Compositions and methods for gene editing

Abstract

To provide compositions and methods for manipulation of genomes of living organisms.SOLUTION: The present invention provides a fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease enzyme, a Kruppel associated box domain, and a DNA methyltransferase domain.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 62 / 660,023, filed April 19, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] STATEMENT OF RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. HR0011-17-2-0043 awarded by the Defense Advanced Research Projects Agency and Grant No. R01DA036858 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Although considered a promising therapeutic approach for the treatment of diseases, genome editing carries inherent risks due to the potential for genotoxicity through double-strand breaks. Moreover, genome editing is often associated with an all-or-nothing effect on the target gene (i.e., generating a complete knockout). In contrast, targeted epigenetic engineering does not carry the risk of DSB-induced genotoxicity, and moreover, it offers the opportunity to generate more graded effects on gene expression, thus working from complete silencing to less pronounced effects.

[0004] Provided herein are solutions to these and other needs in the art. Summary of the Invention

[0005] In one embodiment, a fusion protein is provided that includes a nuclease-deficient RNA-guided DNA endonuclease enzyme, a Kruppel-associated box (KRAB) domain, and a DNA methyltransferase domain. In one embodiment, the fusion protein is any one of SEQ ID NOs: 1 to 15.

[0006] In one aspect, there is provided a nucleic acid sequence encoding a fusion protein as described herein, including embodiments and aspects thereof.

[0007] In one aspect, there is provided a complex comprising a fusion protein as described herein, including embodiments and aspects thereof, and a polynucleotide comprising (1) a DNA targeting sequence complementary to a target polynucleotide sequence and (2) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence.

[0008] In one aspect, there is provided a vector comprising the nucleic acid sequence of a fusion protein described herein, including embodiments and aspects thereof.

[0009] In one aspect, there is provided a cell comprising a fusion protein as described herein, including embodiments and aspects thereof, a nucleic acid as described herein, including embodiments and aspects thereof, a complex as described herein, including embodiments and aspects thereof, or a vector as described herein, including embodiments and aspects thereof.

[0010] In one aspect, a method of silencing a target nucleic acid sequence in a cell is provided, comprising: delivering a first polynucleotide encoding a fusion protein as described herein, including embodiments and aspects thereof, to a cell containing the target nucleic acid; and delivering a second polynucleotide to the cell, the second polynucleotide comprising (i) a DNA target sequence complementary to the target nucleic acid sequence, and (ii) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme. Without intending to be bound by any theory, it is believed that the fusion protein silences the target nucleic acid sequence in the cell by methylating the chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark to the chromatin containing the target nucleic acid sequence. Thus, in an aspect, the fusion protein silences the target nucleic acid sequence in the cell by methylating the chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark to the chromatin containing the target nucleic acid sequence.

[0011] In one aspect, a method for silencing a target nucleic acid sequence in a cell is provided, comprising delivering a complex described herein, including embodiments and aspects thereof, to a cell comprising a target nucleic acid, and the complex silences the target nucleic acid sequence in the cell.Without intending to be bound by any theory, it is believed that the complex silences the target nucleic acid sequence in the cell by methylating the chromatin comprising the target nucleic acid sequence and / or by introducing a repressive chromatin mark to the chromatin comprising the target nucleic acid sequence.Thus, in an aspect, the complex silences the target nucleic acid sequence in the cell by methylating the chromatin comprising the target nucleic acid sequence and / or by introducing a repressive chromatin mark to the chromatin comprising the target nucleic acid sequence. [Brief description of the drawings]

[0012] [Figure 1A]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 1B]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 1C]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 1D]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 1E]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 1F]The engineering of an all-in-one protein for long-term gene silencing is described. Figure 1A is a schematic diagram of the all-in-one protein (SEQ ID NO: 1) of the present disclosure, with a KRAB domain fused to the -N terminus of dCas9 (SEQ ID NO: 23) separated by a GGSGGGS (SEQ ID NO: 17) linker and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C terminus of dCas9. Figure 1B provides a schematic diagram of the dCas9-fused epigenetic modulators tested for persistent gene silencing. The dCas9-KRAB protein is adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications. The dCas9-Dnmt3A-Dnmt3L fusion is adopted from Stepper et al., Nucleic Acids Research, 2016. We designed a novel all-in-one protein that integrates KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), Dnmt3A-Dnmt3L (SEQ ID NO: 33, where Dnmt3A is SEQ ID NO: 26 and Dnmt3L is SEQ ID NO: 28) into one polypeptide. Figure 1C shows a schematic diagram of the methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) that was used to evaluate long-term silencing by the all-in-one protein. Figures 1D-1E provide an illustration and results of the hit-and-run experimental workflow in HEK293T cells expressing the GFP reporter shown in Figure 3. Figure 1D shows that plasmids were co-transfected into the cells, one encoding the hit-and-run protein and the other a plasmid encoding the sgRNA. Figure 1E shows the results of the hit-and-run assay sorted for cells co-transfected with the all-in-one plasmid and the sgRNA plasmid. FIG. 1F shows that the outcome of silencing of the GFP reporter is dependent on the sgRNA sequence. [Figure 2A]Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 2B] Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 2C]Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 2D] Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 2E]Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 2F] Long-term silencing of endogenous genes is illustrated. Figures 2A-2C are representative flow cytometry data acquired 22 days after transfection following gene (CD29, CD81, CD151) targeting for long-term silencing using the all-in-one protein. Quadrant IV represents cells with genes turned off, shown as the percentage of cells with genes turned off (i.e., 45%, 66%, 53%, respectively). Figure 2D provides quantification of CD29, CD81, and CD151 silencing by three different sgRNAs. Figure 2E provides quantification of silencing two or three genes simultaneously to show that all-in-one protein can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. Figure 2F provides plots representing time points acquired 9 months after transfection of all-in-one protein and sgRNA targeting the CLTA gene, implying that the majority of cells have stably turned off the CLTA gene. [Figure 3A]Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3B] Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3C]Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3D] Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3E]Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3F] Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3G]Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3H] Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 3I]Long-term silencing of endogenous genes is described. Figures 3A-3C show that collected cells lost expression of CD29 (Figure 3A), CD81 (Figure 3B), and CD151 (Figure 3C) 36 days after transfection, as determined by their RNA expression profiles. Figures 3D-3F are volcano plots showing that the target genes CD29 (Figure 3D), CD81 (Figure 3E), and CD151 (Figure 3F) were the only significant genes knocked down in each experiment, implying high specificity of gene silencing. Figures 3G-3I provide quantification of the transcript levels of CD151 (Figure 3G), CD81 (Figure 3H), and CD29 (Figure 3I), showing greater than 96% knockdown of each of the target genes. [Figure 4A] Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4B]Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4C] Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4D]Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4E] Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4F]Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4G] Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Figure 4H]Long-term gene silencing in different mammalian cell lines is described. Figures 4A-4F are flow cytometry plots showing BFP expression (fused to all-in-one protein) in HeLa (cervical) (Figure 4A), U2OS (bone) (Figure 4B), and human induced pluripotent cells (iPSCs) (Figure 4C). Figures 4D-4F are the untransfected controls of Figures 4A-4C, respectively. Figure 4G shows that stable silencing of endogenous genes was achieved in HeLa and U2OS cells, measured 18 days after transfection with all-in-one protein. In Figures 4A-4F, the x-axis is BFP (fused to all-in-one protein) and the y-axis is mCherry. Figure 4H shows that gene silencing was detected 14 days after transfection by qPCR in the AML12 mouse hepatic cell line when targeting Pcsk9, Npc1, Spcs1, and Cd81. [Diagram 5] Schematic diagrams of fusion proteins p76, p90 to p102, and p112, corresponding to SEQ ID NOs: 1 to 15, respectively, are provided. [Figure 6A] We illustrate the gene silencing activity of all-in-one protein variants. Figures 6A-6B show gene silencing results 18 days after transfection of fusion proteins of SEQ ID NO:1-15 transfected into HEK293T cells for targeted silencing of the CLTA gene. The dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L designs showed transient and less efficient long-term silencing. Figures 6C-6D provide a comparison of SEQ ID NO:1 (p76) and SEQ ID NO:15 (p112) for silencing the HIST2H2BE (H2B) endogenous gene (Figure 6C) and the synthetic Snrpn-GFP reporter gene (Figure 6D) stably expressed in HEK293T cells. Figure 6E provides plots of protein expression (dotted lines) of p76 and p112 over a 50-day time course for turning off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the All-in-One protein. [Figure 6B] We illustrate the gene silencing activity of all-in-one protein variants. Figures 6A-6B show gene silencing results 18 days after transfection of fusion proteins of SEQ ID NO:1-15 transfected into HEK293T cells for targeted silencing of the CLTA gene. The dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L designs showed transient and less efficient long-term silencing. Figures 6C-6D provide a comparison of SEQ ID NO:1 (p76) and SEQ ID NO:15 (p112) for silencing the HIST2H2BE (H2B) endogenous gene (Figure 6C) and the synthetic Snrpn-GFP reporter gene (Figure 6D) stably expressed in HEK293T cells. Figure 6E provides plots of protein expression (dotted lines) of p76 and p112 over a 50-day time course for turning off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the All-in-One protein. [Figure 6C] We illustrate the gene silencing activity of all-in-one protein variants. Figures 6A-6B show gene silencing results 18 days after transfection of fusion proteins of SEQ ID NO:1-15 transfected into HEK293T cells for targeted silencing of the CLTA gene. The dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L designs showed transient and less efficient long-term silencing. Figures 6C-6D provide a comparison of SEQ ID NO:1 (p76) and SEQ ID NO:15 (p112) for silencing the HIST2H2BE (H2B) endogenous gene (Figure 6C) and the synthetic Snrpn-GFP reporter gene (Figure 6D) stably expressed in HEK293T cells. Figure 6E provides plots of protein expression (dotted lines) of p76 and p112 over a 50-day time course for turning off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the All-in-One protein. [Figure 6D]We illustrate the gene silencing activity of all-in-one protein variants. Figures 6A-6B show gene silencing results 18 days after transfection of fusion proteins of SEQ ID NO:1-15 transfected into HEK293T cells for targeted silencing of the CLTA gene. The dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L designs showed transient and less efficient long-term silencing. Figures 6C-6D provide a comparison of SEQ ID NO:1 (p76) and SEQ ID NO:15 (p112) for silencing the HIST2H2BE (H2B) endogenous gene (Figure 6C) and the synthetic Snrpn-GFP reporter gene (Figure 6D) stably expressed in HEK293T cells. Figure 6E provides plots of protein expression (dotted lines) of p76 and p112 over a 50-day time course for turning off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the All-in-One protein. [Figure 6E] We illustrate the gene silencing activity of all-in-one protein variants. Figures 6A-6B show gene silencing results 18 days after transfection of fusion proteins of SEQ ID NO:1-15 transfected into HEK293T cells for targeted silencing of the CLTA gene. The dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L designs showed transient and less efficient long-term silencing. Figures 6C-6D provide a comparison of SEQ ID NO:1 (p76) and SEQ ID NO:15 (p112) for silencing the HIST2H2BE (H2B) endogenous gene (Figure 6C) and the synthetic Snrpn-GFP reporter gene (Figure 6D) stably expressed in HEK293T cells. Figure 6E provides plots of protein expression (dotted lines) of p76 and p112 over a 50-day time course for turning off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the All-in-One protein. [Figure 7A]Western blots of all-in-one protein variants are provided. Figure 7A is a Western blot analysis of all-in-one protein variants p76 and p90-p102 using an antibody against Cas9 of Steptococcus pyogenes. The upper band represents the full-length protein, and the smaller size bands represent the proteolysis of the all-in-one protein. Figure 7B is a Western blot analysis of all-in-one protein variants to detect free Dnmt3A cleaved from the fusion protein. [Figure 7B] Western blots of all-in-one protein variants are provided. Figure 7A is a Western blot analysis of all-in-one protein variants p76 and p90-p102 using an antibody against Cas9 of Steptococcus pyogenes. The upper band represents the full-length protein, and the smaller size bands represent the proteolysis of the all-in-one protein. Figure 7B is a Western blot analysis of all-in-one protein variants to detect free Dnmt3A cleaved from the fusion protein. [Figure 8A] A pooled screen to determine optimal sgRNAs is described. Figure 8A is a schematic of a pooled screen to determine optimal sgRNAs that result in long-term gene silencing. Figures 8B-8E are flow cytometry histograms of the percent of cells undergoing gene silencing 4 weeks after transfection. Four HEK293T cell lines, each harboring a different gene with a GFP tag, were used, including CLTA (Figure 8B), VIM (Figure 8C), HIST2H2BE (H2B) (Figure 8D), and RAB11A (Figure 8E). [Figure 8B]A pooled screen to determine optimal sgRNAs is described. Figure 8A is a schematic of a pooled screen to determine optimal sgRNAs that result in long-term gene silencing. Figures 8B-8E are flow cytometry histograms of the percent of cells undergoing gene silencing 4 weeks after transfection. Four HEK293T cell lines, each harboring a different gene with a GFP tag, were used, including CLTA (Figure 8B), VIM (Figure 8C), HIST2H2BE (H2B) (Figure 8D), and RAB11A (Figure 8E). [Figure 8C] A pooled screen to determine optimal sgRNAs is described. Figure 8A is a schematic of a pooled screen to determine optimal sgRNAs that result in long-term gene silencing. Figures 8B-8E are flow cytometry histograms of the percent of cells undergoing gene silencing 4 weeks after transfection. Four HEK293T cell lines, each harboring a different gene with a GFP tag, were used, including CLTA (Figure 8B), VIM (Figure 8C), HIST2H2BE (H2B) (Figure 8D), and RAB11A (Figure 8E). [Figure 8D] A pooled screen to determine optimal sgRNAs is described. Figure 8A is a schematic of a pooled screen to determine optimal sgRNAs that result in long-term gene silencing. Figures 8B-8E are flow cytometry histograms of the percent of cells undergoing gene silencing 4 weeks after transfection. Four HEK293T cell lines, each harboring a different gene with a GFP tag, were used, including CLTA (Figure 8B), VIM (Figure 8C), HIST2H2BE (H2B) (Figure 8D), and RAB11A (Figure 8E). [Figure 8E]A pooled screen to determine optimal sgRNAs is described. Figure 8A is a schematic of a pooled screen to determine optimal sgRNAs that result in long-term gene silencing. Figures 8B-8E are flow cytometry histograms of the percent of cells undergoing gene silencing 4 weeks after transfection. Four HEK293T cell lines, each harboring a different gene with a GFP tag, were used, including CLTA (Figure 8B), VIM (Figure 8C), HIST2H2BE (H2B) (Figure 8D), and RAB11A (Figure 8E). [Figure 9A] Figure 9A is a map of sgRNA functionality spanning transcription start sites of target genes, including CLTA (Figure 9A), H2B (Figure 9B), RAB11 (Figure 9C), and VIM (Figure 9D). Transcription start sites (TSSs) and CpG islands are annotated above each plot. Each dot represents one sgRNA, and its efficacy in long-term gene silencing is plotted as the log2 fold change in sgRNA abundance. Nucleosome occupancy (bottom plot) is plotted from the MNase signal. [Figure 9B] Figure 9A is a map of sgRNA functionality spanning transcription start sites of target genes, including CLTA (Figure 9A), H2B (Figure 9B), RAB11 (Figure 9C), and VIM (Figure 9D). Transcription start sites (TSSs) and CpG islands are annotated above each plot. Each dot represents one sgRNA, and its efficacy in long-term gene silencing is plotted as the log2 fold change in sgRNA abundance. Nucleosome occupancy (bottom plot) is plotted from the MNase signal. [Figure 9C]Figure 9A is a map of sgRNA functionality spanning transcription start sites of target genes, including CLTA (Figure 9A), H2B (Figure 9B), RAB11 (Figure 9C), and VIM (Figure 9D). Transcription start sites (TSSs) and CpG islands are annotated above each plot. Each dot represents one sgRNA, and its efficacy in long-term gene silencing is plotted as the log2 fold change in sgRNA abundance. Nucleosome occupancy (bottom plot) is plotted from the MNase signal. [Figure 9D] Figure 9A is a map of sgRNA functionality spanning transcription start sites of target genes, including CLTA (Figure 9A), H2B (Figure 9B), RAB11 (Figure 9C), and VIM (Figure 9D). Transcription start sites (TSSs) and CpG islands are annotated above each plot. Each dot represents one sgRNA, and its efficacy in long-term gene silencing is plotted as the log2 fold change in sgRNA abundance. Nucleosome occupancy (bottom plot) is plotted from the MNase signal. [Figure 10A] We describe functional sgRNAs for long-term gene silencing. Figure 10A is the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA targeting locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). Figures 10B-10E are representative plots showing the growth phenotypes of four genes, including ARL1 (Figure 10B), EIF6 (Figure 10C), SMC3 (Figure 10D), and HEATR1 (Figure 10E), along with the all-in-one proteins (bottom plots), from an existing dCas9-KRAB / CRISPRi dataset in K562 cells (Gilbert, Horlbeck et al., Cell 2014). Each dot represents a sgRNA. The TSS and annotated CpG islands are shown for each gene. [Figure 10B]We describe functional sgRNAs for long-term gene silencing. Figure 10A is the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA targeting locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). Figures 10B-10E are representative plots showing the growth phenotypes of four genes, including ARL1 (Figure 10B), EIF6 (Figure 10C), SMC3 (Figure 10D), and HEATR1 (Figure 10E), along with the all-in-one proteins (bottom plots), from an existing dCas9-KRAB / CRISPRi dataset in K562 cells (Gilbert, Horlbeck et al., Cell 2014). Each dot represents a sgRNA. The TSS and annotated CpG islands are shown for each gene. [Figure 10C] We describe functional sgRNAs for long-term gene silencing. Figure 10A is the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA targeting locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). Figures 10B-10E are representative plots showing the growth phenotypes of four genes, including ARL1 (Figure 10B), EIF6 (Figure 10C), SMC3 (Figure 10D), and HEATR1 (Figure 10E), along with the all-in-one proteins (bottom plots), from an existing dCas9-KRAB / CRISPRi dataset in K562 cells (Gilbert, Horlbeck et al., Cell 2014). Each dot represents a sgRNA. The TSS and annotated CpG islands are shown for each gene. [Figure 10D]We describe functional sgRNAs for long-term gene silencing. Figure 10A is the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA targeting locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). Figures 10B-10E are representative plots showing the growth phenotypes of four genes, including ARL1 (Figure 10B), EIF6 (Figure 10C), SMC3 (Figure 10D), and HEATR1 (Figure 10E), along with the all-in-one proteins (bottom plots), from an existing dCas9-KRAB / CRISPRi dataset in K562 cells (Gilbert, Horlbeck et al., Cell 2014). Each dot represents a sgRNA. The TSS and annotated CpG islands are shown for each gene. [Figure 10E] We describe functional sgRNAs for long-term gene silencing. Figure 10A is the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA targeting locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). Figures 10B-10E are representative plots showing the growth phenotypes of four genes, including ARL1 (Figure 10B), EIF6 (Figure 10C), SMC3 (Figure 10D), and HEATR1 (Figure 10E), along with the all-in-one proteins (bottom plots), from an existing dCas9-KRAB / CRISPRi dataset in K562 cells (Gilbert, Horlbeck et al., Cell 2014). Each dot represents a sgRNA. The TSS and annotated CpG islands are shown for each gene. [Figure 11A]A comparison of the growth phenotype and nucleosome positioning (from MNase signal) of VPS53 (Figure 11A) and VPS54 (Figure 11B) is provided, showing the location of functional sgRNAs in nucleosome-depleted regions. [Figure 11B] A comparison of the growth phenotype and nucleosome positioning (from MNase signal) of VPS53 (Figure 11A) and VPS54 (Figure 11B) is provided, showing the location of functional sgRNAs in nucleosome-depleted regions. [Figure 12A] Figure 12A shows the delivery of all-in-one proteins by mRNA expression. Figure 12A shows the in vitro transcription of two all-in-one variants (p102 and p112) showing the full-length synthesis of each design. Figure 12B provides flow cytometry plots showing the expression of p102 and p112 one day after transfection of mRNA into HEK293T cells. Figure 12C provides a time course of CLTA endogenous gene silencing in HEK293T cells after transfection with mRNA expressing p102 and p112 all-in-one variants. [Figure 12B] Figure 12A shows the delivery of all-in-one proteins by mRNA expression. Figure 12A shows the in vitro transcription of two all-in-one variants (p102 and p112) showing the full-length synthesis of each design. Figure 12B provides flow cytometry plots showing the expression of p102 and p112 one day after transfection of mRNA into HEK293T cells. Figure 12C provides a time course of CLTA endogenous gene silencing in HEK293T cells after transfection with mRNA expressing p102 and p112 all-in-one variants. [Figure 12C]Figure 12A shows the delivery of all-in-one proteins by mRNA expression. Figure 12A shows the in vitro transcription of two all-in-one variants (p102 and p112) showing the full-length synthesis of each design. Figure 12B provides flow cytometry plots showing the expression of p102 and p112 one day after transfection of mRNA into HEK293T cells. Figure 12C provides a time course of CLTA endogenous gene silencing in HEK293T cells after transfection with mRNA expressing p102 and p112 all-in-one variants. [Figure 13A] Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13B]Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13C] Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13D]Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13E] Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13F]Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. [Figure 13G] Illustrating the regulated expression of all-in-one protein by doxycycline induction. Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding all-in-one protein under a doxycycline-inducible promoter. The dotted line represents baseline median BFP fluorescence without doxycycline administration. Figure 13B provides western blots of cells to detect expression of all-in-one protein before and after doxycycline treatment. Figures 13C-13F are flow cytometry plots of CD81 (Figures 13C-13D) and CD151 (Figures 13E-13F) knockdown after 14 days of doxycycline treatment of K562 cells. Figure 13G shows quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The technology described herein allows, among other things, for the permanent silencing of genes in mammalian cells without generating double-stranded DNA breaks in the host genome. In embodiments, the central component is a single-chain polypeptide composed of catalytically inactive Cas9 (dCas9) fused to Dnmt3A, Dnmt3L, and KRAB domains (referred to herein as "all-in-one protein"). This fusion protein provided herein can be directed to a specific site in the mammalian genome using a single guide RNA (sgRNA) and can add DNA methylation and / or repressive chromatin marks to the site. In embodiments, the result is gene silencing that is heritable over subsequent cell divisions. In embodiments, the fusion proteins provided herein (and sgRNA) are expressed only transiently, avoiding the use of viral delivery methods to induce permanent silencing.

