RNA-induced transcriptional regulation

Abstract

Provided is a method for controlling expression of target DNA using a DNA-binding protein-guide RNA complex. [Solution] A method for inserting a donor nucleic acid into a cell by homologous recombination includes: causing a cell to contain a spacer sequence, a tracr mate sequence, and two types of guide RNAs having a tracr sequence, a portion of the tracr sequence hybridizing to the tracr mate sequence, the tracr mate sequence and the tracr sequence being connected by a linker nucleic acid sequence, and each of the spacer sequences being complementary to adjacent sites in a DNA target nucleic acid; and causing a Cas9 protein nickase to be contained in the cell, wherein each of the two types of guide RNAs co-localizes with the Cas9 protein nickase in the DNA target nucleic acid, and the donor nucleic acid is inserted into the DNA target nucleic acid by homologous recombination with an offset nick.

Description

[Technical Field]

[0001] Related application data This application claims priority to U.S. Provisional Patent Application No. 61 / 830,787, filed June 4, 2013, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Explanation of government interests This invention was made with government support under National Institutes of Health Grant No. P50 HG005550 and U.S. Department of Energy Grant No. DE-FG02-02ER63445. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs complexed with Cas proteins to direct the degradation of complementary sequences present within invading foreign nucleic acids. Deltcheva, E. et al., CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, Vol. 471, pp. 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V., Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, pp. E2579-2586 (2012); Jinek, M. et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, Vol. 337, pp. 816-821 (2012); Sapranauskas, R. et al., The Streptococcus thermophilus CRISPR / Cas See, e.g., “A system provides immunity in Escherichia coli.” Nucleic Acids Research, Vol. 39, pp. 9275-9282 (2011); and Bhaya, D., Davison, M., and Barrangou, R., “CRISPR-Cas system in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.” Annual Review of Genetics, Vol. 45, pp. 273-297 (2011).Recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that crRNA ("CRISPR RNA") fused to the normally trans-encoded tracrRNA ("trans-activating CRISPR RNA") is sufficient to direct the Cas9 protein to sequence-specifically cleave the target DNA sequence corresponding to the crRNA. Expression of a gRNA homologous to the target site triggers Cas9 recruitment and target DNA degradation. See H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology, 190:1390 (February 2008). Summary of the Invention [Problem to be solved by the invention]

[0004] An embodiment of the present disclosure relates to a complex between a guide RNA, a DNA-binding protein, and a double-stranded DNA target sequence. According to a specific embodiment, the DNA-binding protein within the scope of the present disclosure includes a protein that forms a complex with a guide RNA, and a protein that forms a complex with a guide RNA that guides the complex to a double-stranded DNA sequence and binds to the DNA sequence. This embodiment of the present disclosure can be referred to as co-localization of the RNA and the DNA-binding protein to the double-stranded DNA or co-localization with the double-stranded DNA. In this way, the DNA-binding protein-guide RNA complex can be used to localize a transcription factor protein or domain to the target DNA to control the expression of the target DNA. [Means for solving the problem]

[0005] According to a specific embodiment, there is provided a method for controlling expression of a target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid) comprising the target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding an RNA-guided nuclease-null DNA-binding protein that binds to the DNA and is guided by the one or more RNAs; and introducing into the cell a third exogenous nucleic acid encoding a transcription factor protein or domain, wherein the one or more RNAs, the RNA-guided nuclease-null DNA-binding protein, and the transcription factor protein or domain are expressed, the one or more RNAs, the RNA-guided nuclease-null DNA-binding protein, and the transcription factor protein or domain are co-localized with the DNA, and the transcription factor protein or domain controls the expression of the target nucleic acid.

[0006] According to one embodiment, the exogenous nucleic acid encoding the RNA-guided nuclease-deficient DNA-binding protein further encodes the transcription factor protein or domain fused to the RNA-guided nuclease-deficient DNA-binding protein. According to one embodiment, the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain, and the exogenous nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain.

[0007] According to one embodiment, the cell is a eukaryotic cell. According to one embodiment, the cell is a yeast cell, a plant cell, or an animal cell. According to one embodiment, the cell is a mammalian cell.

[0008] In one embodiment, the RNA is between about 10 nucleotides and about 500 nucleotides. In one embodiment, the RNA is between about 20 nucleotides and about 100 nucleotides.

[0009] According to one embodiment, the transcription factor protein or domain is a transcription activator. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse health condition. According to one embodiment, the target nucleic acid is associated with a disease or adverse health condition.

[0010] According to one embodiment, the one or more RNAs are guide RNAs. According to one embodiment, the one or more RNAs are tracrRNA-crRNA fusions. According to one embodiment, the guide RNA comprises a spacer sequence and a tracer mate sequence. The guide RNA may comprise a tracr sequence, a portion of which hybridizes to the tracr mate sequence. The guide RNA may comprise a linker nucleic acid sequence that links the tracer mate sequence and the tracr sequence to create a tracrRNA-crRNA fusion. The spacer sequence binds to the target DNA, for example, by hybridization.

[0011] According to one embodiment, the guide RNA comprises a shortened spacer sequence. According to one embodiment, the guide RNA comprises a shortened spacer sequence with a 5'-terminal truncation of 1 base. According to one embodiment, the guide RNA comprises a shortened spacer sequence with a 5'-terminal truncation of 2 bases. According to one embodiment, the guide RNA comprises a shortened spacer sequence with a 5'-terminal truncation of 3 bases. According to one embodiment, the guide RNA comprises a shortened spacer sequence with a 5'-terminal truncation of 4 bases. Thus, the spacer sequence may have a 5'-terminal truncation of 1 to 4 bases in its sequence.

[0012] In certain embodiments, the spacer sequence may comprise between about 16 and about 20 nucleotides that hybridize to the target nucleic acid sequence. In certain embodiments, the spacer sequence may comprise about 20 nucleotides that hybridize to the target nucleic acid sequence.

[0013] According to one particular embodiment, the linker nucleic acid sequence may comprise between about 4 and about 6 nucleic acids.

[0014] In certain embodiments, the tracr sequence may comprise between about 60 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 64 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 65 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 66 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 67 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 68 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 69 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 70 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 80 and about 500 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 90 and about 500 nucleic acids. According to one particular embodiment, the tracr sequence may comprise between about 100 and about 500 nucleic acids.

[0015] In certain embodiments, the tracr sequence may comprise between about 60 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 64 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 65 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 66 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 67 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 68 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 69 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 70 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 80 and about 200 nucleic acids. In certain embodiments, the tracr sequence may comprise between about 90 and about 200 nucleic acids. According to one particular embodiment, the tracr sequence may comprise between about 100 and about 200 nucleic acids.

[0016] An exemplary guide RNA is shown in Figure 5B.

[0017] According to one embodiment, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0018] According to a particular aspect, there is provided a method for controlling expression of a target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid) comprising the target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding an RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system that binds to the DNA and is guided by the one or more RNAs; and introducing into the cell a third exogenous nucleic acid encoding a transcription regulator protein or domain, wherein the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system, and the transcription regulator protein or domain are expressed, the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system, and the transcription regulator protein or domain are co-localized with the DNA, and the transcription regulator protein or domain controls the expression of the target nucleic acid.

[0019] According to one embodiment, the exogenous nucleic acid encoding an RNA-guided, nuclease-deficient DNA-binding protein of a Type II CRISPR system further encodes the transcription regulator protein or domain fused to the RNA-guided, nuclease-deficient DNA-binding protein of a Type II CRISPR system. According to one embodiment, the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain, and the exogenous nucleic acid encoding the transcription regulator protein or domain further encodes an RNA-binding domain fused to the transcription regulator protein or domain.

[0020] According to one embodiment, the cell is a eukaryotic cell. According to one embodiment, the cell is a yeast cell, a plant cell, or an animal cell. According to one embodiment, the cell is a mammalian cell.

[0021] In one embodiment, the RNA is between about 10 nucleotides and about 500 nucleotides. In one embodiment, the RNA is between about 20 nucleotides and about 100 nucleotides.

[0022] According to one embodiment, the transcription factor protein or domain is a transcription activator. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse health condition. According to one embodiment, the target nucleic acid is associated with a disease or adverse health condition.

[0023] According to one embodiment, the one or more RNAs are guide RNAs. According to one embodiment, the one or more RNAs are tracrRNA-crRNA fusions.

[0024] According to one embodiment, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0025] According to a specific aspect, there is provided a method for controlling expression of a target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding one or more RNAs (ribonucleic acids) complementary to DNA (deoxyribonucleic acid) comprising the target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding a nuclease-deficient Cas9 protein that binds to the DNA and is guided by the one or more RNAs; and introducing into the cell a third exogenous nucleic acid encoding a transcription regulator protein or domain, wherein the one or more RNAs, the nuclease-deficient Cas9 protein, and the transcription regulator protein or domain are expressed, the one or more RNAs, the nuclease-deficient Cas9 protein, and the transcription regulator protein or domain are co-localized with the DNA, and the transcription regulator protein or domain controls the expression of the target nucleic acid.

[0026] According to one embodiment, the exogenous nucleic acid encoding the nuclease-deficient Cas9 protein further encodes the transcription regulator protein or domain fused to the nuclease-deficient Cas9 protein. According to one embodiment, the exogenous nucleic acid encoding one or more RNAs further encodes a target of an RNA-binding domain, and the exogenous nucleic acid encoding the transcription regulator protein or domain further encodes an RNA-binding domain fused to the transcription regulator protein or domain.

[0027] According to one embodiment, the cell is a eukaryotic cell. According to one embodiment, the cell is a yeast cell, a plant cell, or an animal cell. According to one embodiment, the cell is a mammalian cell.

[0028] In one embodiment, the RNA is between about 10 nucleotides and about 500 nucleotides. In one embodiment, the RNA is between about 20 nucleotides and about 100 nucleotides.

[0029] According to one embodiment, the transcription factor protein or domain is a transcription activator. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse health condition. According to one embodiment, the target nucleic acid is associated with a disease or adverse health condition.

[0030] According to one embodiment, the one or more RNAs are guide RNAs. According to one embodiment, the one or more RNAs are tracrRNA-crRNA fusions.

[0031] According to one embodiment, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0032] According to one embodiment, there is provided a cell comprising a first exogenous nucleic acid encoding one or more RNAs complementary to DNA comprising a target nucleic acid, a second exogenous nucleic acid encoding an RNA-guided nuclease-deficient DNA-binding protein, and a third exogenous nucleic acid encoding a transcription factor protein or domain, wherein the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein, and the transcription factor protein or domain are components of a co-localized complex with the target nucleic acid.

[0033] According to one embodiment, the exogenous nucleic acid encoding the RNA-guided nuclease-deficient DNA-binding protein further encodes the transcription factor protein or domain fused to the RNA-guided nuclease-deficient DNA-binding protein. According to one embodiment, the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain, and the exogenous nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain.

[0034] According to one embodiment, the cell is a eukaryotic cell. According to one embodiment, the cell is a yeast cell, a plant cell, or an animal cell. According to one embodiment, the cell is a mammalian cell.

[0035] In one embodiment, the RNA is between about 10 nucleotides and about 500 nucleotides. In one embodiment, the RNA is between about 20 nucleotides and about 100 nucleotides.

[0036] According to one embodiment, the transcription factor protein or domain is a transcription activator. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid. According to one embodiment, the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse health condition. According to one embodiment, the target nucleic acid is associated with a disease or adverse health condition.

[0037] According to one embodiment, the one or more RNAs are guide RNAs. According to one embodiment, the one or more RNAs are tracrRNA-crRNA fusions.

[0038] According to one embodiment, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0039] According to a particular embodiment, the RNA-guided nuclease-deficient DNA-binding protein is a RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system. According to a particular embodiment, the RNA-guided nuclease-deficient DNA-binding protein is a nuclease-deficient Cas9 protein.

[0040] According to one embodiment, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more types of RNA, each of which is complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one type of RNA-guided DNA-binding protein nickase guided by the two or more types of RNA, wherein the two or more types of RNA and the at least one type of RNA-guided DNA-binding protein nickase are expressed, and the at least one type of RNA-guided DNA-binding protein nickase co-localizes with the two or more types of RNA in the DNA target nucleic acid, thereby nicking the DNA target nucleic acid to generate two or more adjacent nicks.

[0041] According to one aspect, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase of a type II CRISPR system guided by the two or more RNAs, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase of the type II CRISPR system are expressed, and the at least one RNA-guided DNA-binding protein nickase of the type II CRISPR system co-localizes with the two or more RNAs to the DNA target nucleic acid, thereby nicking the DNA target nucleic acid to create two or more adjacent nicks.

[0042] According to one aspect, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one Cas9 nickase protein having an inactive nuclease domain and guided by the two or more RNAs, wherein the two or more RNAs and the at least one Cas9 nickase protein are expressed, and the at least one Cas9 nickase protein co-localizes with the two or more RNAs to the DNA target nucleic acid, thereby nicking the DNA target nucleic acid to generate two or more adjacent nicks.

