Compositions and methods for effecting epigenetic modifications

Abstract

In various embodiments, the present disclosure provides fusion proteins comprising a DNA-binding domain, a DNMT3 A-binding domain, and H3K4me0, as well as polynucleotides and vectors encoding one or more of the fusion proteins. In various embodiments, the present disclosure also provides gene delivery systems, cells, compositions (e.g., pharmaceutical compositions), and kits comprising one or more of the fusion protein polynucleotides or vectors, methods of epigenetically modifying genomic loci in cells, and methods of treating a subject (e.g., a human) in need thereof.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 485,860, filed February 17, 2023, and claims the benefit of U.S. Provisional Application No. 63 / 520,594, filed August 18, 2023, the entire teachings of which are incorporated herein by reference.

[0002] Government support This invention was made with government support under grant number 5U19NS132315 from the National Institutes of Health and NIH 1RM1 HG009490-06 CEGS. The government has certain rights in this invention.

[0003] Including material by reference in XML This application incorporates by reference the sequence listing contained in the following extensible markup language (XML) file filed concurrently herewith: a) File name: 03992071-002.xml, created on February 16, 2024, size 1,470,492 bytes. [Background technology]

[0004] Programmable gene regulation is a promising therapeutic approach that exhibits several advantages over genetic engineering, including tunability, reversibility, and a lack of DNA cleavage-related cytotoxicity. Epigenetic silencing works by introducing epigenetic modifications that reduce the expression of target genes rather than introducing mutations into the target gene. Therefore, there is no risk of toxicity from chronic expression of mutant messages encoding damaged, truncated proteins that strain the cell's nonsense-mediated decay machinery. However, therapeutic applications of current programmable gene regulation designs face challenges. For example, overexpression of potentially toxic enzymes, such as DNA methyltransferases, can result in off-target effects. Furthermore, available constructs often exceed the packaging capacity of certain delivery vectors, such as adeno-associated virus (AAV) vectors. Therefore, there is a need for more compact and less toxic epigenetic editors. Summary of the Invention

[0005] The disclosure provided herein is based in part on the discovery that a fusion protein comprising the DNMT3L C-terminal domain and an unmethylated H3 tail can recruit endogenous DNMT3A methyltransferase to a predetermined genomic locus in a cell. The disclosure generally relates to compositions, such as fusion proteins, polynucleotides, vectors, gene delivery systems, cells, pharmaceutical compositions, kits, and methods useful for making epigenetic modifications.

[0006] In one aspect, the present disclosure provides a fusion protein comprising a DNA-binding domain, a DNMT3 methyltransferase-binding domain, and H3K4me0.

[0007] In another aspect, the present disclosure provides a fusion protein comprising a nuclease sequence, a DNMT3 methyltransferase binding domain, and H3K4me0. In some embodiments, the nuclease sequence lacks nuclease activity (e.g., is nuclease-deficient).

[0008] In some embodiments, the fusion protein lacks nuclease activity. In some embodiments, the fusion protein further comprises a histone methyltransferase. In some embodiments, the fusion protein lacks a DNA methyltransferase catalytic domain.

[0009] In another aspect, the disclosure provides a polynucleotide encoding any one or more of the fusion proteins disclosed herein. In some embodiments, the polynucleotide is about 4.7 kb or less in length.

[0010] In another aspect, the present disclosure provides a vector comprising any one or more of the polynucleotides disclosed herein. In some embodiments, the vector is about 4.7 kb or less in length.

[0011] In another aspect, the present disclosure provides a gene delivery system comprising any one of the polynucleotides or vectors disclosed herein. In some embodiments, the gene delivery system comprises an adeno-associated viral vector (AAV).

[0012] In another aspect, the present disclosure provides a composition comprising any one or more of the fusion proteins, polynucleotides, vectors, or gene delivery systems disclosed herein, or any combination of the foregoing. In some embodiments, the composition is a pharmaceutical composition.

[0013] In another aspect, the disclosure provides a kit comprising a container and optionally instructions for use, wherein the container comprises any one or more of the fusion proteins, polynucleotides, vectors, gene delivery systems, compositions, or pharmaceutical compositions disclosed herein, or any combination of the foregoing.

[0014] In another aspect, the present disclosure provides a cell comprising any one or more of the fusion proteins, polynucleotides, vectors, or gene delivery systems disclosed herein, or any combination of the foregoing.

[0015] In another aspect, the disclosure provides progeny cells, wherein the progeny cells are derived from a cell that comprises any one or more of the fusion proteins, polynucleotides, vectors, or gene delivery systems disclosed herein, or any combination of the foregoing.

[0016] In another aspect, the present disclosure provides a method of epigenetically modifying a genomic locus in a cell, the method comprising delivering to the cell any one or more of the fusion proteins, polynucleotides, or vectors disclosed herein, or any combination of the foregoing.

[0017] In another aspect, the present disclosure provides an epigenetically modified cell or progeny thereof produced by any one or more of the methods disclosed herein.

[0018] In another aspect, the disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject any one or more of the fusion proteins, polynucleotides, gene delivery systems, compositions, pharmaceutical compositions, or cells disclosed herein. In some embodiments, the subject is a human.

[0019] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] The foregoing will become apparent from the following more detailed description of exemplary embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. [Brief explanation of the drawings]