[0014] In embodiments, the fusion proteins provided herein provide robust long-term or permanent silencing of endogenous gene expression by epigenome editing rather than genome editing. Both alleles of a gene can be targeted, or a single pathogenic allele can be selectively targeted. In embodiments, the advantage of the fusion proteins provided herein is that epigenetic editing is reversible and therefore inherently safer than genome editing. Thus, in embodiments, the fusion proteins provided herein are useful in preventative applications. For example, gene silencing allows acute protection from infection / biological toxins and can be reversed after the risk of infection or poisoning has passed. Thus, in embodiments, the fusion proteins provided herein are useful for viruses or toxins that enter cells via interactions with proteins required for long-term organ function or homeostasis. In embodiments, the fusion proteins provided herein are useful in genome editing-based therapies.

[0015] In embodiments, durable gene silencing in mammalian cells can be achieved with two components: a single polypeptide composed of dCas9 fused to three epigenetic modulators, and a single guide RNA that directs the protein to a specific site in the host genome. In embodiments, the components are only transiently expressed in the host cell, thus reducing toxicity and off-target events.

[0016] In embodiments, the fusion proteins provided herein do not induce DNA breaks in host cells for permanent gene silencing.In embodiments, the epigenetic marks added to the genomic site of interest are reversible, thus allowing for the removal of off-target events that may occur.

[0017] definition While various embodiments and aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be used.

[0018] The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated by reference in their entirety for all purposes.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.The following references provide one of the skills with the general definitions of many terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Cambridge Dictionary of Science and Technology (Walker ed.,1988), The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991), and Hale&Marham, The Harper Collins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0020] The use of singular indefinite or definite articles (e.g., "a," "an," "the," etc.) in this disclosure and the claims that follow follows the conventional approach in patents to mean "at least one," unless it is clear from the context that the term is specifically intended to mean only one in a particular instance. Similarly, the term "comprising" is open-ended, without excluding additional items, features, components, etc. References identified herein are expressly incorporated herein by reference in their entirety, unless otherwise noted.

[0021] The terms "comprise," "include," "have," and their derivatives are used interchangeably herein as inclusive, open-ended terms. For example, the use of "comprising," "including," or "having" means that the elements encompassed by the subject of the clause containing the verb are not just the elements comprised, have, or included.

[0022] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, in either single-stranded, double-stranded, or multiple-stranded form, or their complements. Terms such as "polynucleotide," "oligonucleotide," "oligo," and the like refer to a linear sequence of nucleotides in the usual and customary sense. The term "nucleotide" refers to a single polynucleotide unit, i.e., monomer, in the usual and customary sense. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides, contemplated herein include, but are not limited to, any type of RNA, e.g., mRNA, siRNA, miRNA, sgRNA, and guide RNA, as well as any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragment thereof. In an embodiment, the nucleic acid is messenger RNA. In an embodiment, the messenger RNA is a messenger ribonucleoprotein (RNP). The term "double-stranded" in the context of polynucleotide refers to double-stranded in the usual and customary sense. Nucleic acid can be linear or branched. For example, nucleic acid can be a linear chain of nucleotides, or nucleic acid can be branched, for example, such that the nucleic acid comprises one or more arms or branches of nucleotides. Optionally, branched nucleic acid is repeatedly branched to form higher order structures, such as dendrimers.

[0023] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid oligomer", "oligonucleotide", "nucleic acid sequence", "nucleic acid fragment" and "polynucleotide" are used interchangeably and are intended to include, but are not limited to, polymeric forms of nucleotides covalently linked to one another that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of sequences, isolated RNA of sequences, sgRNA, guide RNA, nucleic acid probes, and primers. Polynucleotides useful in the methods of the present disclosure may include naturally occurring nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of such sequences.

[0024] The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (uracil (U) is substituted for thymine if the polynucleotide is RNA). Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule, or the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. A polynucleotide may optionally contain one or more non-standard nucleotide(s), nucleotide analog(s), and / or modified nucleotides.

[0025] For example, nucleic acids, including nucleic acids with phosphothioate backbones, may contain one or more reactive moieties.As used herein, the term reactive moiety includes any group that can react with another molecule, such as a nucleic acid or a polypeptide, via covalent, non-covalent, or other interaction.As an example, a nucleic acid may contain an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide via covalent, non-covalent, or other interaction.

[0026] This term also includes nucleic acids that contain known nucleotide analogs or modified backbone residues or bonds, synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as reference nucleic acids, and are metabolized in a similar manner as reference nucleotides.Examples of such analogs include, but are not limited to, for example, phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate, which replaces the oxygen in phosphoric acid with double bond sulfur), phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boron phosphonate, or O-methyl phosphoramidite bonds (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and modifications to nucleotide bases such as 5-methylcytidine or pseudouridine, and phosphodiester derivatives that contain peptide nucleic acid backbones and bonds. Other analog nucleic acids include those with cationic backbones, non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) as known in the art), including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modifications of the ribose-phosphate backbone can be made for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0027] Nucleic acid may contain non-specific sequences. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary or only partially complementary to any other nucleic acid sequence. By way of example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0028] The term "complementary" or "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick or other non-conventional methods. For example, the sequence AGT is complementary to the sequence TCA. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, 100% complementary, respectively). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions).

[0029] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize particularly at higher temperatures. An extensive guide to nucleic acid hybridization is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). In general, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, 50% of the probes are occupied at equilibrium at Tm). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are as follows: 50% formamide, 5xSSC, and 1% SDS, incubated at 42°C; or 5xSSC, 1% SDS, incubated at 65°C, washed with 0.2xSSC and 0.1% SDS at 65°C.

[0030] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when copies of nucleic acids are made using the maximum codon degeneracy permitted by the genetic code. In such cases, nucleic acids usually hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 1X SSC at 45°C. Positive hybridization is at least 2 times background. Those skilled in the art will readily recognize that alternative hybridization and washing conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in many references, such as Current Protocols in Molecular Biology, ed. Ausubel, et al., supra.

[0031] The term "gene" refers to a segment of DNA involved in the production of a protein, including the regions preceding and following the coding region (leader and trailer), and the intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. In addition, a "protein gene product" is the protein expressed from a particular gene.

[0032] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of a non-coding nucleic acid molecule (e.g., sgRNA) can be detected by standard PCR or Northern blotting methods well known in the art. See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0033] The term "transcriptional regulatory sequence" as provided herein refers to a segment of DNA that can increase or decrease the transcription (e.g., expression) of a particular gene in an organism. Non-limiting examples of transcriptional regulatory sequences include promoters, enhancers, and silencers.

[0034] The terms "transcription start site" and "transcription initiation site" may be used interchangeably herein to refer to the 5' end of a genetic sequence (e.g., a DNA sequence) at which an RNA polymerase (e.g., a DNA-directed RNA polymerase) begins synthesis of an RNA transcript. A transcription initiation site may be the first nucleotide of a transcribed DNA sequence at which an RNA polymerase begins synthesis of an RNA transcript. One of skill in the art can determine a transcription initiation site through routine experimentation and analysis, for example, by performing a run-off transcription assay or by definition according to the FANTOM5 database.

[0035] The term "promoter" as used herein refers to a region of DNA that initiates transcription of a particular gene. Promoters are usually located near the transcription start site of the gene, upstream of the gene, and on the same strand of DNA (i.e., 5' of the sense strand). Promoters can be from about 100 to about 1000 base pairs in length.

[0036] The term "enhancer" as used herein refers to a region of DNA that can be bound by a protein (e.g., a transcription factor) to increase the likelihood that transcription of a gene will occur. Enhancers can be from about 50 to about 1500 base pairs in length. Enhancers can be located downstream or upstream of the transcription start site that they regulate, and can be several hundred base pairs away from the transcription start site.

[0037] The term "silencer" as used herein refers to a DNA sequence that can bind to a transcriptional regulator known as a repressor, thereby negatively affecting the transcription of a gene. Silencer DNA sequences can be found in many different locations throughout DNA, including, but not limited to, upstream of a target gene, where they act to suppress the transcription of the gene (e.g., silencer gene expression).

[0038] "Guide RNA" or "gRNA" as provided herein refers to any polynucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and have direct sequence-specific binding of CRISPR complex to the target sequence. In an embodiment, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50% or more, about 60% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more, when optimally aligned using a suitable alignment algorithm.

[0039] In embodiments, the polynucleotide (e.g., gRNA) is a single-stranded ribonucleic acid. In aspects, the polynucleotide (e.g., gRNA) is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In aspects, the polynucleotide (e.g., gRNA) is 10-30 nucleic acid residues in length. In aspects, the polynucleotide (e.g., gRNA) is 20 nucleic acid residues in length. In embodiments, the length of a polynucleotide (e.g., gRNA) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleic acid or sugar residues in length. In embodiments, the polynucleotide (e.g., gRNA) may be 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 5-75, 10-75, 15-75, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, In some embodiments, the polynucleotide (e.g., gRNA) is 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-110, 10-120, 10-130, 10-140, 10-150, 10-20, 10-30, 10-40, 10-5 ...150, 10-20, 10-30, 10-40, 10-50, 10-150, 10-150, 10-20, 10-30, 10-40, 10-50, 10-150, 10-150, 10-20,

[0040] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that differs from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid. The terms "unnatural amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0041] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0042] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which in embodiments may be conjugated to a moiety not composed of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0043] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that code for the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, many nucleic acid sequences code for any given protein. For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that codes for a polypeptide is inherent in each sequence described.