[0043] According to the method for modifying a DNA target nucleic acid, the two or more adjacent nicks are present on the same strand of double-stranded DNA. According to one embodiment, the two or more adjacent nicks are present on the same strand of double-stranded DNA, causing homologous recombination. According to one embodiment, the two or more adjacent nicks are present on different strands of double-stranded DNA. According to one embodiment, the two or more adjacent nicks are present on different strands of double-stranded DNA, causing double-stranded breaks. According to one embodiment, the two or more adjacent nicks are present on different strands of double-stranded DNA, causing double-stranded breaks and non-homologous end joining. According to one embodiment, the two or more adjacent nicks are present on different strands of double-stranded DNA, and are offset relative to one another. According to one embodiment, the two or more adjacent nicks are present on different strands of double-stranded DNA, and are offset relative to one another, causing double-stranded breaks. According to one embodiment, the two or more adjacent nicks are on different strands of double-stranded DNA, are staggered relative to one another, and create double-strand breaks that result in non-homologous end joining. According to one embodiment, the method further comprises introducing into the cell a third exogenous nucleic acid encoding a donor nucleic acid sequence, wherein the two or more nicks cause homologous recombination of the target nucleic acid with the donor nucleic acid sequence.

[0044] According to one embodiment, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase guided by the two or more RNAs, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase are expressed, the at least one RNA-guided DNA-binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid, and nicks the DNA target nucleic acid to create two or more adjacent nicks, the two or more adjacent nicks being on different strands of double-stranded DNA, thereby creating a double-strand break and causing fragmentation of the target nucleic acid, thereby disrupting expression of the target nucleic acid.

[0045] According to one aspect, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase of a type II CRISPR system guided by the two or more RNAs, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase of the type II CRISPR system are expressed, and the at least one RNA-guided DNA-binding protein nickase of the type II CRISPR system co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid to create two or more adjacent nicks, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, creating double-strand breaks and causing fragmentation of the target nucleic acid, thereby disrupting expression of the target nucleic acid.

[0046] According to one aspect, there is provided a method for modifying a DNA target nucleic acid in a cell, the method comprising: introducing into the cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and introducing into the cell a second exogenous nucleic acid encoding at least one Cas9 protein nickase having an inactive nuclease domain and guided by the two or more RNAs, wherein the two or more RNAs and the at least one Cas9 protein nickase are expressed, the at least one Cas9 protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid, and nicks the DNA target nucleic acid to create two or more adjacent nicks, the two or more adjacent nicks being on different strands of double-stranded DNA, creating double-strand breaks and causing fragmentation of the target nucleic acid, thereby disrupting expression of the target nucleic acid.

[0047] According to one embodiment, there is provided a cell comprising a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in a DNA target nucleic acid, and a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase are components of a co-localized complex with the DNA target nucleic acid.

[0048] According to one embodiment, the RNA-guided DNA-binding protein nickase is a RNA-guided DNA-binding protein nickase of a type II CRISPR system. According to one embodiment, the RNA-guided DNA-binding protein nickase is a Cas9 protein nickase having one inactive nuclease domain.

[0049] According to one embodiment, the cell is a eukaryotic cell. According to one embodiment, the cell is a yeast cell, a plant cell, or an animal cell. According to one embodiment, the cell is a mammalian cell.

[0050] According to one embodiment, the RNA comprises between about 10 and about 500 nucleotides. According to one embodiment, the RNA comprises between about 20 and about 100 nucleotides.

[0051] According to one embodiment, the target nucleic acid is associated with a disease or adverse health condition.

[0052] According to one embodiment, the two or more RNAs are guide RNAs. According to one embodiment, the two or more RNAs are tracrRNA-crRNA fusions.

[0053] According to one embodiment, the DNA target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0054] Other features and advantages of certain embodiments of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.

[0055] The patent or patent application file contains color drawings. Copies of this patent or patent application publication with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The above and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0056] [Figure 1-1] Figure 1A shows a schematic diagram of RNA-induced transcriptional activation. Figure 1B shows a schematic diagram of RNA-induced transcriptional activation. Figure 1C shows the design of the reporter construct. [Figure 1-2] Figure ID shows data demonstrating that Cas9N-VP64 fusions exhibit RNA-induced transcriptional activation when assayed by both fluorescence-activated cell sorting (FACS) and immunofluorescence analysis (IF). [Figure 1-3]Figure ID shows data demonstrating that Cas9N-VP64 fusions exhibit RNA-induced transcriptional activation when assayed by both fluorescence-activated cell sorting (FACS) and immunofluorescence analysis (IF). [Figure 1-4] Figure 1E shows FACS and IF assay data demonstrating gRNA sequence-specific transcriptional activation of a reporter construct in the presence of Cas9N, MS2-VP64, and a gRNA with the appropriate MS2 aptamer binding site. [Figure 1-5] Figure 1E shows FACS and IF assay data demonstrating gRNA sequence-specific transcriptional activation of a reporter construct in the presence of Cas9N, MS2-VP64, and a gRNA with the appropriate MS2 aptamer binding site. [Figure 1-6] Figure 1F shows data representing transcription induction by individual gRNAs and multiple gRNAs.

[0057] [Figure 2-1] Figure 2A shows a method for assessing the targeting landscape by Cas9-gRNA complexes and TALEs. [Figure 2-2] Figure 2B shows data demonstrating that the Cas9-gRNA complex tolerates, on average, one to three mutations in its target sequence, and Figure 2C shows data demonstrating that the Cas9-gRNA complex is largely insensitive to point mutations except those located in the PAM sequence. [Figure 2-3] Figure 2D shows heat plot data demonstrating that the introduction of two mismatches significantly impaired Cas9-gRNA complex activity, and Figure 2E shows data demonstrating that 18-mer TALEs tolerate, on average, one to two mutations in their target sequence. [Figure 2-4] Figure 2F shows data demonstrating that the 18-mer TALE, like the Cas9-gRNA complex, is largely insensitive to a single-base mismatch in its target. Figure 2G shows heat plot data demonstrating that the introduction of a two-base mismatch significantly impairs the activity of the 18-mer TALE.

[0058] [Figure 3-1] Figure 3A is a schematic diagram of guide RNA design. [Figure 3-2] Figure 3B presents data showing the percentage of non-homologous end joining for offset nicks that create 5' overhangs and offset nicks that create 3' overhangs. [Figure 3-3] Figure 3C presents data showing the percentage of targeting for offset nicks that create 5' overhangs and offset nicks that create 3' overhangs.

[0059] [Figure 4-1] Figure 4A shows a schematic diagram of the metal-coordinating residue at position D7 of RuvC with PDB ID: 4EP4 (blue) (left), a schematic diagram of the HNH endonuclease domain with PDB IDs: 3M7K (orange) and 4H9D (cyan), including the coordinating Mg ion (gray box) and DNA (purple) of 3M7K (center), and a list of the analyzed mutants (right). [Figure 4-2] Figure 4B presents data showing undetectable nuclease activity of Cas9 mutants m3 and m4, and also of their respective fusions with VP64. [Figure 4-3] Figure 4C is a sensitive examination of the data in Figure 4B.

[0060] [Figure 5-1] Figure 5A is a schematic diagram of the homologous recombination assay for measuring Cas9-gRNA activity. [Figure 5-2] Figure 5B shows the percentage of homologous recombination with guide RNAs containing random sequence inserts. [Figure 5-3] Figure 5B shows the percentage of homologous recombination with guide RNAs containing random sequence inserts.

[0061] [Figure 6-1]Figure 6A shows a schematic diagram of the guide RNA for the OCT4 gene, and Figure 6B shows transcriptional activation of the promoter-luciferase reporter construct. [Figure 6-2] Figure 6C shows transcriptional activation of endogenous genes by qPCR.

[0062] [Figure 7-1] Figure 7A shows a schematic diagram of the guide RNA for the REX1 gene, and Figure 7B shows transcriptional activation of the promoter-luciferase reporter construct. [Figure 7-2] Figure 7C shows transcriptional activation of endogenous genes by qPCR.

[0063] [Figure 8-1] FIG. 8A is a schematic diagram of the high-level specificity analysis process flow for calculation of normalized expression levels. [Figure 8-2] Figure 8B shows data on the distribution of the percentage of binding sites per number of mismatches generated in the biased construct library. Left: theoretical distribution. Right: distribution observed from the actual TALE construct library. Figure 8C shows data on the distribution of the percentage of tags recruited to binding sites per number of mismatches. Left: distribution observed from the positive control sample. Right: distribution observed from the non-control TALE-guided sample.

[0064] [Figure 9-1] Figure 9A shows the targeting landscape analysis data of the Cas9-gRNA complex, demonstrating its tolerance of one to three mutations in the target sequence, and Figure 9B shows the targeting landscape analysis data of the Cas9-gRNA complex, demonstrating its insensitivity to point mutations except those located in the PAM sequence. [Figure 9-2] Figure 9C shows heat plot data from an analysis of the targeting landscape of the Cas9-gRNA complex, demonstrating that the introduction of two mismatches significantly impaired activity. [Figure 9-3]Figure 9D shows data from a nuclease-mediated HR assay confirming that the predicted PAM of S. pyogenes Cas9 is NGG and also NAG.

[0065] [Figure 10-1] Figure 10A shows data from a nuclease-mediated HR assay confirming that 18-mer TALEs tolerate multiple mutations in their target sequences. [Figure 10-2] Figure 10A shows data from a nuclease-mediated HR assay confirming that 18-mer TALEs tolerate multiple mutations in their target sequences. [Figure 10-3] Figure 10B shows the analysis data of the targeting landscape of TALEs of three different sizes (18-mer, 14-mer, and 10-mer). [Figure 10-4] Figure 10C shows data from a 10-mer TALE demonstrating near-single-base mismatch resolution. Figure 10D shows heat plot data from a 10-mer TALE demonstrating near-single-base mismatch resolution.

[0066] [Figure 11-1] Figure 11A shows the designed guide RNAs. [Figure 11-2] Figure 11B shows the percentage of non-homologous end joining for various guide RNAs. [Figure 12-1] Figure 12A shows 10 gRNAs designed for the SOX2 gene that target an approximately 1 kb long DNA strand upstream of the transcription start site. [Figure 12-2] Figure 12B shows ten gRNAs targeted to an approximately 1 kb long DNA strand upstream of the transcription start site for the NANOG gene. [Figure 13-1] Figure 13A shows the targeting landscape of Cas9N-VP64+gRNA2, and Figure 13B shows the single-base mismatch plot of Cas9N-VP64+gRNA2. [Figure 13-2]Figure 13C shows the two-base mismatch plot of Cas9N-VP64+gRNA2, and Figure 13D shows the targeting landscape of Cas9N-VP64+gRNA3. [Figure 13-3] Figure 13E shows a single-base mismatch plot for Cas9N-VP64+gRNA3, and Figure 13F shows a two-base mismatch plot for Cas9N-VP64+gRNA3. [Figure 14] Figures 14A-C show specificity data using two different sgRNA:Cas9 complexes. [Figure 15-1] Figure 15A shows testing using a nuclease assay for gRNA2 carrying one or two base mismatches. [Figure 15-2] Figure 15B-1 shows a test using a nuclease assay for gRNA2 carrying one or two base mismatches. [Figure 15-3] Figure 15B-2 shows a test using a nuclease assay for gRNA2 carrying one or two base mismatches. [Figure 15-4] Figure 15C and Figure 15D-1 show tests using a nuclease assay for gRNA3 carrying one or two mismatches. [Figure 15-5] Figure 15D-2 shows a test using a nuclease assay for gRNA3 carrying one or two mismatches. [Figure 16-1] Figure 16A shows a test using a nuclease assay for gRNA1 with a truncated 5' end of the spacer. [Figure 16-2] Figure 16B-1 shows a test using a nuclease assay for gRNA1 with a truncated 5' end of the spacer. [Figure 16-3] Figure 16B-2 shows a test using a nuclease assay for gRNA1 with a shortened 5' end of the spacer. [Figure 16-4] Figure 16C shows a test using a nuclease assay for gRNA3 with a truncated 5' end of the spacer. [Figure 16-5] Figure 16D-1 shows a test using a nuclease assay for gRNA3 with a shortened 5' end of the spacer. [Figure 16-6] Figure 16D-2 shows a test using a nuclease assay for gRNA3 with a shortened 5' end of the spacer. [Figure 17-1] Figure 17A shows a study of the PAM of S. pyogenes Cas9. [Figure 17-2] Figure 17B shows testing of PAM for S. pyogenes Cas9. [Figure 18-1] Figure 18A shows a targeting experiment in a nuclease assay for TALE mutations. [Figure 18-2] Figure 18B shows a targeting experiment in a nuclease assay for TALE mutations. [Figure 19-1] Figure 19A shows the testing approach for the comparison between TALE monomer specificity and TALE protein specificity. [Figure 19-2] Figure 19B-1 shows a comparison between TALE monomer specificity and TALE protein specificity. [Figure 19-3] Figure 19B-2 shows a comparison between TALE monomer specificity and TALE protein specificity. [Figure 19-4] Figure 19C-1 shows a comparison between TALE monomer specificity and TALE protein specificity. [Figure 19-5] Figure 19C-2 shows a comparison between TALE monomer specificity and TALE protein specificity. [Figure 20-1] Figure 20A shows targeting of the AAVS1 locus with offset nicking. [Figure 20-2] Figure 20B shows data on offset nick formation. [Figure 21-1] Figure 21A shows the offset nicking and NHEJ profiles. [Figure 21-2]Figure 21B shows the offset nicking and NHEJ profiles. [Figure 21-3] Figure 21C shows the offset nicking and NHEJ profiles. DETAILED DESCRIPTION OF THE INVENTION

[0067] Embodiments of the present disclosure are based on the use of DNA-binding proteins to co-localize transcription factor proteins or domains to DNA in methods for regulating target nucleic acids. Such DNA-binding proteins are known to those skilled in the art for binding to DNA for a variety of purposes. Such DNA-binding proteins may be naturally occurring. DNA-binding proteins within the scope of the present disclosure include those that can be guided by RNA, referred to herein as guide RNA. According to this aspect, the guide RNA and the RNA-guided DNA-binding protein form a co-localized complex at DNA. According to certain aspects, the DNA-binding protein may be a nuclease-deficient DNA-binding protein. According to this aspect, the nuclease-deficient DNA-binding protein may result from the alteration or modification of a DNA-binding protein with nuclease activity. Such DNA-binding proteins with nuclease activity are known to those skilled in the art and include, for example, naturally occurring DNA-binding proteins with nuclease activity, such as the Cas9 protein present in type II CRISPR systems. Such Cas9 proteins and type II CRISPR systems are well described in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477, including all additional information, which is incorporated herein by reference in its entirety.

