucleic acid (e.g. , a guide RNA). The association can be, for example, via a direct or indirect (e.g. , via a linker) covalent linkage. In some embodiments, the association is covalent. In some embodiments, two molecules are conjugated via a linker connecting both molecules. For example, in some embodiments where two portions of RNA are conjugated to each other, e.g., an aptamer (or nucleic acid sensing domain) and a gRNA, the two RNAs may be conjugated via a polynucleotide linker, e.g., a nucleotide sequence connecting 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.

The term "effective amount," as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a desired target site specifically bound and cleaved by the nuclease, preferably with minimal or no off- target cleavage. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a nuclease, a hybrid protein, a fusion protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, the specific allele, genome, target site, cell, or tissue being targeted, and the agent being used.

The term "engineered," as used herein refers to a nucleic acid molecule, a protein molecule, complex, substance, or entity that has been designed, produced, prepared, synthesized, and/or manufactured by a human. Accordingly, an engineered product is a product that does not occur in nature.

The term "linker," as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties, e.g. , an aptazyme (e.g., an aptazyme comprising a blocking sequence) and a gRNA. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is a nucleic acid 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, at least 30, at least 40, or at least 50 nucleotides. In some embodiments, the linker comprises 40, 41, 42, 43, 44, or 45 nucleotides. In some embodiments, the linker comprises 42 nucleotides. In some embodiments, the nucleic acid linker is an RNA linker. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some

embodiments, the linker connects two RNA sequences. In some embodiments, the linker is connected to the 5'-end of a sgRNA. In some embodiments, the linker is connected to the 3 '-end of a sgRNA. In some embodiments, the linker is connected to the 5 '-end of a tracrRNA sequence of the sgRNA. In some embodiments, the linker is connected to the 3 '-end of a crRNA sequence of the sgRNA. In some embodiments, the linker is connected covalently to the gRNA.

The term "guide RNA" also referred to as "gRNA", refers to an RNA molecule (e.g., in the case of a sgRNA), or molecules (e.g., in the case where separate crRNA and tracrRNA molecules are non-covalently bound) that has (1) a domain that shares homology to a target nucleic acid, which is referred to as a spacer sequencing or targeting sequence (e.g. , and directs binding of a nucleic acid programmable DNA binding protein (napDNAbp), such as a Cas9, to a target DNA sequence); and (2) a domain that binds a napDNAbp, such as Cas9, (e.g. , may be referred to as the backbone sequence). In some embodiments, the guide is a single-guide RNA

(sgRNA), which is an engineered version of the naturally occurring two-piece guide RNA complex engineered into a single, continuous sequence. The simplified single- guide RNA can be used to direct the Cas9 protein to bind and/or cleave a particular DNA sequence, e.g., for genome editing, gene modification, or transcriptional regulation.

The structure of gRNAs (e.g., sgRNAs) are known in the art and have several have been described, for example, in Briner et ah, "Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality." Molecular Cell. 56, 333-339, October 23, 2014; and Nowak et ah, "Guide RNA engineering for versatile Cas9 functionality." Nucleic Acids Res. 2016 Nov 16; 44(20): 9555-9564; the contents of each of which are incorporated herein by reference. Typically, sgRNA sequences contain six modules, which include a "spacer" sequence that targets the sgRNA to a nucleic acid sequence (e.g., DNA sequence); the "upper stem", "bulge", and "lower stem" sequences that are formed by the CRISPR repeat (crRNA): tracrRNA duplex and the "nexus" and "hairpins" from the 3 ' end of the tracrRNA. The term "gRNA backbone" or "backbone sequence" refers to a portion of the gRNA sequence that does not include the spacer sequence. Targeting and cleavage by Cas9 systems rely on a RNA duplex consisting of CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). This native complex can be replaced by a synthetic single guide RNA (sgRNA) chimera that mimics the crRNA:tracrRNA duplex. sgRNAs in combination with Cas9 make convenient, compact, and portable sequence-specific targeting systems that are amenable to engineering and heterologous transfer into a variety of model systems of industrial and translational interest. See, e.g., Deltcheva, E., et ah, "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. (2011) 471, 602-607; and Jinek, M., et al, "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (2012) 337, 816-821 ; the contents of each of which are hereby incorporated by reference. It should be appreciated that a skilled artisan would be able to identify the gRNA modules that are derived from any species {Streptococcus pyogenes) encoding CRISPR-Cas systems. Accordingly, the exemplary gRNA {e.g., sgRNA) sequences provided herein are not meant to be limiting and a blocking sequence may be designed to target a portion of any gRNA.

As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence (a guide sequence is also referred to as a spacer sequence), which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. In some embodiments, the guide RNA comprises a structure 5'-[guide sequence]- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3' (SEQ ID NO: 46), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. In some

embodiments, the guide sequence {i.e. spacer sequence) 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, or 30 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9 fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of a target nucleotide to be modified, or a target gene to modulate {e.g., increase or decrease) the expression of, for example by targeting a gene promoter sequence. Some exemplary guide RNA sequences suitable for targeting Cas9 fusion proteins to specific target sequences are provided below. Exemplary guide RNA structures, including guide RNA backbone sequences, are described, for example, in Jinek M, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-812; Mali P, et al. (2013) Cas9 as a versatile tool for engineering biology. Nature Methods, 10, 957-963; Li JF, et al. (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotech, 31, 688-691; Hwang WY, et al. (2013) Efficient in vivo genome editing using RNA- guided nucleases. Nat Biotechnol, 31, 227-229; Cong L, et al. (2013) Multiplex genome engineering using CRIPSR/Cas systems. Science, 339, 819-823; Cho SW, et al. (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 31, 230-232; Jinek MJ, et al. (2013) RNA- programmed genome editing in human cells. eLIFE, 2:e00471; DiCarlo JE, et al. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucl Acids Res, 41, 4336-4343; Qi LS, et al. (2013) Repruposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183; and Briner AE, et al. (2014) Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell, 56, 333-339; each of which is incorporated herein by reference.

Novel gRNAs have been readily discovered and engineered in recent years. Systematic mutations of the gRNAs from Streptococcus pyogenes have resulted in several unique gRNAs (See, Briner et al "Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality" Molecular Cell, 2014, 56:2, 333-339; hereby incorporated by reference.) Additionally, gRNAs associated with other organisms such as Staphylococcus aureus, Streptococcus thermophiles, Neisseria meningitides, Acidaminococcus sp., and Leptotrichia shahii have been described. See, e.g.

Nishimasu et al "Crystal structure of Staphylococcus aureus Cas9." Cell, 2015, 162, 1113-1126.; Horvath et al. "Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus." J. BacterioL, 2008, 190, 1401-1412.; Ma et al. "Single- stranded DNA cleavage by divergent CRISPR-Cas9 enzymes." Mol. Cell, 2015, 60, 398-407.; Hou et al. "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis." Proc. Natl. Acad. Sci. U.S.A., 2013, 110, 15644-15649.; Zetsche et al. "Cpfl is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell, 2015, 163, 759-771.; Shmakov et al. "Discovery and functional characterization of diverse class 2 CRISPR-Cas systems." Mol. Cell, 2015, 60, 385-397. The term "mutation," as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Methods for making the amino acid substitutions (mutations) provided herein are known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

The term "nuclease," as used herein, refers to an agent, for example, a protein or a small molecule, capable of cleaving a phosphodiester bond connecting nucleotide residues in a nucleic acid molecule. In some embodiments, a nuclease is a protein, e.g., an enzyme that can bind a nucleic acid molecule and cleave a phosphodiester bond connecting nucleotide residues within the nucleic acid molecule. A nuclease may be an endonuclease, cleaving a phosphodiester bonds within a polynucleotide chain, or an exonuclease, cleaving a phosphodiester bond at the end of the

polynucleotide chain. In some embodiments, a nuclease is a site-specific nuclease, binding and/or cleaving a specific phosphodiester bond within a specific nucleotide sequence, which is also referred to herein as the "recognition sequence," the "nuclease target site," or the "target site." In some embodiments, a nuclease is a RNA-guided (i.e., RNA-programmable) nuclease, which complexes with (e.g., binds with) an RNA (e.g., a guide RNA, "gRNA") having a sequence that complements a target site, thereby providing the sequence specificity of the nuclease. In some embodiments, a nuclease recognizes a single stranded target site. In other embodiments, a nuclease recognizes a double- stranded target site, for example, a double-stranded DNA target site. The target sites of many naturally occurring nucleases, for example, many naturally occurring DNA restriction nucleases, are well known to those of skill in the art. In many cases, a DNA nuclease, such as EcoRI, Hindlll, or BamHI, recognize a palindromic, double- stranded DNA target site of 4 to 10 base pairs in length, and cut each of the two DNA strands at a specific position within the target site. Some endonucleases cut a double- stranded nucleic acid target site symmetrically, i.e., cutting both strands at the same position so that the ends comprise base-paired nucleotides, also referred to herein as blunt ends. Other endonucleases cut a double- stranded nucleic acid target sites asymmetrically, i.e., cutting each strand at a different position so that the ends comprise unpaired nucleotides. Unpaired nucleotides at the end of a double- stranded DNA molecule are also referred to as "overhangs," e.g., as "5'-overhang" or as "3 '-overhang," depending on whether the unpaired nucleotide(s) form(s) the 5' or the 5' end of the respective DNA strand. Double-stranded DNA molecule ends ending with unpaired nucleotide(s) are also referred to as sticky ends, as they can "stick to" other double- stranded DNA molecule ends comprising complementary unpaired nucleotide(s). A nuclease protein typically comprises a "binding domain" that mediates the interaction of the protein with the nucleic acid substrate, and also, in some cases, specifically binds to a target site, and a "cleavage domain" that catalyzes the cleavage of the phosphodiester bond within the nucleic acid backbone. In some embodiments a nuclease protein can bind and cleave a nucleic acid molecule in a monomeric form, while, in other embodiments, a nuclease protein has to dimerize or multimerize in order to cleave a target nucleic acid molecule. Binding domains and cleavage domains of naturally occurring nucleases, as well as modular binding domains and cleavage domains that can be fused to create nucleases binding specific target sites, are well known to those of skill in the art. For example, the binding domain of RNA-programmable nucleases (e.g., Cas9), or a Cas9 protein having an inactive DNA cleavage domain, can be used as a binding domain (e.g., that binds a gRNA to direct binding to a target site) to specifically bind a desired target site, and fused or conjugated to a cleavage domain, for example, the cleavage domain of Fokl, to create an engineered nuclease cleaving the target site. The terms "nucleic acid" and "nucleic acid molecule," as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used

interchangeably to refer to a polymer of nucleotides (e.g. , a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non- naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including 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, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides {e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs {e.g. , 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine);

chemically modified bases; biologically modified bases {e.g. , methylated bases); intercalated 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).