[0044] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" in which the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present disclosure. Each of the following eight groups contains amino acids that are conservative substitutions for one another: (1) alanine (A), glycine (G), (2) aspartic acid (D), glutamic acid (E), (3) asparagine (N), glutamine (Q), (4) arginine (R), lysine (K), (5) isoleucine (I), leucine (L), methionine (M), valine (V), (6) phenylalanine (F), tyrosine (Y), tryptophan (W), (7) serine (S), threonine (T), and (8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0045] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to give the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0046] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a certain percentage of amino acid residues or nucleotides that are identical or identical (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity for a particular region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, for example, the NCBI website ncbi.nlm.nih.gov / BLAST / , etc.). Such sequences are then said to be "substantially identical". This definition can also refer to or apply to the complement of the test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As explained below, preferred algorithms are capable of calculating gaps and the like. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, and more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0047] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, as determined by simply counting from the N-terminus, is generally not necessarily the same as the number of corresponding positions in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there will be no amino acid in the variant that corresponds to the position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0048] When used in the context of the numbering of a given amino acid or polynucleotide sequence, the term "numbered with reference to" or "corresponding to" refers to the numbering of the residues of a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0049] For certain proteins described herein (e.g., KRAB, dCas9, Dnmt3A, Dnmt3L), the designated protein includes either the naturally occurring form of the protein, or a variant or homolog that maintains the protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In embodiments, a variant or homolog has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring form. In embodiments, the protein is a protein identified by its NCBI sequence reference. In embodiments, the protein is one identified by its NCBI sequence reference or a functional fragment or homolog thereof.

[0050] The term "Kruppel-associated box domain" or "KRAB domain" as provided herein refers to a category of transcriptional repression domain present in about 400 human zinc finger protein-based transcription factors. KRAB domains typically contain about 45 to about 75 amino acid residues. Descriptions of KRAB domains, including their functions and uses, can be found, for example, in Ecco, G., Imbeault, M., Trono, D., KRAB zinc finger proteins, Development 144, 2017; Lambert et al. The human transcription factors, Cell 172, 2018; Gilbert et al., Cell (2013); and Gilbert et al., Cell (2014), all of which are incorporated herein by reference in their entireties. In an embodiment, the KRAB domain is the KRAB domain of Kox 1. In an embodiment, the KRAB domain comprises the sequence set forth by SEQ ID NO: 16. In embodiments, the KRAB domain is the sequence of SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. In embodiments, the KRAB domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:16.

[0051] The term "DNA methyltransferase" as provided herein refers to an enzyme that catalyzes the transfer of methyl groups to DNA. Non-limiting examples of DNA methyltransferases include Dnmt1, Dnmt3A, Dnmt3B, and Dnmt3L. In embodiments, the DNA methyltransferase is a bacterial cytosine methyltransferase and / or a bacterial non-cytosine methyltransferase. Depending on the particular DNA methyltransferase, different regions of DNA are methylated. For example, Dnmt3A usually targets CpG dinucleotides for methylation. Through DNA methylation, DNA methyltransferases can modify the activity (such as gene expression) of a DNA segment without altering the DNA sequence. In embodiments, DNA methylation results in the repression of gene transcription and / or the regulation of methylation-sensitive transcription factors or CTCF. As described herein, a fusion protein can include one or more (e.g., two) DNA methyltransferases. When the DNA methyltransferase is included as part of a fusion protein, the DNA methyltransferase may be referred to as a "DNA methyltransferase domain." In embodiments, the DNA methyltransferase domain comprises one or more DNA methyltransferases. In embodiments, the DNA methyltransferase domain comprises two DNA methyltransferases. In embodiments, the DNA methyltransferase domain is Dnmt3A. In embodiments, the DNA methyltransferase domain has an amino acid sequence of SEQ ID NO:26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:26.In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:26. In embodiments, the DNA methyltransferase domain is Dnmt3L. In embodiments, the DNA methyltransferase domain has an amino acid sequence of SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:28. In embodiments, the DNA methyltransferase domain comprises Dnmt3A and Dnmt3L. In embodiments, the DNA methyltransferase domain has the amino acid sequence of SEQ ID NO:33. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:33.In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 33. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 33. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. A description of the structure and uses of Dnmt3A-3L domains can be found, for example, in Siddique et al., Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity, J. Mol. Biol. 425, 2013 and Stepper et al., Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase, Nucleic Acids Res. 45, 2017, which are incorporated by reference in their entireties and for all purposes.

[0052] "Dnmt3A", "Dnmt3a", "DNA (cytosine-5)-methyltransferase 3A" or "DNA methyltransferase 3a" proteins referred to herein include any recombinant or naturally occurring form of the Dnmt3A enzyme or a variant or homologue thereof that maintains Dnmt3A enzyme activity (e.g., within at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Dnmt3A). In an embodiment, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring Dnmt3A protein. In an embodiment, the Dnmt3A protein is substantially identical to the protein identified by UniProt reference number Q9Y6K1, or a variant or homolog having substantial identity thereto. In an embodiment, the Dnmt3A polypeptide is encoded by a nucleic acid sequence identified by NCBI reference sequence accession number NM_022552, a homolog or functional fragment thereof. In an embodiment, the Dnmt3A comprises a sequence as set forth by SEQ ID NO:26. In an embodiment, the Dnmt3A is a sequence as set forth by SEQ ID NO:26. In embodiments, Dnmt3A has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 26. In embodiments, the DNA methyltransferase domain has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 26.In embodiments, Dnmt3A has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26. In embodiments, Dnmt3A has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:26.

[0053] "Dnmt3L," "DNA (cytosine-5)-methyltransferase 3L" or "DNA methyltransferase 3L" protein referred to herein includes any recombinant or naturally occurring form of the Dnmt3L enzyme, or a variant or homologue thereof that maintains the Dnmt3L enzyme (e.g., within at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Dnmt3L). In an embodiment, the variant or homolog has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to a naturally occurring Dnmt3L protein. In an embodiment, the Dnmt3L protein is substantially identical to the protein identified by UniProt reference number Q9CWR8, or a variant or homolog having substantial identity thereto. In an embodiment, the Dnmt3L protein is identical to the protein identified by UniProt reference number Q9CWR8. In an embodiment, the Dnmt3L protein has at least 75% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9CWR8. In an embodiment, the Dnmt3L protein has at least 80% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9CWR8. In an embodiment, the Dnmt3L protein has at least 85% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9CWR8. In an embodiment, the Dnmt3L protein has at least 95% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9CWR8.

[0054] In an embodiment, the Dnmt3L protein is substantially identical to the protein identified by UniProt reference number Q9UJW, or a variant or homolog having substantial identity thereto. In an embodiment, the Dnmt3L protein is identical to the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 50% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 55% sequence identity to the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 60% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 65% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 70% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 75% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 80% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 85% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 90% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW. In an embodiment, the Dnmt3L protein has at least 95% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q9UJW.In an embodiment, the Dnmt3L polypeptide is encoded by a nucleic acid sequence identified by NCBI Reference Sequence Accession Number NM_001081695, or a homolog or functional fragment thereof. In an embodiment, the Dnmt3L comprises a sequence as set forth by SEQ ID NO:28. In an embodiment, the Dnmt3L is a sequence as set forth by SEQ ID NO:28. In an embodiment, the Dnmt3L has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28. In an embodiment, the Dnmt3L has an amino acid sequence having at least 50% sequence identity to SEQ ID NO:28. In an embodiment, the Dnmt3L has an amino acid sequence having at least 55% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 60% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 65% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 97% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28. In embodiments, Dnmt3L has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:28.

[0055] Terms like "RNA-guided DNA endonuclease" refer in their normal and customary sense to an enzyme that cleaves phosphodiester bonds in a DNA polynucleotide strand, where recognition of the phosphodiester bond is facilitated by a separate RNA sequence (e.g., a single guide RNA).

[0056] The term "class II CRISPR endonucleases" refers to endonucleases that have endonuclease activity similar to Cas9 and are involved in class II CRISPR systems. An example of a class II CRISPR system is the type II CRISPR locus of Streptococcus pyogenes SF370, which contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA, and a characteristic array of repeat sequences (direct repeats) spaced by short stretches of non-repetitive sequences (spacers, approximately 30 bp each). The Cpf1 enzyme belongs to a putative type V CRISPR-Cas system. Both type II and type V systems are included in class II of the CRISPR-Cas systems.

[0057] A "nuclear localization sequence" or "nuclear localization signal" or "NLS" is a peptide that targets a protein to the nucleus. In an embodiment, the NLS comprises five basic positively charged amino acids. The NLS can be located anywhere in the peptide chain. In an embodiment, the NLS is an NLS from SV40. In an embodiment, the NLS comprises a sequence set forth by SEQ ID NO:25. In an embodiment, the NLS is a sequence set forth by SEQ ID NO:25. In an embodiment, the NLS has an amino acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25. In an embodiment, the NLS has an amino acid sequence that has at least 75% sequence identity to SEQ ID NO:25. In embodiments, the NLS has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has the amino acid sequence of SEQ ID NO: 25.

[0058] As used herein, "cell" refers to a cell that performs sufficient metabolic or other functions to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian cells, insect cells (e.g., Spodoptera), and human cells. Cells can be useful when they are naturally non-adherent or have been treated (e.g., by trypsinization) to prevent them from adhering to a surface.

[0059] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions.In addition, some viral vectors can target specific cell types specifically or non-specifically.Replication-incompetent or replication-defective viral vectors refer to viral vectors that can infect their target cells and deliver their viral payload, but cannot continue the typical lytic pathway that leads to cell lysis and death.

[0060] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as the process of introducing a nucleic acid molecule and / or a protein into a cell. Non-viral or virus-based methods can be used to introduce a nucleic acid into a cell. The nucleic acid molecule can be a sequence encoding a complete protein or a functional portion thereof. Typically, it is a nucleic acid vector that contains the necessary elements for protein expression (e.g., promoter, transcription start site, etc.). Non-viral methods of transfection include any suitable method that does not use viral DNA or viral particles as a delivery system for introducing a nucleic acid molecule into a cell. Exemplary non-viral transfection methods include nanoparticle encapsulation of nucleic acid encoding a fusion protein (e.g., lipid nanoparticles, gold nanoparticles, etc.), calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetifection and electroporation. In the case of virus-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In an embodiment, nucleic acid molecules are introduced into cells using retroviral vectors according to standard procedures well known in the art. The term "transfection" or "transduction" also refers to introducing proteins into cells from the external environment. Usually, protein transduction or transfection relies on the attachment of a peptide or protein that can cross the cell membrane to the protein of interest. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat.Methods 4:119-20.

[0061] A "peptide linker" as provided herein is a linker that includes a peptide moiety. In embodiments, a peptide linker is a bivalent peptide, e.g., an amino acid sequence attached at the N-terminus and C-terminus to the remainder of a compound (e.g., a fusion protein provided herein). A peptide linker may be a cleavable peptide moiety (bivalent peptide moiety) (e.g., a P2A cleavable polypeptide). A peptide linker as provided herein may also be interchangeably referred to as an amino acid linker. In an embodiment, a peptide linker includes 1 to about 80 amino acid residues. In an embodiment, a peptide linker includes 1 to about 70 amino acid residues. In an embodiment, a peptide linker includes 1 to about 60 amino acid residues. In an embodiment, a peptide linker includes 1 to about 50 amino acid residues. In an embodiment, a peptide linker includes 1 to about 40 amino acid residues. In an embodiment, a peptide linker includes 1 to about 30 amino acid residues. In an embodiment, a peptide linker includes 1 to about 25 amino acid residues. In an embodiment, the peptide linker comprises 1 to about 20 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 20 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 19 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 18 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 17 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 16 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 15 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 14 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 13 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 12 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 11 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 10 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 9 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 8 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 7 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 6 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 5 amino acid residues.In an embodiment, the peptide linker comprises about 2 to about 4 amino acid residues. In an embodiment, the peptide linker comprises about 2 to about 3 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 19 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 18 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 17 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 16 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 15 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 14 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 13 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 12 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 11 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 10 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 9 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 8 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 7 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 6 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 5 amino acid residues. In an embodiment, the peptide linker comprises about 3 to about 4 amino acid residues. In an embodiment, the peptide linker comprises about 10 to about 20 amino acid residues. In an embodiment, the peptide linker comprises about 15 to about 20 amino acid residues. In an embodiment, the peptide linker comprises about 2 amino acid residues. In an embodiment, the peptide linker comprises about 3 amino acid residues. In an embodiment, the peptide linker comprises about 4 amino acid residues. In an embodiment, the peptide linker comprises about 5 amino acid residues. In an embodiment, the peptide linker comprises about 6 amino acid residues. In embodiments, the peptide linker comprises about 7 amino acid residues. In embodiments, the peptide linker comprises about 8 amino acid residues. In embodiments, the peptide linker comprises about 9 amino acid residues. In embodiments, the peptide linker comprises about 10 amino acid residues. In embodiments, the peptide linker comprises about 11 amino acid residues. In embodiments, the peptide linker comprises about 12 amino acid residues.In embodiments, the peptide linker comprises about 13 amino acid residues. In embodiments, the peptide linker comprises about 14 amino acid residues. In embodiments, the peptide linker comprises about 15 amino acid residues. In embodiments, the peptide linker comprises about 16 amino acid residues. In embodiments, the peptide linker comprises about 17 amino acid residues. In embodiments, the peptide linker comprises about 18 amino acid residues. In embodiments, the peptide linker comprises about 19 amino acid residues. In embodiments, the peptide linker comprises about 20 amino acid residues. In embodiments, the peptide linker comprises about 21 amino acid residues. In embodiments, the peptide linker comprises about 22 amino acid residues. In embodiments, the peptide linker comprises about 23 amino acid residues. In embodiments, the peptide linker comprises about 24 amino acid residues. In embodiments, the peptide linker comprises about 25 amino acid residues.

[0062] In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 17. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 17. In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 18. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 18. In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 19. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 19. In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 20. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 20. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 21. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 21. In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 22. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 22. In an embodiment, the peptide linker comprises the sequence set forth by SEQ ID NO: 27. In an embodiment, the peptide linker is the sequence set forth by SEQ ID NO: 27. In an embodiment, the peptide linker comprises a sequence as set forth by SEQ ID NO: 24. In an embodiment, the peptide linker is a sequence as set forth by SEQ ID NO: 24. In an embodiment, the peptide linker comprises a sequence as set forth by SEQ ID NO: 29. In an embodiment, the peptide linker is a sequence as set forth by SEQ ID NO: 29. In an embodiment, the peptide linker is an XTEN polypeptide. In an embodiment, the peptide linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 24, 27, or 29. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:17, 18, 19, 20, 21, 22, 24, 27, or 29.

[0063] In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 24, 27, or 29. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 18. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 19. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 20. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 21. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 24. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:27.In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:29.

[0064] As used herein, the term "XTEN", "XTEN linker", or "XTEN polypeptide" refers to a recombinant polypeptide (e.g., an unstructured recombinant peptide) that lacks hydrophobic amino acid residues. The development and use of XTEN can be found, for example, in Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009), which is incorporated herein by reference in its entirety and for all purposes. In an embodiment, the XTEN linker comprises a sequence set forth in SEQ ID NO: 31. In an embodiment, the XTEN linker is a sequence set forth in SEQ ID NO: 31. In an embodiment, the XTEN linker comprises a sequence set forth in SEQ ID NO: 32. In an embodiment, the XTEN linker is a sequence set forth in SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31. In embodiments, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31. In embodiments, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31. In embodiments, the XTEN linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32.

