US 2015 0071901A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0071901 A1 Liu et al. (43) Pub. Date: Mar. 12, 2015 (54) MRNA-SENSING SWITCHABLE GRNAS (52) U.S. Cl. (71) Applicant: President and Fellows of Harvard CPC .............. CI2N 15/01 (2013.01); C12N 15/I 13 College, Cambridge, MA (US) (2013.01); C12N 9/22 (2013.01); C12N 23 10/10 (2013.01) (72) Inventors: JohnnyDavid R. Hao Liu, Hu, Lexington, Cambridge, MA MA(US); (US) USPC ........ 424/94.3: 536/24. 1; 435/188: 435/91.1; 435/320.1; 435/325; 536/23.1 (73) Assignee: President and Fellows of Harvard College, Cambridge, MA (US) (57) ABSTRACT (21) Appl. No.: 14/326,340 Some aspects of this disclosure provide compositions, meth (22) Filed: Jul. 8, 2014 ods, systems, and kits for controlling the activity and/or Related U.S. Application Data improving the specificity of RNA-programmable endonu (60) Provisional application No. 61/874,682, filed on Sep. cleases, such as Cas9. For example, provided are guide RNAs 6, 2013. (gRNAs) that are engineered to exist in an “on” or “off” state, Publication Classification which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this (51) y;5/0 (2006.01) disclosure provide mRNA-sensing gRNAs that modulate the CI2N 9/22 (2006.01) activity of RNA-programmable endonucleases based on the CI2N IS/II3 (2006.01) presence or absence of a target mRNA. Patent Application Publication Mar. 12, 2015 Sheet 1 of 12 US 2015/0071901 A1 SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS Patent Application Publication Mar. 12, 2015 Sheet 2 of 12 US 2015/0071901 A1 Patent Application Publication Mar. 12, 2015 Sheet 3 of 12 US 2015/0071901 A1 s: s DI‘ROHH 3. Patent Application Publication Mar. 12, 2015 Sheet 4 of 12 US 2015/0071901 A1 (II‘50IH Patent Application Publication Mar. 12, 2015 Sheet 5 of 12 US 2015/0071901 A1 VZ'5018 Patent Application Publication Mar. 12, 2015 Sheet 6 of 12 US 2015/0071901 A1 Patent Application Publication Mar. 12, 2015 Sheet 7 of 12 US 2015/0071901 A1 OZ."?INH Patent Application Publication Mar. 12, 2015 Sheet 8 of 12 US 2015/0071901 A1 WWW ww. yA. Mar. 12, 2015 Sheet 9 of 12 US 2015/0071901 A1 Patent Application Publication Mar. 12, 2015 Sheet 10 of 12 US 2015/0071901 A1 Patent Application Publication Mar. 12, 2015 Sheet 11 of 12 US 2015/0071901 A1 Patent Application Publication Mar. 12, 2015 Sheet 12 of 12 US 2015/0071901 A1 *3.§§§§§§§§ {} US 2015/007 1901 A1 Mar. 12, 2015 MRNA-SENSING SWITCHABLE GRNAS ligand bound to the aptamer; and (ii) a Cas9 protein. In some embodiments, the aptamer is bound by a ligand. In some RELATED APPLICATIONS aspects, the ligand is any molecule. In some aspects, the 0001. This application claims priority under 35 U.S.C. ligand is a small molecule, a metabolite, a carbohydrate, a S119(e) to U.S. provisional application, U.S. Ser. No. 61/874, peptide, a protein, or a nucleic acid. In some embodiments, 682, filed Sep. 6, 2013, which is incorporated herein by ref the gRNA:ligand:Cas9 complex binds to and mediates cleav CCC. age of a target nucleic acid. See, e.g., FIG. 1. 0006. According to another embodiment, gRNAs com BACKGROUND OF THE INVENTION prising an aptamer are provided. In some embodiments, the gRNA does not hybridize to a target nucleic acid in the 0002 Site-specific endonucleases theoretically allow for absence of a ligand bound to the aptamer. Such gRNAS may the targeted manipulation of a single site within a genome and be referred to as “switchable gRNAs. For example, in some are useful in the context of gene targeting as well as for aspects, the gRNA does not bind Cas9 in the absence of a therapeutic applications. In a variety of organisms, including ligand bound to the aptamer. See, e.g., FIGS. 1A-B. In some mammals, site-specific endonucleases have been used for embodiments, the gRNA binds Cas9 when the aptamer is genome engineering by stimulating either non-homologous bound by a ligand specific to the aptamer. In some embodi end joining or homologous recombination. In addition to ments, the gRNA binds Cas9 in the absence or presence of a providing powerful research tools, site-specific nucleases ligand bound to the aptamer but binds to a target nucleic acid also have potential as gene therapy agents, and two site only in the presence of a ligand bound to the aptamer. In some specific endonucleases have recently entered clinical trials: aspects, the ligand is any molecule. In some aspects, the one, CCR5-2246, targeting a human CCR-5 allele as part of ligand is a small molecule, a metabolite, a carbohydrate, a an anti-HIV therapeutic approach (NCT00842634, peptide, a protein, or a nucleic acid. In some embodiments, NCT01044654, NCT01252641), and the other one, the aptamer is an RNA aptamer, for example, an RNA VF24684, targeting the human VEGF-A promoter as part of aptamer derived from a