The term "pharmaceutical composition," as used herein, refers to a

composition that can be administrated to a subject in the context of treatment of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, e.g. , a nuclease or a nucleic acid encoding a nuclease, and a pharmaceutically acceptable excipient. The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation,

functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term "fusion protein" as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and

purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. The term "RNA -programmable nuclease," and "RNA-guided nuclease" are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though "gRNA" is used interchangeabley to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise at least two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein (e.g., the backbone sequence). In some

embodiments, domain (2) is the "sgRNA Backbone" of SEQ ID NO: 46, or a variant thereof that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide changes as compared to SEQ ID NO: 46. As one example, domain (2) is homologous to a tracrRNA as depicted in Figure IE of Jinek et ah, Science 337:816- 821(2012), the entire contents of which is incorporated herein by reference. In some embodiments, domain 2 is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the "sgRNA backbone" of SEQ ID NO: 46 or the tracrRNA as described by Jinek et ah, Science 337:816-821(2012). In some embodiments, domain 2 is at least 1, 2, 3, 4, 5, 6, 7, 9, or 10 nucleotides longer or shorter as compared to SEQ ID NO: 46. The sequence of a gRNA that binds a target nucleic acid (e.g., spacer sequence) can comprise any sequence that complements a region of the target and is suitable for a nuclease:RNA complex to bind. In some embodiments, the RNA-programmable nuclease is the (CRIS PR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L. expand/collapse author list McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 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 Z.A., Eckert M.R., 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 J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference.

Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to determine target DNA cleavage sites, these proteins are able to cleave, in principle, any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see 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, W.Y. 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, J.E. 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 contents of each of which are incorporated herein by reference). The terms "small molecule" and "organic compound" are used

interchangeably herein and refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, an organic compound contains carbon. An organic compound may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, or heterocyclic rings). In some embodiments, organic compounds are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol. In certain embodiments, the small molecule is a drug, for example, a drug that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. In certain embodiments, the small molecule is known to bind an aptamer. In some embodiments, the organic compound is an antibiotic drug, for example, an anticancer antibiotic such as dynemicin, neocarzinostatin, calicheamicin, esperamicin, bleomycin, or a derivative thereof. The term "subject," as used herein, refers to an individual organism, 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, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a 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 sex and at any stage of

development.

The terms "target nucleic acid," and "target genome," as used herein in the context of nucleases, refer to a nucleic acid molecule or a genome, respectively, that comprises at least one target site of a given gRNA or complex comprising a gRNA (e.g., a gRNA:Cas9 complex, or a gRNA:Cas9 fusion protein complex).

The term "target site," refers to a sequence within a nucleic acid molecule that is contacted, or modified by a napDNAbp (e.g., Cas9) that is bound to a gRNA, which targets the napDNAbp to the target site. A target site may be single- stranded or double-stranded. In the context of RNA-guided (e.g. , RNA-programmable) nucleases (e.g., a protein dimer comprising a Cas9 gRNA binding domain and an active Cas9 DNA cleavage domain), a target site typically comprises a nucleotide sequence that is complementary to a 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, the target site may be, in some embodiments, 20 base pairs plus a 3 base pair PAM (e.g. , NNN, wherein N represents any nucleotide). Typically, the first nucleotide of a PAM can be any nucleotide, while the two downstream nucleotides are specified depending on the specific RNA-guided nuclease. Exemplary target sites for RNA-guided nucleases, such as Cas9, are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In addition, Cas9 nucleases from different species (e.g. , S. thermophilus instead of S. pyogenes) recognize a PAM that comprises the sequence: NGGNG. Additional PAM sequences are known, including, but not limited to, NNAGAAW and NAAR (see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013), the entire contents of which are incorporated herein by reference). For example, the target site of an RNA-guided nuclease, such as, e.g., Cas9, may comprise the structure [Nz]-[PAM], 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" may also refer to a sequence within a nucleic acid molecule that is bound but not cleaved by a nuclease. For example the target site may be part of a gene, or a promoter of a gene. In some embodiments, a napDNAbp, such as Cas9, is bound to a transcription factor and modulates expression of a gene.

The terms "treatment," "treat," and "treating," refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms "treatment," "treat," and "treating" refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed 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 onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

The term "vector" refers to a polynucleotide comprising one or more recombinant polynucleotides of the present invention, e.g. , those encoding a gRNA provided herein and/or a Cas9 protein. Vectors include, but are not limited to, plasmids, viral vectors, cosmids, artificial chromosomes, and phagemids. The vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut and into which a desired nucleic acid sequence may be inserted. Vectors may contain one or more marker sequences suitable for use in the identification and/or selection of cells which have or have not been transformed or genomically modified with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics (e.g. , kanamycin, ampicillin) or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g. , β-galactosidase, alkaline phosphatase or luciferase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. Any vector suitable for the transformation of a host cell, (e.g. , E. coli, mammalian cells such as CHO cell, insect cells, etc.) as embraced by the present invention, for example vectors belonging to the pUC series, pGEM series, pET series, pBAD series, pTET series, or pGEX series. In some embodiments, the vector is suitable for transforming a host cell for recombinant protein production. Methods for selecting and engineering vectors and host cells for expressing gRNAs and/or proteins (e.g., those provided herein), transforming cells, and expressing/purifying

recombinant proteins are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridinzing to the target nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.

The term "recombinant" as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, or at least 30 mutations as compared to any naturally occurring sequence.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Some aspects of the disclosure are based on the discovery that guide RNAs can be engineered to provide ligand-dependent regulation of Cas9 binding to a target DNA. Such ligand-dependent regulation has been used to modify (e.g., cleave) a DNA target sequence, and has also been used to modulate gene expression.

Accordingly, the disclosure provides compositions and methods for ligand-dependent regulation of Cas9 binding to specific DNA targets.

Methods and compositions of the present disclosure represent, in some aspects, an improvement over previous methods and compositions by providing means to control the temporal activity of nucleic acid programmable DNA binding proteins (e.g., Cas9). For example, RNA-guided nucleases such as Cas9 are known in the art and include both naturally-occurring and engineered proteins, which typically bind to and cleave DNA upon forming a complex with an RNA (e.g. , a gRNA) that complements the target. Aspects of the present invention relate to the recognition that having temporal control over the timing of the binding of a napDNAbp: RNA complex to its target will decrease the likelihood of off-target effects by minimizing or controlling the amount of time a complex is able to bind to and cleave the target. Additionally, engineering gRNAs that only bind the target site, can be useful for temporal regulation of Cas9 mediated gene editing and gene regulation, for example by regulating the binding of base editors or transcription factors to target DNA sequences via Cas9. The strategies, methods, compositions, kits, and systems provided herein can be used to control the activity and/or improve the specificity of any RNA-guided protein (e.g., Cas9). Suitable nucleic acid programmable DNA binding proteins for use with the gRNAs as described herein will be apparent to those of skill in the art based on this disclosure.

In certain embodiments, the strategies, methods, compositions, kits, and systems provided herein are utilized to control the timing of RNA-guided (e.g., RNA- programmable) binding activity of a napDNAbp to a target sequence. It should be appreciated that napDNAbp can be fused to an effector domain that is capable of modifying a nucleic acid or is capable of regulating an activity, such as transcriptional activity of a gene. In some embodiments, the napDNAbp (e.g., Cas9) is fused to a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. Whereas typical RNA-guided nucleases recognize and cleave a target sequence upon forming a nuclease:RNA complex, the gRNAs provided herein allow for control over target binding and/or cleavage. Other aspects provide gRNAs engineered to bind a target site only in the presence of the appropriate ligand (e.g., a small molecule), thereby allowing temporal control of the RNA-guided nuclease activity. While Cas9:gRNA complexes have been successfully 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, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature

Biotechnology 31, 227-229 (2013)), a study using Cas9:guide RNA complexes to modify zebrafish embryos observed toxicity (e.g., off-target effects) at a rate similar to that of ZFNs and TALENs (Hwang, W.Y. et al. Nature Biotechnology 31, 227-229 (2013)). Accordingly, aspects of the present disclosure aim to reduce the chances for Cas9 off-target effects using novel gRNA platforms that control for the timing of target binding and cleavage and/or improve the specificity of RNA-guided nucleases.

While of particular relevance 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 can be applied to any nucleic acid:napDNAbp system utilizing nucleic acid templates such as RNA to direct binding to a target nucleic acid.

Ligand-dependent regulation guide RNAs (gRNAs)

Some aspects of this disclosure provide gRNAs engineered to have both an

"on" and "off state. In some aspects, the gRNAs provided herein comprise an aptazyme comprising a blocking sequence that undergoes self-cleavage in the presence of a ligand, thereby releasing the blocking sequence from the gRNA, which allows the gRNA to bind to a napDNAbp (e.g., Cas9) and a target DNA sequence. For example, a gRNA is said to be in an "off state when the gRNA is in a structural state that prevents binding of the gRNA to a target nucleic acid or to a napDNAbp (e.g., Cas9). In some aspects, a gRNA in an "off state can bind to its cognate RNA- guided protein (e.g., Cas9), however, the Cas9:gRNA complex (when the gRNA is in an "off state) is unable to bind the target nucleic acid to mediate cleavage. In other aspects, a gRNA that is in an "off state is unable to bind its target sequence or an

RNA-guided protein, such as Cas9. Conversely, a gRNA is said to be in an "on" state when the gRNA is in a structural state that allows binding of the gRNA to a target nucleic acid (e.g., as a complex with an RNA-guided protein such as Cas9). Some embodiments of this disclosure provide complexes comprising an inventive gRNA associated with an RNA-guided protein, such as Cas9, and methods of their use.

Some embodiments of this disclosure provide nucleic acids encoding such gRNAs and/or RNA-guided proteins (e.g., Cas9), which may be fused to other effector domains that are capable of modifying nucleic acids, proteins, or transcriptional activity. Some embodiments of this disclosure provide expression constructs comprising such encoding nucleic acids. gRNAs associated with aptazymes that comprise a blocking sequence.