[0065] A "detectable agent" or "detectable moiety" is a composition detectable by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 225Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, 32 P, fluorophore (such as a fluorescent dye), electron-dense reagent, enzyme (e.g., those commonly used in ELISA), biotin, digoxigenin, paramagnetic molecule, paramagnetic nanoparticle, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticle, USPIO nanoparticle aggregate, superparamagnetic iron oxide ("SPIO") nanoparticle, SPIO nanoparticle aggregate, single crystal iron oxide nanoparticle, single crystal iron oxide, nanoparticle contrast agent, liposome, or other delivery vehicle containing gadolinium chelate ("Gd-chelate") molecule, gadolinium, radioisotope, radionuclide (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma ray emitting radionuclide, positron emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloid. , microbubbles (e.g., microbubble shells comprising albumin, galactose, lipids, and / or polymers, microbubble gas cores comprising air, heavy gas(es), perfluorocarbon, nitrogen, octafluoropropane, perflexane lipid microspheres, perflutren, etc.), iodine contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins, or other entities that can be made detectable, for example, by incorporating a radioactive label into a peptide or antibody that specifically reacts with a target peptide.

[0066] The detectable moiety is a detectable agent capable of forming a bond with a monovalent detectable agent or another composition. In an embodiment, the detectable agent is an HA tag. In an embodiment, the HA tag comprises a sequence as set forth by SEQ ID NO:24. In an embodiment, the HA tag is a sequence as set forth by SEQ ID NO:24. In an embodiment, the HA tag has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:24. In an embodiment, the HA tag has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:24. In an embodiment, the HA tag has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:24. In an embodiment, the HA tag has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:24. In an embodiment, the detectable agent is a blue fluorescent protein (BFP). In an embodiment, the BFP comprises a sequence as set forth by SEQ ID NO:30. In an embodiment, the BFP is a sequence as set forth by SEQ ID NO:30. In an embodiment, the BFP has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:30. In embodiments, the BFP has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 30. In embodiments, the BFP has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30. In embodiments, the BFP has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30.

[0067] Radioactive substances (e.g., radioisotopes) that can be used as imaging and / or labeling agents according to embodiments of the present disclosure include, but are not limited to, 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr,94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Paramagnetic ions that can be used as additional imaging agents according to embodiments of the present disclosure include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals with atomic numbers 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu.

[0068] "Contacting" is used according to its simple ordinary meaning to refer to a process that allows at least two different species to come into sufficient proximity to react, interact, or come into physical contact. However, it should be understood that the resulting reaction product may be produced directly from the reaction between the added reagents, or may be produced from an intermediate derived from one or more of the added reagents that may be produced in the reaction mixture.

[0069] The term "contacting" can include allowing two species to react, interact, or come into physical contact, where the two species can be, for example, a fusion protein provided herein and a nucleic acid sequence (e.g., a target DNA sequence).

[0070] As defined herein, the terms "inhibit", "inhibiting", "suppressing", "repressing", "silencing", "silence", and the like, when used in reference to compositions (e.g., fusion proteins, complexes, nucleic acids, vectors) provided herein, refer to negatively affecting (e.g., decreasing) the activity (e.g., transcription) of a nucleic acid sequence (e.g., decreasing transcription of a gene) compared to the activity (e.g., transcription of a gene) of the nucleic acid sequence in the absence of the composition (e.g., fusion protein, complex, nucleic acid, vector). In an embodiment, inhibition refers to a reduction in a disease or a symptom of a disease (e.g., cancer). Thus, inhibition includes at least partially, partially or completely blocking activation (e.g., transcription) or reducing, preventing, or delaying activation (e.g., transcription) of a nucleic acid sequence. Inhibited activity (e.g., transcription) may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less compared to that of a control. In embodiments, inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to a control.

[0071] A "control" sample or value refers to a sample that is a standard, usually a known standard, for comparison with a test sample. For example, a test sample can be taken from a test condition, for example, in the presence of a test compound, and compared to a sample from a known condition, for example, in the absence of a test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value collected from several tests or results. A person skilled in the art will recognize that a control can be designed for the evaluation of any number of parameters. For example, a control can be devised to compare the efficacy of treatment based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). A person skilled in the art will understand whether a control is valuable in a given situation and will be able to analyze data based on comparison with a control value. A control can also be useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control, the variation in the test sample is not considered significant.

[0072] Fusion proteins Provided herein are, among other things, fusion proteins that can use CRISPR-based epigenetic editing to permanently (e.g., irreversibly) and reversibly turn off genes in mammalian cells. In embodiments, the fusion protein comprises a single polypeptide fusion of four proteins (e.g., catalytically inactive Cas9 (e.g., dCas9), KRAB domain, Dnmt3A and Dnmt3L) that can be expressed transiently in cells. The fusion protein can be directed to a specific site in the mammalian genome using a polynucleotide complementary to the target nucleic acid sequence (e.g., DNA sequence), which further comprises a sequence (i.e., a binding sequence) that can bind to the fusion protein. When appropriately positioned, and without intending to be bound by theory, the fusion protein adds DNA methylation and / or repressive chromatin marks to the target nucleic acid, resulting in inheritable gene silencing over subsequent cell divisions. In this way, the fusion protein can perform epigenetic editing that avoids the need to generate DNA double-strand breaks in the host genome, making it a safe and reversible method of manipulating the genome of an organism.

[0073] In an embodiment, the fusion protein comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, a KRAB domain, and a DNA methyltransferase domain. In an aspect, the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a KRAB domain. In an aspect, the fusion protein comprises, from N-terminus to C-terminus, a KRAB domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a DNA methyltransferase domain. In an embodiment, the fusion protein further comprises one or more peptide linkers. In an aspect, the fusion protein further comprises one or more detectable tags. In an aspect, the fusion protein further comprises one or more nuclear localization sequences. In an aspect, the fusion protein further comprises one or more peptide linkers, one or more detectable tags, one or more nuclear localization sequences, or a combination of two or more of the foregoing. When the fusion protein comprises one or more peptide linkers, each peptide linker may be the same or different. When a fusion protein includes one or more detectable tags, each detectable tag may be the same or different. In embodiments, the fusion protein includes 1-10 detectable tags. In embodiments, the fusion protein includes 1-9 detectable tags. In embodiments, the fusion protein includes 1-8 detectable tags. In embodiments, the fusion protein includes 1-7 detectable tags. In embodiments, the fusion protein includes 1-6 detectable tags. In embodiments, the fusion protein includes 1-5 detectable tags. In embodiments, the fusion protein includes 1-4 detectable tags. In embodiments, the fusion protein includes 1-3 detectable tags. In embodiments, the fusion protein includes 1-2 detectable tags. In embodiments, the fusion protein includes 1 detectable tag. In embodiments, the fusion protein includes 2 detectable tags. In embodiments, the fusion protein includes 3 detectable tags. In embodiments, the fusion protein includes 4 detectable tags. In embodiments, the fusion protein comprises five detectable tags.

[0074] In embodiments, the fusion protein comprises the following structure: ABC, or BAC, or CAB, or CBA, or BCA, or ACB, in which A comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, B comprises a KRAB domain, and C comprises a DNA methyltransferase domain, in which the left-hand component is the N-terminus and the right-hand component is the C-terminus. In embodiments, the fusion protein further comprises one or more peptide linkers and one or more detectable tags. In embodiments, AB, BA, BC, CB, AC, and CA are each independently linked via a covalent bond, a peptide linker, a detectable tag, a nuclear localization sequence, or a combination of two or more thereof. The peptide linker may be any known in the art (e.g., P2A cleavable peptide, XTEN linker, etc.). In embodiments, the fusion protein comprises other components such as detectable tags (e.g., HA tag, blue fluorescent protein, etc.).

[0075] In embodiments, the fusion protein comprises the following structure: A-L1-B-L2-C, where A comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, B comprises a KRAB domain, C comprises a DNA methyltransferase domain, L1 is a covalent bond or a peptide linker, L2 is a covalent bond or a peptide linker, and where A is at the N-terminus and C is at the C-terminus. In embodiments, A is covalently linked to B via a peptide linker. In embodiments, A is covalently linked to B via a covalent bond. In embodiments, B is covalently linked to C via a peptide linker. In embodiments, B is covalently linked to C via a covalent bond. The peptide linker may be any known in the art (e.g., P2A cleavable peptide, XTEN linker, etc.). In embodiments, the fusion protein comprises other components such as a detectable tag, a nuclear localization sequence, etc. In embodiments, L1 is a covalent bond, a peptide linker, a detectable tag, a nuclear localization sequence, or a combination thereof. In embodiments, L2 is a covalent bond, a peptide linker, a detectable tag, a nuclear localization sequence, or a combination thereof.

[0076] In embodiments, the fusion protein comprises the following structure: B-L1-A-L2-C, where A comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, B comprises a KRAB domain, C comprises a DNA methyltransferase domain, L1 is a covalent bond or a peptide linker, and L2 is a covalent bond or a peptide linker, where B is at the N-terminus and C is at the C-terminus. In embodiments, L1 is a peptide linker. In embodiments, L1 is a covalent bond. In embodiments, L2 is a peptide linker. In embodiments, L2 is a covalent bond. The peptide linker can be any known in the art or described herein (e.g., P2A cleavable peptide, XTEN linker, etc.). In embodiments, the fusion protein comprises other components, such as a detectable tag. In embodiments, L1 is a covalent bond, a peptide linker, a detectable tag, or a combination thereof. In embodiments, L2 is a covalent bond, a peptide linker, a detectable tag, or a combination thereof. In embodiments, the fusion protein further comprises a nuclear localization sequence. Exemplary fusion proteins comprising the structure: B-L1-A-L2-C include p76, p90, p91, p92, p93, p94, p95, p96, p97, p98, p99, p100, p101, and p102 (Figure 5).

[0077] In an embodiment, the fusion protein comprises the following structure: B-L3-A-L4-C-L5-D, where A comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, B comprises a KRAB domain, C comprises a DNA methyltransferase domain, D is absent or D comprises one or more detectable tags, L3 comprises a covalent bond, a peptide linker, a detectable tag, or a combination of two or more thereof, L4 comprises a covalent bond, a peptide linker, a detectable tag, or a combination of two or more thereof, L5 is absent or L5 comprises a covalent bond or a peptide linker, where B is at the N-terminus and D is at the C-terminus. In an embodiment, L3 is a peptide linker. In an embodiment, L3 is a covalent bond. In an embodiment, L3 comprises a peptide linker and a detectable tag. In an embodiment, L3 comprises a detectable tag. In an embodiment, L4 is a peptide linker. In an embodiment, L4 comprises a peptide linker and a detectable tag. In an embodiment, L4 is a covalent bond. In an embodiment, L4 comprises a detectable tag. In an embodiment, L5 is a peptide linker. In an embodiment, L5 is a covalent bond. In an embodiment, D comprises one or more detectable tags. In an embodiment, D comprises one detectable tag. In an embodiment, D comprises two detectable tags. In an embodiment, D comprises three detectable tags. In an embodiment, D comprises multiple detectable tags. D can be any detectable tag known in the art and / or described herein (e.g., HA tag, blue fluorescent protein, etc.). In one embodiment, L5 and D are absent. When L3, L4, L5, and D comprise two or more detectable tags, each detectable tag is the same or different. The peptide linker can be any known in the art and / or described herein (e.g., P2A cleavable peptide, XTEN linker, etc.). In an embodiment, the fusion protein further comprises a nuclear localization sequence. Exemplary fusion proteins comprising the structure: B-L3-A-L4-C-L5-D include p76, p90, p91, p92, p93, p94, p95, p96, p97, p98, p99, p100, p101, and p102, as shown in FIG.

[0078] In an embodiment, the fusion protein comprises the following structure: C-L3-A-L4-B-L5-D, where A comprises a nuclease-deficient RNA-guided DNA endonuclease enzyme, B comprises a KRAB domain, C comprises a DNA methyltransferase domain, D is absent or D comprises one or more detectable tags, L3 comprises a covalent bond, a peptide linker, a detectable tag, or a combination of two or more thereof, L4 comprises a covalent bond, a peptide linker, a detectable tag, or a combination of two or more thereof, L5 is absent or L5 comprises a covalent bond or a peptide linker, where C is at the N-terminus and D is at the C-terminus. In an embodiment, L3 is a peptide linker. In an embodiment, L3 is a covalent bond. In an embodiment, L3 comprises a detectable tag. In an embodiment, L3 comprises a peptide linker and a detectable tag. In an embodiment, L4 is a peptide linker. In an embodiment, L4 is a covalent bond. In an embodiment, L4 comprises a detectable tag. In an embodiment, L4 comprises a peptide linker and a detectable tag. In an embodiment, L5 is a peptide linker. In an embodiment, L5 is a covalent bond. In an embodiment, D comprises one or more detectable tags. In an embodiment, D comprises one detectable tag. In an embodiment, D comprises two detectable tags. In an embodiment, D comprises three detectable tags. In an embodiment, D comprises multiple detectable tags. D can be any detectable tag known in the art and / or described herein (e.g., HA tag, blue fluorescent protein, etc.). In one embodiment, L5 and D are absent. When L3, L4, L5, and D comprise two or more detectable tags, each detectable tag is the same or different. The peptide linker can be any known in the art and / or described herein (e.g., P2A cleavable peptide, XTEN linker, etc.). In an embodiment, the fusion protein further comprises a nuclear localization sequence. An exemplary fusion protein comprising the structure: C-L3-A-L4-B-L5-D contains p112, as shown in FIG.

[0079] Terms like "nuclease-deficient RNA-guided DNA endonuclease enzyme" refer in their normal and customary sense to an RNA-guided DNA endonuclease (e.g., a mutant of a naturally occurring RNA-guided DNA endonuclease) that targets a specific phosphodiester bond within a DNA polynucleotide, where recognition of the phosphodiester bond is driven by a separate polynucleotide sequence (e.g., an RNA sequence (e.g., a single guide RNA (sgRNA)) but is unable to cleave the targeted phosphodiester bond to any significant extent (e.g., a biotinylated nucleotide sequence). (There is no measurable cleavage of phosphodiester bonds under physical conditions.) Thus, the nuclease-deficient RNA-guided DNA endonuclease retains DNA binding ability (e.g., specific binding to a target sequence) when complexed with a polynucleotide (e.g., sgRNA), but lacks significant endonuclease activity (e.g., a detectable amount of endonuclease activity). In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9, ddCpf1, a nuclease-deficient Cas9 variant, or a nuclease-deficient class II CRISPR endonuclease.

[0080] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9. The term "dCas9" or "dCas9 protein" referred to herein is a Cas9 protein that is defective or lacks activity in both catalytic sites of endonuclease activity. In an embodiment, the dCas9 protein has mutations at positions corresponding to D10A and H840A in S.pyogenesCas9. In an embodiment, the dCas9 protein lacks endonuclease activity due to point mutations at both endonuclease catalytic sites (RuvC and HNH) of wild-type Cas9. The point mutations can be D10A and H840A. In an embodiment, the dCas9 has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In an embodiment, the dCas9 comprises the amino acid sequence of SEQ ID NO:23. In an embodiment, the dCas9 has the amino acid sequence of SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23. In embodiments, dCas9 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.

[0081] As referred to herein, "CRISPR associated protein 9", "Cas9", "Csn1" or "Cas9 protein" includes any recombinant or native form of Cas9 endonuclease, or variants or homologs thereof that maintain Cas9 endonuclease enzymatic activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Cas9). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring Cas9 protein. In embodiments, the Cas9 protein is substantially identical to a protein identified by UniProt reference number Q99ZW2, or a variant or homolog having substantial identity thereto. In embodiments, the Cas9 protein has at least 75% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 80% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 85% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 90% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In embodiments, the Cas9 protein has at least 95% sequence identity to the amino acid sequence of the protein identified by UniProt reference number Q99ZW2.