[0068] Exemplary DNA-binding proteins with nuclease activity function to nick or cleave double-stranded DNA. Such nuclease activity can result from DNA-binding proteins having one or more polypeptide sequences that exhibit nuclease activity. Such exemplary DNA-binding proteins may have two distinct nuclease domains, each responsible for cleaving or nicking a specific strand of double-stranded DNA. Exemplary polypeptide sequences with nuclease activity known to those skilled in the art include the McrA-HNH nuclease-associated domain and the RuvC-like nuclease domain. Thus, exemplary DNA-binding proteins are proteins that naturally contain one or more of the McrA-HNH nuclease-associated domain and the RuvC-like nuclease domain. According to certain embodiments, the DNA-binding proteins are modified or engineered to inactivate the nuclease activity. Such modifications or alterations include altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. Such modifications include removing one or more polypeptide sequences exhibiting nuclease activity, i.e., nuclease domains, such that the polypeptide sequence(s) exhibiting nuclease activity, i.e., nuclease domains, are absent from the DNA-binding protein. Other modifications for inactivating nuclease activity will be readily apparent to those skilled in the art based on the present disclosure. Thus, a nuclease-deficient DNA-binding protein includes a polypeptide sequence modified to inactivate nuclease activity or the removal of one or more polypeptide sequences to inactivate nuclease activity. The nuclease-deficient DNA-binding protein retains DNA-binding ability even when nuclease activity is inactivated. Thus, the DNA-binding protein may contain one or more polypeptide sequences necessary for DNA binding but lack one or more or all of the nuclease sequences exhibiting nuclease activity.Thus, the DNA binding protein contains one or more polypeptide sequences necessary for DNA binding, but may also contain one or more or all of the nuclease sequences that exhibit inactivating nuclease activity.

[0069] In one embodiment, a DNA-binding protein having two or more nuclease domains may be modified or altered so that all but one of the nuclease domains are inactivated. Such modified or altered DNA-binding proteins are called DNA-binding protein nickases, as long as the DNA-binding protein nickases only cut or nick one strand of double-stranded DNA. When guided to DNA by RNA, the DNA-binding protein nickases are called RNA-guided DNA-binding protein nickases.

[0070] Exemplary DNA binding proteins include the RNA-guided DNA binding proteins of type II CRISPR systems that lack nuclease activity.Exemplary DNA binding proteins include nuclease-deficient Cas9 proteins.Exemplary DNA binding proteins include Cas9 protein nickase.

[0071] In S. pyogenes, Cas9 creates a blunt-ended double-stranded break 3 bp upstream of the protospacer adjacent motif (PAM) through processing mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinke et al., Science 337:816-821 (2012), incorporated herein by reference in its entirety. The Cas9 protein is known to be present in a number of type II CRISPR systems, including those found in supplementary information in Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis strain C7; Corynebacterium diphtheriae; Corynebacterium efficiens strain YS-314; Corynebacterium glutamicum ATCC 13032 strain Kitasato; and Corynebacterium glutamicum ATCC 13032. Bielefeld strain; Corynebacterium glutamicum strain R; Corynebacterium kloppenstettii strain DSM44385; Mycobacterium abscessus strain ATCC19977; Nocardia farcinica strain IFM10152; Rhodococcus erythropolis strain PR4; Rhodococcus jostii strain RHA1; Rhodococcus opacus B4 strain uid36573; Acidothermus cellulolyticus strain 11B; Arthrobacter chlorophenolicus strain A6; Kluyvera flavida DSM17836 strain uid43465; Thermonospora curbata strain DSM43183; Bifidobacterium dentium strain Bd1; Bifidobacterium longum strain DJO10A; Slacchia heliotrinireducens strain DSM20476; Persephonera marina EX H1; Bacteroides fragilis strain NCTC9434; Capnocytophaga ochracea strain DSM7271; Flavobacterium psychrophilum strain JIP02 86; Akkermansia muciniphila ATCC BAA835; Roseiflexus castenholzii strain DSM13941; Roseiflexus RS1; Synechocystis strain PCC6803; Yersinia minutum strain Pei191;Uncultivable termite group 1 bacteria phylotype Rs strain D17; Fibrobacter succinogenes strain S85; Bacillus cereus strain ATCC10987; Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus strain GG; Lactobacillus salivarius strain UCC118; Streptococcus agalactiae strain A909; Streptococcus agalactiae strain NEM316; Streptococcus agalactiae strain 2603; Streptococcus dysgalactiae subsp. equisimilis strain GGS124; Streptococcus equi subsp. zooepidemicus strain MGCS10565; Streptococcus gallolyticus strain UCN34 uid46061; Streptococcus gordonii Challis subst CH1 strain; Streptococcus mutans NN2025 uid46353 strain; Streptococcus mutans; Streptococcus pyogenes M1 GAS strain; Streptococcus pyogenes MGAS5005 strain; Streptococcus pyogenes MGAS2096 strain; Streptococcus pyogenes MGAS9429 strain; Streptococcus pyogenes MGAS10270 strain; Streptococcus pyogenes MGAS6180 strain; Streptococcus pyogenes MGAS315 strain; Streptococcus pyogenes SSI-1 strain; Streptococcus pyogenes MGAS10750 strain; Streptococcus pyogenes NZ131 strain; Streptococcus thermophilus (Streptococcus thermophiles strain CNRZ1066; Streptococcus thermophiles strain LMD-9; Streptococcus thermophiles strain LMG18311; Clostridium botulinum A3 strain Loch Maree; Clostridium botulinum B strain Eklund 17B; Clostridium botulinum Ba4 strain 657; Clostridium botulinum F strain Langeland; Clostridium cellulolyticum strain H10; Finegoldia magna strain ATCC29328; Eubacterium rectare strain ATCC33656; Mycoplasma gallisepticum;Mycoplasma mobile strain 163K; Mycoplasma penetrans; Mycoplasma synoviae strain 53; Streptobacillus moniliformis strain DSM12112; Bradyrhizobium strain BTAi1; Nitrobacter hamburgensis strain X14; Rhodopseudomonas palustris strain BisB18; Rhodopseudomonas palustris strain BisB5; Barbibacrum rubamentivorans strain DS-1; Dinoroseobacter shibae strain DFL12; Gluconacetobacter diazotrophicus Pal 5 strain FAPERJ; Gluconacetobacter diazotrophicus Pal 5 strain JGI; Azospirillum B510 strain uid46085; Rhodospirillum rubrum strain ATCC11170; Diafolobacter TPSY strain uid29975; Ferminephorobacter eiseniae strain EF01-2; Neisseria meningitides strain 053442; Neisseria meningitides strain alpha14; Neisseria meningitides strain Z2491; Desulfovibrio salexigens strain DSM2638; Campylobacter jejuni subsp. doylei strain 26997; Campylobacter jejuni strain 81116; Campylobacter jejuni; Campylobacter lari strain RM2100; Helicobacter hepaticus; Wolinella succinogenes; Tormonas auensis strain DSM9187; Pseudoalteromonas atlantica T strain 6c; Shewanella pareana strain ATCC700345; Legionella pneumophila strain Paris; Actinobacillus succinogenes strain 130Z; Pasteurella multocida strain; Francisella tularensis subsp. novicida strain U112; Francisella tularensis subsp. holarctica; Francisella tularensis strain FSC198; Francisella tularensis subsp. tularensis; Francisella tularensis strain WY96-3418;and Treponema denticola strain ATCC 35405. Accordingly, aspects of the present disclosure relate to Cas9 proteins present in Type II CRISPR systems that have been rendered nuclease-deficient or nickase-modified as described herein.

[0072] The Cas9 protein may be referred to in the literature by those skilled in the art as Csn1. The S. pyogenes Cas9 protein sequence, the subject of the experiments described herein, is shown below. See Deltcheva et al., Nature 471:602-607 (2011), incorporated herein by reference in its entirety.

[0073] [ka]

[0074] According to certain embodiments of the RNA-guided genome regulation methods described herein, Cas9 is modified to reduce, substantially reduce, or eliminate nuclease activity. According to one embodiment, Cas9 nuclease activity is reduced, substantially reduced, or eliminated by modifying the RuvC nuclease domain or the HNH nuclease domain. According to one embodiment, the RuvC nuclease domain is inactivated. According to one embodiment, the HNH nuclease domain is inactivated. According to one embodiment, the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to additional embodiments, Cas9 proteins are provided in which the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to additional embodiments, nuclease-deficient Cas9 proteins are provided in which the RuvC nuclease domain and the HNH nuclease domain are inactivated. According to additional embodiments, Cas9 nickases are provided in which either the RuvC nuclease domain or the HNH nuclease domain is inactivated, thereby leaving the remaining nuclease domain active for nuclease activity, such that only one strand of double-stranded DNA is cleaved or nicked.

[0075] According to additional embodiments, nuclease-deficient Cas9 proteins are provided in which one or more amino acids in Cas9 are modified or removed to provide a nuclease-deficient Cas9 protein. According to one embodiment, these amino acids include D10 and H840. See Jinke et al., Science 337:816-821 (2012). According to additional embodiments, these amino acids include D839 and N863. According to one embodiment, one or more or all of D10, H840, D839, and H863 are substituted with amino acids that reduce, substantially reduce, or eliminate nuclease activity. According to one embodiment, one or more or all of D10, H840, D839, and H863 are substituted with alanine. According to one embodiment, a Cas9 protein in which one or more or all of D10, H840, D839, and H863 have been substituted with an amino acid that reduces, substantially reduces, or eliminates nuclease activity, such as alanine, is referred to as a nuclease-deficient Cas9 or Cas9N, and the protein has reduced or eliminated nuclease activity, or no or substantially no nuclease activity within the level of detection. According to this embodiment, the nuclease activity of Cas9N may not be detectable using known assays, i.e., may be below the level of detection of known assays.

[0076] According to one embodiment, the nuclease-deficient Cas9 protein includes homologs and orthologs thereof that retain the protein's ability to bind DNA and be guided by RNA. According to one embodiment, the nuclease-deficient Cas9 protein includes the sequence shown for native Cas9 from Streptococcus pyogenes (S. pyogenes), in which one or more or all of D10, H840, D839, and H863 are substituted with alanine, and a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity thereto, wherein the protein is a DNA-binding protein, such as an RNA-guided DNA-binding protein.

[0077] According to one embodiment, the nuclease-deficient Cas9 protein includes the sequence set forth for native Cas9 from S. pyogenes, excluding the protein sequences of the RuvC and HNH nuclease domains, and also includes a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology thereto, wherein the protein sequence is a DNA-binding protein, such as an RNA-guided DNA-binding protein. Thus, embodiments of the present disclosure include protein sequences responsible for DNA binding, e.g., responsible for colocalization with a guide RNA and DNA binding, and protein sequences homologous thereto, and need not include the protein sequences of the RuvC and HNH nuclease domains (unless required for DNA binding), since these domains can be either inactivated or removed from the protein sequence of the native Cas9 protein to generate the nuclease-deficient Cas9 protein.