Some aspects of the disclosure provide ligand-regulatable guide RNAs

(gRNAs). Such regulatable guide RNAs can be used to direct nucleic acid

programmable binding proteins (napDNAbp) to specific nucleic acid (e.g., DNA) target sequences. In some embodiments, the gRNAs are associated with an aptazyme comprising a blocking sequence that prevents the gRNA from binding to a target sequence and/or prevents the gRNA from associating with a napDNAbp, such as Cas9, when the aptazyme is not bound by a ligand. In some embodiments, the aptazyme undergoes self-cleavage in the presence of a ligand, thereby releasing the blocking sequence from the gRNA. Once the blocking sequence is released from the gRNA, the gRNA is capable of binding to a napDNAbp (e.g., Cas9) and targeting the napDNAbp to a target nucleic acid sequence (e.g., a target DNA sequence).

Some aspects of the disclosure provide RNAs that comprise an aptazyme that comprises a blocking sequence and a guide RNA that is associated with the aptazyme. Targeting and cleavage by Cas9 systems rely on a RNA duplex consisting of CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, the guide RNA comprises two RNA sequences that are non-covalently bound. In some embodiments, the two RNA sequences include a crRNA and a tracrRNA. In some embodiments, the two RNA sequences are bound via complementary base pairing. In some embodiments, the two RNA sequences are bound via 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 complementary base pairs. Alternatively, the native crRNA: tracrRNA complex can be replaced by a synthetic single guide RNA (sgRNA) chimera that mimics the crRNA: tracrRNA duplex. sgRNAs in combination with Cas9 make convenient, compact, and portable sequence- specific targeting systems that are amenable to engineering and heterologous transfer into a variety of model systems of industrial and translational interest. Accordingly, in some embodiments, the guide RNA is a sgRNA. Guide RNAs are known in the art and have been described in the literature, for example, in Deltcheva, E., et al., "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. (2011) 471, 602-607. Exemplary guide RNA structures, including guide RNA backbone sequences, are described, for example, in Jinek M, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-812; Mali P, et al. (2013) Cas9 as a versatile tool for engineering biology. Nature Methods, 10, 957-963; Li JF, et al. (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotech, 31, 688-691; Hwang WY, et al. (2013) Efficient in vivo genome editing using RNA-guided nucleases. Nat Biotechnol, 31, 227-229; Cong L, et al. (2013) Multiplex genome engineering using CRIPSR/Cas systems. Science, 339, 819-823; Cho SW, et al. (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 31, 230- 232; Jinek MJ, et al. (2013) RNA-programmed genome editing in human cells.

eLIFE, 2:e00471; DiCarlo JE, et al. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucl Acids Res, 41, 4336-4343; Qi LS, et al. (2013) Repruposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183; and Briner AE, et al. (2014) Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell, 56, 333-339.

In some embodiments, any of the gRNAs provided herein are derived from one or more naturally-occurring gRNAs. It should be appreciated that such gRNAs can be used to engineer sgRNAs. In some embodiments, the gRNAs are derived from and/or contain a sgRNA backbone sequence that is derived from any naturally- occurring gRNA of a prokaryote. For example, the gRNA may comprise a gRNA backbone that is derived from a naturally-occurring gRNA from Streptococcus pyogenes, Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophiles, Listeria innocua, Campylobacter jejuni, or Neisseria meningitides. However, it should be appreciated that gRNAs, including sgRNAs, may comprise backbone sequences that are derived from other naturally-occurring organisms, and the examples provided herein are not meant to be limiting.

In some embodiments, the gRNA comprises a backbone sequence that is derived from a naturally-occurring Streptococcus pyogenes gRNA. In some embodiments, the gRNA comprises a backbone sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a naturally-occurring gRNA sequence, or the gRNA backbone sequence of 5'- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3' (SEQ ID NO: 46). In some embodiments, the gRNA comprises a backbone sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences {e.g., substitutions, insertions, or deletions) relative to a naturally-occurring gRNA sequence, or the gRNA backbone sequence of 5'- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3' (SEQ ID NO: 46). In some embodiments, alternatively or in addition, the gRNA comprises a backbone sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides longer or shorter than a naturally-occurring gRNA sequence, or the gRNA backbone sequence of 5'-

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3 ' (SEQ ID NO: 46). In some embodiments, the gRNA comprises the nucleic acid sequence of SEQ ID NO: 46.

Some aspects of the disclosure provide RNAs that comprise an aptazyme that comprises a blocking sequence and a guide RNA that is associated with the aptazyme. Aptazymes are ligand-activatable self-cleaving ribozymes that contain an integrated aptamer domain. In some embodiments, all, or at least a portion of the aptazyme is comprised of RNA. Without wishing to be bound by any particular theory, upon binding ligands of interest, aptazymes undergo structural changes that activate an associated ribozyme domain, which triggers RNA cleavage. In some embodiments, the aptazyme comprises at least one ribozyme (e.g., a self-cleaving ribozyme) and at least one aptamer. In some embodiments, the ribozyme is a site-specific ribozyme. In some embodiments, the ribozyme is a self-cleaving ribozyme. Exemplary ribozymes include, without limitation, a Twister ribozyme from O. sativa, a Twister ribozyme from env9, a Twister ribozyme from env22, Pistol ribozyme, Hepatitis delta virus (HDV) ribozyme, Hammerhead ribozyme, Hairpin ribozyme, Neurospora Varkud satellite ribozyme, glucosamine 6-phosphate synthase (glmS) ribozyme, Twister Sister ribozyme, Pistol ribozyme, and Hatchetribozyme.

In some embodiments, the aptazyme comprises a hammerhead ribozyme. In some embodiments, the aptazyme comprises a ribozyme that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a naturally-occurring ribozyme, such as any of the ribozymes provided herein. In some embodiments, the aptazyme comprises any naturally-occurring ribozyme, such as any of the ribozymes provided herein. In some embodiments, the aptazyme comprises a hammerhead ribozyme. In some embodiments, the aptazyme comprises a ribozyme that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the hammerhead ribozyme from sequence 5'- GGUACAUCCAGCUGAUGAGUCCCAAAUAGGACGAAAUACAUACCAGCC GAAAGGCCCUUGGCAGGUGUCCUGGAUUCCAC-3 ' (SEQ ID NO: 61). In some embodiments, the ribozyme comprises a nucleic acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences (e.g., substitutions, insertions, or deletions) relative to a naturally-occurring ribozyme, or the hammerhead ribozyme from the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, alternatively or in addition, the ribozyme comprises a nucleic acid sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides longer or shorter than a naturally-occurring ribozyme, or the ribozyme from the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the ribozyme comprises the ribozyme from the nucleic acid sequence of SEQ ID NO: 61.

In some embodiments, the aptazyme comprises an aptamer. In some embodiments, the aptamer portion of the aptazyme is configured to regulate the cleaving activity (e.g., self-cleaving activity) of the ribozyme. In some embodiments, the aptamer prevents the ribozyme from cleaving itself in the absence of a ligand that binds to the aptamer. In some embodiments, when a ligand binds to the aptamer, the ribozyme cleaves itself. In some embodiments, the aptamer portion of the aptazyme is a nucleic acid aptamer. In some embodiments, binding of a ligand to the aptamer induces conformational changes in the aptamer, and e.g., other molecules conjugated or linked to the aptamer. In some embodiments, nucleic acid (e.g., DNA or RNA) aptamers are engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, for example, small molecules, macromolecules, metabolites, proteins, proteins, carbohydrates, metals, nucleic acids, cells, tissues and organisms. Methods for engineering aptamers to bind small molecules are known in the art and include those described in 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; the entire contents of each are hereby incorporated by reference. Nucleic acid aptamers are also found in nature, for example, those that form part of a 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., metabolite), and the mRNA undergoes structural changes in response to the changes in the aptamer.

Typically, the structural changes in the mRNA result in a decrease or inhibition of protein expression. Aptamers can be cloned from (e.g. , separated from) riboswitches and used to control the activity of other molecules (e.g., RNA, DNA) linked thereto using routine methods in the art. In some embodiments, the aptamer is from a naturally-occurring riboswitch. Exemplary riboswitches include, without limitation, cobalamin riboswitches (also B 12-element), cyclic di-GMP riboswitches, FMN riboswitches (also RFN-element), GlmS riboswitches, glycine riboswitches, lysine riboswitches (also L-box), PreQl riboswitches, purine riboswitches, guanine riboswitches, SAH riboswitches, SAM riboswitches, tetrahydrofolate riboswitches, theophylline riboswitches, and TPP riboswitches (also THI-box). In some embodiments, the aptazyme comprises an aptamer from a theophylline riboswitches, or a guanine riboswitch.

In some embodiments, the aptazyme comprises an aptamer that is at least

80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to an aptamer from a naturally-occurring riboswitch, such as any of the riboswitches provided herein. In some embodiments, the aptazyme comprises an aptamer from any naturally-occurring riboswitch, such as any of the riboswitches provided herein. In some embodiments, the aptazyme comprises an aptamer from a theophylline riboswitch or a guanine riboswitch. In some embodiments, the aptazyme comprises an aptamer that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the aptamer nucleic acid sequence UACAUACCAGCCGAAAGGCCCUUGGCAGGUG (SEQ ID NO: 66) or

UAUAAUCGCGUGGAUAUGGCACGCAAGUUUCUACCGGGCACCGUAAAU GUCCGACUA (SEQ ID NO: 67). In some embodiments, the aptazyme comprises an aptamer that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences (e.g. , substitutions, insertions, or deletions) relative to a naturally-occurring aptamer or relative to SEQ ID NO: 66 or 67. In some

embodiments, alternatively or in addition, the aptazyme comprises an aptamer that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides longer or shorter than a naturally-occurring aptamer or SEQ ID NO: 66 or 67. In some embodiments, the aptazyme comprises the amino acid sequence of SEQ ID NO: 66 or 67. It should be appreciated that a skilled artisan could readily engineer ligand- activatable self-cleaving ribozymes that contain an integrated aptamer domain by combining aptamers with ribozymes. Indeed, Exemplary aptazymes are known in the art and include aptamers that bind theophylline, guanine, tetracycline, and thiamine pyrophosphate; and include ribozymes from the Pistol, Hammerhead, Hepatitis delta virus (HDV) and Twister families. See, e.g. Wieland, M., and Hartig, J. S. "Improved aptazyme design and in vivo screening enable riboswitching in bacteria." Angew. Chem., Int. Ed. 2008 47, 2604-2607.; Zhong et al. "Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells." eLife 2016 5, el8858.; Wieland et al. "Artificial ribozyme switches containing natural riboswitch aptamer domains." Angew. Chem., Int. Ed. 2009 48, 2715-2718.; Nomura et al. "Synthetic mammalian riboswitches based on guanine aptazyme." Chem. Commun. 2012, 48, 7215-7217.; Nomura et al. "Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes." ACS Synth. Biol, 2013 2, 684-689.;

Felletti et al. "Twister ribozymes as highly versatile expression platforms for artificial riboswitches." Nat. Commun. 7, 12834.; Kobori et al. "Deep Sequencing Analysis of Aptazyme Variants Based on a Pistol Ribozyme" ACS Synth. Biol. 2017, 6, 1283- 1288.