[0082] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is "ddCpf1" or "ddCas12a". The term "DNAse-deadCpf1" or "ddCpf1" refers to a mutated Acidaminococcus genus Cpf1 (AsCpf1) that results in inactivation of Cpf1 DNAse activity. In aspects, ddCpf1 comprises an E993A mutation in the RuvC domain of AsCpf1. In aspects, ddCpf1 has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In aspects, ddCpf1 comprises the amino acid sequence of SEQ ID NO:34. In aspects, ddCpf1 has the amino acid sequence of SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:34. In embodiments, ddCpf1 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:34.

[0083] In an embodiment, the nuclease-deficient RNA-guided DNA endonuclease enzyme is dLbCpf1. The term "dLbCpf1:" refers to a Lachnospiraceae bacterium that lacks DNAse activity. Refers to a mutated Cpf1 from ND2006 (LbCpf1). In an embodiment, dLbCpf1 comprises a D832A mutation. In an embodiment, dLbCpf1 has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In an embodiment, dLbCpf1 comprises the amino acid sequence of SEQ ID NO: 35. In an embodiment, dLbCpf1 has the amino acid sequence of SEQ ID NO: 35. In an embodiment, dLbCpf1 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO: 35. % sequence identity to SEQ ID NO:35. In an embodiment, dLbCpf1 has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:35. In an embodiment, dLbCpf1 has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:35. In an embodiment, dLbCpf1 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35. In an embodiment, dLbCpf1 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:35. In an embodiment, dLbCpf1 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:35.

[0084] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is dFnCpf1. The term "dFnCpf1" refers to a mutant Cpf1 from Francisella novicida U112 (FnCpf1) that lacks DNAse activity. In an aspect, dFnCpf1 comprises a D917A mutation. In an aspect, dFnCpf1 has substantially no detectable endonuclease (e.g., endodeoxyribonuclease) activity. In an aspect, dFnCpf1 comprises the amino acid sequence of SEQ ID NO: 36. In an aspect, dFnCpf1 has the amino acid sequence of SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36. In embodiments, dFnCpf1 has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:36.

[0085] "Cpf1" or "Cpf1 protein" as referred to herein includes either recombinant or naturally occurring forms of Cpf1 (CRISPR from Prevotella and Francisella 1) endonuclease or variants or homologs thereof that maintain Cpf1 endonuclease enzymatic activity (within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cpf1), in embodiments where the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to a naturally occurring Cpf1 protein. In an embodiment, the Cpf1 protein is substantially identical to the protein identified by UniProt reference number U2UMQ6, or a variant or homolog having substantial identity thereto. In an embodiment, the Cpf1 protein is identical to the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein has at least 75% sequence identity to the amino acid sequence of the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein has at least 80% sequence identity to the amino acid sequence of the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein is identical to the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein has at least 85% sequence identity to the amino acid sequence of the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein is identical to the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein has at least 90% sequence identity to the amino acid sequence of the protein identified by UniProt reference number U2UMQ6. In an embodiment, the Cpf1 protein is identical to the protein identified by UniProt reference number U2UMQ6.In embodiments, the Cpf1 protein has at least 95% sequence identity to the amino acid sequence of the protein identified by UniProt reference number U2UMQ6.

[0086] In an embodiment, the nuclease-deficient RNA-guided DNA endonuclease enzyme is a nuclease-deficient Cas9 variant. The term "nuclease-deficient Cas9 variant" refers to a Cas9 protein that has one or more mutations that enhance binding specificity to the PAM compared to wild-type Cas9, and further includes protein mutations that render the protein incapable of or significantly impair endonuclease activity. Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif), i.e., a short sequence that is recognized by the CRISPR complex. The exact sequence and length requirements of the PAM vary depending on the CRISPR enzyme used, but the PAM is usually a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). The binding specificity of the nuclease-deficient Cas9 variant to the PAM can be determined by any method known in the art. Descriptions and uses of known Cas9 variants are described, for example, in Shmakov et al., Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 2017 and Cebrian-Serrano et al., CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools Mamm. Genome 7-8, 2017, which are incorporated by reference herein in their entirety and for all purposes. Exemplary Cas9 variants are shown in Table 4 below. [Table 1]

[0087] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is a nuclease-deficient class II CRISPR endonuclease.As used herein, the term " nuclease-deficient class II CRISPR endonuclease " refers to any class II CRISPR endonuclease that has a mutation that causes reduced, impaired or inactive endonuclease activity.

[0088] In an embodiment, the DNA methyltransferase domain is a Dnmt3A-3L domain. As provided herein, "Dnmt3A-3L domain" refers to a protein that includes both Dnmt3A and Dnmt3L. In an embodiment, Dnmt3A and Dnmt3L are covalently linked. In an embodiment, Dnmt3A is covalently linked to Dnmt3L via a peptide linker. In an embodiment, the peptide linker comprises a sequence as set forth in SEQ ID NO:27. In an embodiment, the peptide linker is a sequence as set forth in SEQ ID NO:27. In an embodiment, the peptide linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27. In embodiments, the peptide linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:27. In embodiments, the peptide linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:27. In embodiments, the Dnmt3A-3L domain comprises a sequence set forth by SEQ ID NO:33. In embodiments, the Dnmt3A-3L domain is a sequence set forth by SEQ ID NO:33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In embodiments, the Dnmt3A-3L domain has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33.

[0089] In embodiments, the peptide linker is an XTEN linker. In aspects, the XTEN linker comprises about 16 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 17 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 18 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 19 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 20 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 30 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 40 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 50 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 60 to about 80 amino acid residues. In aspects, the XTEN linker comprises about 70 to about 80 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 70 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 60 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 50 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 40 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 35 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 30 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 25 amino acid residues. In an embodiment, the XTEN linker comprises about 16 to about 20 amino acid residues. In an embodiment, the XTEN linker comprises about 16 amino acid residues. In an embodiment, the XTEN linker comprises about 17 amino acid residues. In an embodiment, the XTEN linker comprises about 18 amino acid residues. In embodiments, the XTEN linker comprises about 19 amino acid residues. In embodiments, the XTEN linker comprises about 20 amino acid residues.

[0090] In embodiments, the XTEN linker comprises a sequence as set forth by SEQ ID NO:31. In an aspect, the XTEN linker is a sequence as set forth by SEQ ID NO:31. In an aspect, the XTEN linker comprises a sequence as set forth by SEQ ID NO:32. In an aspect, the XTEN linker is a sequence as set forth by SEQ ID NO:32. In an aspect, the XTEN linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:31. In embodiments, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31. In embodiments, the XTEN linker has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32.

[0091] The fusion protein may comprise an amino acid sequence useful for targeting the fusion protein to a specific region of a cell (e.g., cytoplasm, nucleus). Thus, in an embodiment, the fusion protein further comprises a nuclear localization signal (NLS) peptide. In an embodiment, the NLS comprises a sequence as set forth by SEQ ID NO:25. In an embodiment, the NLS is a sequence as set forth by SEQ ID NO:25. In an embodiment, the NLS has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25. In an embodiment, the NLS has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25. In an embodiment, the NLS has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:25. In embodiments, the NLS has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25. In embodiments, the NLS has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 25.

[0092] In embodiments, the fusion protein comprises, from N-terminus to C-terminus, a KRAB domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a DNA methyltransferase domain.

[0093] In embodiments, the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0094] In embodiments, dCas9 is covalently linked to the KRAB domain via a peptide linker, where dCas9 is covalently linked to the Dnmt3A-3L domain via a peptide linker.

[0095] In embodiments, the peptide linker is an XTEN linker. In an aspect, the XTEN linker comprises a sequence set forth by SEQ ID NO:31. In an aspect, the XTEN linker is a sequence set forth by SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 75% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 85% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31. In an aspect, the XTEN linker comprises a sequence set forth by SEQ ID NO:32. In an aspect, the XTEN linker is a sequence set forth by SEQ ID NO:32. In embodiments, the XTEN linker has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32. In embodiments, the XTEN linker has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32.

[0096] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:1. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:1. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:2. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:2. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:3. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:3. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:4. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:4. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:5. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:6. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:6. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:7. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:7. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:8. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:8. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:9. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:9. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:10. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:10. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:11. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:11. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:12. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:12. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:13. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:13. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:14. In an embodiment, the fusion protein is the amino acid sequence of SEQ ID NO:14. In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 15. In embodiments, the fusion protein is the amino acid sequence of SEQ ID NO:15.

[0097] In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In aspects, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:13.In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In embodiments, the fusion protein comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15.

[0098] In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:2. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:4. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:5. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:6. In an aspect, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:7. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:9. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:10. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:11. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:12. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:13. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:14. In embodiments, the fusion protein comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:15.

[0099] In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:3. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:4. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:6. In an aspect, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:7. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:9. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:10. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:11. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:12. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:13. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:14. In embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:15.

[0100] In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:4. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:5. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:6. In an aspect, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:7. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:9. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:11. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:12. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:13. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:14. In embodiments, the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:15.

[0101] In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6. In an aspect, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:13. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14. In embodiments, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15.

[0102] In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6. In an aspect, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:8. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:10. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:11. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:12. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:13. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:14. In embodiments, the fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:15.

[0103] Complex For the fusion protein to perform epigenetic editing, the fusion protein interacts with (e.g., non-covalently binds to) a polynucleotide (e.g., sgRNA) that is complementary to a target polynucleotide sequence (e.g., a target DNA sequence to be edited) and further comprises a sequence (i.e., a binding sequence) to which the nuclease-deficient RNA-guided DNA endonuclease enzyme of the fusion protein described herein can bind. By forming this complex, the fusion protein is appropriately positioned to perform epigenetic editing. The term "complex" refers to a composition that includes two or more components, where the components bind together to form a functional unit. In an embodiment, the complex described herein includes a fusion protein described herein and a polynucleotide described herein. Thus, in one aspect, there is provided a fusion protein as described herein, including embodiments and aspects thereof, and a polynucleotide comprising: (1) a DNA targeting sequence complementary to a target polynucleotide sequence; and (2) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme, where the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence (e.g., an amino acid sequence capable of binding to a DNA target sequence).

[0104] A DNA targeting sequence refers to a polynucleotide that comprises a nucleotide sequence that is complementary to a target polynucleotide sequence (DNA or RNA). In embodiments, a DNA targeting sequence can be a single RNA molecule (single RNA polynucleotide) that can comprise a "single guide RNA" or "sgRNA". In embodiments, a DNA targeting sequence comprises two RNA molecules (e.g., linked together via hybridization at a binding sequence (e.g., dCas9 binding sequence)). In embodiments, a DNA targeting sequence (e.g., sgRNA) is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to a target polynucleotide sequence. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) binds to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 75% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 80% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) binds to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 85% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) binds to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 90% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) binds to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) is at least 95% complementary to a sequence of a cellular gene. In embodiments, the DNA targeting sequence (e.g., sgRNA) binds to a sequence of a cellular gene.

[0105] The "target polynucleotide sequence" provided herein is a nucleic acid sequence present in or expressed by a cell, and the guide sequence (or DNA targeting sequence) is designed to have a complementarity such that hybridization between the target sequence and the guide sequence (or DNA targeting sequence) promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. In an embodiment, the target polynucleotide sequence is an exogenous nucleic acid sequence. In an embodiment, the target polynucleotide sequence is an endogenous nucleic acid sequence.

[0106] The target polynucleotide sequence can be any region of a polynucleotide (e.g., a DNA sequence) suitable for epigenome editing. In an embodiment, the target polynucleotide sequence is a part of a gene. In an embodiment, the target polynucleotide sequence is a part of a transcriptional regulatory sequence. In an embodiment, the target polynucleotide sequence is a part of a promoter, an enhancer, or a silencer. In an embodiment, the target polynucleotide sequence is a part of a promoter. In an embodiment, the target polynucleotide sequence is a part of an enhancer. In an embodiment, the target polynucleotide sequence is a part of a silencer.

[0107] In an embodiment, the target polynucleotide sequence is a hypomethylated nucleic acid sequence. "Hypomethylated nucleic acid sequence" is used herein according to its standard meaning in the art and refers to the loss or absence of methyl groups on 5-methylcytosine nucleotides (e.g., at CpG). The loss or absence of methyl groups can occur compared to a standard control. Hypomethylation can occur, for example, in senescent cells or cancer (e.g., early stage of neoplasia) compared to younger cells or non-cancerous cells, respectively. Thus, this complex can be useful for re-establishing normal (e.g., non-disease non-aging) methylation levels.

[0108] In embodiments, the target polynucleotide sequence is within about 3000 base pairs (bp) adjacent to the transcription start site. In aspects, the target polynucleotide sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) adjacent to the transcription start site.

[0109] In an embodiment, the target polynucleotide sequence is located in, near, or within a promoter sequence. In an aspect, the target polynucleotide sequence is located within a CpG island. In an aspect, the target polynucleotide sequence is known to be associated with a disease or condition characterized by DNA hypomethylation.

[0110] In embodiments, exemplary target polynucleotide sequences include those set forth in Tables 1 and 2. In aspects, a target polynucleotide sequence comprises the sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95. In embodiments, the target polynucleotide sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95. In embodiments, the target polynucleotide sequence is SEQ ID NO:37. In embodiments, the target polynucleotide sequence is SEQ ID NO:39. In embodiments, the target polynucleotide sequence is SEQ ID NO:41. In embodiments, the target polynucleotide sequence is SEQ ID NO:43. In embodiments, the target polynucleotide sequence is SEQ ID NO:45. In embodiments, the target polynucleotide sequence is SEQ ID NO:47. In embodiments, the target polynucleotide sequence is SEQ ID NO:49. In embodiments, the target polynucleotide sequence is SEQ ID NO:51. In embodiments, the target polynucleotide sequence is SEQ ID NO:53. In embodiments, the target polynucleotide sequence is SEQ ID NO:55. In embodiments, the target polynucleotide sequence is SEQ ID NO:57. In embodiments, the target polynucleotide sequence is SEQ ID NO:59. In embodiments, the target polynucleotide sequence is SEQ ID NO:61. In embodiments, the target polynucleotide sequence is SEQ ID NO:63. In embodiments, the target polynucleotide sequence is SEQ ID NO:65. In embodiments, the target polynucleotide sequence is SEQ ID NO:67. In embodiments, the target polynucleotide sequence is SEQ ID NO:69. In embodiments, the target polynucleotide sequence is SEQ ID NO:71. In embodiments, the target polynucleotide sequence is SEQ ID NO:73. In embodiments, the target polynucleotide sequence is SEQ ID NO:75. In embodiments, the target polynucleotide sequence is SEQ ID NO:77. In embodiments, the target polynucleotide sequence is SEQ ID NO:79.In embodiments, the target polynucleotide sequence is SEQ ID NO: 81. In embodiments, the target polynucleotide sequence is SEQ ID NO: 83. In embodiments, the target polynucleotide sequence is SEQ ID NO: 85. In embodiments, the target polynucleotide sequence is SEQ ID NO: 87. In embodiments, the target polynucleotide sequence is SEQ ID NO: 89. In embodiments, the target polynucleotide sequence is SEQ ID NO: 91. In embodiments, the target polynucleotide sequence is SEQ ID NO: 93. In embodiments, the target polynucleotide sequence is SEQ ID NO: 95.

[0111] In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43. In embodiments, the target polynucleotide sequence is SEQ ID NO: 45. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 49. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 51. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 55.In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 57. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 61. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 65. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 67. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 69. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 71. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 73. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:75.In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 79. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:81. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 83. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 85. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 91. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 93. In embodiments, the target polynucleotide sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:95.