[0078] For purposes of this disclosure, metal-coordinating residues in known protein structures with homology to Cas9 are shown in Figure 4A. Residues are labeled based on their position in the Cas9 sequence. On the left is the RuvC structure (blue) from PDB ID: 4EP4. Position D7, corresponding to D10 in the Cas9 sequence, is highlighted among the Mg ion-coordinating positions. In the center are structures of the HNH endonuclease domains from PDB IDs: 3M7K (orange) and 4H9D (turquoise), including the coordinating Mg ion of 3M7K (gray box) and DNA (purple). Residues D92 and N113 in 3M7K and positions D53 and N77 in 4H9D share sequence homology with amino acids D839 and N863 of Cas9 and are shown as sticks. On the right is a list of mutants that were generated and analyzed for nuclease activity: wild-type Cas9, Cas9 with an alanine substitution for D10, and m1 Cas9 with alanine substitutions at D10 and H840 m2Cas9 with alanine substitutions at D10, H840, and D839 m3 and Cas9 with alanine substitutions at D10, H840, D839, and N863. m4 .

[0079] As shown in Figure 4B, when deep sequencing of the targeted locus was performed, Cas9 mutants m3 and m4 and their respective VP64 fusions showed undetectable nuclease activity. Mutation frequency is plotted against genomic position, with red lines indicating gRNA targets. Figure 4C shows a sensitive examination of the data in Figure 4B, confirming that the mutational landscape exhibits a similar profile to the unmodified locus.

[0080] According to one embodiment, a genetically engineered Cas9-gRNA system is provided that enables RNA-guided genome regulation in human cells by linking a transcription activation domain to either a nuclease-deficient Cas9 or a guide RNA. According to one embodiment of the present disclosure, one or more transcriptional regulatory proteins or domains (such terms are used interchangeably) are linked or otherwise connected to the nuclease-deficient Cas9 or one or more guide RNAs (gRNAs). These transcriptional regulatory domains correspond to target loci. Accordingly, embodiments of the present disclosure include methods and materials for localizing transcriptional regulatory domains to target loci by fusing, linking, or linking such domains to either Cas9N or gRNA.

[0081] In one embodiment, a Cas9N fusion protein capable of transcriptional activation is provided. In one embodiment, a VP64 activation domain (see Zhang et al., Nature Biotechnology 29:149-153 (2011), incorporated herein by reference in its entirety) is linked, fused, connected, or otherwise tethered to the C-terminus of Cas9N. In one method, the Cas9N protein provides a transcriptional regulatory domain at a target genomic DNA site. In one method, Cas9N fused to a transcriptional regulatory domain is provided intracellularly with one or more guide RNAs. Cas9N with the fused transcriptional regulatory domain binds to or near the target genomic DNA. One or more guide RNAs bind to or near the target genomic DNA. The transcriptional regulatory domain controls expression of a target gene. According to a particular embodiment, the Cas9N-VP64 fusion, when combined with a gRNA targeting a sequence near the promoter, activated transcription of the reporter construct, thereby activating RNA-guided transcription.

[0082] According to one embodiment, a gRNA fusion protein capable of transcriptional activation is provided. According to one embodiment, a VP64 activation domain is linked, fused, connected, or otherwise tethered to a gRNA. According to one method, the gRNA provides a transcriptional regulatory domain that targets a genomic DNA site. According to one method, the gRNA fused to the transcriptional regulatory domain is provided intracellularly along with a Cas9N protein. The Cas9N binds to or near the target genomic DNA. One or more guide RNAs fused to a transcriptional regulatory protein or domain bind to or near the target genomic DNA. The transcriptional regulatory domain controls expression of a target gene. According to a particular embodiment, the Cas9N protein and the gRNA fused to the transcriptional regulatory domain activated transcription of a reporter construct, thereby activating RNA-guided transcription.

[0083] Transcriptionally regulatable gRNA conjugates were created by inserting random sequences into the gRNA and assaying for Cas9 function to identify which regions of the gRNA were permissive for modification. gRNAs with random sequence insertions at either the 5' end of the crRNA portion or the 3' end of the tracrRNA portion of the chimeric gRNA retained functionality, whereas insertions into the tracrRNA scaffold portion of the chimeric gRNA abolished function. See Figures 5A-B for a summary of gRNA flexibility toward random base insertions. Figure 5A is a schematic diagram of the homologous recombination (HR) assay for measuring Cas9-gRNA activity. As shown in Figure 5B, gRNAs with random sequence insertions at either the 5' end of the crRNA portion or the 3' end of the tracrRNA portion of the chimeric gRNA retained functionality, whereas insertions into the tracrRNA scaffold portion of the chimeric gRNA abolished function. The location of the insertion in the gRNA sequence is indicated by red nucleotides. Without wishing to be bound by scientific theory, the increased activity due to random base insertion at the 5' end may be due to the increased half-life of the gRNA due to its longer length.

[0084] To bind VP64 to the gRNA, two copies of the MS2 bacteriophage coat protein-binding RNA stem-loop were attached to the 3' end of the gRNA. See Fusco et al., Current Biology: CB13, 161-167 (2003), incorporated herein by reference in its entirety. These chimeric gRNAs were co-expressed with Cas9N and the MS2-VP64 fusion protein. Sequence-specific transcriptional activation of the reporter construct was observed in the presence of all three elements.

[0085] Figure 1A shows a schematic diagram of RNA-induced transcriptional activation. To generate a transcriptionally activatable Cas9N fusion protein, a VP64 activation domain was directly linked to the C-terminus of Cas9N. To generate a transcriptionally activatable gRNA conjugate, two copies of an MS2 bacteriophage coat protein-binding RNA stem-loop were attached to the 3' end of the gRNA. These chimeric gRNAs were co-expressed with Cas9N and the MS2-VP64 fusion protein. Figure 1C shows the design of the reporter constructs used to assay transcriptional activation. The two reporters have distinct gRNA target sites and share a control TALE-TF target site. As shown in Figure 1D, the Cas9N-VP64 fusion exhibits RNA-induced transcriptional activation when assayed by both fluorescence-activated cell sorting (FACS) and immunofluorescence analysis (IF). Specifically, while the control TALE-TF activated both reporters, the Cas9N-VP64 fusion activated the reporter in a gRNA sequence-specific manner. As shown in Figure 1E, gRNA sequence-specific transcriptional activation of the reporter construct was observed by both FACS and IF only when all three elements, i.e., Cas9N, MS2-VP64, and a gRNA with the appropriate MS2 aptamer binding site, were present.

[0086] According to certain embodiments, methods are provided for regulating endogenous genes using Cas9N, one or more gRNAs, and a transcriptional regulatory protein or domain. According to one embodiment, the endogenous gene can be any desired gene (referred to herein as a target gene). According to one exemplary embodiment, the genes targeted for regulation included ZFP42 (REX1) and POU5F1 (OCT4), both tightly regulated genes involved in maintaining pluripotency. As shown in Figure 1F, 10 gRNAs were designed for the REX1 gene, targeting a 5-kb stretch of DNA upstream of the transcription start site (DNase hypersensitive sites are highlighted in green). Transcriptional activation was assayed using a promoter-luciferase reporter construct (see Takahashi et al., Cell 131:861-872 (2007), incorporated herein by reference in its entirety) or directly by qPCR of the endogenous gene.

[0087] Figures 6A-C show RNA-guided OCT4 regulation using Cas9N-VP64. As shown in Figure 6A, 21 gRNAs targeting a 5-kb DNA stretch upstream of the transcription start site were designed for the OCT4 gene. DNase-hypersensitive sites are highlighted in green. Figure 6B demonstrates transcriptional activation using a promoter-luciferase reporter construct. Figure 6C demonstrates direct transcriptional activation via qPCR of the endogenous gene. While introduction of individual gRNAs moderately stimulated transcription, multiple gRNAs acted synergistically to promote robust, multifold transcriptional activation.

[0088] Figures 7A-C show RNA-guided REX1 regulation using Cas9N, MS2-VP64, and gRNA + 2xMS2 aptamer. As shown in Figure 7A, 10 gRNAs were designed for the REX1 gene, targeting a 5-kb DNA stretch upstream of the transcription start site. DNase hypersensitive sites are highlighted in green. Figure 7B shows transcriptional activation using a promoter-luciferase reporter construct. Figure 7C shows direct transcriptional activation by qPCR of the endogenous gene. While introduction of individual gRNAs moderately stimulated transcription, multiple gRNAs acted synergistically to promote robust, multifold transcriptional activation. In one embodiment, the absence of the 2xMS2 aptamer on the gRNA prevented transcriptional activation. See Maeder et al., Nature Methods, 10:243-245 (2013) and Perez-Pinera et al., Nature Methods, 10:239-242 (2013), each of which is incorporated by reference in its entirety.

[0089] Thus, the methods involve using a Cas9N protein and a transcriptional regulatory protein or domain and multiple guide RNAs to control expression of a target gene.

[0090] Both the Cas9-ligation approach and the gRNA-ligation approach were effective, with the former demonstrating approximately 1.5- to 2-fold higher potency. This difference appears to be due to the requirement for binary complex assembly as opposed to ternary complex assembly. However, the gRNA-ligation approach, in principle, allows for the recruitment of different effector domains by separate gRNAs, as long as each gRNA utilizes a different RNA-protein interaction pair. See Karyer-Bibens et al., Biology of the Cell, Vol. 100, pp. 125-138 (2008), in support of the European Society for Cell Biology, incorporated herein by reference in its entirety. According to one embodiment of the present disclosure, specific guide RNAs and generic Cas9N proteins, i.e., identical or similar Cas9N proteins for different target genes, can be used to regulate different target genes. According to one embodiment, a method for multiplexed gene regulation using identical or similar Cas9N proteins is provided.

[0091] The disclosed methods also relate to editing target genes using the Cas9N protein and guide RNAs described herein to provide multiplexed genetic and epigenetic manipulation of human cells. Because Cas9-gRNA targeting is an issue (see Jiang et al., Nature Biotechnology 31:233-239 (2013), incorporated herein by reference in its entirety), they provide a method for thoroughly investigating Cas9 affinity for a wide variety of target sequence variations. Thus, embodiments of the disclosure provide a direct, high-throughput readout of Cas9 targeting in human cells while avoiding the complications posed by dsDNA break toxicity and mutation repair posed by specificity testing using native, nuclease-active Cas9.

[0092] Other embodiments of the present disclosure generally relate to the use of DNA-binding proteins or systems for transcriptional regulation of target genes. Those skilled in the art will readily identify exemplary DNA-binding systems based on this disclosure. Such DNA-binding systems do not need to possess any nuclease activity, as found in native Cas9 proteins. Therefore, such DNA-binding systems do not need to inactivate nuclease activity. One exemplary DNA-binding system is a TALE. TALE-FokI dimers are commonly used as genome editing tools, and TAEL-VP64 fusions have been shown to be highly effective for genome regulation. According to one embodiment, the specificity of TALEs was evaluated using the method depicted in Figure 2A. A construct library was designed in which each library element contained a minimal promoter expressing the dTomato fluorescent protein. A 24-bp (A / C / G) random transcript tag was inserted downstream of the transcription start site m, while two TF binding sites were placed upstream of the promoter. One is a constant DNA sequence shared by all library elements, and the second is a variable feature with a "biased" binding site library designed to span a large collection of sequences representing numerous mutation combinations away from the target sequence to which the programmable DNA targeting complex is designed to bind. This is achieved using degenerate oligonucleotides designed to have nucleotide frequencies at each position such that the target sequence nucleotide occurs 79% of the time and each of the other nucleotides occurs 7% of the time. See Patwardhan et al., Nature Biotechnology 30:265-270 (2012), incorporated herein by reference in its entirety. The reporter library is then sequenced to reveal the relationship between the 24-bp dTomato transcript tags and their corresponding "biased" target sites in the library elements.The large diversity of their transcript tags confirms that tag sharing between different targets is extremely rare, while the biased composition of the target sequences means that sites with few mutations bind more tags than sites with more mutations. Transcription of the dTomato reporter gene is then stimulated by either a control TF designed to bind to a shared DNA site or a target TF designed to bind to a target site. The amount of each expressed transcript tag is measured in each sample by performing RNA-seq analysis on stimulated cells, and the transcript tag is then mapped to its corresponding binding site using a previously generated association table. Because the binding site for the control TF is shared across all library elements, the control TF is expected to stimulate all library components equally, while the target TF is expected to skew the distribution of expressed components toward the component that the target TF preferentially targets. This assumption is utilized in step 5 to calculate a normalized expression level for each binding site by dividing the number of tags obtained for the target TF by the number of tags obtained for the control TF.

[0093] As shown in Figure 2B, the targeting landscape of the Cas9-gRNA complex reveals that it tolerates, on average, one to three mutations in its target sequence. As shown in Figure 2C, the Cas9-gRNA complex is also largely insensitive to point mutations, except for those located in the PAM sequence. Notably, the data reveal that the predicted PAM of S. pyogenes Cas9 is not only NGG but also NAG. However, as shown in Figure 2D, the introduction of two mismatches significantly impaired Cas9-gRNA complex activity only when these mismatches were located an additional 8–10 bases closer to the 3' end of the gRNA target sequence (the target sequence positions are labeled 1–23 from the 5' end in the heat plot).