In some embodiments, the aptazyme is theophylline-dependent. In some embodiments, the aptazyme is a theophylline-dependent hammerhead aptazyme. In some embodiments, the aptazyme is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the aptazyme of sequence 5'- GGUACAUCCAGCUGAUGAGUCCCAAAUAGGACGAAAUACAUACCAGCC GAAAGGCCCUUGGCAGGUGUCCUGGAUUCCAC-3 ' (SEQ ID NO: 61). In some embodiments, the aptazyme comprises a nucleic acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences (e.g., substitutions, insertions, or deletions) relative to the aptazyme of SEQ ID NO: 61. In some embodiments, alternatively or in addition, the aptazyme comprises a nucleic acid sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides longer or shorter than the aptazyme of SEQ ID NO: 61. In some embodiments, the aptazyme comprises the nucleic acid sequence of SEQ ID NO: 61.

In some embodiments, the aptazyme is guanine-dependent. In some embodiments, the aptazyme is a guanine-dependent hammerhead aptazyme. In some embodiments, the aptazyme is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the aptazyme of sequence 5'- GUACAUCCAGCUGAUGAGUCCCAAAUAGGACGAAAUACUAUAAUCGCG UGGAUAUGGCACGCAAGUUUCUACCGGGCACCGUAAAUGUCCGACUAG UGUCCUGGAUUCCAC-3 ' (SEQ ID NO: 62). In some embodiments, the aptazyme comprises a nucleic acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences (e.g., substitutions, insertions, or deletions) relative to the aptazyme of SEQ ID NO: 62. In some embodiments, alternatively or in addition, the aptazyme comprises a nucleic acid sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides longer or shorter than the aptazyme of SEQ ID NO: 62. In some embodiments, the aptazyme comprises the nucleic acid sequence of SEQ ID NO: 62.

In some embodiments, the aptazyme comprises a blocking sequence. In some embodiments, the blocking sequence comprises a nucleic acid sequence (e.g., RNA) that is complementary to a portion of a nucleic acid sequence within a guide RNA. The blocking sequence may be designed to hybridize (e.g., via complementary base pairing) with a portion of a guide RNA in order to inhibit the guide RNA from binding to a Cas9 protein (e.g., by binding to a portion of the gRNA backbone), and/or to inhibit the guide RNA from binding to its target sequence (e.g., by binding to the spacer sequence of the gRNA). In some embodiments, the blocking sequence has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that are complementary to a nucleic acid sequence of a gRNA, for example, a spacer sequence, a backbone sequence, an upper stem sequence, a bulge sequence, a lower stem sequence, a nexus sequence, a hairpin sequence, or a sequence that disrupts the crRNA:tracrRNA portion of the gRNA.

In some embodiments, the blocking sequence comprises a nucleic acid sequence that has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that are complementary to (SEQ ID NO: 46). In some embodiments, the blocking sequence comprises a nucleic acid sequence that has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides that are complementary to any of the nucleic acid sequences of SEQ ID NOs: 47-60. In some embodiments, the blocking sequence comprises a nucleic acid sequence that is perfectly complementary to any of the nucleic acid sequences of SEQ ID NOs: 47-60. In some embodiments, the blocking sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are complementary to a spacer sequence, which guides the gRNA to a target DNA sequence. In some embodiments, the blocking sequence binds to the first 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides at the 5' end of the spacer sequence. In some embodiments, the blocking sequence binds to the first 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides at the 3' end of the spacer sequence. It should be appreciated that a skilled artisan would be able to design a nucleic acid sequence that is capable of hybridizing with a gRNA sequence and preventing its ability to bind a target DNA and/or a Cas9 protein, e.g., by disrupting its structure.

In some embodiments, any of the guide RNAs provided herein are associated with any of the aptazymes provided herein (e.g., aptazymes comprising a blocking sequence). In some embodiments, the guide RNA is covalently associated with the aptazyme. In some embodiments, the guide RNA is covalently associated with the 5' end of the aptazyme. In some embodiments, the guide RNA is covalently associated with the 3' end of the aptazyme. In some embodiments, the aptazyme is covalently bound to the tetraloop of the gRNA, which links the crRNA and tracrRNA portions of the gRNA. In some embodiments, the gRNA and the aptazyme are fused via a linker. In some embodiments, the linker is a nucleic acid linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 467, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the guide RNA is non-covalently associated with the aptazyme. For example, in some embodiments, the guide RNA is associated with the aptazyme via complementary base pairing. In some embodiments, the guide RNA is associated with the aptazyme via 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 complementary base pairs.

Some aspects of the disclosure provide RNAs that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any of the amino acid sequences of SEQ ID NOs: 1-33. In some embodiments, the disclosure provides RNAs that comprise the any of the amino acid sequences of SEQ ID NOs: 1-33. In some embodiments, the disclosure provides RNAs that consist of the any of the amino acid sequences of SEQ ID NOs: 1-33. Some aspects of the disclosure provide RNAs that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any of the amino acid sequences of SEQ ID NOs: 1-33 without the sequence of SEQ ID NO: 47. In some embodiments, the disclosure provides RNAs that comprise the any of the amino acid sequences of SEQ ID NOs: 1-33 without the sequence of SEQ ID NO: 47. In some embodiments, the disclosure provides RNAs that consist of the any of the amino acid sequences of SEQ ID NOs: 1-33 without the sequence of SEQ ID NO: 47.

In some embodiments, any of the RNAs provided herein are bound to a ligand. Exemplary ligands include, without limitation, guanine, S-adenosylcobalamin, cyclic di-GMP, flavin mononucleotide (FMN), glucosamine-6-phosphate, glycine, lysine, pre-queuosinei, S-adenosyl methionine (SAM), tetrahydrofolate, theophylline, and thiamin pyrophosphate (TPP). In some embodiments, an aptamer binds its ligand with a K _d between about 1 nM - 10 μΜ, between about 1 nM - 1 μΜ, between about 1 nM - 500 nM, or between about 1 nM - 100 nM. With RNA-based aptamers, for example, those found in riboswitches of mRNAs, binding of the ligand to the aptamer domain results in conformational changes that control expression (e.g., translation) of the mRNA. RNA aptamers have been successfully cloned and adapted to other molecules, for example, to control gene expression, or have been engineered/selected for particular ligands using SELEX (See, e.g., Dixon et al., "Reengineering orthogonally selective riboswitches." PNAS 2010; 107 (7): 2830-2835; Suess et al, "A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo." Nucleic Acids Res. 2004; 32(4): 1610-1614; 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; the entire contents of each are hereby incorporated by reference). Ligands that bind aptamers include, but are not limited to, small molecules, metabolites, carbohydrates, proteins, peptides, or nucleic acids.

Complexes

In some embodiments, the disclosure provides complexes comprising any of the RNAs provided herein {e.g., RNAs comprising a gRNA linked to an aptazyme comprising a blocking sequence) and a napDNAbp. In some aspects, a complex comprises any of the RNAs provided herein and a nucleic acid programmable DNA binding protein. In some embodiments, the napDNAbp is Cas9, a variant of Cas9, a fragment of Cas9, or a Cas9 fusion protein. In some embodiments, the napDNAbp {e.g., Cas9) is a fusion protein that comprises a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments, the fusion protein is a base editor. In some embodiments, the fusion protein comprises a transcription factor. In some embodiments, the fusion protein comprises a transcriptional activator or a transcriptional repressor. Exemplary transcription factors include, without limitation, basic helix-loop-helix transcription factors, basic-leucine zipper transcription factors, C-terminal effector domain of the bipartite response regulators, AP2/ERF/GCC box transcription factors, helix-turn-helix transcription factors, lambda repressor-like transcription factors, and zinc finger transcription factors. In some embodiments, the napDNAbp is any of the Cas9 protein as provided in International PCT Application, PCT/US2014/054291, filed November 3, 2014, entitled "Cas9 Variants And Uses Thereof," and International PCT Application, PCT/US2014/054247, filed September 5, 2014, entitled "Delivery System For Functional Nucleases," the entire contents of each are hereby incorporated by reference in their entirety.

In some embodiments, the complex further comprises a ligand, e.g., a ligand that binds the aptamer of the RNA associated with the napDNAbp (e.g., Cas9), as described herein. Exemplary ligands include, without limitation, guanine, S- adenosylcobalamin, cyclic di-GMP, flavin mononucleotide (FMN), glucosamine-6- phosphate, glycine, lysine, pre-queuosinei, S-adenosyl methionine (SAM), tetrahydrofolate, theophylline, and thiamin pyrophosphate (TPP). In some embodiments, any of the complexes provided herein {e.g., comprising a provided RNA (gRNA):Cas9 protein) binds to and optionally modifies, or cleaves a target nucleic acid, or modulates transcription of one or more genes.

Pharmaceutical compositions

In some embodiments, any of the gRNAs described herein are 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 a gRNA provided herein. For example, some embodiments provide pharmaceutical compositions comprising a gRNA and an RNA-guided nuclease as provided herein, or a nucleic acid encoding such gRNAs and/or nuclease, and a pharmaceutically acceptable excipient. In some embodiments, any of the

pharmaceutical compositions provided herein comprise a ligand that binds to the aptazyme of any of the RNAs provided herein. In some embodiments, the ligand is any of the ligands provided herein. A skilled artisan would understand which ligands to administer based on the specific aptazyme that is associated with the RNA.

Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a provided gRNA associated with an RNA-guided nuclease or nucleic acid(s) encoding such ex vivo. In some embodiments, cells removed from a subject and contacted ex vivo with an inventive gRNA:nuclease complex are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising 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 disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for

administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any,

experimentation. Subjects to which administration of the pharmaceutical

compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

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 the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 ^st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical

compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication Number WO2011053982, filed

November 2, 2010), incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

In some embodiments, compositions in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g., diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. , arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections (e.g. , HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g. , Alzheimer's disease, Huntington' s disease; autism;

Duchenne muscular dystrophy); cardiovascular disorders (e.g., atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g., cancer, benign neoplasms);

respiratory disorders (e.g. , chronic obstructive pulmonary disease); digestive disorders (e.g., inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. , fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. , diabetes, osteoporosis); urological 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. Methods for site-specific nucleic acid cleavage

In another embodiment of this disclosure, methods for site-specific nucleic acid (e.g. , DNA) modification and transcriptional regulation are provided. In some embodiments, the methods comprise contacting a DNA with any of the napDNAbp (e.g., Cas9):RNA complexes provided herein. For example, in some embodiments, the method comprises contacting a DNA with a complex comprising (i) a gRNA linked to an aptazyme that has a blocking sequence as described herein, such as any of the RNAs provided herein, and (ii) a napDNAbp (e.g. , Cas9). The complex of (i) and (ii) is contacted with a ligand that binds to the aptazyme, thereby activating the RNA, which targets the napDNAbp (e.g., Cas9) to a target DNA sequence. In some embodiments, the Cas9 is a Cas9 nuclease, a dCas9, an nCas9 or a Cas9 fusion protein. In some embodiments, the Cas9 fusion protein comprises a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments, the method includes modifying (e.g., cleaving or deaminating) a target nucleic acid or nucleotide. In some embodiments, the method includes increasing the transcription of one or more genes, e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the method includes decreasing the transcription of one or more genes, e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%.

In some embodiments, methods for inducing site-specific DNA cleavage in a cell are provided. In some embodiments, the method comprises: (a) contacting a cell or expressing within a cell any of the RNAs provided herein, wherein the gRNA comprises a sequence capable of binding to a DNA target sequence; (b) contacting a cell or expressing within a cell a napDNAbp (e.g., a Cas9 protein); and (c) contacting the cell with a specific ligand that binds the aptazyme of the RNA, resulting in the formation of a gRNA:ligand:Cas9 complex, thereby removing the blocking sequence from the gRNA and promoting the targeting of Cas9 to a target DNA sequence. In some embodiments, the method comprises: (a) contacting the cell with a complex comprising a Cas9 protein and a gRNA comprising an aptazyme as described herein, wherein the gRNA comprises a sequence capable of binding to a DNA target sequence; and (b) contacting the cell with a specific ligand that binds the aptazyme of the gRNA, resulting in the formation of a gRNA:ligand:Cas9 complex, thereby cleaving a portion of the RNA that releases the blocking sequence from the gRNA. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, steps (a) and (b) are performed sequentially. Thus, in some

embodiments, the cell is contacted with the ligand subsequent to the cell being contacted with the RNA and napDNAbp, and targeting of the napDNAbp to a DNA target sequence is achieved since the ligand induces cleavage of and releases the blocking sequence from the RNA. In some embodiments of these methods, the ligand is not delivered to the cell, but is produced internally by the cell, for example as part of a physiological or pathophysiological process.

In some embodiments, the method is performed in vitro, for example in cultured cells or in a reaction tube. In some embodiments, the method is performed in vivo, for example in a subject. In some embodiments, the methods include administering a ligand to a human subject that has been administered any of the RNAs, complexes, or nucleic acids provided herein. 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 a RNA-comprising complex as 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.

Polynucleotides, Vectors, Cells, Kits

In another embodiment of this disclosure, polynucleotides are provided that encode any of the RNAs (and optionally any Cas9 protein or Cas9 fusion protein) described herein. For example, polynucleotides encoding any of the RNAs and/or Cas9 proteins described herein are provided, e.g., for recombinant expression and purification of inventive RNAs, or complexes comprising such, e.g., complexes comprising inventive RNAs and an RNA-guided protein (e.g., a Cas9 protein). In some embodiments, provided polynucleotides comprise one or more sequences encoding a RNA, alone or in combination with a sequence encoding any of the napDNAbp described herein.

In some embodiments, vectors encoding any of the RNAs (and optionally any Cas9 protein or Cas9 fusion protein) described herein are provided, e.g., for recombinant expression and purification of inventive RNAs, or complexes comprising inventive RNAs and a napDNAbp (e.g., a Cas9 protein). In some embodiments, the vector comprises or is engineered to include a polynucleotide, e.g., those described herein. In some embodiments, the vector comprises one or more sequences encoding a RNA and/or any Cas9 protein (e.g., as described herein). Typically, the vector comprises a sequence encoding an RNA provided herein operably linked to a promoter, such that the RNA is expressed in a host cell.

In some embodiments, cells are provided for recombinant expression and purification of any of the RNAs (and optionally any Cas9 protein or Cas9 fusion protein) described herein. The cells include any cell suitable for recombinant RNA expression and optionally protein expression, for example, cells comprising a genetic construct expressing or capable of expressing an inventive gRNA (e.g., cells that have been transformed with one or more vectors described herein, or cells having genomic modifications that express an inventive RNA and optionally any Cas9 protein or Cas9 fusion protein provided herein from an allele that has been incorporated in the cell's genome). Methods for transforming cells, genetically modifying cells, and expressing genes and proteins in such cells are well known in the art, and include those provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)) and Friedman and Rossi, Gene Transfer: Delivery and Expression ofDNA and RNA, A Laboratory Manual (1 ^st ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2006)).

Some aspects of this disclosure provide kits comprising any of the inventive RNAs or complexes provided herein and optionally any Cas9 protein or Cas9 fusion protein described herein. In some embodiments, the kit comprises any of the polynucleotides encoding a provided RNAs, and optionally any Cas9 protein or Cas9 fusion protein. In some embodiments, the kit comprises a vector for recombinant expression of any inventive RNA and optionally any Cas9 protein or Cas9 fusion protein. In some embodiments, the kit comprises a cell that comprises a genetic construct for expressing any of the inventive RNAs, complexes, and optionally any Cas9 protein or Cas9 fusion protein provided herein. In some embodiments, the kit comprises an excipient and instructions for contacting any of the inventive compositions with the excipient to generate a composition suitable for contacting a nucleic acid with e.g., a complex of a RNA provided herein and a RNA-guided protein, such as Cas9. In some embodiments, the composition is suitable for contacting a nucleic acid within a genome. In some embodiments, the composition is suitable for delivering an inventive composition (e.g., a RNA, complexes thereof with Cas9) to a cell. In some embodiments, the composition is suitable for delivering an inventive composition (e.g., a RNA, complexes thereof with Cas9) to a subject. In some embodiments, the kit provides one or more ligands, e.g., any of the ligands provided herein, that may be used to induce RNA activity. In some embodiments, the excipient is a pharmaceutically acceptable excipient. EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic examples described in this application are offered to illustrate the compounds and methods provided herein and are not to be construed in any way as limiting their scope.

Example 1

Blocking guide RNA activity

To inactivate a guide RNA in the absence of ligand, the length of the guide RNA was extended to install a "blocking sequence" complementary to a region of the guide RNA. Two key regions of the Streptococcus pyogenes single guide RNA

(sgRNA) were tested as the targets of the blocking sequences: (1) the spacer and (2) the annealing region between the crRNA and tracrRNA (Fig. la) ¹ . The crystal structure of the guide RNA bound to Cas9 suggests that an elongated 5' end and insertions in the tetraloop should not significantly interfere with guide RNA

function ¹⁹ ' ²⁰ . Therefore, a 42-nucleotide linker followed by a 6-nucleotide blocking sequence that complements nucleotides 1-6 in the spacer was appended to the 5' end of the sgRNA (Fig. la). Eventually replacing the linker with a ligand -responsive RNA was envisioned and an elongated linker was chosen to test if the size and flexibility of the RNA would disturb intramolecular hybridization of the blocking sequence to the spacer. To block the interaction between the crRNA and tracrRNA, appended to the 3' end of the crRNA or to the 5' end of the tracrRNA was the 42- nucleotide linker and a 10-nucleotide blocking sequence that is complementary to the annealing region in the crRNA or tracrRNA, respectively (Fig. la).

The blocker-linked guide RNAs were prepared by in vitro transcription and incubated in vitro with Cas9 protein and a target dsDNA. Target DNA cleavage was followed by gel electrophoresis (Fig. lb). The activity of the 5' spacer-blocked sgRNA was very low and no cleavage product could be detected by gel

electrophoresis, while cleavage catalyzed by the unmodified sgRNA approached 100% efficiency under the same conditions (Fig. lb). Although the blocked crRNA and tracrRNA were less active than the unmodified guide RNAs, they both resulted in partial cleavage of the target DNA, indicating that the strategy for blocking the crRNA and tracrRNA was not as effective as the use of a spacer-blocked sgRNA (bsgRNA).

Together, these findings demonstrate an effective strategy for using RNA to occlude a region of a sgRNA, thereby abrogating Cas9 activity. Based on these results, the bsgRNA architecture was chosen as the starting point for efforts to engineer ligand-induced restoration of guide RNA function. Guide RNA activation by removal of the blocking sequence

Next, molecular strategies to restore guide RNA activity by removing the blocking sequence in situ were developed. A fast-cleaving hammerhead ribozyme 21 with low magnesium dependence was integrated between the blocking sequence and the guide RNA (HHR-bsgRNA, Fig. 2a). Once transcribed, the hammerhead ribozyme should spontaneously fold and undergo self-cleavage to remove most of the ribozyme as well as the blocking sequence. Self-cleavage transforms the

intramolecular base pairs between the spacer and the blocking sequence in the HHR- bsgRNA into less favorable intermolecular interactions, promoting the release of the blocking sequence from the guide RNA. The post-cleavage sgRNA contained 11 extra nucleotides at the 5' end that are remnants from the hammerhead ribozyme and maintained most of its activity compared to the unmodified guide RNA (Fig. 2b). This result establishes that the ribozyme self-cleavage of the blocking region restores sgRNA activity.

To compare the activities of the guide RNAs with the blocking region either present or removed, a non-cleaving "dead" variant of HHR-bsgRNA (dHHR-bsgRNA) containing a single A to G point mutation in the catalytic loop of the hammerhead ribozyme was produced. To optimize the restoration of guide RNA activity by self- cleavage of the hammerhead ribozyme, the length of the blocking sequence was varied. Three sets of HHR-bsgRNAs and dHHR-bsgRNAs with blocking sequences of 10 to 17 nucleotides were constructed. The genome editing activities of these sgRNA variants in the presence of Cas9 in human embryonic kidney cells harboring genomic GFP genes (HEK293-GFP) were assayed by targeting the endogenous GFP locus (Fig. 2a).