[0112] In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:37. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:39. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:41. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:43. In embodiments, the target polynucleotide sequence is SEQ ID NO:45. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:47. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:49. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:51. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:53. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:55. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:57. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:59. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:61. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:63. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:65. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:67. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:69. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:71. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:73. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:75. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:77.In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:79. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:81. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:83. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:85. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:87. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:89. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:91. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:93. In embodiments, the target polynucleotide sequence has at least 90% sequence identity to SEQ ID NO:95.

[0113] In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:37. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:39. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:41. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:43. In embodiments, the target polynucleotide sequence is SEQ ID NO:45. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:47. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:49. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:51. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:53. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:55. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:57. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:59. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:61. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:63. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:65. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:67. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:69. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:71. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:73. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:75. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:77.In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:79. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:81. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:83. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:85. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:87. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:89. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:91. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:93. In embodiments, the target polynucleotide sequence has at least 95% sequence identity to SEQ ID NO:95.

[0114] In embodiments, the complex comprises dCas9 that binds to a polynucleotide by binding to a binding sequence of the polynucleotide, thereby forming a ribonucleoprotein complex. In aspects, the binding sequence forms a hairpin structure. In aspects, the binding sequence is 30-100 nt, 35-50 nt, 37-47 nt, or 42 nt in length.

[0115] In embodiments, the binding sequence (e.g., Cas9 binding sequence) interacts with or binds to the Cas9 protein (e.g., dCas9 protein), and together they bind to the target polynucleotide sequence recognized by the DNA targeting sequence. The binding sequence (e.g., Cas9 binding sequence) comprises two complementary stretches of nucleotides that hybridize with each other to form a double-stranded RNA duplex (dsRNA duplex). These two complementary stretches of nucleotides can be covalently linked by an intervening nucleotide (e.g., in the case of a single molecule polynucleotide) known as a linker or linker nucleotide, and hybridize to form a double-stranded RNA duplex (dsRNA duplex, or "Cas9-binding sequence binding hairpin") of the binding sequence (e.g., Cas9 binding sequence), thus resulting in a stem-loop structure. Alternatively, in some aspects, the two complementary stretches of nucleotides may not be covalently linked, but instead are held together by hybridization between the complementary sequences (e.g., two molecule polynucleotides).

[0116] The binding sequence (e.g., Cas9 binding sequence) can have a length of 10 nucleotides to 100 nucleotides, e.g., 10 nucleotides (nt) to 20 nt, 20 nt to 30 nt, 30 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, or 90 nt to 100 nt. In embodiments, the binding sequence has a length of 15 nucleotides (nt) to 80 nt. In embodiments, the binding sequence has a length of 15 nt to 50 nt. In embodiments, the binding sequence has a length of 15 nt to 40 nt. In embodiments, the binding sequence has a length of 15 nt to 30 nt. In embodiments, the binding sequence has a length of 37 nt to 47 nt (e.g., 42 nt). In embodiments, the binding sequence has a length of 15 nt to 25 nt.

[0117] The dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) can have a length of 6 base pairs (bp) to 50 bp. For example, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) can have a length of 6 bp to 40 bp, 6 bp to 30 bp, 6 bp to 25 bp, 6 bp to 20 bp, 6 bp to 15 bp, 8 bp to 40 bp, 8 bp to 30 bp, 8 bp to 25 bp, 8 bp to 20 bp, or 8 bp to 15 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 8 bp to 10 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 10 bp to 15 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 15 bp to 18 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 18 bp to 20 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 20 bp to 25 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 25 bp to 30 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 30 bp to 35 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 35 bp to 40 bp. In embodiments, the dsRNA duplex of the binding sequence (e.g., Cas9 binding sequence) has a length of 40 bp to 50 bp.

[0118] In embodiments, exemplary polynucleotides that form complexes with the fusion proteins described herein include those set forth as sgRNAs in Tables 1 and 2. In aspects, polynucleotides that form complexes with the fusion proteins described herein include the sequences of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, or 94, or their corresponding RNA sequences. In embodiments, polynucleotides that form complexes with fusion proteins described herein comprise sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, or 94 or their corresponding RNA sequences. In embodiments, polynucleotides that form complexes with fusion proteins described herein comprise sequences of SEQ ID NO: 38. In embodiments, polynucleotides that form complexes with fusion proteins described herein comprise sequences of SEQ ID NO: 40. In embodiments, polynucleotides that form complexes with fusion proteins described herein comprise sequences of SEQ ID NO: 42. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 44. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 46. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 48. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 50. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 52. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 54. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 56.In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:58. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:60. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:62. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:64. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:66. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:68. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:70. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:72. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:74. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:76. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises a sequence of SEQ ID NO:78. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 80. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 82. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 84. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 86. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 88. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 90. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 92. In an embodiment, a polynucleotide that forms a complex with a fusion protein described herein comprises the sequence of SEQ ID NO: 94.

[0119] Nucleic acids and vectors The fusion proteins described herein, including embodiments and aspects thereof, may be provided as nucleic acid sequences encoding the fusion proteins. Thus, in one aspect, nucleic acid sequences are provided that encode the fusion proteins described herein, including embodiments and aspects thereof. In one aspect, nucleic acid sequences (including DNA targeting sequences) are provided that encode the fusion proteins described herein, including embodiments and aspects thereof. In aspects, the nucleic acid sequences encode the fusion proteins described herein, including fusion proteins having amino acid sequences with a particular % sequence identity as described herein. In aspects, the nucleic acid is RNA. In aspects, the nucleic acid is messenger RNA. In aspects, the messenger RNA is messenger RNP. In aspects, the nucleic acid sequences encode the fusion proteins described herein, including embodiments and aspects thereof. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:1. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:2. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:3. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:4. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:5. In aspects, the nucleic acid sequences encode the fusion proteins of SEQ ID NO:6. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:7. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:8. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:9. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:10. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:11. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:12. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:13. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:14. In embodiments, the nucleic acid sequence encodes a fusion protein of SEQ ID NO:15.

[0120] It is further contemplated that the nucleic acid sequence encoding the fusion protein described herein, including embodiments and aspects thereof, may be included in a vector. Thus, in one aspect, a vector is provided that includes a nucleic acid sequence described herein, including embodiments and aspects thereof. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein described herein, including a fusion protein having an amino acid sequence with a particular % sequence identity as described herein. In an aspect, the nucleic acid is messenger RNA. In an aspect, the messenger RNA is a messenger RNP. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:1. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:2. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:3. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:4. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:5. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:6. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:7. In an aspect, the vector includes a nucleic acid sequence encoding a fusion protein of SEQ ID NO:8. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 9. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 10. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 11. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 12. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 13. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 14. In embodiments, the vector comprises a nucleic acid sequence encoding a fusion protein of SEQ ID NO: 15.

[0121] In an embodiment, the vector further comprises a polynucleotide, the polynucleotide comprising (1) a DNA targeting sequence complementary to the target polynucleotide sequence, and (2) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme. Thus, one or more vectors may comprise all the components necessary to perform epigenetic editing.

[0122] cell The compositions described herein can be incorporated into cells. Within the cells, the compositions described herein, including embodiments and aspects thereof, can perform epigenome editing. Thus, in one aspect, a cell is provided that includes a fusion protein described herein, including embodiments and aspects thereof, a nucleic acid described herein, including embodiments and aspects thereof, a complex described herein, including embodiments and aspects thereof, or a vector described herein, including embodiments and aspects thereof. In one aspect, a cell is provided that includes a fusion protein described herein, including embodiments and aspects thereof. In one aspect, a cell is provided that includes a nucleic acid described herein, including embodiments and aspects thereof. In one aspect, a cell is provided that includes a complex described herein, including embodiments and aspects thereof. In one aspect, a cell is provided that includes a vector described herein, including embodiments and aspects thereof. In an aspect, the cell is a eukaryotic cell. In an aspect, the cell is a mammalian cell.

[0123] method It is contemplated that the compositions described herein can be used for epigenetic editing, more specifically epigenetic editing that results in suppression or silencing of a target nucleic acid sequence (e.g., gene). Without intending to be bound by any theory, silencing can result from methylation and / or introduction of repressive chromatin markers (e.g., mono-, di-, or trimethylation, deacetylation, acetylation, phosphorylation, ubiquitination of specific histones (e.g., H3K9, H3K27)) on the chromatin that contains the target nucleic acid sequence. Without intending to be bound by any theory, this method can be used to change epigenetic state, for example, by closing chromatin via methylation or introducing repressive chromatin markers into the chromatin that contains the target nucleic acid sequence (e.g., gene). Without intending to be bound by any theory, it is believed that the Dnmt3A-3L fusion functions to add methyl marks to CG DNA sites found in CpG islands, and the KRAB domain recruits epigenetic factors that modify histones by introducing repressive marks. Without intending to be bound by any theory, DNA is methylated at C nucleotides of CG sequences found in CpG islands (i.e., adding methyl marks to C nucleotides of CG DNA sites found in CpG islands).

[0124] In one aspect, a method of silencing a target nucleic acid sequence in a cell is provided, comprising delivering a first polynucleotide encoding a fusion protein described herein, including embodiments and aspects thereof, to a cell containing the target nucleic acid, and delivering a second polynucleotide to the cell, the second polynucleotide comprising (i) a DNA targeting sequence complementary to the target nucleic acid sequence, and (ii) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme. Without intending to be bound by any theory, the fusion protein silences the target nucleic acid sequence in the cell by methylating the chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark to the chromatin containing the target nucleic acid sequence. Without intending to be bound by any theory, methylating chromatin means that DNA is methylated at the C nucleotide of the CG sequence found in the CpG island (i.e., adding a methyl mark to the C nucleotide of the CG DNA site found in the CpG island). In an aspect, a sequence within about 3000 base pairs of the target nucleic acid sequence is methylated. In embodiments, sequences within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs of the target nucleic acid sequence are methylated.

[0125] As used herein, the term "repressive chromatin marker" refers to a modification made to chromatin that results in silencing (e.g., reducing or inhibiting transcription) of a target nucleic acid sequence (e.g., gene). Examples of repressive chromatin markers include, but are not limited to, monomethylation, dimethylation, and / or trimethylation, acetylation / deacetylation, phosphorylation, and ubiquitination of histones (e.g., H3K9, H3K27, H3K79, H2BK5).

[0126] In embodiments, silencing refers to a complete suppression of transcription. In aspects, silencing refers to a significant reduction in transcription compared to a control level of transcription.

[0127] In an embodiment, the first polynucleotide is comprised in a first vector. In an aspect, the first polynucleotide is comprised in a second vector. In an aspect, the first vector and the second vector are the same. In an aspect, the first vector is different from the second vector.

[0128] In embodiments, the polynucleotides described herein are delivered to cells by any method known in the art, for example, by transfection, electroporation, or transduction.

[0129] Alternatively, in one aspect, a method for silencing a target nucleic acid sequence in a cell is provided, comprising delivering the complex described herein, including embodiments and aspects thereof, to a cell that contains the target nucleic acid.Without intending to be bound by any theory, the complex silences the target nucleic acid sequence in a cell by methylating the chromatin that contains the target nucleic acid sequence and / or by introducing repressive chromatin marks into the chromatin that contains the target nucleic acid sequence.

[0130] In an embodiment, the cell is a mammalian cell.

[0131] In an embodiment, the method has a specificity that is 2 times higher than the specificity for non-target nucleic acid sequences. In an aspect, the method has a specificity that is at least 2 times (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times) higher than the specificity for non-target nucleic acid sequences. Methods for determining specificity are well known in the art and include, but are not limited to, RNA sequencing (RNA-seq), bisulfite sequencing, chromatin immunoprecipitation, flow cytometry, and qPCR. Thus, in an aspect, the specificity is determined by RNA sequencing. In an aspect, the specificity is determined by bisulfite sequencing. In an aspect, the specificity is determined by chromatin immunoprecipitation. In an aspect, the specificity is determined by flow cytometry. In an aspect, the specificity is determined by qPCR.

[0132] In embodiments, the complex is delivered to the cell via any method known in the art, e.g., ribonucleoprotein (RNP) delivery.

[0133] Embodiments N1 to N41 Embodiment N1. A fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease enzyme, a Krüppel-associated box domain, and a DNA methyltransferase domain.

[0134] Embodiment N2. The fusion protein of embodiment N1, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9, ddCpf1, a nuclease-deficient Cas9 variant, or a nuclease-deficient class II CRISPR endonuclease.

[0135] Embodiment N3. The fusion protein of embodiment N1 or N2, wherein the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0136] Embodiment N4. The fusion protein of embodiment N1, wherein the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel-associated box domain.

[0137] Embodiment N5. The fusion protein of embodiment N4, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0138] Embodiment N6. The fusion protein of embodiment N5, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0139] Embodiment N7. The fusion protein of embodiment N6, wherein the peptide linker is an XTEN linker.

[0140] Embodiment N8. The fusion protein of embodiment N1, wherein the fusion protein comprises, from N-terminus to C-terminus, a Kruppel-associated box, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a DNA methyltransferase domain.

[0141] Embodiment N9. The fusion protein of embodiment N8, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0142] Embodiment N10. A fusion protein according to embodiment N9, wherein the dCas9 is covalently linked to the Dnmt3A-3L domain via a peptide linker and the Krüppel-associated box domain is covalently linked to the dCas9 via a peptide linker.

[0143] Embodiment N11. The fusion protein of embodiment N10, wherein the peptide linker is an XTEN linker.

[0144] Embodiment N12. The fusion protein of any one of embodiments N1 to N3, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the Krüppel-associated box domain via a peptide linker.

[0145] Embodiment N13. The fusion protein of any one of embodiments N1 to N3, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the DNA methyltransferase domain via a peptide linker.

[0146] Embodiment N14. The fusion protein of any one of embodiments N1 to N3, wherein the Kruppel-associated box domain is covalently linked to the DNA methyltransferase domain via a peptide linker.

[0147] Embodiment N15. A fusion protein according to any one of embodiments N12 to N14, wherein the peptide linker is an XTEN linker.

[0148] Embodiment N16. A fusion protein according to embodiment N15, wherein the XTEN linker comprises between about 16 and 80 amino acid residues.

[0149] Embodiment N17. The fusion protein of any one of embodiments N1 to N16, further comprising a nuclear localization signal peptide.

[0150] Embodiment N18. The fusion protein of embodiment N1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0151] Embodiment N19. A nucleic acid sequence encoding a fusion protein according to any one of embodiments N1 to N18.

[0152] Embodiment N20. The nucleic acid sequence of embodiment N19, wherein the nucleic acid sequence is messenger RNA.

[0153] Embodiment N21. A complex comprising: (i) a fusion protein according to any one of embodiments N1 to N18; and (ii) a polynucleotide comprising: (a) a DNA targeting sequence complementary to a target polynucleotide sequence; and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence.

[0154] Embodiment N22. The complex of embodiment N21, wherein the target polynucleotide sequence is part of a gene.

[0155] Embodiment N23. The complex of embodiment N21, wherein the target polynucleotide sequence is part of a transcriptional regulatory sequence.

[0156] Embodiment N24. The complex of embodiment N21, wherein the target polynucleotide sequence is part of a promoter, enhancer, or silencer.

[0157] Embodiment N25. The complex of embodiment N21, wherein the target polynucleotide sequence is within about 3000 bp adjacent to the transcription start site.

[0158] Embodiment N26. A vector comprising a nucleic acid sequence according to embodiment N19 or N20.

[0159] Embodiment N27. The vector of embodiment N26, further comprising a polynucleotide, the polynucleotide comprising (a) a DNA targeting sequence complementary to a target polynucleotide sequence, and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme.

[0160] Embodiment N28. A cell comprising a fusion protein according to any one of embodiments N1 to N18, a nucleic acid according to embodiment N19 or N20, a complex according to any one of embodiments N21 to N25, or a vector according to embodiment N26 or N27.