[0021] [Figure 1A] Assay and epigenetic editor construct. Monoclonal HEK293T cell lines were generated by tagging the CLTA gene with the mScarlet reporter, followed by single-cell sorting and delivery of a lentiviral vector containing mU6-sgRNA targeting the CLTA transcription start site. Transfected cells were sorted 2 days posttransfection and monitored for mScarlet silencing. [Figure 1B] Assays and epigenetic editor constructs. Schematic diagram of the dCas9 epigenetic editor fusion proteins tested for silencing activity. D3L indicates the Dnmt3l C-terminal domain. [Figure 2A] Time course of reporter silencing. Histogram of mScarlet fluorescence in cells of the mScarlet-CLTA monoclonal reporter cell line transiently transfected with the indicated epieditor constructs. D3L, Dnmt3l C-terminal domain; PT, post-transfection. [Figure 2B] Time course of reporter silencing. Time course plot of mScarlet fluorescence corresponding to the transfected cell lines in Figure 2A. Data are mean ± SD of n=2 replicates. [Figure 3A] The editor was not dose- or expression-limited. Percent silencing of the mScarlet-CLTA reporter 18 days after transfection with different amounts of DNA. [Figure 3B]The editor was not dose- or expression-limited. Percent silencing of the mScarlet-CLTA reporter at 16 days post-transfection after sorting at different levels of protein expression from I (low) to IV (high). H3, H3K4me0.D3L, Dnmt3l C-terminal domain. Data are means ± SD of n=2 replicates. [Figure 4A] Linker optimization. Percent silencing of the mScarlet-CLTA reporter over a 3-week period using different standard fusion protein linkers. [Figure 4B] Linker optimization. Percent silencing of the mScarlet-CLTA reporter over 3 weeks using the N-term bpNLS signal and double H3 fusion. [Figure 4C] Linker optimization. Percent silencing of the mScarlet-CLTA reporter at 15 days post-transfection tiling different length linkers connecting H3 to D3L. H3, H3K4me0. H3mut, H3A4me0 mutant. D3L, Dnmt3l C-terminal domain. N-term bpNLS signal, N-terminal bipartite nuclear localization signal. Data are mean ± SD of n=2 replicates. [Figure 5A] Improved genome mining for DNMT3L. Phylogenetic tree of DNMT3L orthologues. Proteins with silencing activity of >5% of the reporter are labeled. [Figure 5B] Genome mining for improved DNMT3L. Percent silencing of the mScarlet-CLTA reporter at 16 days post-transfection using D3L-dCas9 fusions. ASR, ancestral reconstruction. D3L, Dnmt3l C-terminal domain. Data are mean ± SD of n=2 replicates. [Figure 6] H3-D3L-ZFP-KRAB is equivalent to CRISPRoff. Percent silencing of the CD55 cell surface marker (stained with APC-conjugated antibody) at day 6 posttransfection. D3L, Dnmt3l C-terminal domain. H3, H3K4me0. [Figure 7] Non-limiting example of an H3 epieditor. Schematic of a histone tail-based epigenetic editor through multiple rounds of optimization. Drug candidates are designed to be below the AAV vector packaging limit of 4.7 kilobases of DNA. D3L, Dnmt3l C-terminal domain. H3, H3K4me0. Mm, Mus musculus. As, Apodemus sylvaticus. [Figure 8A] Non-limiting example of an H3 epieditor. Schematic of a histone tail-based epigenetic editor through multiple rounds of optimization. [Figure 8B] Non-limiting examples of H3 epieditors. Percent silencing of the mScarlet-CLTA reporter in CLTA reporter cell lines transiently transfected with each epieditor indicated. Data are mean ± SD of n=2 replicates. [Figure 9] In vivo knockdown of PrP. ELISA of prion protein from right hemisphere brain homogenates harvested from mice 6 weeks after AAV injection with each indicated transgene. Data are mean ± SD of n=8 replicates. [Figure 10] In vivo methylation of the Prnp promoter. Nanopore long-read sequencing of native DNA extracted from coronal sections of the left mouse brain hemisphere after 6 weeks of treatment. Each column represents a single molecule from an independent cell covering a 5-kilobase region surrounding the Prnp transcription start site (TSS). [Figure 11] Knockdown of Prnp transcripts in neurons by HCR-FISH. For HCR-FISH, 100 μm-thick coronal brain sections were used simultaneously from both mock-treated mice (top) and mice treated with H3V4-ZFP-KRAB AAV (bottom) for Rbfox3 (neuron-specific) and Prnp transcripts. DAPI is a nuclear marker. Scale bar is 100 μm. [Figure 12A] Non-limiting examples of self-silencing epieditors. Experimental design. [Figure 12B]Non-limiting example of a self-silencing epieditor. Flow cytometry data assessing epieditor and target gene expression 6, 14, and 60 days after lentiviral transduction of a self-silencing epieditor in Neuro-2a cells. Epieditor expression was measured via TagBFP fluorescence, and target gene expression was measured using a fluorescently conjugated antibody against the target gene product. [Figure 13] Split intein effectors function well in HEK293T cells. HEK cells transduced with lentiviruses packaging direct fusions of the H3V4 Krabby Patty effector and a ZFP targeting the HsPRNP gene promoter were as effective at silencing the target as effectors separated from the ZFP by a T2A-P2A sequence. The histogram represents flow cytometry measurements of cells stained with an APC-conjugated PrP antibody (BioLegend antibody 6D11). ZFPs r96a and r99a were custom designed and purchased from Sigma-Aldrich's zinc finger design pipeline (licensed from Sangamo Therapeutics). The method was the same as in Figure 12A, except HEK293T cells were used instead of N2a cells. [Figure 14] Schematic of a split-intein-based multiplexing strategy in an AAV vector. Using a split-intein strategy, multiple DNA-binding domains can be multiplexed with a single effector. The ERT2 engineered estrogen receptor domain has been used in synthetic biology applications requiring drug-controllable nuclear accumulation. Self-binding ZFPs can be combined with target-binding ZFPs to enable the AAV to turn off its transgene cassette after the target gene has been silenced. [Figure 15A]PRNP is a viable target for epigenetic silencing, but existing technologies are not suitable for therapeutic use. HEK293T cell lines were generated by incorporating a lentiviral vector containing mU6-sgRNA targeting the PRNP transcription start site (TSS). Transfected cells were sorted by FACS (TagBFP) 2 days after transfection and monitored for PRNP silencing by Alexa Fluor 647 anti-PRNP staining and flow cytometry. [Figure 15B] Although PRNP is a viable target for epigenetic silencing, existing technologies are not suitable for therapeutic use. Time course of PRNP and effector expression in HEK293T cells transiently transfected with plasmids encoding CRISPRi and CRISPRoff effectors. Data are mean ± SEM of n=2 replicates. [Figure 15C] PRNPs are viable targets for epigenetic silencing, but existing technologies are not suitable for therapeutic applications. DNA methylation was assessed by targeted nanopore long-read sequencing of native genomic DNA extracted from HEK293T cells 50 days after transfection. [Figure 15D] Prnp is a viable target for epigenetic silencing, but existing technologies are not suitable for therapeutic use. Mouse N2a cells co-transfected with a plasmid encoding CRISPRi / CRISPRoff and three sgRNAs targeting the TSS of Prnp were evaluated for Prnp expression and DNA methylation. Data are the mean ± SEM of n = 2 replicates. [Figure 15E] PRNPs are viable targets for epigenetic silencing, but existing technologies are not suitable for therapeutic applications. Schematic depicting, to scale, the AAV genome packaging constraints with CRISPRoff and ZFPoff. [Figure 15F]Although PRNP is a viable target for epigenetic silencing, existing technologies are not suitable for therapeutic use. HEK293T cells were transiently transfected with ZFPoff and D3L-ZFP-KRAB and imaged 6 days later. [Figure 15G] Although PRNP is a viable target for epigenetic silencing, existing technologies are not suitable for therapeutic use. HEK293T cells were transiently transfected with ZFPoff and D3L-ZFP-KRAB and imaged 6 days later. [Figure 16A] Figure 1. Reproducibility of in vitro nanopore analysis and DNMT3A recruitment. Nanopores were performed in two biological replicates and Pearson correlation coefficients are shown. [Figure 16B] Figure 1. Reproducibility of in vitro nanopore analysis and DNMT3A recruitment. Nanopores were performed in two biological replicates and Pearson correlation coefficients are shown. [Figure 16C] Figure 1. Reproducibility of in vitro nanopore analysis and DNMT3A recruitment. Nanopores were performed in two biological replicates and Pearson correlation coefficients are shown. [Figure 16D] Figure 1. Reproducibility of in vitro nanopore analysis and DNMT3A recruitment. Nanopores were performed in two biological replicates and Pearson correlation coefficients are shown. [Figure 16E] In vitro nanopore analysis and reproducibility of DNMT3A recruitment. HEK293T cells containing the mScarlet-CLTA reporter and constitutively expressing sgRNAs targeting the CLTA TSS and full-length DNMT3A-EGFP fusion were transfected with effector fusions of dCas9, D3L, KRAB, and GFP nanobody (GNb). Cells were monitored for reporter silencing by flow cytometry. Data are the mean ± SEM of n=2 replicates. [Figure 16F]In vitro nanopore analysis and reproducibility of DNMT3A recruitment. HEK293T cells containing the mScarlet-CLTA reporter and constitutively expressing sgRNAs targeting the CLTA TSS and full-length DNMT3A-EGFP fusion were transfected with effector fusions of dCas9, D3L, KRAB, and GFP nanobody (GNb). Cells were monitored for reporter silencing by flow cytometry. Data are the mean ± SEM of n=2 replicates. [Figure 17A] Schematic of CHARM dose titration and optimization. Schematic of DNMT3A1 domains. [Figure 17B] Schematic of CHARM dose titration and optimization. Schematic of the DNMT3L domain. [Figure 17C] Schematic of CHARM dose titration and optimization. HEK293T cells containing the mScarlet-CLTA reporter and sgRNA against the CLTA TSS were targeted with effectors containing full-length (FL) or catalytic-only (D3A) fusions of DNMT3A and D3L-dCas9 and monitored for silencing by flow cytometry. Data are the mean ± SEM of n=2 replicates. [Figure 17D] Schematic of CHARM dose titration and optimization. mScarlet-CLTA reporter HEK293T cells were transiently transfected with different nanogram (ng) amounts of plasmid DNA encoding D3L-dCas9 or CHARM effectors. Data are mean ± SEM of n=2 replicates. [Figure 17E] Schematic of CHARM dose titration and optimization. mScarlet-CLTA reporter HEK293T cells were transiently transfected with plasmids encoding D3L-dCas9 or CHARM effectors and sorted into four vessels based on transgene expression levels. Data are mean ± SEM of n=2 replicates. [Figure 17F]Schematic of CHARM dose titration and optimization. mScarlet-CLTA reporter HEK293T cells were transiently transfected with plasmids encoding D3L-dCas9 or CHARM effectors and sorted into four vessels based on transgene expression levels. Data are mean ± SEM of n=2 replicates. [Figure 17G] Schematic of CHARM dose titration and optimization. mScarlet-CLTA reporter HEK293T cells were transiently transfected with different plasmids encoding D3L-dCas9 or CHARM effectors with various N-terminal appendages. Data are the mean ± SEM of n=2 replicates. [Figure 17H] Schematic diagram of CHARM dosage titration and optimization. Schematic diagram of the complete optimization history and nomenclature for non-limiting example of CRISPRcharm effector. The term " kv1 " is added when CRISPRcharm effector contains KRAB domain at its C-terminus. CRISPRcharm3 is also called CRISPRcharm, CRISPRcharm3 Kv1 is also called CRISPRcharm Kv1, and CRISPRcharm3 Kv2 is also called CRISPRcharm Kv2. [Figure 17I] Schematic of CHARM dose titration and optimization. To improve the dynamic range of silencing, mScarlet-CLTA reporter HEK293T cells harboring a mismatched sgRNA against the CLTA TSS were transiently transfected with plasmids encoding either the CRISPRcharm Kv1 or CRISPRcharm Kv2 effector. Data are mean ± SEM of n=2 replicates. [Figure 18A] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Cartoon depiction of endogenous DNMT3A recruitment and activation with an unrestricted example of the CHARM system. [Figure 18B]Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Time course of effector (TagBFP) and mScarlet-CLTA reporter expression after transient transfection with effector-containing plasmids. Data are mean ± SEM of n=2 replicates. [Figure 18C] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. First-pass histone H3 tail fusion studies on the mScarlet-CLTA reporter using different linkers for D3L. [Figure 18D] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Improved linker sequence between H3 tail and D3L. Data are means ± SEM of n=2 replicates. [Figure 18E] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Phylogenetic tree of DNMT3L orthologs and ancestral reconstructed nodes. Orthologs with >5% measured silencing activity by day 14 are labeled. [Figure 18F] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Repression of the mScarlet-CLTA reporter 2 weeks after transfection by different DNMT3L ortholog C-terminal domains fused to dCas9. Data are mean ± SEM of n=2 replicates. [Figure 18G]The histone H3 tail fused to the Dnmt3l C-terminal domain functions as a potent mediator of DNA methylation and transcriptional silencing. Transient transfection and silencing of the mScarlet-CLTA reporter with