As expected, the activities of both non-cleaving blocked guide RNAs (dHHR- bsgRNAs) and self-cleaving blocked guide RNAs (HHR-bsgRNAs) decreased when the length of the blocking sequence increased (Fig. 2b). A 17-nucleotide blocking sequence was chosen for further engineering because it resulted in minimal

background activity in the off state that was comparable to a negative control lacking any guide RNA, but showed substantial restoration of activity once the blocking sequence was removed by ribozyme self-cleavage (Fig. 2b).

Theophylline-dependent genome editing

Next, ligand dependence was imparted in an HHR-bsgRNA by replacing the hammerhead ribozyme with an aptazyme that contains a ligand-binding aptamer that transduces the presence of a small molecule into ribozyme self-cleavage. The hammerhead ribozyme was replaced with the theophylline-dependent hammerhead aptazyme 22 ' 23. To ensure that the theophylline-dependent hammerhead aptazyme uses the same cleavage site as the unmodified ribozyme when fused to the guide RNA, the theophylline- agRN A was transcribed in vitro and the cleaved products were analyzed by mass spectrometry. The observed masses were in good agreement with the expected masses for the 5' fragment and the 3' fragment (Fig. 5), confirming that appending the guide RNA did not alter the cleavage site preference of the

hammerhead ribozyme. The genome-editing activity of theophylline- agRN A was further assayed in HEK293-GFP cells (Fig. 3a). While control cells expressing Cas9 but lacking guide RNAs resulted in 9.6% loss of GFP fluorescence, cells that were co- transfected with both Cas9 and theophylline- agRN A exhibited 22% GFP loss in the absence of theophylline (Fig. 3b). This off-state background activity of 12% additional GFP loss is consistent with reports that the activity of the hammerhead ribozyme in the theophylline aptazyme, while substantially decreased, is detectable even in the absence of theophylline 23. In addition, incorporation of the theophylline aptamer increases the overall bulkiness of the ribozyme domain and the additional steric hindrance may affect the interaction between the blocking sequence and the spacer, resulting in less efficient inhibition.

Importantly, in the presence of theophylline, the activity of the agRNA increased 4-fold, resulting in 58% GFP fluorescence loss (48% additional loss beyond the off-state) (Fig. 3b). By comparison, wild-type Cas9 and a canonical sgRNA resulted in 72% GFP loss (62% loss beyond the off-state), only 1.3-fold that of the activated agRNA. The activity of the theophylline- agRNA was dependent on the concentration of theophylline, reaching a maximum at 2 mM (Fig. 3b). These results show that ligand-induced agRNA activation can result in genome modification levels that approach those of canonical Cas9:sgRNA complexes.

To rule out the possibility that the observed loss of GFP fluorescence was a consequence of theophylline toxicity rather than genome editing at endogenous GFP sites, cells transfected without any guide RNA plasmid, or with a control plasmid producing the dHHR-bsgRNA were treated with the highest concentration of theophylline used in the above experiments (4 mM). Neither control sample exhibited significant GFP loss in the presence of theophylline compared to the same cells that were not treated with theophylline (Fig. 3b), suggesting that theophylline did not negatively impact cell fluorescence and the observed GFP loss resulted from disruption of the endogenous GFP gene. To confirm that activation of the

theophylline- agRNA was due to the ligand-responsive self-cleavage of the aptazyme, rather than to binding of theophylline to the aptamer domain without cleavage, an A to G mutation that inactivates the hammerhead ribozyme into the theophylline- agRNA while maintaining theophylline binding activity 23 was installed and the genome editing activity of the resulting "dead" (d)theophylline-agRNA was assayed. This point mutation abolished the ligand response of theophylline- agRNA and resulted in only basal levels of GFP loss in the presence or absence of theophylline, indicating that ribozyme self-cleavage, and not merely ligand binding, is required for agRNA activation (Fig. 6). Together, these findings establish that small molecule- induced aptazyme self-cleavage can activate guide RNAs and enable ligand- dependent genome editing.

Guanine-dependent transcriptional activation

In addition to genome editing, transcriptional regulation is another widely used application of CRISPR-Cas9 ⁴ . To further explore the utility of agRNAs, we sought to develop an agRNA that responds to a different small-molecule ligand and activates the expression of a guide RNA-specified target gene, rather than editing a target gene through DNA cleavage. A previously reported guanine-aptazyme 24 that contains a naturally occurring guanine aptamer 25 was incorporated into the guide RNA and the resulting guanine-agRNA was tested for its ability to activate transcription of a GFP gene in HEK293T cells (Fig. 3c). The guanine-agRNA targeted the sequence upstream of a modified promoter for the GFP gene and recruited the fusion of catalytically inactive dCas9 with tandem mammalian transcriptional activators VP64-p65-Rta (dCas9-VPR) ⁴ . In the presence of an active guide RNA, dCas9-VPR initiates transcription of GFP, ultimately resulting in cell fluorescence.

Similar to the theophylline-agRNA, the guanine-agRNA also exhibited detectable off-state activity, resulting in elevated GFP fluorescence in the absence of guanine compared to control cells lacking any guide RNA (Fig. 3d and Fig. 7). The elevated off-state activity can be attributed to the presence of endogenous guanine, as well as basal self-cleaving activity of the guanine aptazyme in the absence of the small molecule 24. When guanine was added to media at a concentration of 50 μΜ, however, a 5-fold activation of GFP fluorescence was observed in cells that were transfected with guanine-agRNA expression plasmid. In contrast, no GFP activation was detected in control cells lacking guide RNA or in control cells expressing non- cleaving dHHR-bsgRNA (Fig. 3d and Fig. 7). In addition, the non-splicing mutant (d)guanine-agRNA resulted in only a low level of GFP expression that could no longer be activated by guanine (Fig. 8). Indeed, increasing the concentration of guanine in the medium from 0 to 200 μΜ slightly reduced GFP fluorescence (Fig. 8), suggesting that guanine might have a modest negative effect on cell fluorescence and the actual activation ratio of the guanine-agRNA could be higher than was calculated from GFP fluorescence.

Additional optimization of the blocking sequence

Controlling RNA activity by intramolecular blocking sequences has been achieved in other functional RNAs and different architectures of blocking sequences have been explored ²³ ' ²⁶ . In the "riboregulators" that enable post-transcriptional control of gene expression in E. coli by regulating the accessibility of the ribosomal binding site (RBS), blockers containing bulges exhibit stronger activation than blockers that are fully complementary to the RBS, since the bulges destabilize the RNA duplex and facilitate the activation process ²⁶ . Inspired by this observation, we further optimized the blocking sequence in the guanine-agRNA by increasing the blocking sequence to 18 nucleotides and incorporating three bulges.

The newly constructed guanine-agRNA with the optimized blocking sequence exhibited a similar off-state activity to the original guanine-agRNA design but resulted in a slightly higher GFP fluorescence when activated by the presence of guanine (Fig. 9). To further generalize this strategy, we tested the guanine-agRNA with three bulges for RFP activation in HEK293T cells with a different spacer sequence that is unrelated to the spacer used to target GFP. Consistent with above results, guanine-dependent RFP activation was observed in the presence of dCas9- VPR with an activation ratio of ~3-fold (Fig. 10), suggesting that the agRNA architecture is generally applicable on different spacer sequences as well as different target genes.

Application of agRNAs to endogenous sites for genome editing

To further define the application scope of agRNAs, we tested them on four additional guide RNAs that target endogenous HEK-3, FANCF, EMX-1 and HEK-4 loci in the human genome. To establish the dynamic range of the responsive guide RNAs, the activities of HHR-bsgRNA and dHHR-bsgRNA with a 17-nucleotide blocking sequence was first measured for gene disruption on these four loci. In the presence of Cas9, 1.2% to 16% insertion or deletion (indel) formation was observed with dHHR-bsgRNA, whereas a 3- to 13-fold higher activity could be achieved when the HHR-bsgRNA was introduced (Fig. 4a, 4b, and Fig. 11), defining a potential dynamic range for ligand-activated genome editing at these four endogenous sites.

Base editing is a novel technology that enables programmable single-base pair modification in the human genome without inducing double-stranded DNA breaks 7 ' 27- ²⁹ . We tested the activities of HHR-bsgRNAs and dHHR-bsgRNA for base editing of endogenous HEK-3, FANCF, EMX-1 and HEK-4 loci in HEK293 cells in the presence of the BE3 base editor . Similar to the case with nuclease-mediated indel formation, base editing occurred with different efficiencies at the four loci (Fig. 4c, 4d, and Fig. 11). The active HHR-bsgRNAs resulted in 5- to 8-fold higher levels of base editing than the inactive dHHR-bsgRNA on all four sites (Fig. 4c, 4d, and Fig. 11), defining the potential dynamic range for ligand-activated base editing.

Encouraged by these observations, we engineered the theophylline-responsive hammerhead ribozyme into the guide RNAs targeting the HEK-3 and FANCF sites. Indeed, these agRNAs resulted in small molecule-controlled base editing. The FANCF site exhibited 1.0-1.2% base editing with the theophylline- agRN A in the absence of theophylline (Fig. 4e). The addition of 4 mM theophylline increased the editing to 3.6-3.9% (Fig. 4e). In contrast, a much lower dose of guide RNA plasmid was required for the HEK-3 site to achieve base editing. With 2 ng guide RNA plasmid and 200 ng BE3 plasmid, 1.7%-2.1% base editing was observed at the HEK-3 locus (Fig. 4f). The presence of 4 mM theophylline raised base editing efficiency to 4.2-5.8% (Fig. 4f). By increasing the dose of guide RNA and BE3 plasmid, as high as 36% base editing efficiency could be achieved in the presence of theophylline with 2- to 3-fold ligand dependence (Fig. 4g and 4h). To test the ligand specificity of small molecule-controlled base editing, we measured the response of theophylline-agRNA- mediated base editing on the FANCF site in the presence of 3-methylxanthine, a close analogue of theophylline lacking one methyl group. At a ligand concentration of 4 mM, the theophylline- agRN A responded 2-fold less strongly to 3-methylxanthine than to theophylline (Fig. 12). This lower response to 3-methylxanthine is consistent with the known preference of the theophylline-dependent ribozyme for theophylline over 3-methylxanthine 30. Collectively, these results demonstrate the ability of agRNAs to confer small-molecule-dependence on base editing at endogenous loci in human cells. DISCUSSION

Aptazymes have been applied to control biological processes in various contexts, such as gene expression in prokaryotes and miRNA processing in mammalian cells 23 ' 24. By using aptazymes to remove blocking sequences that abrogate essential guide RNA structures, we have developed a set of small molecule- responsive agRNAs that enable exogenous control over genome engineering in mammalian cells using strategies that are complementary to other conditional Cas9 variants. In addition, small molecule-dependent guide RNAs have been reported in CRISPR "signal conductors" to control gene expression for constructing logic gates and genetic circuits 31. Our work serves as the first example to our knowledge of small molecule-controlled genome editing in mammalian cells achieved through guide RNA engineering. The non-cleavable theophylline- agRN A ((d)theophylline-agRNA) did not show any response to the small molecule for both genome editing and gene activation, strongly suggesting that the cleavage activity of the ribozyme is crucial for guide RNA activation. Because the most widely used sgRNA, that derived from Streptococcus pyogenes, has a length > 100 nt and contains multiple regions susceptible to secondary structural changes ¹⁹ , our strategy of triggering the self- cleavage of the blocking region from the guide RNA minimizes the possible of interference with guide RNA activity from the regulatory element since the activated agRNA closely resembles that of a canonical sgRNA.