[0094] The transcription specificity assay described herein was used to determine the mutation tolerance of TALE domains, another widely used genome editing tool. As shown in Figure 2E, TALE off-targeting data for 18-mer TALEs reveals that 18-mer TALEs can tolerate, on average, one to two mutations in their target sequence and are unable to activate the vast majority of three-base mismatch mutants in their target. As shown in Figure 2F, 18-mer TALEs, like the Cas9-gRNA complex, are largely insensitive to a single-base mismatch in their target. As shown in Figure 2G, the activity of 18-mer TALEs is significantly impaired by the introduction of two-base mismatches. TALE activity is more sensitive to mismatches located closer to the 5' end of their target sequence (the target sequence positions are labeled 1-18 from the 5' end in the heat plot).

[0095] We confirmed our results using nuclease targeting assays, the subject of Figures 10A-C, which evaluate the targeting landscape of TALEs of various sizes. As shown in Figure 10A, 18-mer TALEs were confirmed to tolerate multiple mutations in their target sequences using a nuclease-mediated HR assay. As shown in Figure 10B, we analyzed the targeting landscapes of TALEs of three different sizes (18-mer, 14-mer, and 10-mer) using the approach described in Figure 2. As TALEs become shorter (14-mer and 10-mer), their targeting becomes increasingly specific and their activity decreases by approximately one order of magnitude. As shown in Figures 10C and 10D, 10-mer TALEs exhibit sensitivity to approximately one mismatch and lose almost all activity against targets with two mismatches (target sequence positions are labeled 1–10 from the 5' end in the heat plots). Taken together, these data indicate that higher specificity can be achieved by designing shorter TALEs in genome engineering applications, while the requirement for FokI dimerization is important to avoid off-target effects in TALE nuclease applications. See Kim et al., Proceedings of the National Academy of Sciences of the United States of America 93:1156-1160 (1996) and Pattanayak et al., Nature Methods 8:765-770 (2011), each of which is incorporated by reference in its entirety.

[0096] Figures 8A-C show a high-level specificity analysis process flow for calculating normalized expression levels, using experimental data as an example. As shown in Figure 8A, a construct library is constructed with biased binding site sequences and random 24-bp tags incorporated into reporter gene transcripts (top row). Because the transcribed tags are highly degenerate, many will correspond to a single Cas9 or TALE binding sequence. The construct library is sequenced (row 3, left) to determine which tags correspond to which binding sites, and a table of associations between binding sites and transcribed tags is generated (row 4, left). Multiple construct libraries constructed for different binding sites can be sequenced using library barcodes (shown here in light blue and light yellow; rows 1-4, left). The construct library is then transfected into a cell population, and a set of different Cas9 / gRNA or TALE transcription factors is introduced into the population samples (row 2, right). One sample is always guided by a specific TALE activator targeting a specific binding site sequence within the construct (top row, green box). This sample serves as a positive control (green sample, also represented by a + sign). cDNA generated from reporter mRNA molecules within these guide samples is then sequenced and analyzed to obtain the tag count for each tag in the sample (rows 3 and 4, right). Similar to construct library sequencing, multiple samples, including the positive control, are sequenced and analyzed together by adding sample barcodes. Here, bright red represents one non-control sample that was sequenced and analyzed together with the positive control (green). Because only the transcribed tag appears in each read, not the construct's binding site, the association table between binding sites and tags obtained from construct library sequencing is then used to calculate the total number of tags expressed from each binding site in each sample (row 5). The total count for each non-positive control sample is then converted to a normalized expression level for each binding site by dividing the total count for each non-positive control sample by the total count obtained in the positive control sample. Examples of plots of normalized expression levels by number of mismatches are provided in Figures 2B and 2E, and Figures 9A and 10B.Several levels of filtering for error-prone tags, tags that cannot be bound by the construct library, and tags that are clearly shared by multiple binding sites are not included in this overall processing flow. Figure 8B shows an exemplary distribution of the percentage of binding sites per number of mismatches generated in the biased construct library. Left: Theoretical distribution. Right: Distribution observed from an actual TALE construct library. Figure 8C shows an exemplary distribution of the percentage of tags recruited to binding sites per number of mismatches. Left: Distribution observed from a positive control sample. Right: Distribution observed from a non-control TALE-guided sample. Because the positive control TALE binds to a fixed site within the construct, the distribution of recruited tags closely mirrors the distribution of binding sites in Figure 8B, while the distribution is skewed to the left for the non-control TALE sample because sites with fewer mismatches guide higher expression levels. Bottom: The relationship between the average expression level and the number of mutations at the target site is revealed by dividing the number of tags obtained for the target TF by the number of tags obtained for the control TF to calculate the relative enrichment between them.

[0097] These results are further confirmed by specificity data generated using different Cas9-gRNA complexes. As shown in Figure 9A, different Cas9-gRNA complexes tolerate one to three mutations in their target sequence. As shown in Figure 9B, the Cas9-gRNA complexes are largely insensitive to point mutations, except those located in the PAM sequence. However, as shown in Figure 9C, the introduction of two mismatches significantly impaired activity (the target sequence positions are labeled 1–23 from the 5′ end in the heat plot). As shown in Figure 9D, the predicted PAM of S. pyogenes Cas9 was confirmed to be NGG and NAG using a nuclease-mediated HR assay.

[0098] According to certain embodiments, binding specificity is increased according to the methods described herein. Because synergistic interactions between multiple complexes are responsible for target gene activation by Cas9N-VP64, and the effects of individual off-target binding events should be minimal, Cas9N transcriptional regulatory applications are naturally highly specific. According to one embodiment, offset nicks are used in genome editing methods. Because the majority of nicks rarely result in NHEJ events (see Certo et al., Nature Methods, 8:671-676 (2011), incorporated herein by reference in its entirety), the effects of off-target nick formation are minimized. In contrast, inducing offset nicks to create double-strand breaks (DSBs) is highly effective in inducing gene disruption. According to certain embodiments, 5' overhangs induce more significant NHEJ events than 3' overhangs. Similarly, although the total number of HR events is significantly lower than when 5' overhangs are generated, 3' overhangs favor HR events over NHEJ events. Taken together, we provide a method that uses a nick for homologous recombination and an offset nick to generate a double-strand break, minimizing the effects of off-target Cas9-gRNA activity.

[0099] Figures 3A–C describe a method for reducing multiple offset nicking and off-target binding with guide RNAs. As shown in Figure 3A, we simultaneously assayed HR and NHEJ events during targeted nicking or targeted cleavage using a traffic light reporter. DNA cleavage events resolved by the HDR pathway restore the GFP sequence, while mutagenic NHEJ causes a frameshift that removes GFP from the reading frame and brings the downstream mCherry sequence into reading frame. For this assay, we designed 14 gRNAs spanning 200 bp of DNA: seven targeting the sense strand (U1–U7) and seven targeting the antisense strand (D1–D7). A Cas9D10A mutant that nicks the complementary strand was used to induce a series of planned 5' or 3' overhangs using different dual gRNA combinations (nicking sites for the 14 gRNAs are shown). As shown in Figure 3B, inducing an offset nick to create a double-strand break (DSB) is highly effective for inducing gene disruption. In particular, offset nicks that create 5' overhangs result in more NHEJ events than 3' overhangs. As shown in Figure 3C, although the generation of 3' overhangs favors the rate of HR events over NHEJ events, the total number of HR events is significantly lower than when 5' overhangs are created.

[0100] Figures 11A-B show Cas9D10A nickase-mediated NHEJ. As shown in Figure 11A, we used a traffic light reporter to assay NHEJ events upon the introduction of a targeted nick or a targeted break. Briefly, upon the introduction of a DNA break, the GFP gene becomes out of frame and the downstream mCherry sequence becomes in frame, resulting in red fluorescence, when the break induces mutagenic NHEJ. We designed 14 gRNAs spanning 200 bp of DNA strand length: seven targeting the sense strand (U1-7) and seven targeting the antisense strand (D1-7). As shown in Figure 11B, unlike wild-type Cas9, which induces DSBs and robust NHEJ for all targets, we observed that (using the Cas9D10A mutant) the majority of nicks rarely result in NHEJ events. All 14 sites were located within a continuous 200 bp long DNA stretch, and a >10-fold difference in targeting efficiency was observed.

[0101] According to certain embodiments, the present disclosure provides methods for regulating expression of a target nucleic acid in a cell, the methods comprising introducing one or more, two or more, or multiple exogenous nucleic acids into the cell. The exogenous nucleic acids introduced into the cell encode a guide RNA or multiple guide RNAs, a nuclease-deficient Cas9 protein or multiple nuclease-deficient Cas9 proteins, and a transcription factor protein or domain. The guide RNA, nuclease-deficient Cas9 protein, and transcription factor protein or domain are referred to as a co-localized complex, as understood by those skilled in the art, so long as the guide RNA, nuclease-deficient Cas9 protein, and transcription factor protein or domain bind to DNA and regulate expression of the target nucleic acid. According to certain additional embodiments, the exogenous nucleic acid introduced into the cell encodes a guide RNA or multiple guide RNAs and a Cas9 nickase protein. The guide RNA and Cas9 nickase protein are referred to as a co-localized complex, as understood by those skilled in the art, so long as the guide RNA and Cas9 nickase protein bind to DNA and nick the target nucleic acid.

[0102] Cells according to the present disclosure include any cell into which an exogenous nucleic acid can be introduced and expressed as described herein. It should be understood that the basic concept of the present disclosure described herein is not limited by cell type. Cells according to the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, eubacterial cells, etc. Cells include eukaryotic cells such as yeast cells, plant cells, and animal cells. Specific cells include mammalian cells. Furthermore, cells include any cell in which it is beneficial or desirable to control a target nucleic acid. Such cells may include cells with defects in the expression of a specific protein that causes a disease or adverse health condition. Such diseases or adverse health conditions are readily apparent to those skilled in the art. According to the present disclosure, nucleic acids responsible for the expression of a specific protein can be targeted using the methods described herein and transcriptional activators that cause the upregulation of the target nucleic acid and the corresponding expression of the specific protein. In this case, the methods described herein provide a therapeutic treatment.

[0103] Target nucleic acids include any nucleic acid sequence for which the colocalization complexes described herein may be useful for either regulation or nick formation. Target nucleic acids include genes. For purposes of this disclosure, DNA, such as double-stranded DNA, can comprise a target nucleic acid, and a colocalization complex can bind to, be adjacent to, or be near the target nucleic acid, or otherwise colocalize with the DNA, such that the colocalization complex can have a desired effect on the target nucleic acid. Such target nucleic acids can include endogenous (or natural) nucleic acids and exogenous (or foreign) nucleic acids. Based on this disclosure, one skilled in the art can easily identify or design guide RNAs and Cas9 proteins that colocalize with DNA containing a target nucleic acid. One skilled in the art can further identify transcription factor proteins or domains that similarly colocalize with DNA containing a target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0104] Foreign nucleic acid (i.e., nucleic acid that is not part of the cell's natural nucleic acid composition) may be introduced into a cell using any method for introduction known to those of skill in the art, including transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, cytoplasmic conversion, conjugation, etc. Those of skill in the art will readily understand and apply such methods using readily identifiable literature.

[0105] Transcriptional regulator proteins or domains that are transcriptional activators include VP16, VP64, and others readily identifiable by one of skill in the art based on the present disclosure.

[0106] Diseases and adverse health conditions are characterized by the abnormal loss of expression of specific proteins. Such diseases or adverse health conditions can be treated by upregulating specific proteins. Accordingly, provided are methods for treating diseases or adverse health conditions, in which a colocalization complex described herein associates or otherwise binds to DNA containing a target nucleic acid, and a transcriptional activator of the colocalization complex upregulates expression of the target nucleic acid. For example, upregulation of PRDM16 and other genes that promote brown fat differentiation and increased metabolic uptake can be used to treat metabolic syndrome or obesity. Activation of anti-inflammatory genes is useful in autoimmune and cardiovascular diseases. Activation of tumor suppressor genes is useful in the treatment of cancer. Those skilled in the art will readily identify such diseases and adverse health conditions based on this disclosure.