different lengths of histone H3 domain (FL, full-length). To improve the dynamic range, a mismatched sgRNA against the CLTA TSS is used. Data are the mean ± SEM of n=2 replicates. [Figure 18H] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Time course of effector expression and mScarlet-CLTA silencing comparing CRISPRoff and CRISPRi with a series of optimized CHARM constructs. Data are mean ± SEM of n=2 replicates. [Figure 18I] Histone H3 tails fused to the Dnmt3l C-terminal domain function as potent mediators of DNA methylation and transcriptional silencing. Comparison of CRISPRi, CRISPRoff, and optimized CRISPRcharm effectors in silencing cell surface markers. Plasmids were transiently co-transfected with vectors encoding mU6-sgRNA. Data are mean ± SEM of n=2 replicates. [Figure 19A] CHARMs are flexible and specific. Mouse N2a cells were transiently transfected with plasmids encoding ZFcharm Kv1 constructed with mouse Prnp-targeting ZFP81187, ZFP81201, or a non-targeting ZFP and stained with Alexa Fluor 647 anti-PRNP. Data are the mean ± SEM of n = 2 replicates. [Figure 19B] CHARMs are flexible and specific. Mouse N2a cells were transiently transfected with TALEcharm, consisting of engineered TALE proteins targeting mouse Prnp TSS or non-targeted TALEs, and TALEcharm Kv2, and then measured using Alexa Fluor 647 anti-PRNP. Data are the mean ± SEM of n=2 replicates. [Figure 19C] CHARM is both flexible and specific. Schematic of potential AAV packaging strategies using space-saving technologies such as split inteins19 or self-silencing approaches involving the tamoxifen-inducible engineered estrogen receptor ERT221. WPRE3 is a structured 3' element for mRNA stability56. [Figure 19D] CHARM is both flexible and specific. HEK293T cells were transiently transfected with plasmids encoding ZFP81187 alone or the fusions ZFPoff, D3L-ZFP-KRAB, and ZFcharm Kv1, and then counted by flow cytometry after cell viability staining with LIVE / DEAD near-infrared dye. Data are the mean ± SEM of n=3 replicates. [Figure 19E] CHARMs are flexible and specific: ZFcharm Kv1, which uses ZFP81201 targeting the mouse Prnp TSS, was introduced into N2a cells by lentiviral transduction and assessed for knockdown and specificity by RNA sequencing 4 weeks later. [Figure 19F] CHARM is flexible and specific: CRISPRcharm Kv1 carrying sgRNAs targeting the mouse Prnp TSS or a non-targeting sgRNA was introduced into N2a cells by lentiviral transduction, and specificity was assessed by Prnp knockdown and RNA sequencing 4 weeks later. [Figure 20A] CHARM variations using split inteins or small Cas effectors. HEK293T cells were transduced with lentivirus encoding ZFcharm Kv1, which carries a ZFP targeting the human PRNP TSS. Direct fusion and split intein CHARMs are equally effective at suppressing PRNP, as measured by Alexa Fluor 647 anti-PRNP. [Figure 20B]CHARM variations using split inteins or small Cas effectors. HEK293T cells containing the mScarlet-CLTA reporter and SaCas9 sgRNA against the CLTA TSS were transiently transfected with CRISPRcharm constructs using dSaCas9 as the DNA-binding domain. These were monitored for CLTA silencing by flow cytometry. [Figure 20C] CHARM variations using split inteins or small Cas effectors. Quantified Prnp RNA levels in N2a cells transduced with ZFcharm Kv1 or CRISPRcharm Kv1 lentiviral constructs corresponding to CHARM variations using split inteins or small Cas effectors (Figures 19E-19F). Data are mean ± SEM of n=2 replicates. [Figure 21A] AAV-delivered ZFcharm represses and methylates Prnp in vivo. Schematic of experimental design. [Figure 21B] AAV-delivered ZFcharm represses and methylates Prnp in vivo. PRNP ELISA and Prnp RT-qPCR data generated from brain hemisphere homogenates 6 weeks after injection of 1.5e13vg / kg AAV. [Figure 21C] AAV-delivered ZFcharm represses and methylates Prnp in vivo. AAV dose-response analysis using KRAB-containing ZFcharm. [Figure 21D] AAV-delivered ZFcharm represses and methylates Prnp in vivo. Quantification of DNA methylation at the Prnp promoter in treated and untreated brains 6 weeks after injection. 5mCpGs were detected by targeted enrichment nanopore sequencing. [Figure 21E]AAV-delivered ZFcharm represses and methylates Prnp in vivo. Visualization of Prnp (yellow) and pan-neuronal marker Uchl1 (magenta) expression in 10 μm coronal brain sections via HCR RNA-FISH (blue DAPI staining). Representative maximum intensity projection of a coronal brain hemispheric tile scan. The white box indicates the brain region shown in panel F. Scale bar, 1 mm. [Figure 21F] AAV-delivered ZFcharm represses and methylates Prnp in vivo. Visualization of Prnp (yellow) and the pan-neuronal marker Uchl1 (magenta) expression in 10 μm coronal brain sections via HCR RNA-FISH (blue DAPI staining). Zoom-in views of the cortex (CTX), hippocampus (HP), and thalamus (TH). Scale bar, 100 μm. [Figure 21G] AAV-delivered ZFcharm represses and methylates Prnp in vivo. Single-cell identification of Prnp+ (yellow) and Prnp- (magenta) neurons in the coronal brain hemisphere shown in panel E. Prnp-expressing Uchl1+ neurons were identified via machine learning classification using QuPath software. Cell boundaries represent a 4 μm extension from the DAPI-detected nucleus. [Figure 21H] AAV-delivered ZFcharm represses and methylates Prnp in vivo. Representative histogram of mean Prnp intensity in neurons. [Figure 21I] 1 is a bar graph showing % Prnp+ neurons in treated and untreated brains based on QuPath classification. [Figure 22A] CHARMs can be programmed for transient expression through self-silencing. Schematic of the experimental approach used to develop self-silencing ZFcharms. [Figure 22B] CHARM can be programmed for transient expression through self-silencing. Quantification of self-silencing kinetics by measuring ZFcharm and Prnp expression over time following lentiviral transduction of N2a cells. [Figure 22C]CHARM can be programmed for transient expression through self-silencing. Flow cytometry histograms of ZFcharm (orange, TagBFP) and PRNP (navy, Alexa Fluor 647 anti-CD230) expression at days 6, 14, and 60 post-infection. [Figure 22D] CHARM can be programmed for transient expression through self-silencing. Clonal bisulfite sequencing of the EFS promoter driving expression of ZFcharm-SCR and ZF-charm-SPM at 5 and 25 days postinfection. The percent 5mCpG (black) was calculated for each CpG site across PCR clones and depicted as a pie chart. The locations of the CAAT box, EFP1, EFP2, TATA box, initiator element, and TSS sites within the EFS promoter are shown in the schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 22E] CHARM can be programmed for transient expression through self-silencing. A 60-day flow cytometry time course monitoring ZFcharm and PRNP expression across ZF-SPM constructs. [Figure 22F] CHARM can be programmed for transient expression through self-silencing. ZFcharm-SPM and PRNP expression quantified by flow cytometry 6 months after infection. [Figure 22G] CHARMs can be programmed for transient expression through self-silencing. Schematic of the experimental strategy for designing modular self-silencing ZFcharms using two different ZF domains. [Figure 22H]CHARM can be programmed for transient expression through self-silencing. Tuning self-silencing kinetics using an allelic series of ZF3 backbone RtoA mutations. ZFcharm (orange, mCherry) and PRNP (navy, Alexa Fluor 647 anti-CD230) expression was quantified by flow cytometry at 9 and 22 days post-infection. [Figure 23A] Confirmation of ZFcharm self-silencing efficacy. ZFcharm and Prnp expression over time after transfection of piggyBac donor and transposase plasmids in N2a cells. [Figure 23B] Confirmation of ZFcharm self-silencing efficacy. Clonal bisulfite sequencing of the EFS promoter driving ZFcharm-SCR and ZF-charm-SPM expression 5 and 25 days after infection. Each strain is an individual PCR clone. Circles depict methylated (black) and unmethylated (white) CpG sites. Sequence elements within the EFS promoter are shown in a schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray. [Figure 23C] Validation of ZFcharm self-silencing efficacy. Bar graph showing the mean % of 5mCpGs between the TATA box and the TSS. [Figure 23D] Confirmation of ZFcharm self-silencing efficacy. Flow cytometry histograms of ZF editor and PRNP expression 6 and 60 days after infection across ZF-SPM constructs. [Figure 24A] Optimization of modular self-silencing ZFcharm constructs. As shown above for dCas9-based targeting, placing a KRAB domain in the linker region between the H3 tail and D3L is permissive when using ZF-mediated DNA binding (Figure S2H). [Figure 24B] Optimization of modular self-silencing ZFcharm constructs. ZFcharms with a ZF3 DNA binding domain can rapidly silence themselves. [Figure 24C] Optimization of modular self-silencing ZFcharm constructs. Placing NLS sequences in both the N and C exteins improves self-silencing. [Figure 24D] Optimization of modular self-silencing ZFcharm constructs. Schematic of an alternative approach to tailoring modular self-silencing ZFcharm. [Figure 24E] Optimization of the modular self-silencing ZFcharm construct. Introduction of a point mutation into the ZF3 binding site upstream of the EFS promoter slows the rate of self-silencing. All flow cytometry data shown in this figure represent two technical replicates and were collected on the indicated days after lentiviral transduction of N2a cells. [Figure 25A] Self-silencing ZFcharm is functional in vivo. Schematic of experimental design. [Figure 25B] Self-silencing ZFcharm is functional in vivo. PRNP ELISA and Prnp RT-qPCR data generated from brain hemisphere homogenates 6 weeks after injection of 1.5e13vg / kg AAV. AAV capsids were packaged with self-silencing ZFcharm constructs containing (Figure 25B) or lacking (Figure 25C) the KRAB domain. [Figure 25C] Self-silencing ZFcharm is functional in vivo. PRNP ELISA and Prnp RT-qPCR data generated from brain hemisphere homogenates 6 weeks after injection of 1.5e13vg / kg AAV. AAV capsids were packaged with self-silencing ZFcharm constructs containing (Figure 25B) or lacking (Figure 25C) the KRAB domain. [Figure 25D]The self-silencing ZF charm is functional in vivo. Clonal bisulfite sequencing of the EFS promoter driving expression of the self-silencing ZF charm construct containing (Figure 25D) or lacking (Figure 25E) the KRAB domain. The percent 5mCpG (black) was calculated for each CpG site across PCR clones and depicted as a pie chart. Sequence elements within the EFS promoter are shown in the schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 25E] The self-silencing ZF charm is functional in vivo. Clonal bisulfite sequencing of the EFS promoter driving expression of the self-silencing ZF charm construct containing (Figure 25D) or lacking (Figure 25E) the KRAB domain. The percent 5mCpG (black) was calculated for each CpG site across PCR clones and depicted as a pie chart. Sequence elements within the EFS promoter are shown in the schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 26A] Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Each line is an individual PCR clone. Circles depict methylated (black) and unmethylated (white) CpG sites. Sequence elements within the EFS promoter are shown in a schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 26B]Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Each line is an individual PCR clone. Circles depict methylated (black) and unmethylated (white) CpG sites. Sequence elements within the EFS promoter are shown in a schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 26C] Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Bar graph showing the average 5mCpG percentage between the TATA box and the TSS. [Figure 26D] Clonal bisulfite sequencing of the EFS promoter driving the self-silencing ZFcharm construct in vivo. Clonal bisulfite sequencing of the EFS promoter in single-stranded AAV genomic DNA extracted from ZFcharm-SPM virus. [Figure 27A] Prnp silencing is stable after ZFcharm self-silencing in vivo. Schematic of experimental design. [Figure 27B] Prnp silencing is stable after ZFcharm self-silencing in vivo. PRNP ELISA and Prnp RT-qPCR data generated from brain hemisphere homogenates 13 weeks after injection of 1.5e13vg / kg AAV. [Figure 27C]Prnp silencing is stable after ZFcharm self-silencing in vivo. Clonal bisulfite sequencing of the EFS promoter driving expression of self-silencing ZFcharm 13 weeks after AAV infection. Each strain is an individual PCR clone. Circles depict methylated (black) and unmethylated (white) CpG sites. Sequence elements within the EFS promoter are shown in the schematic diagram below the data. The CpG between the TATA box and TSS is highlighted in gray, and the ZF binding site is highlighted in orange. [Figure 27D] Prnp silencing is stable after ZFcharm self-silencing in vivo. Bar graph showing the average 5mCpG percentage between the TATA box and the TSS. [Figure 28A] Self-silencing ZF charm methylates the Prnp promoter in vivo. Targeted enrichment nanopore sequencing of the Prnp promoter in brains harvested 13 weeks after AAV injection. [Figure 28B] Self-silencing ZF charm methylates the Prnp promoter in vivo. 5mCpG quantification via nanopore sequencing is reproducible. For each condition, 5mCpG percent is compared between two biological replicates. Pearson correlations are shown on each graph. DETAILED DESCRIPTION OF THE INVENTION