[0161] Embodiment N29. The cell of embodiment N28, wherein the cell is a eukaryotic cell.

[0162] Embodiment N30. The cell of embodiment N28, wherein the cell is a mammalian cell.

[0163] Embodiment N31. A method for silencing a target nucleic acid sequence in a cell, comprising: (i) delivering to a cell containing the target nucleic acid a first polynucleotide encoding a fusion protein described in any one of embodiments N1 to N18; and (ii) delivering to the cell a second polynucleotide, the second polynucleotide comprising (a) a DNA targeting sequence complementary to the target nucleic acid sequence and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme.

[0164] Embodiment N32. The method of embodiment N31, wherein the fusion protein silences a target nucleic acid sequence in a cell by methylating chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark into chromatin containing the target nucleic acid sequence.

[0165] Embodiment N33. The method of embodiment N31 or N32, wherein the first polynucleotide is contained within a first vector.

[0166] Embodiment N34. The method of any one of embodiments N31 to N33, wherein the first polynucleotide is contained within a second vector.

[0167] Embodiment N35. The method of embodiment N34, wherein the first vector and the second vector are the same.

[0168] Embodiment N36. The method of embodiment N34, wherein the first vector is different from the second vector.

[0169] Embodiment N37. The method of embodiment N31, wherein the cell is a mammalian cell.

[0170] Embodiment N38. The method of embodiment N31, wherein the method has a specificity that is two-fold higher than the specificity for non-target nucleic acid sequences.

[0171] Embodiment N39. A method for silencing a target nucleic acid sequence in a cell, comprising delivering a complex described in any one of embodiments N21 to N25 to a cell containing the target nucleic acid.

[0172] Embodiment N40. The method of embodiment N39, wherein the complex silences a target nucleic acid sequence in the cell by methylating chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark into chromatin containing the target nucleic acid sequence.

[0173] Embodiment N41. The method of embodiment N39 or N40, wherein the cell is a mammalian cell.

[0174] Embodiment N42. The method of any one of embodiments N39 to N41, wherein the method has a specificity that is 2-fold higher than the specificity for non-target nucleic acid sequences.

[0175] Embodiments 1 to 36 Embodiment 1. A fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease enzyme, a Krüppel-associated box (KRAB) domain, and a DNA methyltransferase domain.

[0176] Embodiment 2. The fusion protein of embodiment 1, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9, ddCpf1, a nuclease-deficient Cas9 variant, or a nuclease-deficient class II CRISPR endonuclease.

[0177] Embodiment 3 The fusion protein of embodiment 1 or 2, wherein the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0178] Embodiment 4. The fusion protein of any one of embodiments 1 to 3, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the KRAB domain via a peptide linker.

[0179] Embodiment 5. The fusion protein of any one of embodiments 1 to 4, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the DNA methyltransferase domain via a peptide linker.

[0180] Embodiment 6. A fusion protein according to any one of embodiments 1 to 5, wherein the KRAB domain is covalently linked to the DNA methyltransferase domain via a peptide linker.

[0181] Embodiment 7. A fusion protein according to any one of embodiments 4 to 6, wherein the peptide linker is an XTEN linker.

[0182] Embodiment 8. The fusion protein of embodiment 7, wherein the XTEN linker comprises about 16 to 80 amino acid residues.

[0183] Embodiment 9. A fusion protein according to any one of embodiments 1 to 8, further comprising a nuclear localization signal peptide.

[0184] Embodiment 10. The fusion protein of any one of embodiments 1 to 9, wherein the fusion protein comprises, from N-terminus to C-terminus, a KRAB domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a DNA methyltransferase domain.

[0185] Embodiment 11. A fusion protein according to any one of embodiments 1 to 10, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

[0186] Embodiment 12. The fusion protein of embodiment 11, wherein dCas9 is covalently linked to the KRAB domain via a peptide linker and dCas9 is covalently linked to the Dnmt3A-3L domain via a peptide linker.

[0187] Embodiment 13 The fusion protein of embodiment 12, wherein the peptide linker is an XTEN linker.

[0188] Embodiment 14. A fusion protein according to any one of embodiments 1 to 13, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0189] Embodiment 15. A nucleic acid sequence encoding a fusion protein according to any one of embodiments 1 to 14.

[0190] Embodiment 16. A complex comprising: (i) the fusion protein of any one of embodiments 1-18; and (ii) a polynucleotide comprising: (a) a DNA targeting sequence complementary to a target polynucleotide sequence; and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence.

[0191] Embodiment 17 The complex of embodiment 21, wherein the target polynucleotide sequence is part of a gene.

[0192] Embodiment 18 The complex of embodiment 21, wherein the target polynucleotide sequence is part of a transcriptional regulatory sequence.

[0193] Embodiment 19. The complex of embodiment 21, wherein the target polynucleotide sequence is part of a promoter, enhancer, or silencer.

[0194] Embodiment 20. The complex of embodiment 21, wherein the target polynucleotide sequence is a hypomethylated nucleic acid sequence.

[0195] Embodiment 21. The complex of embodiment 21, wherein the target polynucleotide sequence is within about 3000 bp adjacent to the transcription start site.

[0196] Embodiment 22. A vector comprising the nucleic acid sequence of embodiment 19.

[0197] Embodiment 23. The vector of embodiment 26, further comprising a polynucleotide, the polynucleotide comprising: (a) a DNA targeting sequence complementary to a target polynucleotide sequence; and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme.

[0198] Embodiment 24. A cell comprising a fusion protein according to any one of embodiments 1 to 14, a nucleic acid according to embodiment 15, a complex according to any one of embodiments 16 to 21, or a vector according to embodiment 22 or 23.

[0199] Embodiment 25. The cell of embodiment 28, wherein the cell is a eukaryotic cell.

[0200] Embodiment 26 The cell of embodiment 28, wherein the cell is a mammalian cell.

[0201] Embodiment 27. A method for silencing a target nucleic acid sequence in a cell, comprising: (i) delivering to a cell containing the target nucleic acid a first polynucleotide encoding a fusion protein described in any one of embodiments 1 to 14; and (ii) delivering to the cell a second polynucleotide, the second polynucleotide comprising (a) a DNA targeting sequence complementary to the target nucleic acid sequence and (b) a binding sequence for a nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the fusion protein silences the target nucleic acid sequence in the cell by methylating chromatin containing the target nucleic acid sequence and / or introducing a repressive chromatin mark into chromatin containing the target nucleic acid sequence.

[0202] Embodiment 28 The method of embodiment 27, wherein the first polynucleotide is contained within a first vector.

[0203] Embodiment 29. The method of embodiment 27, wherein the first polynucleotide is contained within a second vector.

[0204] Embodiment 30. The method of embodiment 28 or 29, wherein the first vector and the second vector are the same.

[0205] Embodiment 31 The method of embodiment 28 or 29, wherein the first vector is different from the second vector.

[0206] Embodiment 32. The method of any one of embodiments 27 to 31, wherein the cell is a mammalian cell.

[0207] Embodiment 33. The method of any one of embodiments 27 to 32, wherein the method has a specificity that is 2-fold higher than the specificity for non-target nucleic acid sequences.

[0208] Embodiment 34. A method for silencing a target nucleic acid sequence in a cell, the method comprising delivering a complex described in any one of embodiments 16 to 20 to a cell containing the target nucleic acid, wherein the complex silences the target nucleic acid sequence in the cell by (i) methylating chromatin containing the target nucleic acid sequence, (ii) introducing a repressive chromatin mark to the chromatin containing the target nucleic acid sequence, or (iii) methylating chromatin containing the target nucleic acid sequence and introducing a repressive chromatin mark to the chromatin containing the target nucleic acid sequence.

[0209] Embodiment 35 The method of embodiment 34, wherein the cell is a mammalian cell.

[0210] Embodiment 36 The method of embodiment 34 or 35, wherein the method has a specificity that is 2-fold higher than the specificity for non-target nucleic acid sequences. EXAMPLES

[0211] The embodiments and aspects of the present specification are further illustrated by the following examples, which are intended merely to illustrate the embodiments and aspects and should not be construed as limiting the scope of the present specification.

[0212] Example 1 dCas9-fused epigenetic modulators were tested for persistent gene silencing. The initial version (V1, p76 (SEQ ID NO: 1)) of the all-in-one protein (Figure 1A) has a KRAB domain fused to the N-terminus of dCas9 (SEQ ID NO: 23), separated by a GGSGGGS (SEQ ID NO: 17) linker, and Dnmt3A-Dnmt3L (separated by an EASGSGRASPGIPGSTR (SEQ ID NO: 19) linker) at the C-terminus of dCas9. Another all-in-one protein (Figure 1B) combines the KRAB domain (SEQ ID NO: 16), dCas9 (D10A, H208A), and Dnmt3A-Dnmt3L (SEQ ID NO: 33, where SEQ ID NO: 26 is Dnmt3A and SEQ ID NO: 28 is Dnmt3L) into one polypeptide. Referring to FIG. 1B, the dCas9-KRAB protein was adopted from Gilbert et al., Cell 2013 for CRISPR interference (CRISPRi) applications, and the dCas9-Dnmt3A-Dnmt3L fusion was adopted from Stepper et al., Nucleic Acids Research, 2016.

[0213] To evaluate long-term silencing by the all-in-one protein, the activity of the V1 epigenetic editor was tested in HEK293T cells using a DNA methylation-sensitive GFP reporter (adopted from Stelzer et al., Cell 2015) (Figure 1C). To prevent background silencing of lentiviral vectors in mammalian cells, a ubiquitous chromatin opening element (UCOE) was added upstream of the GAPDH CpG island (CGI). When the GAPDH CGI is methylated, the gfp gene is turned off. A, B, and C indicate the positions in the promoter where the targeting single guide RNA (sgRNA) was encoded. These target sequences and the corresponding sgRNA sequences are shown in Table 1 below. Two plasmids were co-transfected into the cells, one encoding the Hit-and-Run protein and the other a plasmid encoding the sgRNA (Figure 1D). Two days after transfection, the cells expressing the Hit-and-Run protein and the sgRNA expression vector are sorted. GFP fluorescence is assessed over time by flow cytometry. When the all-in-one protein was expressed with sgRNA, a population of cells undergoing long-term silencing of the GFP reporter was observed (Figure 1E). The number of cells undergoing long-term silencing was greater with dCas9-Dnmt3A-Dnmt3L (lacking the KRAB domain). [Table 2]

[0214] Silencing of the GFP reporter was dependent on the sgRNA sequence, with guide C resulting in the highest level of silencing among the three sgRNA sequences tested. Pooling of sgRNAs encoding different sequences did not significantly alter gene silencing.

[0215] Example 2 Three genes (CD29, CD81, CD151) were targeted for long-term silencing using Hit-and-Run fusion proteins. All three proteins were localized to the cell surface, and knockdown was assessed by cell surface antibody staining of cells followed by flow cytometry. Representative flow cytometry data taken 22 days after transfection are shown in Figure 2A-2C. Quadrant IV represents cells in which the gene is turned off, shown as the percentage of cells in which the gene is turned off. The absence of cells in quadrants I and II means that the Hit-and-Run protein (marked by BFP) is no longer present in the cells. Figure 2D provides quantification of the silencing of CD29, CD81 and CD151 using three different sgRNA sequences or a pool of all three sgRNAs. The target DNA sequences and their sgRNAs used in this experiment are summarized in Table 2. [Table 3]

[0216] Two or three genes were targeted simultaneously to demonstrate that all-in protein expression can be multiplexed by simultaneous delivery of sgRNAs targeting different genes. NT sgRNA refers to a non-targeting sgRNA control. The results are shown in Figure 2E.

[0217] Gene silencing of cells initiated as single clones was tracked and the majority of cells were observed to keep the targeted CLTA gene turned off (37 out of 39 clones). The plot in Figure 2F represents a time point taken 9 months after transfection of the all-in-one protein and sgRNA targeting the CLTA gene.

[0218] The system described herein can target any gene in mammalian genome, especially genes that contain CpG islands in gene promoters.Dnmt3A-Dnmt3L canonically target CpG dinucleotides.Examples of genes that can be targeted include, but are not limited to, CXCR4, CD4, CD8, CD45, PD-1, CLTA-4, TGFBR, TCRa, TCRb, B2M.

[0219] Example 3 Cells that lost expression of ITGB1 (CD29), CD81 and CD151 36 days after transfection were collected and their RNA expression profiles were analyzed. As shown in Figures 3A-3C, successful knockdown of the targeted genes was detected compared to non-targeting sgRNA controls. Figures 3D-3F are volcano plots showing that the targeted genes were the only significant genes knocked down in each experiment, indicating high specificity of gene silencing. Figures 3G-3I are quantifications of transcript levels showing greater than 96% knockdown of the targeted genes.

[0220] Example 4 The all-in-one protein can be transfected and expressed in HeLa (cervical), U2OS (bone), and human induced pluripotent stem cells (iPSCs). Flow cytometry plots in Figure 4A-4F show BFP expression fused to the protein. Three endogenous genes (i.e., CD29, CD81, and CD151) in HeLa and U2OS cells were targeted. As shown in Figure 4G, stable silencing was detected, measured 18 days after transfection. Gene silencing was detected in the AML12 mouse hepatic cell line when Pcsk9, Npc1, Spcs1, and Cd81 were targeted. Silencing was detected by qPCR, measured 14 days after transfection, as shown in Figure 4H. The sgRNA sequences used in this experiment are summarized in Table 3. [Table 4]

[0221] Example 5 FIG. 5 provides a schematic of the all-in-one protein constructs designed and tested for gene silencing. The initial design of SEQ ID NO:1 (p76, V1) was modified to encode an XTEN linker (e.g., 16 amino acids (SEQ ID NO:31) or 80 amino acids (SEQ ID NO:32)) at either the N- or C-terminus of dCas9 (SEQ ID NO:29). All vectors contain an HA tag (SEQ ID NO:24) at the C-terminus of dCas9. In embodiments, for example, a CAG promoter is used to provide good expression in constructs p76, and p90-102, p112 (V2). With reference to FIG. 5, the protein constructs p90-p102 correspond to SEQ ID NOs:2-14, respectively, and protein construct p112 corresponds to SEQ ID NO:15.

[0222] Example 6 The protein constructs shown in Figure 5 were tested for silencing of the CLTA gene in HEK293T cells 18 days after transfection (Figures 6A-6B). Variable levels of gene silencing activity were detected, including a panel of variants such as p99 (SEQ ID NO: 11), p100 (SEQ ID NO: 12), and p112 (SEQ ID NO: 15) with more durable gene silencing compared to the p76(V1) design. Figures 6A and 6B show that the dCas9-KRAB and dCas9-Dnmt3A-Dnmt3L constructs demonstrated less efficient transient and long-term silencing.

[0223] p76 (SEQ ID NO:1), p112 (SEQ ID NO:15) were tested for silencing of the HIST2H2BE (H2B) endogenous gene and synthetic Snrpn-GFP reporter gene stably expressed in HEK293T cells (Figure 6C-6D). Cells were tracked for 50 days after transfection. The p112 variant maintained gene silencing with higher efficiency than the p76(V1) design. The dCas9-Dnmt3A-Dnmt3L and dCas9-KRAB fusion proteins are transient and have longer-term silencing with lower efficiency. Figure 6E provides plots of protein expression of p76 and p112 over a 50-day time course to turn off the HIST2H2BE (H2B) gene. Protein levels were measured by flow cytometric detection of BFP co-expressed with the all-in-one protein.