Both nature and researchers have evolved hundreds of RNA aptamers that bind a wide variety of ligands 32 ' 33. By incorporating these aptamers into the agRNA architecture, agRNAs could potentially be constructed to respond to many different molecules of interest. As ligand specificities of aptamers are generally high, agRNAs have the potential to be multiplexed for complicated applications in which precise temporal and spatial control of multiple genome engineering events is required. The successful engineering of artificial guide RNAs to be ligand-responsive raises the possibility that naturally occurring CRISPR components may already use small- molecule regulation.

Various parameters including communication between the aptamer and the ribozyme, interaction between the aptazyme and the guide RNA, hybridization of the blocking sequence and the spacer, as well as the binding affinity of guide RNA backbone to Cas9 in the presence or absence of the regulatory domain may be optimized in future generations of agRNAs.

Methods

Construction of guide RNA and reporter plasmids. Plasmids that overexpress Cas9 (pJDS246) and dCas9-VPR (pAWG-dCas9-VPR ⁴ ) were used for mammalian cell experiments. The guide RNA plasmids were constructed based on the previously reported pFYF1320 plasmid ³⁴ and sequences of the guide RNAs were provided in Table 1. The GFP and RFP reporter plasmids were constructed by swapping the promoter and the reporter gene from a previously reported plasmid (TF reporter for gRNA-AAVS l_Tl, Addgene #47320 ³ ) and the modified sequences were listed in Table 2. Plasmids constructed in this study will be available from Addgene.

In vitro DNA cleavage assay. SpCas9 protein was purified as previously described 1 ' 35. Guide RNAs were transcribed using a T7 High Yield RNA Synthesis Kit (NEB) following the manufacturer's protocol, and purified with the E.Z.N. A. PF miRNA Isolation Kit (Omega Bio-tek, Inc.). The target dsDNA (full GFP gene) was amplified by polymerase chain reaction and purified using QIAquick PCR Purification Kit (QIAGEN). For the cleavage reaction, 10 nM of the target DNA was incubated with 100 nM of the guide RNA in the presence of 100 nM Cas9 protein in a Cas9 DNA cleavage buffer (150 mM KC1, 10 mM MgCl ₂ , 0.5 mM DTT, 0.1 mM EDTA, 20 mM HEPES pH 7.5). In vitro editing reactions were incubated at 37 °C for 1 h for sgRNA and its variants and the reactions for crRNA and tracrRNA and their variants were incubated at 37 °C for 5 h before being stopped with 6X DNA loading buffer and analyzed by non-denaturing agarose gel electrophoresis.

Editing of GFP gene in HEK293 cells. HEK293 cells with integrated GFP genes (GenTarget Inc.) were cultured in 48-well plates (-40,000 cells seeded per well) in Dulbecco's Modified Eagle's Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS. Plasmids were transfected -20 h after plating when cells reached -70% confluence. To edit the GFP genes in HEK293 cells, 100 ng of Cas9 plasmid and 2 ng of guide RNA plasmid were transfected in each well using 1.2 Lipofectamine 2000 (Life Technologies) following the manufacturer's protocol. To distinguish transfected cells from non-transfected cells, 2 ng of an iRFP expression plasmid was co-transfected. A stock solution of 100 niM theophylline was made in 60 niM NaOH and was supplied to cells 6 h after plasmid transfection at designed concentrations. The cells were incubated for additional 7 days before analysis and media was changed at day 3, 5 and 6. Fresh theophylline was supplied immediately at the same

concentration as in the initial condition each time the media was changed. GFP fluorescence was quantified by flow cytometry and the transfected live population was gated by iRFP signal.

Nuclease-mediated genome editing and base editing on endogenous sites in HEK293T cells. Similar cell culture procedures were used for HEK293T cells as described above for HEK293-GFP cells. A modified guide RNA plasmid containing an iRFP gene was used to differentiate transfected cells from non-transfected ones when necessary. To each well in a 48-well plate, 400 ng of Cas9 or BE3 plasmid and 40 ng of guide RNA plasmid were transfected using 1.25 μΐ _^ Lipofectamine 2000. Cells were harvested 3 days after transfection. To test the small molecule response, HEK293T cells were plated in a 24-well plate (100,000 cells seeded in each well) and BE3 and guide RNA plasmids were transfected 24 h after plating. For the FANCF site, 800 ng BE3 and 80 ng guide RNA plasmids were transfectedl. For the HEK-3 site, 200, 400, or 800 ng of BE3 and 2, 10, or 80 ng of guide RNA plasmids were transfected, respectively. 2.5 μΐ _^ Lipofectamine 2000 was used for each transfection. Stock solutions of 100 mM theophylline and 100 mM 3-methylxanthine were made in 100 mM NaOH and supplied to cells 6 h after plasmid transfection at specified concentrations. Cells were cultured for 3 days after transfection before sorting by flow cytometry. Indel formation and base editing were quantified by PCR amplification of the endogenous genomic loci of interest and analysis by high-throughput DNA sequencing.

Transcriptional activation in HEK293T cells. Similar cell culture procedures were used for HEK293T cells as described above for HEK293-GFP cells. To each well in a 48-well plate, 200 ng of dCas9-VPR plasmid, 0.5 ng of guide RNA plasmid and 60 ng of reporter plasmid were transfected using 1.2 μL· Lipofectamine 2000. To distinguish transfected cells from non-transfected cells, 0.2 ng of an iRFP expression plasmid was co-transfected. A stock solution of 20 mM guanine was made in 60 mM NaOH and was supplied to cells 6 h after plasmid transfection at designed concentrations. Cells were harvested 20 h after transfection. The GFP or RFP fluorescence was quantified by flow cytometry and the transfected live population was gated by iRFP signal.

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Table 1 I Sequences of guide RNAs. The spacer sequences are in bold and the blocking sequences are underlined. Aptamer units in agRNAs are in italics and the catalytic inactivating mutation in the hammerhead ribozyme is marked in bold underline.

Construct Sequence SEQ ID NO Plasmid sgRNA-GFP GGGCACGGGCAGCUUGCCGGGUUUUAGAG 1 pFYF1320

CUAGAAAUAGCAAGUUAAAAUAAGGCUAGU CCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU

5 '-spacer- GGUGCCCGUAGUAGUAGUAGCAACCACGUC 2

blocked GCGUCCGUAGUAGUAGUAGGGCACGGGCA

sgRNA-GFP GCUUGCCGGGUUUUAGAGCUAGAAAUAGCA

AGUUAAAAUAAGGCUAGUCCGUUAUCAACU

UGAAAAAGUGGCACCGAGUCGGUGCUUUUU

uu

3 '-blocked GGGCACGGGCAGCUUGCCGGGUUUUAGAG 3

crRNA-GFP CUAUGCUGUUUUGGAUGGCAACCACGUCGC

GUCCUGUAGUAGUAUGAUAGCUCUAA

5 '-blocked GGAUUUUAACUUUAGUAGUAGUAGGCAACC 4

tracrRNA ACGUCGCGUCCUAGUAGGCAUAGCAAGUUA

AAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUUUUU

processed GCUGGAUUCCACGGGCACGGGCAGCUUGC 5 pWT016g HHR- CGGGUUUUAGAGCUAGAAAUAGCAAGUUAA

sgRNA-GFP AAUAAGGCUAGUCCGUUAUCAACUUGAAAA

AGUGGCACCGAGUCGGUGCUUUUUUU

HHR- GCCCGUGCCCGGUACAUCCAGCUGAUGAGUC 6 pWT016f bsgRNA- CCAAAUAGGACGAAACGCGCUUCGGUGCGU

GFP CCUGGAUUCCACGGGCACGGGCAGCUUGCC

(10 nt GGGUUUUAGAGCUAGAAAUAGCAAGUUAAA

blocker) AUAAGGCUAGUCCGUUAUCAACUUGAAAAA

GUGGCACCGAGUCGGUGCUUUUUUU

dHHR- GCCCGUGCCCGGUACAUCCAGCUGAUGAGUC 7 pWT016e sgRNA-GFP CCAAAUAGGACGAGACGCGCUUCGGUGCGU

(10 nt CCUGGAUUCCACGGGCACGGGCAGCUUGCC

blocker) GGGUUUUAGAGCUAGAAAUAGCAAGUUAAA

AUAAGGCUAGUCCGUUAUCAACUUGAAAAA

GUGGCACCGAGUCGGUGCUUUUUUU

HHR- GCUGCCCGUGCCCGGUACAUCCAGCUGAUGA 8 pWT016a bsgRNA- GUCCCAAAUAGGACGAAACGCGCUUCGGUG

GFP CGUCCUGGAUUCCACGGGCACGGGCAGCUU

(13 nt GCCGGGUUUUAGAGCUAGAAAUAGCAAGUU

blocker) AAAAUAAGGCUAGUCCGUUAUCAACUUGAA

AAAGUGGCACCGAGUCGGUGCUUUUUUU

dHHR- GCUGCCCGUGCCCGGUACAUCCAGCUGAUGA 9 pWT016b sgRNA-GFP GUCCCAAAUAGGACGAGACGCGCUUCGGUG

(13 nt CGUCCUGGAUUCCACGGGCACGGGCAGCUU

blocker) GCCGGGUUUUAGAGCUAGAAAUAGCAAGUU

AAAAUAAGGCUAGUCCGUUAUCAACUUGAA

AAAGUGGCACCGAGUCGGUGCUUUUUUU

HHR- GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 10 pWT016h bsgRNA- AUGAGUCCCAAAUAGGACGAAACGCGCUUC