[0022] A description of exemplary embodiments follows.

[0023] Unless otherwise defined, all technical terms, notations, and other scientific terms or terms used herein are intended to have the meanings that are commonly understood by those skilled in the art to which this disclosure pertains.In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art.It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meanings in the context of the relevant art and / or as otherwise defined herein.

[0024] definition Certain terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0025] As used herein, the terms "a," "an," or "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0026] As used herein, unless the context requires otherwise, the term "comprise," as well as variations such as "comprises" or "comprising," will be understood to imply, for example, the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including."

[0027] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. When used herein in the context of aspects or embodiments of the present disclosure, any of the terms "comprising," "containing," "including," and "having" can be substituted in some embodiments with the terms "consisting of" or "consisting essentially of" to vary the scope of the disclosure.

[0028] As used herein, the conjunction term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. If two or more of the options are simultaneously applicable, it is also understood to be within the meaning and therefore meets the requirements of the term "and / or."

[0029] Where lists are presented, unless otherwise expressly stated, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0030] Unless otherwise indicated, or otherwise apparent from the context and / or understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can assume any specific value or subrange within the stated range, to the tenth of the unit of the lower limit of the range, in different embodiments disclosed herein, unless the context clearly dictates otherwise.

[0031] "About" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art. Typically, the acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation, as practiced in the art. Alternatively, "about" can mean a range of ±20%, e.g., ±10%, ±5%, or ±1% of the given value. It should be understood that the term "about" can precede any specific value specified herein, except for specific values ​​used as examples. When the term "about" precedes the first number in a series of two or more numerical values, the term "about" applies to each of the numerical values ​​in the series. For example, about 1 to 3 is equivalent to about 1 to about 3.

[0032] When the term "at least," "greater than," or "greater than or equal to" precedes a first number in a series of two or more numbers, the term "at least," "greater than," or "greater than or equal to" applies to each number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0033] When the term "not greater than," "less than," or "less than or equal to" precedes the first number in a series of two or more numbers, the term "not greater than," "less than," or "less than or equal to" applies to each number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0034] The term "fusion protein" refers to a synthetic, semi-synthetic, or recombinant single protein molecule. Fusion proteins can contain all or part of two or more different proteins and / or polypeptides attached by covalent bonds (e.g., peptide bonds). The terms "polypeptide," "peptide," or "protein" refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Fusion proteins can contain any suitable L-amino acids and / or D-amino acids, including common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statin), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups in fusion proteins can be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, as well as methods for adding or removing protecting groups, are known in the art and are disclosed, for example, in Green and Wuts, Protecting Groups in Organic Synthesis, John Wiley and Sons, 1991. Functional groups of the fusion protein can be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorogen or hapten) using methods known in the art. The fusion protein can, if desired, contain one or more modifications (e.g., an amino acid linker, acylation, acetylation, amidation, methylation, terminal modification (e.g., cyclization modification), N-methyl-α-amino group substitution). In addition, the fusion protein can be an analog of a known and / or naturally occurring peptide, for example, a peptide analog with conservative amino acid residue substitution(s).

[0035] As used herein, the term "sequence identity" refers to the degree, expressed as a percentage, that two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity.For the purpose of aligning and comparing sequences, typically, one sequence is designated as a reference sequence, and the test sequence is compared to it.The sequence identity of a reference sequence and a test sequence is expressed as the percentage of positions that share the same nucleotide or amino acid throughout the entire length of the reference sequence when the reference sequence and the test sequence are aligned to achieve the maximum level of identity.As an example, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions throughout the entire length of the reference sequence when aligned to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0036] Alignment of sequences for comparison to achieve the maximum level of identity can be readily performed by one skilled in the art using an appropriate alignment method or algorithm, in some instances, the alignment can include the introduction of gaps to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see, e.g., generally, Ausubel et al., Current Protocols in Molecular Biology).

[0037] When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, subsequent coordinates are designated, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence (several) to reference sequence based on designated program parameters.The tool commonly used for determining sequence identity percentage is Protein Basic Local Alignment Search Tool (BLASTP), available from the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (Altschul et al., 1990).

[0038] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative amino acid substitution(s)" or "conservative substitution(s)" refers to an amino acid substitution having a value of 0 or greater in BLOSUM62.

[0039] In some embodiments, the amino acid substitution is a highly conservative substitution. The term "highly conservative amino acid substitution(s)" or "highly conservative substitution(s)" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0040] The term "polynucleotide" refers to a biopolymer comprising naturally occurring deoxyribonucleotide monomers, non-naturally occurring deoxyribonucleotide monomers (e.g., 7-deazaguanosine, inosine, or methylated nucleotides such as 5-methyl dCTP or 5-hydroxymethylcytosine), naturally occurring ribonucleotide monomers, or non-naturally occurring ribonucleotide monomers (e.g., locked nucleic acids (LNA)), or combinations thereof. The polynucleotides described herein can be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotides described herein are DNA molecules. In some embodiments, the polynucleotides described herein are RNA molecules (e.g., linear or circular RNA molecules).

[0041] The term "encode" refers to a specific sequence of nucleotides in a polynucleotide, such as DNA (e.g., cDNA) or RNA (e.g., mRNA), that serves as a template for the synthesis of a protein having a defined sequence of amino acids. Unless otherwise specified, polynucleotides that encode amino acid sequences are degenerate versions of each other and can have any one of all nucleic acid sequences that encode the amino acid sequence.

[0042] The term "vector" refers to a nucleic acid molecule that can be used to introduce nucleic acid sequences or genes into cells, either in vitro, ex vivo, or in vivo.

[0043] The term "ex vivo" refers to methods performed in or on cells or tissues in an artificial environment outside the organism, with minimal alteration of natural conditions.

[0044] The term "in vivo" refers to methods performed within a living organism under normal, intact conditions.

[0045] The term "in vitro" method is taken from its usual biological context and is carried out using isolated components of an organism.

[0046] The term "expression vector" refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into a suitable expression host cell.

[0047] The term "host" cell refers to a cell into which a polynucleotide has been introduced by molecular biology techniques. All techniques that can introduce a polynucleotide into a host cell are contemplated herein, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0048] The term "promoter" refers to a region of DNA to which RNA polymerase binds and initiates transcription of a gene.

[0049] The term "operably linked" means that a nucleic acid is placed within a recombinant polynucleotide, e.g., a vector, in a manner that allows expression of the nucleic acid under the control of elements (e.g., a promoter) to which it is linked.

[0050] The term "selectable marker element" is an element that confers a trait suitable for artificial selection. Selectable marker elements can be negative or positive selectable markers.

[0051] The term "pharmaceutically acceptable" means that the substance or composition it modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, allergic response, and the like, and is commensurate with a reasonable benefit / risk ratio.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are, within the scope of sound medical judgment, suitable for use in contact with mammalian tissues without undue toxicity, irritation, allergic response, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and suitable inorganic and organic bases.

[0053] Examples of salts derived from suitable acids include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy- Examples of the salts include ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0054] Either the mono-, di-, or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated, or substantially anhydrous form.

[0055] Salts derived from appropriate bases include those derived from inorganic bases such as alkali metal, alkaline earth metal, and ammonium bases, as well as salts derived from aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline, or N +((C1-C4) alkyl) salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Additionally, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxyls, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0056] The term "subject" or "patient" refers to a mammal (e.g., a human). In some embodiments, the subject is a mammal selected from a dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, and human. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0057] The terms "therapeutically effective amount," "effective amount," or "effective dosage" are amounts effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treating, curing, inhibiting, or reversing a physiological response or condition). The full therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the mammal, mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual.

[0058] The effective amount of drug to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art. Relevant factors include the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, and the identity (e.g., age, sex, and body weight) of the subject or host being treated. For example, a suitable dosage may be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. Determining the dosage for a particular drug, subject, and disease is well within the capabilities of one of ordinary skill in the art. Preferably, the dosage will cause no or minimal adverse side effects.

[0059] The desired response or desired result can include an effect at the cellular level, tissue level, or clinical outcome. Thus, the term "therapeutically effective amount" or its synonyms depends on the context in which it is applied. For example, in some embodiments, it is an amount of a composition sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition. In other embodiments, it is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition can be easily determined by a skilled artisan using routine methods known in the art. The dosage regimen and route of administration can be adjusted to provide an optimal therapeutic response.

[0060] As used herein, the term "treating" or its synonyms (e.g., "treatment" or "treat") refers to the medical management of a subject with the intent to ameliorate, reverse, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder, such as the specific indications exemplified herein. This term includes active treatment (treatment to ameliorate the disease, pathological condition, or disorder), causal treatment (treatment directed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to alleviate symptoms), prophylactic treatment (treatment designed to minimize or partially or completely prevent the onset of the associated disease, pathological condition, or disorder), and / or preventative treatment (treatment designed to minimize or partially or completely prevent the onset of the associated disease, pathological condition, or disorder). "Treatment" also includes reducing the extent of a disease or condition (e.g., a central nervous system (CNS) disease such as a prion disease), whether detectable or undetectable, preventing the spread of a disease or condition, delaying or slowing the progression of a disease or condition, improving or alleviating, and remission (whether partial or complete) of a disease or condition. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0061] The terms "ameliorating" or "alleviating" a disease or condition means lessening the severity and / or undesirable clinical symptoms of a disease, disorder, or condition and / or slowing or prolonging the time course of progression compared to the severity or time course in the absence of treatment.

[0062] Provided herein, inter alia, is a fusion protein comprising a DNA-binding domain, a DNMT3 methyltransferase-binding domain (e.g., a DNMT3A-binding domain), and H3K4me0.

[0063] Also provided herein are fusion proteins comprising, inter alia, a nuclease sequence, a DNMT3 methyltransferase binding domain (e.g., a DNMT3A binding domain), and H3K4me0. In some embodiments, the nuclease sequence lacks nuclease activity. Such nucleases are referred to herein as "nuclease-deficient" nucleases.