riboswitch. In some embodiments, an anti-cancer therapeutic approach (NCT01082926). the riboswitch from which the aptamer is derived is selected 0003 Specific cleavage of the intended nuclease target site from a theophylline riboswitch, a thiamine pyrophosphate without or with only minimal off-target activity is a prereq (TPP) riboswitch, an adenosine cobalamin (AdoCbl) uisite for clinical applications of site-specific endonuclease, riboswitch, an S-adenosyl methionine (SAM) riboswitch, an and also for high-efficiency genomic manipulations in basic SAH riboswitch, a flavin mononucleotide(FMN) riboswitch, research applications. For example, imperfect specificity of a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine engineered site-specific binding domains has been linked to riboswitch, a purine riboswitch, a GlmS riboswitch, or a cellular toxicity and undesired alterations of genomic loci pre-queosine (PreCR1) riboswitch. In some embodiments, the other than the intended target. Most nucleases available aptamer is derived from a theophylline riboswitch and com today, however, exhibit significant off-target activity, and thus prises SEQ ID NO:3. In other embodiments, the aptamer is may not be suitable for clinical applications. An emerging non-naturally occurring, and in some aspects, is engineered to nuclease platform for use in clinical and research settings are bind a specific ligand using a systematic evolution of ligands the RNA-guided nucleases, such as Cas9. While these by exponential enrichment (SELEX) platform. In some nucleases are able to bind guide RNAs (gRNAs) that direct embodiments, the non-aptamer portion of the gRNA com cleavage of specific target sites, off-target activity is still prises at least 50, at least 60, at least 70, at least 80, at least 90, observed for certain Cas9:gRNA complexes (Pattanayak et at least 100, at least 110, at least 120, at least 130, at least 140, al., “High-throughput profiling of off-target DNA cleavage or at least 150 nucleotides, and the aptamer comprises at least reveals RNA-programmed Cas9 nuclease specificity.” Nat 20, at least 30, at least 40, at least 50, at least 60, at least 70, Biotechnol. 2013; doi:10.1038/nbt.2673). Technology for at least 80, at least 90, at least 100, at least 110, at least 120, engineering nucleases with improved specificity is therefore at least 130, at least 140, at least 150, at least 175, at least 200, needed. at least 250, or at least 300 nucleotides. 0007 According to another embodiment, methods for SUMMARY OF THE INVENTION site-specific DNA cleavage using the inventive Cas9 variants 0004 Some aspects of this disclosure are based on the are provided. For example, in some aspects, the methods recognition that the reported toxicity of some engineered comprise contacting a DNA with a complex comprising (i) a site-specific endonucleases is based on off-target DNA cleav gRNA comprising an aptamer, wherein the gRNA comprises age. Further, the activity of existing RNA-guided nucleases a sequence that binds to a portion of the DNA, (ii) a specific generally cannot be controlled at the molecular level, for ligand bound to the aptamer of the gRNA, and (iii) a Cas9 example, to switch a nuclease from an “off to an “on” state. protein, under conditions in which the Cas9 protein cleaves Controlling the activity of nucleases could decrease the like the DNA. lihood of incurring off-target effects. Some aspects of this 0008 According to another embodiment, methods for disclosure provide strategies, compositions, systems, and inducing site-specific DNA cleavage in a cell are provided. methods to control the binding and/or cleavage activity RNA For example, in Some embodiments, the methods comprise: programmable endonucleases, such as Cas9 endonuclease. (a) contacting a cell or expressing within a cell a gRNA 0005 Accordingly, one embodiment of the disclosure pro comprising an aptamer, wherein the gRNA comprises a vides RNA-guided nuclease complexes comprising a “Swit sequence capable of binding to a DNA target sequence; (b) chable' guide RNA (gRNA). For example, in some embodi contacting a cell or expressing within a cell a Cas9 protein; ments, the invention provides a complex comprising: (i) a and (c) contacting the cell with a ligand that binds the aptamer gRNA comprising an aptamer, wherein the gRNA does not of the gRNA, resulting in the formation of a gRNA:ligand: hybridize to a target nucleic acid in the absence of a specific Cas9 complex that cleaves the DNA target. In some embodi US 2015/007 1901 A1 Mar. 12, 2015 ments, the cell produces the ligand intracellularly, for at least 5, at least 10, at least 15, at least 20, at least 25, at least example as a part of physiological or pathophysiological 30, at least 40, at least 50, at least 75, or at least 100 nucle process.