[0107] The following examples are presented as representative of the present disclosure and should not be construed as limiting the scope of the disclosure, as these and other equivalent embodiments will be apparent in light of the disclosure, drawings, and appended claims. [Example]

[0108] [Example 1] Cas9 mutants To identify candidate mutations in Cas9 that could eliminate the native activity of the RuvC and HNH domains of Cas9, we searched for sequences homologous to Cas9s with known structures. The full-length Cas9 sequence was used as a query sequence against the Full-Length Protein Data Bank (January 2013) using HHpred (World Wide Website Toolkit, tuebingen.mpg.de / hhpred). This search returned two distinct HNH endonucleases, a Pad endonuclease and a putative endonuclease (PDB IDs: 3M7K and 4H9D, respectively), with significant sequence homology to the Cas9 HNH domain. These proteins were examined to identify residues involved in magnesium ion coordination. These corresponding residues were then identified in sequence alignments to Cas9. Two Mg-coordinating side chains in each structure were identified that aligned with the same amino acids in Cas9: D92 and N113 in 3M7K and D53 and N77 in 4H9D. These residues corresponded to D839 and N863 in Cas9. It has also been reported that mutation of Pad residues D92 and N113 to alanine renders the nuclease catalytically defective. Based on this analysis, we generated the Cas9 mutations D839A and N863A. Furthermore, HHpred predicts homology between Cas9 and the N-terminus of Thermus thermophilus RuvC (PDB ID: 4EP4). This sequence alignment includes the previously reported mutation D10A, which eliminates the function of the RuvC domain in Cas9. To confirm this mutation as an appropriate mutation, we determined the metal-binding residues as previously described. In 4EP4, D7 functions to coordinate a magnesium ion. This position shares sequence homology with Cas9 D10, confirming that this mutation serves to eliminate metal binding and, consequently, catalytic activity from the RuvC domain of Cas9.

[0109] [Example 2] Plasmid construction Cas9 mutants were generated using the Quikchange kit (Agilent Technologies). Target gRNA expression constructs were either (1) ordered directly from IDT as individual gBlocks and cloned into the pCR-BluntII-TOPO vector (Invitrogen), (2) custom synthesized by Genewiz, or (3) assembled into a gRNA cloning vector using Gibson assembly of oligonucleotides (plasmid no. 41824). A vector for the HR reporter assay with a truncated GFP was constructed by PCR construction of the appropriate fragment with a stop codon in the EGIP lentivector from Addgene (plasmid no. 26777). These lentivectors were then used to construct GFP reporter stable lines. TALENs used in this study were constructed using standard protocols. See Sanjana et al., Nature Protocols, 7:171–192 (2012), incorporated herein by reference in its entirety. Fusion of Cas9N and MS2 VP64 was performed using standard PCR fusion protocols. Promoter-luciferase constructs for OCT4 and REX1 were obtained from Addgene (Plasmid #17221 and Plasmid #17222).

[0110] [Example 3] Cell culture and transfection HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin / streptomycin (pen / strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). Cells were maintained at 37°C and 5% CO in a humidified incubator.

[0111] Transfection with nuclease assay was as follows: 0.4 × 10 cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol. 6Approximately 1 x 10 cells were transfected with 2 µg of Cas9 plasmid, 2 µg of gRNA, and / or 2 µg of DNA donor plasmid. Three days after transfection, cells were harvested and analyzed by FACS or analyzed using the DNA Easy Kit (Qiagen) for direct assay of genome cleavage. 6 Genomic DNA was extracted from the cells. PCR was performed to amplify the targeting region using genomic DNA from the cells, and the amplicons were deep sequenced using a MiSeq personal sequencer (Illumina) with a coverage of over 200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiency.

[0112] For transfection with transcriptional activation assay, 0.4 × 10 6 Cells were transfected with either (1) 2 μg of Cas9N-VP64 plasmid, 2 μg of gRNA, and / or 0.25 μg of reporter construct, or (2) 2 μg of Cas9N plasmid, 2 μg of MS2-VP64, 2 μg of gRNA-2×MS2 aptamer, and / or 0.25 μg of reporter construct. Cells were harvested 24–48 hours posttransfection and assayed using FACS or immunofluorescence methods, or their total RNA was extracted and subsequently analyzed by RT-PCR. Standard Invitrogen TaqMan probes for OCT4 and REX1 were used, and normalization of each sample was performed against GAPDH.

[0113] 0.4 × 10 for transfection with Cas9-gRNA complexes and transcription activation assay for TALE specificity profile 6Cells were transfected with either (1) 2 μg of Cas9N-VP64 plasmid, 2 μg of gRNA, and 0.25 μg of reporter library, (2) 2 μg of TALE-TF plasmid and 0.25 μg of reporter library, or (3) 2 μg of control TF plasmid and 0.25 μg of reporter library. Cells were harvested 24 hours after transfection (to avoid reporter activation in saturation mode). Total RNA extraction was performed using the RNA Easy Plus Kit (Qiagen), and standard RT-PCR was performed using Superscript-III (Invitrogen). Libraries for next-generation sequencing were generated by targeted PCR amplification of transcript tags.

[0114] [Example 4] Mathematical and sequence analysis for calculation of expression levels of Cas9-TF and TALE-TF reporters A high level logic flow for this process is shown in Figure 8A, and further details are provided herein. For details on the composition of the construct library, see Figures 8A (first row) and 8B.

[0115] Sequencing: For Cas9 experiments, construct libraries (Figure 8A, third row, left) and reporter gene cDNA sequences (Figure 8A, third row, right) were obtained as 150-bp redundant paired-end reads on an Illumina MiSeq, and for TALE experiments, the corresponding sequences were obtained as 51-bp non-redundant paired-end reads on an Illumina HiSeq.

[0116] Construct Library Sequence Processing: Alignment: For Cas9 experiments, novoalign version 2.07.17 (available at the worldwide website novocraft.com / main / index / php) was used to align read pairs to a set of 250-bp reference sequences corresponding to the 234-bp construct flanked by 8-bp library barcode pairs (see Figure 8A, column 3, left). In the reference sequences provided to novoalign, the 23-bp degenerate Cas9 binding site region and the 24-bp degenerate transcript tag region (see Figure 8A, column 1) were designated N, while the construct library barcodes were explicitly defined. For TALE experiments, the same method was used, except that the reference sequences were 203 bp long and the degenerate binding site regions were 18 bp long compared to 23 bp. Validity Check: For the generated file, Novoalign output data aligned the left and right reads of each read pair to the respective reference sequences. Only read pairs that both uniquely align to the reference sequence were subjected to other validity criteria, and only read pairs that passed all of these criteria were retained. (i) Each of the two construct library barcodes must align with the reference sequence barcode at at least four positions, and the two barcodes must align for the same construct library barcode pair. (ii) All bases aligning to the N region of the reference sequence must be called A, C, G, or T by novoalign. Note that for both the Cas9 and TALE experiments, the left and right reads did not overlap in the reference N region, eliminating the possibility of ambiguous novoalign calls of these N bases. (iii) Similarly, these regions must not exhibit any insertions or deletions as called by novoalign. (iv) T must not appear in the transcript tag region (since these random sequences consist only of A, C, and G). Read pairs violating any one of these conditions were collected in a rejected read pair file. We performed these validity checks using a home-written perl script.

[0117] Guided sample reporter gene cDNA sequence processing: Alignment: We first used SeqPrep (downloaded from the worldwide website github.com / jstjohn / SeqPrep) to merge overlapping read pairs down to a 79-bp common segment, then used novoalign (the version described above) to align these 79-bp common segments as unpaired single reads to a set of reference sequences (see Figure 8A, third row, right), in which (for sequence analysis of construct libraries) a 24-bp condensed transcript tag was designated N, while the sample barcode was clearly defined. Both the TALE sequence region and the Cas9 cDNA sequence region corresponded to the same 63-bp cDNA region flanked by 8-bp sample barcode sequence pairs. For validation testing, the same conditions were applied as for sequence analysis of the construct library (see above), except that (a) here, due to the previous SeqPrep merger of read pairs, the validation process did not need to filter for unique alignments of both reads in the read pair, but only for unique alignments of the merged read, and (b) because only transcript tags appear in the cDNA sequence reads, the validation process only applies to these tag regions of the reference sequence and not to distinct binding site regions.

[0118] Construction of association tables between binding sites and transcript tags: We created these tables from validated construct library sequences using a home-written Perl script (Figure 8A, column 4, left). Although 24-bp tag sequences, consisting of A, C, and G bases, should essentially be unique across construct libraries (probability of sharing = approximately 2.8e-11), early analysis of binding site-tag associations revealed that a non-negligible proportion of tag sequences were actually shared by multiple binding sequences, likely due primarily to a combination of sequence errors in the binding sequences or oligo synthesis errors in the oligos used to generate the construct libraries. In addition to tag sharing, tags found to be associated with binding sites in validated read pairs could also be found in construct library read-pair rejection files when it was unclear from which construct library they could have originated due to barcode mismatches. Finally, tag sequences themselves could contain sequence errors. To address these sources of error, tags were classified into three attributes: (i) secure vs. unsecure, where unsecure meant tags that could be found in construct library rejection reads vs. files; shared vs. non-covalent, where shared meant tags found associated with multiple binding site sequences; and (ii) 2+ vs. 1-only, where 2+ meant tags that appeared at least twice in validated construct library sequences and were considered unlikely to contain sequence errors. Combining these three criteria yielded eight classes of tags associated with each binding site, with the most secure (but least abundant) class containing only secure, non-covalent, and 2+ tags, and the least secure (but most abundant) class containing all tags, regardless of security, covalent, or number of occurrences.

[0119] Calculation of normalized expression levels: The steps shown in rows 5-6 of Figure 8A were performed using a custom Perl code. First, the number of tags obtained for each guide sample was tabulated per binding site using a table of binding sites and transcript tags previously calculated for the construct library (see Figure 8C). Next, the number of tags for each binding site tabulated for each sample was divided by the number of tags tabulated for the positive control sample to create a normalized expression level. Other considerations related to these calculations include the following: 1. For each sample, a set of "novel" tags was found in the validated cDNA gene sequence that could not be found in the association table between binding sites and transcript tags. These tags were ignored in subsequent calculations. 2. The above tag counts were calculated for each of the eight classes of tags in the table of associations between binding sites and transcript tags. Because binding sites in the construct library are biased so that sequences similar to the central sequence are generated frequently, while sequences with increasing numbers of mismatches are generated increasingly rarely, binding sites with few mismatches generally constitute the majority of tags, while binding sites with more mismatches constitute fewer tags. Therefore, while it is generally desirable to use the most guaranteed tag class, evaluation of binding sites with two or more mismatches is based on a small number of tags per binding site, and even if the tags themselves are reliable, the guaranteed numbers and ratios may be statistically less reliable. In such cases, all tags were used. A compensation for this consideration is that the number of tags calculated separately for n mismatch positions is multiplied by the number of combinations of mismatch positions (

[0120]

number

[0121] [Example 5] Cas9 N -RNA-induced SOX2 and NANOG regulation using VP64 The aptamer-modified short guide RNA (sgRNA) ligation approach described herein allows for the recruitment of different effector domains by separate sgRNAs, as long as each sgRNA utilizes a different RNA-protein interaction pair, enabling multiple gene regulation using the same Cas9N protein. For the SOX2 gene (Figure 12A) and the NANOG gene (Figure 12B), ten gRNAs were designed to target approximately 1 kb of DNA upstream of the transcription start site. DNase-hypersensitive sites are highlighted in green. Transcriptional activation was assayed by qPCR of the endogenous gene. In both cases, introduction of individual gRNAs moderately stimulated transcription, while multiple gRNAs acted synergistically to promote robust, multifold transcriptional activation. Data are means ± SEM (N=3). As shown in Figures 12A-B, two additional genes, SOX2 and NANOG, were regulated by sgRNAs targeting approximately 1 kb of DNA upstream of the promoter. These sgRNAs near the transcription start site led to robust gene activation.

[0122] [Example 6] Assessing the targeting landscape of Cas9-gRNA complexes Using the approach described in Figure 2, we analyzed the targeting landscapes of two additional Cas9-gRNA complexes (Figures 13A-C) and (Figures 13D-F). The two gRNAs have very different specificity profiles, with gRNA2 tolerating a maximum of 2-3 mismatches and gRNA3 tolerating at most 1 mismatch. These aspects are reflected in both the single-base mismatch plots (Figures 13B, 13E) and the double-base mismatch plots (Figures 13C, 13F). In Figures 13C and 13F, base-mismatch pairs for which insufficient data are available to calculate normalized expression levels are represented by gray boxes containing "x", while mismatch pairs whose normalized expression levels are outliers above the top of the color scale are represented by yellow boxes containing an asterisk "*" to improve data presentation. Statistical significance symbols are: *** P<0.0005 / n, ** P<0.005 / n, * where P<0.05 / n and NS (not significant) where P>=0.05 / n, where n is the number of comparisons (see Table 2).