[0064] In some embodiments, the fusion protein further comprises a Krüppel-associated box (KRAB) domain or a homolog thereof.

[0065] Target sequence As used herein, the term "target sequence" refers to a polynucleotide sequence (e.g., a double-stranded DNA sequence) that is bound, either directly or indirectly (e.g., through an intermediate such as a guide RNA) by a fusion protein disclosed herein.

[0066] In some embodiments, the target sequence is associated with a disease, disorder, or pathological condition.

[0067] In some embodiments, the target sequence is a hypomethylated nucleic acid sequence. In some embodiments, the target sequence is a hypermethylated nucleic acid sequence.

[0068] In some embodiments, the target sequence is an endogenous sequence of an endogenous gene of a cell (e.g., a host cell). In some embodiments, the target sequence is an exogenous sequence.

[0069] In some embodiments, the target sequence is within a region of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within a transcriptional regulatory sequence (e.g., a promoter, enhancer, or silencer) of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within a promoter of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within an enhancer of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within a silencer of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within an exon of a gene targeted for epigenetic editing. In some embodiments, the target sequence is within an intron of a gene targeted for epigenetic editing.

[0070] In some embodiments, the target sequence is within about 3,000 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, or 2,900 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). , 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs. In some embodiments, the target sequence is within about 3,000 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, ...3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, Within 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs.In some embodiments, the target sequence is within about 3,000 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, ...3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, Within 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs.

[0071] In some embodiments, the target sequence is within about 2,500 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0072] In some embodiments, the target sequence is within about 2,000 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0073] In some embodiments, the target sequence is within about 1,500 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0074] In some embodiments, the target sequence is within about 1,000 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0075] In some embodiments, the target sequence is within about 500 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0076] In some embodiments, the target sequence is within about 200 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0077] In some embodiments, the target sequence is within about 100 base pairs adjacent to the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs downstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs upstream of the transcription start point of a gene (e.g., a target gene for epigenetic editing).

[0078] In some embodiments, the target sequence is within about 3,000 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,900, 2,100, 2,200, 3,300, 3,400, 3,500, 4,600, 4,700, 5,800, 5,900, 6,100, 6,100, 7,100, 8,100, 9,100, 10,100, 11,100, 12,100, 13,100, 14,100, 15,100, 16,100, 17,100, 18,100, 19,110, 20,120, 21,100, 22,100, 23,100, 24,100, 25,100, 26,100, 27,100, 28,100, 29,100, 30,110, 31,100, 32,100, 33,100, 34,100, 35,110, 36,100, 37,100, 38,110, 39,120, 41,120, 42,120, 43,120, 44,120, 4 Within 800, 1750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs. In some embodiments, the target sequence is within about 3,000 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, or 1,900 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.In some embodiments, the target sequence is within about 3,000 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, 1,900, 2,950, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,0 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.

[0079] In some embodiments, the target sequence is within about 2,500 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0080] In some embodiments, the target sequence is within about 2,000 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0081] In some embodiments, the target sequence is within about 1,500 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0082] In some embodiments, the target sequence is within about 1,000 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0083] In some embodiments, the target sequence is within about 500 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0084] In some embodiments, the target sequence is within about 200 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0085] In some embodiments, the target sequence is within about 100 base pairs adjacent to the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs downstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs upstream of the promoter sequence of a gene (e.g., a target gene for epigenetic editing).

[0086] In some embodiments, the target sequence is within about 3,000 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,900, 2,100, 2,200, 3,300, 3,400, 3,500, 4,600, 4,700, 5,800, 5,900, 6,100, 6,100, 7,100, 8,100, 9,100, 10,100, 11,100, 12,100, 13,100, 14,100, 15,100, 16,100, 17,100, 18,100, 19,110, 20,120, 21,100, 22,100, 23,100, 24,100, 25,100, 26,100, 27,100, 28,100, 29,100, 30,110, 31,100, 32,100, 33,100, 34,100, 35,110, 36,100, 37,100, 38,110, 39,120, 41,120, 42,120, 43,120, 44,120, Within 800, 1750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs. In some embodiments, the target sequence is within about 3,000 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, or 1,900 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.In some embodiments, the target sequence is within about 3,000 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, or 1,900 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.

[0087] In some embodiments, the target sequence is within about 2,500 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0088] In some embodiments, the target sequence is within about 2,000 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0089] In some embodiments, the target sequence is within about 1,500 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0090] In some embodiments, the target sequence is within about 1,000 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0091] In some embodiments, the target sequence is within about 500 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0092] In some embodiments, the target sequence is within about 200 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0093] In some embodiments, the target sequence is within about 100 base pairs adjacent to an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs downstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs upstream of an enhancer sequence of a gene (e.g., a target gene for epigenetic editing).

[0094] In some embodiments, the target sequence is within about 3,000 base pairs adjacent to the silencer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,900, 2,100, 2,200, 3,300, 3,400, 3,500, 4,600, 4,700, 5,800, 5,900, 6,100, 6,100, 7,100, 8,100, 9,100, 10,100, 11,100, 12,100, 13,100, 14,100, 15,100, 16,100, 17,100, 18,100, 19,110, 20,120, 21,100, 22,100, 23,100, 24,100, 25,100, 26,100, 27,100, 28,100, 29,100, 31,100, 32,100, 33,100, 34,100, 35,110, 36,100, 37,100, 38,110, 39,120, 40,130, 41,130, 42,130, 43,130, 44,130, Within 800, 1750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs. In some embodiments, the target sequence is within about 3,000 base pairs downstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, or 1,900 base pairs downstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing). 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.In some embodiments, the target sequence is within about 3,000 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing), e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,850, or 1,900 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing). 1,000, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs apart.

[0095] In some embodiments, the target sequence is within about 2,500 base pairs adjacent to the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs downstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,500 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0096] In some embodiments, the target sequence is within about 2,000 base pairs adjacent to the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs downstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 2,000 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0097] In some embodiments, the target sequence is within about 1,500 base pairs adjacent to the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs downstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,500 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0098] In some embodiments, the target sequence is within about 1,000 base pairs adjacent to the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs downstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 1,000 base pairs upstream of the silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0099] In some embodiments, the target sequence is within about 500 base pairs adjacent to a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs downstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 500 base pairs upstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0100] In some embodiments, the target sequence is within about 200 base pairs adjacent to a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs downstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 200 base pairs upstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0101] In some embodiments, the target sequence is within about 100 base pairs adjacent to a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs downstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing). In some embodiments, the target sequence is within about 100 base pairs upstream of a silencer sequence of a gene (e.g., a target gene for epigenetic editing).

[0102] The target gene for epigenetic editing can be of or derived from any organism and its genome. In some embodiments, the target gene for epigenetic editing is a prokaryotic gene. In some embodiments, the target gene for epigenetic editing is a eukaryotic gene, for example, an animal gene (e.g., a fish gene or a bird gene) or a plant gene. In some embodiments, the target gene for epigenetic editing is a mammalian gene, for example, a rodent gene (e.g., a mouse gene, a rat gene, a hamster gene, or a guinea pig gene), a horse gene, a pig gene, a sheep gene, a cow gene, or a primate gene (e.g., a chimpanzee gene, a macaque gene, a cynomolgus monkey gene, or a human gene). In some embodiments, the target gene for epigenetic editing is a human gene.