GFP GGUGCGUCCUGGAUUCCACGGGCACGGGCA

(17 nt GCUUGCCGGGUUUUAGAGCUAGAAAUAGCA

blocker) AGUUAAAAUAAGGCUAGUCCGUUAUCAACU

UGAAAAAGUGGCACCGAGUCGGUGCUUUUU

UU dHHR- GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 11 pWT016d sgRNA-GFP AUGAGUCCCAAAUAGGACGAGACGCGCUUC

(17 nt GGUGCGUCCUGGAUUCCACGGGCACGGGCA

blocker) GCUUGCCGGGUUUUAGAGCUAGAAAUAGCA

AGUUAAAAUAAGGCUAGUCCGUUAUCAACU

UGAAAAAGUGGCACCGAGUCGGUGCUUUUU

uu

theophylline GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 12 pWT016i

-agRNA- AUG AGUCCC A A AU AGG ACG A A A UA CA UA CCA

GFP GCCGAAAGGCCCUUGGCAGGUG\5CC\5GGA\5\5

CCACGGGCACGGGCAGCUUGCCGGGUUUU

AGAGCUAGAAAUAGCAAGUUAAAAUAAGGC

UAGUCCGUUAUCAACUUGAAAAAGUGGCAC

CGAGUCGGUGCUUUUUUU

(d)theophyll GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 13 pWT0161 ine-agRNA- AUG AGUCCC A A AU AGG ACG AG A UA CA UA CCA

GFP GCCGAAAGGCCCUUGGCAGGUG\5CC\5GGA\5\5

CCACGGGCACGGGCAGCUUGCCGGGUUUU

AGAGCUAGAAAUAGCAAGUUAAAAUAAGGC

UAGUCCGUUAUCAACUUGAAAAAGUGGCAC

CGAGUCGGUGCUUUUUUU

guanine- GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 14 pWT029c agRNA AUG AGUCCC A A AU AGG ACG A A AU AC UA UAA U

(GFP CGCGUGGAUAUGGCACGCAAGUUUCUACCGG

activation) GCACCGUAAAUGUCCGACUAGUGUCCUGGAU

UCCACGGGCACGGGCAGCUUGCCGGGUUU

UAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCA

CCGAGUCGGUGCUUUUUUU

(d)guanine- GCAAGCUGCCCGUGCCCGGUACAUCCAGCUG 15 pWT029d agRNA AUG AGUCCC A A AU AGG ACG AG AU AC UA UAA U

(GFP CGCGUGGAUAUGGCACGCAAGUUUCUACCGG

activation) GCACCGUAAAUGUCCGACUAG\ G\ CC\ GGh\

UCCACGGGCACGGGCAGCUUGCCGGGUUU

UAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCA

CCGAGUCGGUGCUUUUUUU

guanine- GGCAUGCUCCCCGUGACCGGUACAUCCAGCU 16 pWT029g agRNA GAUGAGUCCC AAAU AGG ACGAAAU AC UA UAA

(GFP UCGCGUGGA UA UGGCA CGCAAGUUUCUA CCG

activation, GGCA CCGUAAA UGUCCGA CK4GUGUCCUGGA

18 nt UUCCACGGGCACGGGCAGCUUGCCGGGUU

blocker UUAGAGCUAGAAAUAGCAAGUUAAAAUAAG

with bulges) GCUAGUCCGUUAUCAACUUGAAAAAGUGGC

ACCGAGUCGGUGCUUUUUUU

(d)guanine- GGCAUGCUCCCCGUGACCGGUACAUCCAGCU 17 pWT029h agRNA GAUGAGUCCC AAAU AGG ACG AG AU AC UA UAA

(GFP UCGCGUGGA UA UGGCA CGCAAGUUUCUA CCG

activation, GGCA CCGUAAA UGUCCGA CK4GUGUCCUGGA

18 nt UUCCACGGGCACGGGCAGCUUGCCGGGUU

blocker UUAGAGCUAGAAAUAGCAAGUUAAAAUAAG

with bulges) GCUAGUCCGUUAUCAACUUGAAAAAGUGGC

ACCGAGUCGGUGCUUUUUUU

guanine- ACUGCGGGAUGGAGGAGACGGUACAUCCAG 18 pWT049d agRNA (19 CUG AUG AGUCCC AAAU AGG ACG AAAU AC UA

nt blocker UAAUCGCGUGGAUAUGGCACGCAAGUUUCUAC

with bulges, CGGGCA CCGUAAA UGUCCGA CK4GUGUCCUG

RFP GAUUCCACGUCCCCUCCACCCCACAGUGGU

activation) UUUAGAGCUAGAAAUAGCAAGUUAAAAUAA

GGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUUUUUUU

(d)guanine- ACUGCGGGAUGGAGGAGACGGUACAUCCAG 19 pWT049e agRNA (19 CUG AUG AGUCCC A A AU AGG ACG AG AU AC UA

nt blocker UAAUCGCGUGGAUAUGGCACGCAAGUUUCUAC

with bulges, CGGGCA CCGUAAA UGUCCGA CK4GUGUCCUG

RFP GAUUCCACGUCCCCUCCACCCCACAGUGGU

activation) UUUAGAGCUAGAAAUAGCAAGUUAAAAUAA

GGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUUUUUUU

sgRNA- GGCCCAGACUGAGCACGUGAGUUUUAGAG 20 pWT055g HEK3 CUAGAAAUAGCAAGUUAAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU

sgRNA- GGAAUCCCUUCUGCAGCACCGUUUUAGAGC 21 pWT055f FANCF UAGAAAUAGCAAGUUAAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU

sgRNA- GAGUCCGAGCAGAAGAAGAAGUUUUAGAG 22 pWT055e EMX1 CUAGAAAUAGCAAGUUAAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU

sgRNA- GGCACUGCGGCUGGAGGUGGGUUUUAGAG 23 pWT055h HEK4 CUAGAAAUAGCAAGUUAAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU

HHR- CGUGCUCAGUCUGGGCCGGUACAUCCAGCUG 24 pWT057e bsgRNA- AUGAGUCCCAAAUAGGACGAAACGCGCUUC

HEK3 GGUGCGUCCUGGAUUCCACGGCCCAGACUG

AGCACGUGAGUUUUAGAGCUAGAAAUAGCA

AGUUAAAAUAAGGCUAGUCCGUUAUCAACU

UGAAAAAGUGGCACCGAGUCGGUGCUUUUU

uu

dHHR- CGUGCUCAGUCUGGGCCGGUACAUCCAGCUG 25 pWT057f bsgRNA- AUGAGUCCCAAAUAGGACGAGACGCGCUUC

HEK3 GGUGCGUCCUGGAUUCCACGGCCCAGACUG

AGCACGUGAGUUUUAGAGCUAGAAAUAGCA

AGUUAAAAUAAGGCUAGUCCGUUAUCAACU

UGAAAAAGUGGCACCGAGUCGGUGCUUUUU

UU

HHR- GCUGCAGAAGGGAUUCCGGUACAUCCAGCU 26 pWT057c bsgRNA- GAUGAGUCCCAAAUAGGACGAAACGCGCUU

FANCF CGGUGCGUCCUGGAUUCCACGGAAUCCCUU

CUGCAGCACCGUUUUAGAGCUAGAAAUAGC

AAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGCUUUU

uuu

dHHR- GCUGCAGAAGGGAUUCCGGUACAUCCAGCU 27 pWT057d bsgRNA- GAUGAGUCCCAAAUAGGACGAGACGCGCUU

FANCF CGGUGCGUCCUGGAUUCCACGGAAUCCCUU

CUGCAGCACCGUUUUAGAGCUAGAAAUAGC

AAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGCUUUU

UUU

HHR- GUUCUUCUGCUCGGACUCGGUACAUCCAGCU 28 pWT057a bsgRNA- GAUGAGUCCCAAAUAGGACGAAACGCGCUU

EMX1 CGGUGCGUCCUGGAUUCCACGAGUCCGAGC

AGAAGAAGAAGUUUUAGAGCUAGAAAUAGC

AAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGCUUUU

UUU dHHR- GUUCUUCUGCUCGGACUCGGUACAUCCAGCU 29 pWT057b bsgRNA- GAUGAGUCCCAAAUAGGACGAGACGCGCUU

EMX1 CGGUGCGUCCUGGAUUCCACGAGUCCGAGC

AGAAGAAGAAGUUUUAGAGCUAGAAAUAGC

AAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGCUUUU

uuu

HHR- GCCUCCAGCCGCAGUGCCGGUACAUCCAGCU 30 pWT057g bsgRNA- GAUGAGUCCCAAAUAGGACGAAACGCGCUU

HEK4 CGGUGCGUCCUGGAUUCCACGGCACUGCGG

CUGGAGGUGGGUUUUAGAGCUAGAAAUAG

CAAGUUAAAAUAAGGCUAGUCCGUUAUCAA

CUUGAAAAAGUGGCACCGAGUCGGUGCUUU

uuuu

dHHR- GCCUCCAGCCGCAGUGCCGGUACAUCCAGCU 31 pWT057h bsgRNA- GAUGAGUCCCAAAUAGGACGAGACGCGCUU

HEK4 CGGUGCGUCCUGGAUUCCACGGCACUGCGG

CUGGAGGUGGGUUUUAGAGCUAGAAAUAG

CAAGUUAAAAUAAGGCUAGUCCGUUAUCAA

CUUGAAAAAGUGGCACCGAGUCGGUGCUUU

UUUU

theophylline CGUGCUCAGUCUGGGCCGGUACAUCCAGCUG 32 pWT055c

-agRNA- AUG AGUCCC A A AU AGG ACG A A A UA CA UA CCA

HEK3 GCCGAAAGGCCCUUGGCAGGUGUCCUGG AUU

CCACGGCCCAGACUGAGCACGUGAGUUUU

AGAGCUAGAAAUAGCAAGUUAAAAUAAGGC

UAGUCCGUUAUCAACUUGAAAAAGUGGCAC

CGAGUCGGUGCUUUUUUU

theophylline GCUGCAGAAGGGAUUCCGGUACAUCCAGCU 33 pWT055b

-agRNA- G AUG AGUCCC A A AU AGG ACG A A A UA CA UA CC

FANCF A GCCGAAA GGCCCUUGGCA GG i/GUCCUGGAU

UCCACGGAAUCCCUUCUGCAGCACCGUUUU

AGAGCUAGAAAUAGCAAGUUAAAAUAAGGC

UAGUCCGUUAUCAACUUGAAAAAGUGGCAC

CGAGUCGGUGCUUUUUUU

Table 2 I Sequences of the engineered promoter and reporter genes. The protospacer sequences are in bold and the engineered promoter is underlined. The GFP and RFP genes are in italic.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or

descriptions that include "or" between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some

embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

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