[0224] Example 7 Western blot analysis was performed on all-in-one protein variants p76, p90-p102 using an antibody against Steptococcus pyogenes Cas9. Referring to FIG. 7A, the upper band represents the full-length protein, while the smaller size bands represent the proteolysis of the all-in-one protein. Variants that showed little proteolysis, such as p99 (SEQ ID NO: 11), p100 (SEQ ID NO: 12), and p102 (SEQ ID NO: 14), showed higher efficiency of gene silencing. Variants with high levels of proteolysis, such as p96 (SEQ ID NO: 8) and p97 (SEQ ID NO: 9), led to less efficient sustained gene silencing.

[0225] To detect free Dnmt3A cleaved from the fusion protein, Western blot analysis was performed using the all-in-one protein variants. As shown in Figure 7B, variants with little or no detectable free Dnmt3, such as p92 (SEQ ID NO: 4), p100 (SEQ ID NO: 12), p101 (SEQ ID NO: 13), and p102 (SEQ ID NO: 14), were more efficient at sustained gene silencing compared with variants with detectable cleaved Dnmt3A, i.e., p76 (SEQ ID NO: 1), p91 ​​(SEQ ID NO: 3), p96 (SEQ ID NO: 8) and p98 (SEQ ID NO: 10).

[0226] Example 8 As shown in Figure 8A, the pooled screen was assayed to determine the optimal sgRNA that resulted in long-term gene silencing. Four HEK293T cell lines, each with a different gene containing a GFP tag (CLTA, VIM, HIST2H2BE (H2B), and RAB11A), were used. A tiling library consisting of sgRNAs spanning + / - 2.5 kb from the transcription start site (TSS) of each gene was stably expressed in cells by lentiviral delivery followed by transient expression plasmid DNA expressing an all-in-one protein. Four weeks after transfection, cells maintaining gene silencing were sorted to determine the identity of the sgRNA. Figures 8B-8E are flow cytometry histograms showing the percent of cells undergoing gene silencing four weeks after transfection.

[0227] Figures 9A-9D are maps of sgRNA functionality spanning transcription start sites of target genes (CLTA, H2B, RAB11, VIM). Transcription start sites (TSS) and CpG islands are annotated above each plot. Each dot represents one sgRNA, and its efficacy in long-term gene silencing is plotted as the log2 fold change in sgRNA abundance. Nucleosome occupancy (bottom plots) is plotted from the MNase signal.

[0228] Example 9 Figure 10A shows the workflow of a pooled screen of HEK293T cells to determine optimal sgRNA target locations for all-in-one proteins, adapted from a conventional lysine tiling screen in K562 cells to determine optimal sgRNAs for dCas9-KRAB (Gilbert, Horlbeck et al., Cell 2014). sgRNAs were first stably expressed in HEK293T cells by lentiviral delivery followed by transient transfection of a plasmid encoding the all-in-one protein (day 0). Cells expressing the all-in-one protein were sorted (day 2) and grown for an additional 3 days. Cells were split on day 5, from which half was collected as the first time point and the other half was passaged for an additional 10 days (day 15) as the final time point. The growth phenotype (γ) was calculated as the log2 sgRNA enrichment divided by the number of cell doublings between T (initial) and T (final).

[0229] Figures 10B-10E are representative plots showing the growth phenotype of four genes (ARL1, EIF6, SMC3, HEATR1) from an existing dCas9-KRAB / CRISPRi dataset (Gilbert, Horlbeck et al., 2014) in K562 cells along with the all-in-one protein (bottom plot). Each dot represents an sgRNA. The TSS and annotated CpG islands are shown for each gene. The functional sgRNA using the all-in-one protein shows broader coverage and more effective targeting than its functional sgRNA.

[0230] Example 10 Figures 11A-11B provide a comparison of the growth phenotype and nucleosome positioning (from MNase signals) of VPS53 and VPS54, showing the location of functional sgRNAs in nucleosome-depleted regions. Furthermore, compared to dCas9-KRAB / CRISPRi, the use of the all-in-one protein broadens the scope of functional sgRNAs.

[0231] Example 11 In vitro transcription of the two all-in-one variants (p102 (SEQ ID NO: 14) and p112 (SEQ ID NO: 15)) shows full-length synthesis of each design (Figure 12A). Figure 12B provides flow cytometry plots showing expression of p102 and p112 one day after transfection of mRNA into HEK293T cells. Figure 12C shows the time course of CLTA endogenous gene silencing in HEK293T cells after transfection with mRNA expressing p102 and p112 all-in-one variants.

[0232] Example 12 Figure 13A provides flow cytometry plots showing induced expression of all-in-one protein by addition of doxycycline in K562 cells stably encoding the all-in-one protein under a doxycycline-inducible promoter. Protein expression was followed for 4 days after doxycycline induction. The dotted line in the panel of Figure 13A represents the baseline median BFP fluorescence without doxycycline administration. Western blots of cells were performed to detect expression of all-in-one protein before and after doxycycline treatment (Figure 13B). The presence of all-in-one protein is not detectable by 96 hours after induction. Flow cytometry plots of CD81 and CD151 knockdown after 14 days of doxycycline treatment of K562 cells are shown in Figures 13C-13F. The percentage of cells in which the target gene was knocked down is shown. There are no cells in the BFP+ quadrant, so there is no detectable expression of all-in-one protein. Quantification of CD81 and CD151 knockdown after 14 days of doxycycline treatment or without doxycycline treatment is shown in Figure 13G.

[0233] References Ecco et al,Development 144,2017.Lambert et al,Cell 172,2018.Siddique et al.,J.Mol.Biol.,425,2013.Stepper et al,Nucleic Acids Res.,45,2017.Shmakov et al.,Nat.Rev.Microbiol.15,2017.Cebrian-Serrano et al, Mamm. Genome 7-8, 2017. Pulecio et al., Cell Stem Cell 21, 2017.

[0234] Unofficial sequence listing SEQ ID NO:1 (p76 (All-in-one protein sequence, version 1): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, residues 612-912, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined)) [ka] [ka]

[0235] SEQ ID NO:2 (p90(KRAB-dCas9-XTEN16-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN16 (uppercase, 16 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0236] SEQ ID NO:3 (p91(KRAB-dCas9-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0237] SEQ ID NO:4 (p92(KRAB-dCas9-XTEN16-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN16 (16 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics and underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0238] SEQ ID NO:5 (p93(KRAB-dCas9-XTEN80-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN80 (80 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0239] SEQ ID NO:6 (p94(KRAB-dCas9-XTEN80-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN80 (80 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka]

[0240] SEQ ID NO:7 (p95(KRAB-XTEN16-dCas9-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN16 (16 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka]

[0241] SEQ ID NO:8 (p96(KRAB-XTEN16-dCas9-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN16 (16 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka]

[0242] SEQ ID NO:9 (p97(KRAB-XTEN80-dCas9-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN80 (80 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0243] SEQ ID NO: 10 (p98(KRAB-XTEN80-dCas9-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN80 (80 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0244] SEQ ID NO:11 (p99(KRAB-XTEN16-dCas9-XTEN80-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), XTEN16 (16 amino acid sequence), dCas9 (italics), HA tag (lowercase), linker (underlined), SV40 NLS (lowercase italics), XTEN80 (lowercase italics bold, 80 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), al., JMB, 2013, Stepper et al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0245] SEQ ID NO: 12 (p100(KRAB-XTEN16-dCas9-XTEN80-Dnmt3A-Dnmt3L-P2A-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), XTEN16 (16 amino acid sequence), dCas9 (italics), HA tag (lowercase), linker (underlined), SV40 NLS (lowercase italics), XTEN80 (lowercase bold italics, 80 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0246] SEQ ID NO: 13 (p101(KRAB-XTEN80-dCas9-XTEN16-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN80 (lowercase bold italics, 80 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN16 (16 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italic underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0247] SEQ ID NO:14 (p102(KRAB-XTEN80-dCas9-XTEN16-Dnmt3A-Dnmt3L-P2A-BFP): KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN80 (lowercase bold italics, 80 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), XTEN16 (16 amino acid sequence), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italic underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), P2A peptide cleavage sequence (lowercase bold), BFP (lowercase underlined) [ka] [ka]

[0248] SEQ ID NO:15 (p112(Dnmt3A-Dnmt3L-XTEN80-dCas9-BFP-KRAB); KRAB (bold, from Gilbert et al., Cell, 2013, 2014), linker (underlined), XTEN80 (lowercase bold italics, 80 amino acid sequence), dCas9 (italics), HA tag (lowercase), SV40 NLS (lowercase italics), Dnmt3A (bold italics, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), 27 amino acid linker (italics underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016), Dnmt3L (bold underlined, from Siddique et al., JMB, 2013, Stepper et al., NAR, 2016) al.,NAR,2016), BFP (lowercase underlined) [ka] [ka]

[0249] SEQ ID NO: 16 (KRAB, from Gilbert et al., Cell, 2013, 2014) DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP

[0250] SEQ ID NO:17 (Linker) GGSGGGS

[0251] SEQ ID NO:18 (Linker) SGS

[0252] SEQ ID NO:19 (linker) EASGSGRASPGIPGSTR

[0253] SEQ ID NO:20 (Linker) SRAD

[0254] SEQ ID NO:21 (Linker) GSG

[0255] SEQ ID NO:22 (linker SPG

[0256] SEQ ID NO:23 (dCas9)

[0257] SEQ ID NO:24 (HA tag) YPYDVPDYA

[0258] SEQ ID NO:25 (SV40 NLS) PKKKRKV

[0259] SEQ ID NO:26 (Dnmt3A, residues 612-912, from Siddique et al., JMB, 2013; Stepper et al., NAR, 2016) NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAM GVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV

[0260] SEQ ID NO:27 (27 amino acid linker, from Siddique et al., JMB, 2013; Stepper et al., NAR, 2016) SSGNSNANSRGPSFSSGLVPLSLRGSH

[0261] SEQ ID NO: 28 (Dnmt3L, from Siddique et al., JMB, 2013; Stepper et al., NAR, 2016) MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQ RPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL

[0262] SEQ ID NO:29 (P2A peptide cleavage sequence) ATNFSLLKQAGDVEENPGP

[0263] SEQ ID NO:30 (BFP) SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIY NVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*

[0264] SEQ ID NO:31 (XTEN16 (16 amino acid sequence)) SGSETPGTSESATPES

[0265] SEQ ID NO: 32 (XTEN80 (80 amino acid sequence)) GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE

[0266] SEQ ID NO: 33 (Dnmt3A-Dnmt3L domain) NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEG DDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR LARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLG SSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL

[0267] SEQ ID NO:34 (ddAsCfp1)

[0268] SEQ ID NO: 35 (ddLbCfp1)

[0269] SEQ ID NO:36 (ddFnCfp1)

Claims

1. A fusion protein comprising a nuclease-deficient RNA-guided DNA endonuclease enzyme, a Kruppel-associated box domain, and a DNA methyltransferase domain.

2. 2. The fusion protein of claim 1, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9, ddCpf1, a nuclease-deficient Cas9 variant, or a nuclease-deficient class II CRISPR endonuclease.

3. The fusion protein of claim 1, wherein the DNA methyltransferase domain is a Dnmt3A-3L domain.

4. 2. The fusion protein of claim 1, comprising, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease enzyme, and the Kruppel-associated box domain.

5. 5. The fusion protein of claim 4, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

6. The fusion protein of claim 5, wherein the dCas9 is covalently linked to the Dnmt3A-3L domain via a peptide linker, and the Dnmt3A-3L domain is covalently linked to the Krüppel-associated box domain via a peptide linker.

7. The fusion protein of claim 6 , wherein the peptide linker is an XTEN linker.

8. 2. The fusion protein of claim 1, wherein the fusion protein comprises, from N-terminus to C-terminus, the Kruppel-associated box, the nuclease-deficient RNA-guided DNA endonuclease enzyme, and the DNA methyltransferase domain.

9. 9. The fusion protein of claim 8, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain is a Dnmt3A-3L domain.

10. The fusion protein of claim 9, wherein the dCas9 is covalently linked to the Dnmt3A-3L domain via a peptide linker, and the Kruppel-associated box domain is covalently linked to the dCas9 via a peptide linker.

11. The fusion protein of claim 10, wherein the peptide linker is an XTEN linker.

12. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the Kruppel-associated box domain via a peptide linker.

13. 2. The fusion protein of claim 1, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the DNA methyltransferase domain via a peptide linker.

14. The fusion protein of claim 1 , wherein the Kruppel-associated box domain is covalently linked to the DNA methyltransferase domain via a peptide linker.

15. The fusion protein of claim 12, wherein the peptide linker is an XTEN linker.

16. The fusion protein of claim 15, wherein the XTEN linker comprises about 16 to 80 amino acid residues.

17. The fusion protein of claim 1 further comprising a nuclear localization signal peptide.

18. The fusion protein of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

19. A nucleic acid sequence encoding the fusion protein of claim 1.

20. 20. The nucleic acid sequence of claim 19, wherein the nucleic acid sequence is messenger RNA.

21. 11. A complex comprising: (i) the fusion protein of claim 1; and (ii) a polynucleotide comprising: (a) a DNA targeting sequence complementary to a target polynucleotide sequence; and (b) a binding sequence for said nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said polynucleotide via said binding sequence.

22. 22. The complex of claim 21 , wherein the target polynucleotide sequence is a portion of a gene.

23. 22. The complex of claim 21, wherein the target polynucleotide sequence is part of a transcriptional regulatory sequence.

24. 22. The complex of claim 21, wherein the target polynucleotide sequence is part of a promoter, enhancer, or silencer.

25. 22. The complex of claim 21, wherein the target polynucleotide sequence is within about 3000 bp adjacent to a transcription start site.

26. A vector comprising the nucleic acid sequence of claim 19.

27. 27. The vector of claim 26, further comprising a polynucleotide, said polynucleotide comprising: (a) a DNA targeting sequence complementary to a target polynucleotide sequence; and (b) a binding sequence for said nuclease-deficient RNA-guided DNA endonuclease enzyme.

28. A cell comprising the fusion protein of claim 1, the nucleic acid of claim 19, the complex of claim 21, or the vector of claim 26.

29. The cell of claim 28 , wherein the cell is a eukaryotic cell.

30. The cell of claim 28 , wherein the cell is a mammalian cell.

31. 11. A method for silencing a target nucleic acid sequence in a cell, comprising: (i) delivering a first polynucleotide encoding the fusion protein of claim 1 to a cell containing the target nucleic acid; and (ii) delivering a second polynucleotide to the cell, the second polynucleotide comprising (a) a DNA targeting sequence complementary to the target nucleic acid sequence and (b) a binding sequence for the nuclease-deficient RNA-guided DNA endonuclease enzyme.

32. 32. The method of claim 31 , wherein the fusion protein silences the target nucleic acid sequence in the cell by methylating chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark into chromatin containing the target nucleic acid sequence.

33. 32. The method of claim 31 , wherein the first polynucleotide is contained within a first vector.

34. 32. The method of claim 31 , wherein the first polynucleotide is contained within a second vector.

35. 35. The method of claim 34, wherein the first vector and the second vector are the same.

36. 35. The method of claim 34, wherein the first vector is different from the second vector.

37. 32. The method of claim 31 , wherein the cell is a mammalian cell.

38. 32. The method of claim 31 , wherein the method has a specificity that is 2-fold higher than the specificity for non-target nucleic acid sequences.

39. 22. A method for silencing a target nucleic acid sequence in a cell, comprising delivering the complex of claim 21 to a cell containing the target nucleic acid.

40. 40. The method of claim 39, wherein the complex silences the target nucleic acid sequence in the cell by methylating chromatin containing the target nucleic acid sequence and / or by introducing a repressive chromatin mark into chromatin containing the target nucleic acid sequence.

41. 40. The method of claim 39, wherein the cell is a mammalian cell.

42. 40. The method of claim 39, wherein the method has a specificity that is 2-fold greater than the specificity for non-target nucleic acid sequences.

Images