[0103] Non-limiting examples of target genes for epigenetic editing include mutS homolog 2 (MSH2), mutL homolog 1 (MLH1), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), erythropoietin (EPO), erb-b2 receptor tyrosine kinase 2 (ErbB2), somatotropin (GH), alpha globin (HBA), beta globin (HBB), gamma globin (HBG1), B-cell lymphoma / leukemia 11A (BCL11A), Krüppel-like factor 1 (KLF1), CC chemokine receptor type 5 (CCR5), and C-X-C chemokine receptor type 2 (CCR6). Protein kinase 4 (CXCR4), protein kinase 1 regulatory subunit 12C (PPP1R12C), hypoxanthine phosphoribosyltransferase (HPRT), albumin, coagulation factor VIII, coagulation factor IX, leucine-rich repeat kinase 2 (LRRK2), huntingtin (Htt), rhodopsin (RHO), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein B (SFTPB), T cell receptor alpha (TRAC), T cell receptor beta (TRBC), programmed cell death 1 (PD1), cytotoxic T lymphocyte antigen 4 (CTLA-4), human leukocyte antigen (HLA) A, HLA B, HLA C, proteasome subunit beta 8 (PSMB8), transporter associated with antigen processing (TAP) 1, TAP2, tapasin (TAPBP), ras II major histocompatibility complex transactivator (CIITA), dystrophin (DMD), glucorticoid receptor (GR), interleukin 2 receptor subunit gamma (IL2RG), regulatory factor X5 (RFX5), fatty acid desaturase (FAD2), fatty acid desaturase 3 (FAD3), ketoacyl acyl esterase (KAC) Carrier protein synthase II (KASII), malate dehydrogenase (MDH), microtubule-associated protein tau (MAPT), apolipoprotein E (APOE) (e.g., apoE2, apoE3, or apoE4 for stroke, concussion, and / or Alzheimer's disease (AD), and epilepsy), alpha-synuclein (SNCA), amyloid precursor protein (APP), presenilin 1 (PSEN1), presenilin 2 (PSEN2) (e.g., AD),Solute carrier family 6 member 4 (SLC6A4), 5-hydroxytryptamine receptor 2A (HTR2A), calcium voltage-gated channel subunit α1C (CACNA1C), calcium voltage-gated channel auxiliary branch beta 2 (CACNB2) (e.g., depression and / or migraine), dystrophy myotonic protein kinase (DMPK) (e.g., myotonic dystrophy), calcium voltage-gated channel subunit α1A (CACNA1A), sodium / potassium transporting ATPase subunit α2 (ATP1A2), sodium channel protein type 1 subunit α (SCN1A) (e.g., migraine), frataxin (FXN) (e.g., Friedreich's ataxia), peripheral myelin protein 22 (PMP22), utrophin (UTRN) (e.g., Duchenne muscular dystrophy), superoxide dismutase type 1 (SOD1), TAR DNA-binding protein (TARDBP), fusion sarcoma (FUS), angiogenin (ANG), arsine Pho guanine nucleotide exchange factor (ALS2), senataxin (SETX), progranulin (GRN), VAMP-associated protein B and C (VAPB) (e.g., in cases of amyotrophic lateral sclerosis (ALS) and / or dementia), fragile X messenger ribonucleoprotein 1 (FMR1) (e.g., in cases of fragile X), hypoxanthine-guanine phosphoribosyltransferase (HPRT) (e.g., in cases of Lesch-Nyhan disease), methyl-CpG-binding protein 2 (MECP2) (e.g., in Rett syndrome), aspartoacylase (ASPA) ) (e.g., Canavan disease), SCN8A (e.g., Dravet syndrome), UDP-glucuronosyltransferase family 1 member A1 (UGT1A1) (e.g., Crigler-Najjar), opioid receptor μ1 (OPRM1) (e.g., borderline personality disorder), opioid receptor kappa 1 (OPRK1), opioid receptor delta 1 (OPRD1) (e.g., opioid use disorder), solute carrier family 6 member 4 (SLC6A4), 5-hydroxytryptamine receptor 2A (HTR2a), tryptophan hydroxylase 2 (TPH2) (e.g., major depressive disorder), dopamine receptor D2 (DRD2), glutamate metabotropic receptor 3 (GRM3),Glutamate ionotropic receptor NMDA-type subunit 2A (GRIN2A), serine racemase (SRR) (e.g., type 2 diabetes), glutamate ionotropic receptor AMPA-type subunit 1 (GRIA1), calcium voltage-gated channel subunit α1C (CACNA1C), calcium voltage-gated channel auxiliary unit β2 (CACNB2), calcium voltage-gated channel subunit α1I (CACN11I), glutamic acid decarboxylase 1 (GAD1), reelin (RELN), brain-derived neurotrophic factor (BDNF), and te Thiomethylmethyltoxin dioxygenase 1 (TET1), dystrobrevin-binding protein 1 (DTNBP1) (e.g., schizophrenia), ankyrin 3 (ANK3), teneurin transmembrane protein 4 (TENM4), tetratricopeptide repeat and ankyrin repeat containing 1 (TRANK1), adenylate cyclase 2 (ADCY2), calcium voltage-gated channel subunit α1C (CACNA1C), brain-derived neurotrophic factor (BDNF) (e.g., bipolar disorder), PR / SET domain 16 (PRDM16), adherens junction-associated protein Adipocyte-associated protein 1 (AJAP1), myocyte enhancer factor 2D (MEF2D), transient receptor potential cation channel subfamily member 8 (TRPM8), transforming growth factor β receptor 2 (TGFBR2), phosphatase and actin reginase 1 (PHACTR1), succinyl-CoA: glutarate-CoA transferase (GA3), matrix metalloptidase 16 (MMP16), astrotactin 2 (ASTN2), tetraspanin 2 (TSPAN2), glial cell line-derived neurotrophic factor family receptor α2 (GFRA2), LDL receptor LRP1 (LRP1) (e.g., migraine), human leukocyte antigen class II histocompatibility-D-related beta chain 1 (HLA-DRB1), interleukin 7 receptor alpha (IL7Ra), interleukin 2 receptor alpha (IL2Ra), cytochrome P450 family 27 subfamily B member 1 (CYP27B1), tyrosine kinase 2 (TYK2) (e.g., multiple sclerosis), neurexin 1 (NRXN1), arylacetamide deacetylase (AADAC), catenin alpha 3 (CTNNA3), fibrillar binding protein CB (FSCB),Regulator of calcineurin 1 (RCAN1) (e.g., Tourette's syndrome), calmodulin-regulated spectrin-related protein 1 (CAMSAP1LK1), NMDA receptor subunit 1, GAMA-A receptor subunit α-1, glutamic acid decarboxylase 2 (GAD65), adenosine kinase, germline nuclear factor (GCNF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), neuropeptide Y, and galanin (e.g., epilepsy).

[0104] In some embodiments, the target gene encodes p2-microglobulin (B2M), BCL11A, KLF1, a chemokine receptor (e.g., CCR5, CXCR4), miRNA (e.g., miR126), PDL1, CTLA4, or collagen, type I, alpha 1 (COL1A1). In some embodiments, the target gene is a globin gene. In some embodiments, the target gene is a TCR gene. In some embodiments, the target gene is HBB, HBA, hMSH2, HMLHI, the growth factor GM-SCF, VEGF, EPO, ErbB2, or hGH.

[0105] For additional non-limiting examples of target genes, see, e.g., U.S. Patent No. 9,970,001, U.S. Patent Application No. 15 / 521,294 (US2019 / 0032049), and U.S. Patent Application No. 16 / 177,403 (US2019 / 0153476, e.g., Tables A-C), the entire contents of which are incorporated herein by reference.

[0106] In some embodiments, the target sequence is within about 3,000 base pairs adjacent to the CpG island, e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,500, 1,650, 1,700, 1,850, 1,900, 1,950, 1,800, 1,750, 1,700, 1,650, 1,600, 1,500, 1,900, 1,950, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,500, 1,750, 1,850, 1,900, 1,950, 2,900, 2,950, 2,950, 2,950, 2,950, 3,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1,000, 1,100, 1,200, 1 Within 50, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs. In some embodiments, the target sequence is within about 3,000 base pairs downstream of the CpG island, e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,550, Within 0, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pair groups. In some embodiments, the target sequence is within about 3,000 base pairs upstream of the CpG island, e.g., within about 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,500, 1,650, 1,700, 1,850, 1,900, 1,950, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,500, 1,650, 1,700, 1,850, 1,850, 1,900, 1,950, 2,900, 2 ...3,000, 3,000, 3,000, 4,000, 4,000, 5,000, 5,000, 6,000, 7,000, 8,000, 9,000, 1,000, 1,000, 1 Within 50, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 base pairs.

[0107] In some embodiments, the target sequence is within about 2,500 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 2,500 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 2,500 base pairs upstream of the CpG island.

[0108] In some embodiments, the target sequence is within about 2,000 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 2,000 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 2,000 base pairs upstream of the CpG island.

[0109] In some embodiments, the target sequence is within about 1,500 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 1,500 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 1,500 base pairs upstream of the CpG island.

[0110] In some embodiments, the target sequence is within about 1,000 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 1,000 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 1,000 base pairs upstream of the CpG island.

[0111] In some embodiments, the target sequence is within about 500 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 500 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 500 base pairs upstream of the CpG island.

[0112] In some embodiments, the target sequence is within about 200 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 200 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 200 base pairs upstream of the CpG island.

[0113] In some embodiments, the target sequence is within about 100 base pairs adjacent to the CpG island. In some embodiments, the target sequence is within about 100 base pairs downstream of the CpG island. In some embodiments, the target sequence is within about 100 base pairs upstream of the CpG island.

[0114] DNA-binding domain The term "DNA-binding domain" refers to a protein or fragment thereof (e.g., a structural motif) that binds to double-stranded and / or single-stranded DNA either directly or indirectly (e.g., through an intermediate such as a guide RNA). In some embodiments, the DNA-binding domain binds to double-stranded DNA. In some embodiments, the DNA-binding domain binds to single-stranded DNA. In some embodiments, the DNA-binding domain binds to a specific DNA sequence (i.e., a target sequence). In some embodiments, the DNA-binding domain has a general affinity for DNA. In some embodiments, the DNA-binding domain (e.g., a zinc finger or transcription activator-like effector domain) binds directly to DNA (e.g., a target sequence). In some embodiments, the DNA-binding domain binds to DNA (e.g., a target sequence) indirectly (e.g., via a guide RNA sequence).

[0115] In some embodiments, the DNA-binding domain comprises the DNA-binding domain of a nuclease (e.g., an endonuclease), such as a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a meganuclease, a homing (HO) endonuclease, a transposon-encoded RNA-guided nuclease (e.g., isrB, iscB, or tnpB), or a eukaryotic programmable RNA-guided endonuclease (e.g., FANZOR), or any combination of the foregoing. In some embodiments, the DNA-binding domain comprises a nuclease (e.g., an endonuclease), such as a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a meganuclease, a homing (HO) endonuclease, or a transposon-encoded RNA-guided nuclease (e.g., isrB, iscB, or tnpB), or any combination of the foregoing. In some embodiments, the nuclease (e.g., an endonuclease) lacks nuclease activity (e.g., is nuclease-deficient).

[0116] In some embodiments, the DNA-binding domain comprises the DNA-binding domain of an RNA-guided nuclease (e.g., endonuclease), such as a Cas (e.g., a Type I, II, III, IV, or V CRISPR-Cas system), a small Cas effector (e.g., CasX, CasMINI, or Casφ), Fanzor (a eukaryotic programmable RNA-guided endonuclease), or a transposon-encoded RNA-guided nuclease (e.g., isrB, iscB, or tnpB), or any combination of the foregoing. In some embodiments, the RNA-guided nuclease (e.g., endonuclease) is nuclease-deficient (e.g., lacks nuclease activity). For additional information regarding Fanzor, see, e.g., Saito et al., Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature 620(7974):660-68(2023), the entire contents of which are incorporated herein by reference.

[0117] In some embodiments, the DNA-binding domain comprises the DNA-binding domain of a nuclease-deficient RNA-guided DNA endonuclease enzyme. The term "nuclease-deficient RNA-guided DNA endonuclease enzyme" refers to an RNA-guided DNA endonuclease (e.g., a variant of a naturally occurring RNA-guided DNA endonuclease) that can target specific phosphodiester bonds within a DNA polynucleotide when complexed with a distinct polynucleotide sequence (e.g., a single guide RNA (sgRNA)), but is unable to significantly cleave said phosphodiester bonds under physiological conditions.

[0118] In some embodiments, the DNA binding domain comprises a Cas, a ZFP, or a TALE.

[0119] In some embodiments, the DNA binding domain comprises a Cas. In some embodiments, the DNA binding domain (e.g., of a nuclease-deficient RNA-guided DNA endonuclease enzyme) comprises a Cas of a Type II-A CRISPR-Cas system (e.g., Cas9).

[0120] In some embodiments, the DNA binding domain comprises a nuclease-deficient Cas9 (dCas9), for example, from any one of the following species: Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacteria, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis audaxviator, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina, or those variants.

[0121] In some embodiments, the DNA-binding domain comprises Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f. In some embodiments, the DNA-binding domain comprises Staphylococcus aureus dCas9. In some embodiments, the DNA-binding domain comprises Streptococcus pyogenes dCas9. In some embodiments, the DNA-binding domain comprises S. aureus dCas9. In some embodiments, the DNA-binding domain comprises dCas12a. In some embodiments, the DNA-binding domain comprises dCas12f.