[0123] [Example 7] Validation and specificity of reporter assays Specificity data were generated using two different sgRNA:Cas9 complexes, as shown in Figures 14A-C. The assay was confirmed to be specific for the sgRNA being evaluated, as the corresponding mutant sgRNA was unable to prime the reporter library. Figure 14A: The specificity profiles of two gRNAs (wild-type and mutant; sequence differences are highlighted in red) were evaluated using a reporter library designed against the wild-type gRNA target sequence. Figure 14B: The assay was confirmed to be specific for the gRNA being evaluated, as the corresponding mutant gRNA was unable to prime the reporter library (data replotted from Figure 13D). Statistical significance symbols are *** P<0.0005 / n, ** P<0.005 / n, * where P<0.05 / n, and NS (not significant) where P>=0.05 / n, where n is the number of comparisons (see Table 2). Different sgRNAs can have different specificity profiles (Figures 13A, 13D); specifically, sgRNA2 tolerates up to three mismatches, while sgRNA3 tolerates only one mismatch. The highest sensitivity to mismatches is located at the 3' end of the spacer, although mismatches at other positions were also observed to affect activity.

[0124] [Example 8] Validation, single and double gRNA mismatches As shown in Figure 15A-D, targeting experiments confirmed that a single-base mismatch within the 12 bp at the 3' end of the spacer in the assayed sgRNAs resulted in detectable targeting. However, a 2-bp mismatch within this region caused a significant loss of activity. Using nuclease assays, we tested two independent gRNAs, gRNA2 (Figure 15A-B) and gRNA3 (Figure 15C-D), which carry either a single-base or a 2-base mismatch (highlighted in red) to the target within the spacer sequence. We confirmed that a single-base mismatch within the 12 bp at the 3' end of the spacer in the assayed gRNAs resulted in detectable targeting, but a 2-bp mismatch within this region caused a rapid loss of activity. These results further highlight the differences in specificity profiles between different gRNAs and are consistent with the results in Figure 13. Data are means ± SEM (N = 3).

[0125] [Example 9] Validation, 5' gRNA shortening As shown in Figure 16A-D, sgRNA activity was maintained even when the 5' portion of the spacer was truncated. Using a nuclease assay, we tested two independent gRNAs, gRNA1 (Figure 16A-B) and gRNA3 (Figure 16C-D), which have truncated 5' ends of the spacer. While 1-3 bp of 5' truncation was well tolerated, larger deletions caused loss of activity. Data are means ± SEM (N=3).

[0126] [Example 10] Validation, PAM for Streptococcus pyogenes (S. pyogenes) As shown in Figures 17A-B, the PAM of S. pyogenes Cas9 is NGG and was also confirmed to be NAG using a nuclease-mediated HR assay. Data are mean ± SEM (N = 3). In additional investigations, a set of approximately 190K engineered Cas9 targets within human exons that did not have an alternative NGG target sharing the last 13 nt of the targeting sequence was scanned for the presence of an alternative NAG site with a mismatch within the previous 13 nt, or for an NGG site. Only 0.4% were found to have no such alternative targets.

[0127] [Example 11] Validation, TALE mutation The tolerance of 18-mer TALEs to multiple mutations in the target sequence was confirmed using a nuclease-mediated HR assay (Figure 18A-B). As shown in Figure 18A-B, certain mutations in the center of the target result in higher TALE activity, as measured by targeting experiments in nuclease assays.

[0128] [Example 12] TALE monomer specificity and TALE protein specificity To isolate the role of individual repetitive variable dinucleotides (RVDs), we confirmed that RVD selection does not contribute to base specificity, but that TALE specificity is also a function of the binding energy of the protein as a whole. Figures 19A-C show a comparison between TALE monomer specificity and TALE protein specificity. Figure 19A: Using a modified version of the approach described in Figure 2, we analyzed the targeting landscapes of two 14-mer TALE-TFs with a series of 6 NI or 6 NH repeats. This approach assayed the specificity of TALE-TFs by creating and using a reduced library of reporters with a central condensed 6-mer sequence. Figures 19B-C: In both examples, enrichment of the expected target sequence (i.e., those with 6 As for the NI repeats and 6 Gs for the NH repeats) was noted. Each of these TALEs still tolerates 1-2 mismatches within the central 6-mer target sequence. While monomer selection does not contribute to base specificity, TALE specificity is also a function of the binding energy of the protein as a whole. According to one embodiment, engineered shortened TALEs or TALEs with a composition of high and low affinity monomers result in higher specificity in genome engineering applications, and the use of short TALEs allows FokI dimerization to further reduce off-target effects in nuclease applications.

[0129] [Example 13] Offset nicking, natural locus Figures 20A-B show data on offset nicking. In the context of genome editing, offset nicks are formed to generate DSBs. The majority of nicks do not result in non-homologous end joining (NHEJ)-mediated indels, so the rate at which off-target single nicking events result in indels when offset nicking is guided is likely very low. Inducing offset nicks to generate DSBs is effective in guiding gene disruption at both the inserted reporter locus and the native AAVS1 genomic locus. Figure 20A: The AAVS1 locus was targeted with eight gRNAs spanning a 200-bp DNA strand: four targeting the sense strand (s1–4) and four targeting the antisense strand (as1–4). A Cas9D10A mutant that nicks the complementary strand was used to induce a series of planned 5' or 3' overhangs using different dual gRNA combinations. Figure 20B: While a single gRNA failed to induce detectable NHEJ events, we observed using a Sanger sequencing-based assay that inducing offset nicks to generate DSBs was highly effective in inducing gene disruption. Notably, offset nicks that generate 5' overhangs result in more NHEJ events than 3' overhangs. The number of Sanger sequencing clones is highlighted above the bars, and the expected overhang lengths are indicated below the corresponding x-axis legend.

[0130] [Example 14] Offset nick formation, NHEJ profile Figures 21A-C show offset nicking and NHEJ profiles. Representative Sanger sequencing results for three different offset nicking combinations are shown, with the location of the targeting gRNA highlighted by a box. Furthermore, consistent with the standard model of homologous recombination (HR)-mediated repair, creating a 5' overhang by offset nicking resulted in more robust NHEJ events than 3' overhangs (Figure 3B). In addition to promoting NHEJ, creating a 5' overhang also strongly induced HR. Creating a 3' overhang did not result in improved HR rates (Figure 3C).

[0131] [Example 15] Table 1 gRNA targeting for endogenous gene regulation Targets in the promoters of REX1, OCT4, SOX2, and NANOG used in Cas9-gRNA-mediated activation experiments are listed.

[0132] [Table 1]

[0133] [Example 16] Table 2 Summary of statistical analysis of Cas9-gRNA and TALE specificity data Table 2(a) P-values ​​for comparisons of normalized expression levels of TALE or Cas9-VP64 activators binding to target sequences with a specific number of target site mutations. Normalized expression levels are represented by boxplots in the figures shown in the figure column, with the boxes representing the distribution of these expression levels by number of mismatches at the target site. P-values ​​were calculated for each consecutive pair of mismatch numbers in each boxplot using a t-test, which was either a one-sample t-test or a two-sample t-test (see Methods). Statistical significance was assessed using a Bonferroni-corrected P-value threshold based on the number of comparisons within each boxplot. Statistical significance symbols are: *** P<0.0005 / n, ** P<0.005 / n,* where p<0.05 / n, and NS (not significant) is p>=0.05 / n, where n is the number of comparisons. Table 2(b) Statistical characterization of the seed region in Figure 2D: log10(P-values) representing the degree of separation between the expression values ​​when Cas9N VP64+gRNA binds to target sequences at the pair of mutation-forming positions within the candidate seed region at the 3' end of the 20-bp target site and all other position pairs. The greatest degree of separation, indicated by the largest -log10(P-value) (highlighted above), is found within the last 8-9 bp of the target site. These positions may be interpreted as representing the beginning of the "seed" region for this target site. For information on how to calculate P-values, see the "Statistical Analysis of the Seed Region" section in the Methods.

[0134] [Table 2]

[0135] [Example 17] Protein and RNA sequences in the examples A. Cas9 based on m4 mutant N The sequences of the -VP64 activator constructs are shown below. m4 VP64 Fusion protein formats and Cas9 m4 VP64 Three versions with N fusion protein formats were constructed. Corresponding vectors for the m3 and m2 mutants (Fig. 4A) were also constructed (the NLS and VP64 domains are highlighted).

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] [ka]

[0144] [ka]

[0145] B. The sequences of the MS2 activator construct and the corresponding gRNA backbone vector with 2x MS2 aptamer domains are provided below (the NLS, VP64, gRNA spacer, and MS2-binding RNA stem-loop domains are highlighted). VP64 Two versions of the former were constructed with N fusion protein formats.

[0146] [ka]

[0147] [ka]

[0148] [ka]

[0149] C. The dTomato fluorescent-based transcriptional activation reporter sequence is described below (ISceI control TF target sequence, gRNA target sequence, minimal CMV promoter sequence and FLAG tag + dTomato sequence are highlighted).

[0150] [ka]

[0151] [ka]

[0152] D. The general format of the reporter library used for TALE and Cas9-gRNA specificity assays is provided below (ISceI control TF target sequence, gRNA / TALE target site sequence (23 bp for gRNA and 18 bp for TALE), minimal CMV promoter sequence, RNA barcode sequence, and dTomato sequence are highlighted).