[0122] In some embodiments, the DNA-binding domain comprises an amino acid sequence having at least 80% sequence identity to Streptococcus pyogenes dCas9 (e.g., SEQ ID NO: 1), e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to Streptococcus pyogenes dCas9 (e.g., SEQ ID NO: 1). In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to Streptococcus pyogenes dCas9 (e.g., SEQ ID NO: 1). In some embodiments, the DNA-binding domain comprises an amino acid sequence that has 100% sequence identity to the entire Streptococcus pyogenes dCas9 (e.g., SEQ ID NO: 1).

[0123] In some embodiments, the DNA binding domain comprises S. aureus dCas9.

[0124] In some embodiments, the DNA-binding domain comprises an amino acid sequence having at least about 80% sequence identity to S. aureus dCas9 (e.g., SEQ ID NO: 489), e.g., an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to S. aureus dCas9 (e.g., SEQ ID NO: 489). In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to S. aureus dCas9 (e.g., SEQ ID NO: 489). In some embodiments, the DNA-binding domain comprises an amino acid sequence that has 100% sequence identity to the entire S. aureus dCas9 (e.g., SEQ ID NO: 489).

[0125] In some embodiments, the DNA-binding domain (e.g., of a nuclease-deficient RNA-guided DNA endonuclease enzyme) comprises a Cas (e.g., Cpf1, also referred to as Cas12a) of a VA-type CRISPR-Cas system. In some embodiments, the Cas12a is AsCas12a (from Acidaminococcus sp.). In some embodiments, the Cas12a is Lb2Cas12a (from Lachnospiraceae sp.). In some embodiments, the DNA-binding domain comprises a nuclease-deficient Cpf1 (e.g., dCas12a, dCpf1, or ddCpf1).

[0126] In some embodiments, the DNA-binding domain (e.g., of a nuclease-deficient RNA-guided DNA endonuclease enzyme) comprises dCas12f. For additional information about dCas12f, see, e.g., Hino et al., An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis, Cell 186(22):4920-35(2023), the entire contents of which are incorporated herein by reference.

[0127] In some embodiments, the DNA-binding domain comprises a CRISPR-Cas protein bound to (i.e., complexed with) a guide polynucleotide, which in some embodiments hybridizes to a target sequence (e.g., a genomic target sequence).

[0128] For more information regarding CRISPR-Cas proteins, see, e.g., PCT Application Nos. PCT / US2021 / 064913 (WO2022 / 140577) and PCT / US2021 / 035244 (WO2021 / 247570), the entire contents of which are incorporated herein by reference.

[0129] In some embodiments, the DNA binding domain comprises the DNA binding domain of a zinc finger protein (ZFP). Non-limiting examples of ZFPs and their DNA binding domains include those disclosed in U.S. Patent No. 7,534,775, the entire contents of which are incorporated herein by reference. For non-limiting examples of ZFP sequences that target the CD55 or PRNP transcription start site, see also SEQ ID NOs: 3-15.

[0130] In some embodiments, the ZFP DNA-binding domain comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 3-15, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs: 3-15. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 3-15. In some embodiments, the ZFP DNA-binding domain comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NO: 3-15.

[0131] In some embodiments, the DNA binding domain comprises a DNA binding domain of a TALEN (e.g., a TALEN DNA binding domain such as a TALE).

[0132] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 454-461 (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the sequences), for example, having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs: 454-461. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 454-461. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 454-461.

[0133] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 462-469 (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the sequences), for example, having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs: 462-469. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 462-469. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 462-469.

[0134] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:454, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:454. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 454. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 454.

[0135] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:455, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:455. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 455. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 455.

[0136] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:456, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:456. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 456. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 456.

[0137] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:457, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:457. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 457. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 457.

[0138] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:458, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:458. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 458. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 458.

[0139] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:459, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:459. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 459. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 459.

[0140] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:460, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:460. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 460. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 460.

[0141] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:461, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:461. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 461. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 461.

[0142] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:462, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:462. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 462. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 462.

[0143] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:463, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:463. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 463. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 463.

[0144] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:464, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:464. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 464. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 464.

[0145] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:465, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:465. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 465. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 465.

[0146] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:466, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:466. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 466. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 466.

[0147] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:467, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:467. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to SEQ ID NO: 467. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 467.

[0148] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:468, such as an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:468. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence of SEQ ID NO: 468. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 468.

[0149] In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:469, for example, an amino acid sequence having at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO:469. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence of SEQ ID NO: 469. In some embodiments, the TALE DNA binding domain comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 469.

[0150] In some embodiments, the DNA binding domain comprises the DNA binding domain of a ZFN (e.g., a nuclease-deficient ZFN, such as a ZFP). Non-limiting examples of TALENs, ZFNs, and their DNA binding domains include those disclosed in Patent Cooperation Treaty (PCT) Application Publication No. WO2016063264 and Gaj et al., "ZFNs, TALENs, and CRISPR / Cas-based methods for genome engineering," Trends Biotechnol. 31(7):397-405 (2013), the entire contents of which are incorporated herein by reference.

[0151] In some embodiments, the DNA-binding domain comprises the DNA-binding domain of a meganuclease (e.g., a nuclease-deficient meganuclease). See, e.g., Silva et al., Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy, Curr Gene Ther. 11(1):11-27 (2011), the entire contents of which are incorporated herein by reference.

[0152] In some embodiments, the DNA-binding domain comprises a DNA-binding domain of a transcriptional regulator (e.g., a tetracycline-controlled repressor (tetR)). In some embodiments, the DNA-binding domain comprises a leucine zipper domain, a winged-helix domain, a helix-turn-helix domain, a helix-loop-helix domain, a chromatin-associated high mobility group (HMG) box domain, a white-opaque regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB)-fold domain, an immunoglobulin domain, or a B3 DNA-binding domain.

[0153] In some embodiments, the DNA binding domain is selected and / or engineered to bind to a desired DNA sequence (eg, a target DNA sequence in, for example, genomic DNA).

[0154] DNMT3 methyltransferase binding domain The term "DNMT3 methyltransferase binding domain" refers to a protein or fragment thereof that binds directly or indirectly (eg, through an intermediate) to DNMT3 methyltransferase.

[0155] In some embodiments, the DNMT3 methyltransferase binding domain binds to DNMT3A (e.g., human DNMT3A), DNMT3B (e.g., human DNMT3B), or DNMT3C (e.g., mouse DNMT3C), or any combination thereof. In some embodiments, the DNMT3 methyltransferase binding domain binds to DNMT3A or DNMT3B, or both. In some embodiments, the DNMT3 methyltransferase binding domain binds to DNMT3A.

[0156] In some embodiments, the DNMT3 methyltransferase binding domain binds to the catalytic domain of a DNMT3 methyltransferase (e.g., human DNMT3 methyltransferase). In some embodiments, the DNMT3 methyltransferase binding domain binds to the catalytic domain of human DNMT3A (e.g., comprising SEQ ID NO: 29).

[0157] In some embodiments, the DNMT3 methyltransferase binding domain is about 300 or less amino acids in length, e.g., about 290, 280, 270, 260, 250, 240, 230, 220, 215, 210, 205, or 200 or less amino acids in length. In some embodiments, the DNMT3 methyltransferase binding domain is about 200-220 amino acids in length.

[0158] In some embodiments, the DNMT3 methyltransferase binding domain comprises a DNA methyltransferase 3-like protein (Dnmt3L) or a fragment thereof. In some embodiments, the DNMT3 methyltransferase binding domain comprises a Dnmt3L C-terminal domain.

[0159] In some embodiments, the Dnmt3L C-terminal domain is a human, monkey, wild boar, mouse, rat, hamster, field mouse, wood mouse, jerboa, vole, lemur, chinchilla, bear, camel, donkey, sloth, panglin, pika, fox, wombat, ancestral alpha, ancestral beta, ancestral delta, ancestral epsilon, or ancestral gamma DNMT3L C-terminal domain.

[0160] In some embodiments, the Dnmt3L C-terminal domain is a rodent (e.g., mouse, rat, hamster, field mouse, wood mouse, jerboa, or bank vole) DNMT3L C-terminal domain. In some embodiments, the Dnmt3L C-terminal domain is a mouse (Mus musculus) DNMT3L C-terminal domain or a field mouse (Apodemus sylvaticus) DNMT3L C-terminal domain. In some embodiments, the Dnmt3L C-terminal domain is a mouse (Mus musculus) DNMT3L C-terminal domain. In some embodiments, the Dnmt3L C-terminal domain is a field mouse (Apodemus sylvaticus) DNMT3L C-terminal domain.

[0161] In some embodiments, the Dnmt3L C-terminal domain is a human DNMT3L C-terminal domain.

[0162] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 31-50 and 71-75, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 31-50 and 71-75. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 31-50 and 71-75. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NOs: 31-50 and 71-75.

[0163] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 31-50, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs: 31-50. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 31-50. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 31-50.

[0164] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:31, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:31. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 31. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 31.

[0165] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:32, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:32. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 32. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 32.

[0166] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:38, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:38. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 38. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 38.

[0167] In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs:71-75, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs:71-75. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 71-75. In some embodiments, the Dnmt3L C-terminal domain comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 71-75.

[0168] In some embodiments, the DNMT3 methyltransferase binding domain comprises a DNMT3 mutant. In some embodiments, the DNMT3 mutant lacks methyltransferase activity (e.g., is catalytically inactive). In some embodiments, the DNMT3 methyltransferase binding domain comprises a catalytically inactive DNMT3 (e.g., catalytically inactive human DNMT3). In some embodiments, the DNMT3 methyltransferase binding domain comprises a catalytically inactive DNMT3A (e.g., catalytically inactive human DNMT3A).

[0169] In some embodiments, the DNMT3 methyltransferase binding domain recruits a DNMT3 methyltransferase (e.g., DNMT3A) to a locus in a cell (e.g., a human cell).

[0170] H3K4me0 As used herein, the term "H3K4me0" refers to the histone 3 (H3) N-terminal tail domain that contains an unmethylated lysine (K) 4 residue and binds to an ADD domain.

[0171] In some embodiments, the H3K4me0 is human H3K4me0.

[0172] In some embodiments, the human H3 protein comprises the amino acid sequence of SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, or SEQ ID NO:396.