[0153] [ka] Aspects of the present invention include the following. Appendix 1 introducing into the cell a first exogenous nucleic acid encoding one or more RNAs complementary to DNA comprising the target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs; introducing into said cell a third exogenous nucleic acid encoding a transcription factor protein or domain; the one or more RNAs, the nuclease-deficient Cas9 protein, and the transcription factor protein or domain are expressed, the one or more RNAs, the nuclease-deficient Cas9 protein, and the transcription factor protein or domain are co-localized with the DNA, and the transcription factor protein or domain controls expression of the target nucleic acid. A method for controlling the expression of a target nucleic acid in a cell. Appendix 2 2. The method of claim 1, wherein the exogenous nucleic acid encoding the nuclease-deficient Cas9 protein further encodes the transcription regulator protein or domain fused to the nuclease-deficient Cas9 protein. Appendix 3 the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain; The method of claim 1, wherein the foreign nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain. Appendix 4 2. The method of claim 1, wherein the cell is a eukaryotic cell. Appendix 5 2. The method of claim 1, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 6 2. The method according to claim 1, wherein the RNA is from about 10 nucleotides to about 500 nucleotides. Appendix 7 2. The method according to claim 1, wherein the RNA is about 20 nucleotides to about 100 nucleotides. Appendix 8 2. The method of claim 1, wherein the transcriptional regulator protein or domain is a transcriptional activator. Appendix 9 2. The method of claim 1, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid. Appendix 10 2. The method of claim 1, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse condition. Appendix 11 2. The method of claim 1, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 12 The method of claim 1, wherein the one or more types of RNA are guide RNAs. Appendix 13 2. The method of claim 1, wherein the one or more RNAs are tracrRNA-crRNA fusions. Appendix 14 2. The method of claim 1, wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 15 introducing into a cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent regions in a DNA target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding at least one Cas9 protein nickase having an inactive nuclease domain and guided by the two or more RNAs; the two or more RNAs and the at least one Cas9 protein nickase are expressed, and the at least one Cas9 protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid to generate two or more adjacent nicks. A method for modifying a DNA target nucleic acid in a cell. Appendix 16 16. The method of claim 15, wherein the two or more adjacent nicks are on the same strand of double-stranded DNA. Appendix 17 16. The method of claim 15, wherein the two or more adjacent nicks are on the same strand of double-stranded DNA to allow homologous recombination. Appendix 18 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of double-stranded DNA. Appendix 19 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, creating a double-stranded break. Appendix 20 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, creating a double-stranded break and inducing non-homologous end joining. Appendix 21 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of the double-stranded DNA and are offset from one another. Appendix 22 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of the double-stranded DNA and are offset from one another, resulting in a double-stranded break. Appendix 23 16. The method of claim 15, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, are offset from one another, and create a double-stranded break resulting in non-homologous end joining. Appendix 24 16. The method of claim 15, further comprising introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence, wherein the two or more nicks cause homologous recombination of the target nucleic acid with the donor nucleic acid sequence. Appendix 25 a first foreign nucleic acid encoding one or more RNAs complementary to the DNA comprising the target nucleic acid; a second exogenous nucleic acid encoding a nuclease-deficient Cas9 protein, and a third exogenous nucleic acid encoding a transcription factor protein or domain, the one or more RNAs, the nuclease-deficient Cas9 protein, and the transcription factor protein or domain are components of a co-localized complex with respect to the target nucleic acid. Appendix 26 26. The cell of claim 25, wherein the exogenous nucleic acid encoding the nuclease-deficient Cas9 protein further encodes the transcription regulator protein or domain fused to the nuclease-deficient Cas9 protein. Appendix 27 the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain; 26. The cell of claim 25, wherein the exogenous nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain. Appendix 28 26. The cell of claim 25, wherein the cell is a eukaryotic cell. Appendix 29 26. The cell of claim 25, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 30 26. The cell of claim 25, wherein the RNA comprises about 10 to about 500 nucleotides. Appendix 31 26. The cell of claim 25, wherein the RNA comprises about 20 to about 100 nucleotides. Appendix 32 26. The cell of claim 25, wherein the transcriptional regulator protein or domain is a transcriptional activator. Appendix 33 26. The cell of claim 25, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid. Appendix 34 26. The cell of claim 25, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse condition. Appendix 35 26. The cell of claim 25, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 36 26. The cell of claim 25, wherein the one or more types of RNA are guide RNAs. Appendix 37 26. The cell of claim 25, wherein the one or more RNAs is a tracrRNA-crRNA fusion. Appendix 38 26. The cell of claim 25, wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 39 a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and a second exogenous nucleic acid encoding at least one Cas9 protein nickase having an inactive nuclease domain; the two or more RNAs and the at least one Cas9 protein nickase are components of a co-localized complex to the DNA target nucleic acid. Appendix 40 40. The cell of claim 39, wherein the cell is a eukaryotic cell. Appendix 41 40. The cell of claim 39, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 42 40. The cell of claim 39, wherein the RNA comprises about 10 to about 500 nucleotides. Appendix 43 40. The cell of claim 39, wherein the RNA comprises about 20 to about 100 nucleotides. Appendix 44 40. The cell of claim 39, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 45 40. The cell of claim 39, wherein the two or more types of RNA are guide RNAs. Appendix 46 40. The cell of claim 39, wherein the two or more types of RNA are a tracrRNA-crRNA fusion. Appendix 47 40. The cell of claim 39, wherein the DNA target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 48 introducing into a cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent regions in a DNA target nucleic acid; introducing into the cell a second exogenous nucleic acid encoding at least one Cas9 protein nickase having an inactive nuclease domain and guided by the two or more RNAs; expressing the two or more RNAs and the at least one Cas9 protein nickase, and co-localizing the at least one Cas9 protein nickase with the two or more RNAs to the DNA target nucleic acid and nicking the DNA target nucleic acid to generate two or more adjacent nicks; A method of modifying a DNA target nucleic acid in a cell, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, causing a double-strand break and fragmenting the target nucleic acid, thereby preventing expression of the target nucleic acid. Appendix 49 introducing into the cell a first exogenous nucleic acid encoding one or more RNAs complementary to DNA comprising the target nucleic acid; introducing into said cell a second exogenous nucleic acid encoding an RNA-guided nuclease-deficient DNA-binding protein; introducing into said cell a third exogenous nucleic acid encoding a transcription factor protein or domain; A method for controlling the expression of a target nucleic acid in a cell, wherein the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein, and the transcription factor protein or domain are expressed, the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein, and the transcription factor protein or domain are co-localized to the DNA, and the transcription factor protein or domain controls the expression of the target nucleic acid. Appendix 50 50. The method of claim 49, wherein the exogenous nucleic acid encoding the RNA-guided nuclease-deficient DNA-binding protein further encodes the transcription factor protein or domain fused to the RNA-guided nuclease-deficient DNA-binding protein. Appendix 51 the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain; 50. The method of claim 49, wherein the foreign nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain. Appendix 52 50. The method of claim 49, wherein the cell is a eukaryotic cell. Appendix 53 50. The method of claim 49, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 54 49. The method of claim 48, wherein the RNA is about 10 nucleotides to about 500 nucleotides. Appendix 55 49. The method of claim 48, wherein the RNA is about 20 nucleotides to about 100 nucleotides. Appendix 56 49. The method of claim 49, wherein the transcriptional regulator protein or domain is a transcriptional activator. Appendix 57 50. The method of claim 49, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid. Appendix 58 50. The method of claim 49, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse condition. Appendix 59 50. The method of claim 49, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 60 50. The method of claim 49, wherein the one or more types of RNA are guide RNAs. Appendix 61 50. The method of claim 49, wherein the one or more RNAs are tracrRNA-crRNA fusions. Appendix 62 50. The method of claim 49, wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 63 introducing into a cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent regions in a DNA target nucleic acid; introducing into said cells a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase; A method for modifying a DNA target nucleic acid in a cell, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase are expressed, and the at least one RNA-guided DNA-binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid to create two or more adjacent nicks. Appendix 64 64. The method of claim 63, wherein the two or more adjacent nicks are on the same strand of double-stranded DNA. Appendix 65 64. The method of claim 63, wherein the two or more adjacent nicks are on the same strand of double-stranded DNA to allow homologous recombination. Appendix 66 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of double-stranded DNA. Appendix 67 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, creating a double-stranded break. Appendix 68 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, creating a double-stranded break and inducing non-homologous end joining. Appendix 69 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of the double-stranded DNA and are offset from one another. Appendix 70 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of double-stranded DNA and are offset from one another, resulting in a double-stranded break. Appendix 71 64. The method of claim 63, wherein the two or more adjacent nicks are on different strands of double-stranded DNA and are offset from one another, and create a double-stranded break resulting in non-homologous end joining. Appendix 72 64. The method of claim 63, further comprising introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence, wherein the two or more nicks cause homologous recombination of the target nucleic acid with the donor nucleic acid sequence. Appendix 73 a first foreign nucleic acid encoding one or more RNAs complementary to the DNA comprising the target nucleic acid; a second exogenous nucleic acid encoding an RNA-guided nuclease-deficient DNA-binding protein, and a third exogenous nucleic acid encoding a transcription factor protein or domain, A cell, wherein the one or more RNAs, the RNA-guided nuclease-deficient DNA-binding protein, and the transcription factor protein or domain are components of a co-localized complex for the target nucleic acid. Appendix 74 74. The cell of claim 73, wherein the exogenous nucleic acid encoding the RNA-guided nuclease-deficient DNA-binding protein further encodes the transcription regulator protein or domain fused to the RNA-guided nuclease-deficient DNA-binding protein. Appendix 75 the exogenous nucleic acid encoding one or more RNAs further encodes a target of the RNA-binding domain; 74. The cell of claim 73, wherein the exogenous nucleic acid encoding the transcription factor protein or domain further encodes an RNA-binding domain fused to the transcription factor protein or domain. Appendix 76 74. The cell of claim 73, wherein the cell is a eukaryotic cell. Appendix 77 74. The cell of claim 73, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 78 74. The cell of claim 73, wherein the RNA comprises about 10 to about 500 nucleotides. Appendix 79 74. The cell of claim 73, wherein the RNA comprises about 20 to about 100 nucleotides. Appendix 80 74. The cell of claim 73, wherein the transcriptional regulator protein or domain is a transcriptional activator. Appendix 81 74. The cell of claim 73, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid. Appendix 82 74. The cell of claim 73, wherein the transcription factor protein or domain upregulates expression of the target nucleic acid to treat a disease or adverse condition. Appendix 83 74. The cell of claim 73, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 84 74. The cell of claim 73, wherein the one or more types of RNA are guide RNAs. Appendix 85 74. The cell of claim 73, wherein the one or more RNAs is a tracrRNA-crRNA fusion. Appendix 86 74. The cell of claim 73, wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 87 a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent sites in the DNA target nucleic acid; and a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase; A cell, wherein the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase are components of a co-localized complex to the DNA target nucleic acid. Appendix 88 88. The cell of claim 87, wherein the cell is a eukaryotic cell. Appendix 89 88. The cell of claim 87, wherein the cell is a yeast cell, a plant cell, or an animal cell. Appendix 90 88. The cell of claim 87, wherein the RNA comprises about 10 to about 500 nucleotides. Appendix 91 88. The cell of claim 87, wherein the RNA comprises about 20 to about 100 nucleotides. Appendix 92 88. The cell of claim 87, wherein the target nucleic acid is associated with a disease or adverse health condition. Appendix 93 88. The cell of claim 87, wherein the two or more types of RNA are guide RNAs. Appendix 94 88. The cell of claim 87, wherein the two or more types of RNA are a tracrRNA-crRNA fusion. Appendix 95 88. The cell of claim 87, wherein the DNA target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Appendix 96 introducing into a cell a first exogenous nucleic acid encoding two or more RNAs, each complementary to adjacent regions in a DNA target nucleic acid; introducing into said cells a second exogenous nucleic acid encoding at least one RNA-guided DNA-binding protein nickase; the two or more RNAs and the at least one RNA-guided DNA-binding protein nickase are expressed, and the at least one RNA-guided DNA-binding protein nickase co-localizes with the two or more RNAs to the DNA target nucleic acid and nicks the DNA target nucleic acid to generate two or more adjacent nicks; A method of modifying a DNA target nucleic acid in a cell, wherein the two or more adjacent nicks are on different strands of double-stranded DNA, causing a double-strand break and fragmenting the target nucleic acid, thereby preventing expression of the target nucleic acid. Appendix 97 50. The method of claim 49, wherein the RNA-guided nuclease-deficient DNA-binding protein is an RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system. Appendix 98 64. The method of claim 63, wherein the at least one RNA-guided DNA-binding protein nickase is an RNA-guided DNA-binding protein nickase of a type II CRISPR system. Appendix 99 74. The cell of claim 73, wherein the RNA-guided nuclease-deficient DNA-binding protein is an RNA-guided nuclease-deficient DNA-binding protein of a type II CRISPR system. Appendix 100 88. The cell of claim 87, wherein the at least one RNA-guided DNA-binding protein nickase is an RNA-guided DNA-binding protein nickase of a type II CRISPR system. Appendix 101 97. The method of claim 96, wherein the at least one RNA-guided DNA-binding protein nickase is an RNA-guided DNA-binding protein nickase of a type II CRISPR system. Appendix 102 A guide RNA containing a spacer sequence with a 5' shortening of 1 to 4 bases. Appendix 103 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA contains approximately 64 to approximately 500 nucleic acids. Appendix 104 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA comprises approximately 65 to approximately 500 nucleic acids. Appendix 105 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA comprises approximately 66 to approximately 500 nucleic acids. Appendix 106 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA comprises approximately 67 to approximately 500 nucleic acids. Appendix 107 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA comprises approximately 68 to approximately 500 nucleic acids. Appendix 108 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA comprises approximately 69 to approximately 500 nucleic acids. Appendix 109 A guide RNA that is a fusion transcript of crRNA and tracrRNA, wherein the tracrRNA contains approximately 70 to approximately 500 nucleic acids. Appendix 110 A guide RNA that is a fusion transcript of crRNA and tracrRNA, wherein the tracrRNA contains approximately 80 to approximately 500 nucleic acids. Appendix 111 A guide RNA that is a fusion transcript of crRNA and tracrRNA, wherein the tracrRNA contains approximately 90 to approximately 500 nucleic acids. Appendix 112 A guide RNA that is a fusion transcript of a crRNA and a tracrRNA, wherein the tracrRNA contains approximately 100 to approximately 500 nucleic acids.

Claims

1. Introducing into cells two or more first exogenous nucleic acids, each encoding a guide RNA complementary to an adjacent site in the DNA target nucleic acid. This involves introducing a second exogenous nucleic acid encoding at least one Cas9 protein niccas having one inactive nuclease domain into the cells. The two or more guide RNAs and the at least one Cas9 protein nickase are expressed, and the at least one Cas9 protein nickase colocalizes with the two or more guide RNAs to the DNA target nucleic acid, thereby introducing nicks into the DNA target nucleic acid and generating two or more adjacent nicks. A method for modifying DNA target nucleic acids within cells.

2. The method according to claim 1, wherein the two or more adjacent nicks are located on the same strand of double-stranded DNA.

3. The method according to claim 1, wherein the two or more adjacent nicks are located on the same strand of double-stranded DNA and induce homologous recombination.

4. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA.

5. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA and cause a double-strand break.

6. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA, causing double-strand breaks and inducing non-homologous end joining.

7. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA and their positions are offset from each other.

8. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA, their positions are offset from each other, and cause a double-strand break.

9. The method according to claim 1, wherein the two or more adjacent nicks are located on different strands of double-stranded DNA, their positions are offset from each other, and they cause double-strand breaks and lead to non-homologous end joining.

10. The method according to claim 1, further comprising introducing a donor nucleic acid into the cells, wherein the two or more adjacent nicks induce homologous recombination of the DNA target nucleic acid with the donor nucleic acid.

11. The method according to claim 1, wherein the cells are eukaryotic cells.

12. The method according to claim 1, wherein the cells are yeast cells, plant cells, or animal cells.

13. The method according to claim 1, wherein the guide RNA is a crRNA-tracrRNA fusion.

14. The method according to claim 1, wherein the DNA target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

15. A first exogenous nucleic acid, each encoding two or more guide RNAs that are complementary to adjacent sites in the DNA target nucleic acid, and It comprises a second exogenous nucleic acid encoding at least one Cas9 protein niccas having one inactive nuclease domain, A cell in which the two or more guide RNAs and the at least one Cas9 protein nickase are components of a colocalization complex for the DNA target nucleic acid.

16. The cell according to claim 15, wherein the cell is a eukaryotic cell.

17. The cell according to claim 15, wherein the cell is a yeast cell, a plant cell, or an animal cell.

18. The cell according to claim 15, wherein the two or more guide RNAs are crRNA-tracrRNA fusions.

19. The cell according to claim 15, wherein the DNA target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

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