[0173] In some embodiments, the human H3 protein comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:393, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:393. In some embodiments, the human H3 protein comprises amino acids having about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 393. In some embodiments, the human H3 protein comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 393. In some embodiments, the H3K4me0 comprises an N-terminal fragment of SEQ ID NO: 393 (e.g., a 12-aa or 30-aa N-terminal fragment).

[0174] In some embodiments, the human H3 protein comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:394, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:394. In some embodiments, the human H3 protein comprises amino acids having about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 394. In some embodiments, the human H3 protein comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 394. In some embodiments, H3K4me0 comprises an N-terminal fragment of SEQ ID NO: 394 (e.g., a 12-aa or 30-aa N-terminal fragment).

[0175] In some embodiments, the human H3 protein comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO: 395, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 395. In some embodiments, the human H3 protein comprises amino acids having about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 395. In some embodiments, the human H3 protein comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 395. In some embodiments, the H3K4me0 comprises an N-terminal fragment (e.g., a 12-aa or 30-aa N-terminal fragment) of SEQ ID NO: 395.

[0176] In some embodiments, the human H3 protein comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:396, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:396. In some embodiments, the human H3 protein comprises amino acids having about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 396. In some embodiments, the human H3 protein comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 396. In some embodiments, the H3K4me0 comprises an N-terminal fragment of SEQ ID NO: 396 (e.g., a 12-aa or 30-aa N-terminal fragment).

[0177] In some embodiments, H3K4me0 comprises an N-terminal fragment (e.g., a 12-aa or 30-aa N-terminal fragment) of SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, or SEQ ID NO: 396. In some embodiments, H3K4me0 comprises an N-terminal fragment (e.g., a 12-aa or 30-aa N-terminal fragment) of SEQ ID NO: 393.

[0178] In some embodiments, H3K4me0 is 4 or more amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acids in length. In some embodiments, H3K4me0 is 12 or more amino acids in length. In some embodiments, H3K4me0 is 57 or less amino acids in length, for example, 55, 52, 50, 48, 45, 42, 40, 38, 35, 34, 33, 32, 31, or 30 or less amino acids in length. In some embodiments, H3K4me0 is 30 or less amino acids in length. In some embodiments, the H3K4me0 is about 4 to 57 amino acids in length, e.g., about 5 to 57, 5 to 55, 5 to 30, 6 to 55, 6 to 50, 6 to 30, 7 to 50, 7 to 45, 7 to 30, 8 to 45, 8 to 40, 8 to 30, 9 to 40, 9 to 35, 9 to 30, 10 to 35, 10 to 32, 10 to 30, 11 to 32, 11 to 30, 12 to 57, 12 to 55, 12 to 50, 12 to 45, 12 to 40, 12 to 35, 12 to 30, 20 to 40, 21 to 39, 22 to 38, 23 to 37, 24 to 36, 25 to 35, 26 to 34, 27 to 33, 28 to 32, or 29 to 31 amino acids in length. In some embodiments, H3K4me0 is about 12 to 30 amino acids in length. In some embodiments, H3K4me0 is about 28 to 32 amino acids in length.

[0179] In some embodiments, the H3K4me0 is about 12 amino acids in length.

[0180] In some embodiments, the H3K4me0 comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO:81, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:81. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 81. In some embodiments, the H3K4me0 comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 81.

[0181] In some embodiments, the H3K4me0 is about 30 amino acids in length.

[0182] In some embodiments, the H3K4me0 comprises amino acids having at least about 80% sequence identity to the sequence set forth in SEQ ID NO: 87, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 87. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 87. In some embodiments, the H3K4me0 comprises amino acids having 100% sequence identity to the sequence set forth in SEQ ID NO: 87.

[0183] In some embodiments, H3K4me0 comprises amino acids having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 81 and 87, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs: 81 and 87. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 81 and 87. In some embodiments, the H3K4me0 comprises amino acids having 100% sequence identity to the sequences set forth in SEQ ID NOs: 81 and 87.

[0184] Linker In some embodiments, the fusion proteins disclosed herein comprise a linker connecting two domains (e.g., a DNMT3 methyltransferase binding domain to H3K4me0, a DNMT3 methyltransferase binding domain to a DNA binding domain, or H3K4me0 to a DNA binding domain). In some embodiments, the linker connects the DNMT3 methyltransferase binding domain to H3K4me0. In some embodiments, the linker connects the DNMT3 methyltransferase binding domain to the DNA binding domain. In some embodiments, the linker connects H3K4me0 to the DNA binding domain.

[0185] In some embodiments, the linker provides increased flexibility, is proteolytically resistant, or is self-cleaving, or any combination thereof.

[0186] In some embodiments, the linker comprises about 15-100 amino acids, e.g., about 16, 18, 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, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids. In some embodiments, the linker comprises about 16-80 amino acids, e.g., about 16-75, 16-70, 16-65, 16-60, 16-55, 16-50, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-65, 20-75, 20-80, 20-90, 20-95, 20-100, 20-120, 20-140, 20-160, 20-180, 20-220, 20-240, 20-260, 20-280, 20-300, 20-320, 20-340, 20-360, 20-380, 20-400, 20-420, 20-440, 20-460, 20-480, 20-500, 20-520, 20-540, 20-560, 20-640, 20-660, 20-700, 20-700, 20-800, 20-800, 20-900, 20-900, 20-1400, 20-1600, 20-1800, 20-2200, 20-2400, 20-3200, 20-3400, 20-4600, 20-54 0~50, 20~45, 20~40, 20~35, 20~30, 20~25, 25~80, 25~75, 25~70, 25~65, 25~60, 25~55, 25~50, 25~45, 25~40, 25~35, 25~30, 30~80, 30~75, 30~70, 30~65, 30~60, 30~55, 30~50, 30~45 , 30~40, 30~35, 35~80, 35~75, 35~70, 35~65, 35~60, 35~55, 35~50, 35~45, 35~40, 40~80, 40~75, 40~70, 40~65, 40~60, 40~55, 40~50, 40~45, 45~80, 45~75, 45~70, 45~65, 45~60, 45 The linker may comprise between 55, 45 and 50, 50 and 80, 50 and 75, 50 and 70, 50 and 65, 50 and 60, 50 and 55, 55 and 80, 55 and 75, 55 and 70, 55 and 65, 55 and 60, 60 and 80, 60 and 75, 60 and 70, 60 and 65, 65 and 80, 65 and 75, 65 and 70, 70 and 80, 70 and 75, or 75 and 80 amino acids. In some embodiments, the linker comprises between about 35 and 45, 36 and 44, 37 and 43, 38 and 42, or 39 and 41 amino acids. In some embodiments, the linker comprises about 40 amino acids.

[0187] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:89, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:89. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 89. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 89.

[0188] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:96, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:96. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 96. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 96.

[0189] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:506, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:506. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 506. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 506.

[0190] In some embodiments, the linker increases the flexibility of the fusion protein or one or more domains thereof.

[0191] In some embodiments, the linker is a glycine-serine linker or a variant thereof, such as (GGGGS) n where n is 1, 2, 3, 4, 5, 6, or 7 or more, or a variant thereof.

[0192] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in, e.g., SEQ ID NOs: 89-100, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in, e.g., SEQ ID NOs: 89-100. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 89-100. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 89-100.

[0193] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in SEQ ID NOs:89-100 and 506, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one sequence set forth in SEQ ID NOs:89-100 and 506. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 89-100 and 506. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOs: 89-100 and 506.

[0194] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:90, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:90. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 90. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 90.

[0195] In some embodiments, the linker is proteolysis resistant. In some embodiments, the linker is an XTEN linker, such as an XTEN16, XTEN24, XTEN32, XTEN34, XTEN36, XTEN38, XTEN40, XTEN42, XTEN44, XTEN46, XTEN48, XTEN50, XTEN52, XTEN54, or XTEN80 linker.

[0196] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to at least one sequence set forth in, for example, SEQ ID NOs: 113-128 (e.g., SEQ ID NOs: 113-127), e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8, or 99.9% sequence identity to at least one sequence set forth in, for example, SEQ ID NOs: 113-128 (e.g., SEQ ID NOs: 113-127). In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one sequence set forth in SEQ ID NOs: 113-128 (e.g., SEQ ID NOs: 113-127). In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to SEQ ID NOs: 113-128 (e.g., SEQ ID NOs: 113-127).

[0197] In some embodiments, the linker (e.g., XTEN) linker comprises a nuclear localization signal (NLS). In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 128, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 128. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 128. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 128.

[0198] In some embodiments, the linker is a self-cleaving linker.

[0199] In some embodiments, the linker is a P2A self-cleaving linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 145, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 145. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 145. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 145.

[0200] In some embodiments, the linker is a T2A self-cleaving linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 147, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 147. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 147. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 147.

[0201] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to at least one of the sequences set forth in, e.g., SEQ ID NOs: 89-100, 113-128, 145, and 147, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one of the sequences set forth in, e.g., SEQ ID NOs: 89-100, 113-128, 145, and 147. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 89-100, 113-128, 145, and 147. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequences set forth in SEQ ID NOs: 89-100, 113-128, 145, and 147.

[0202] In some embodiments, the linker comprises an amino acid sequence having at least about 80% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 96, 100, and 119, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 96, 100, and 119. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 96, 100, and 119. In some embodiments, the linker comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NOs: 96, 100, and 119.

[0203] Histone methyltransferase-recruiting domain In some embodiments, the fusion protein further comprises a domain that recruits a histone methyltransferase.

[0204] In some embodiments, the histone methyltransferase-recruiting domain comprises a Kruppel-associated box (KRAB) domain or a homolog thereof, hi some embodiments, the histone methyltransferase-recruiting domain comprises a human Kruppel-associated box (KRAB) domain or a variant thereof.

[0205] In some embodiments, the KRAB domain is a KRAB domain of a human KRAB-ZFP or a homolog thereof. Non-limiting examples of human KRAB-ZFPs include FPM315, HKr18, HKr19, HZF4, HZF12, KID-1, KOX1, RbaK, RITA, ZBRK1, ZF5128, ZFP1, ZFP14, ZFP28-1, ZFP28-2, ZFP82, Zfp93, ZFP95, ZIM2, ZIM, ZK1, ZNF18, and ZNF3. 0, ZNF33A, ZNF34, ZNF41, ZNF43, ZNF45, ZNF75D, ZNF85, ZNF91, ZNF98, ZNF133, ZNF136, ZNF14 0, ZNF141, ZNF155, ZNF157, ZNF175, ZNF184, ZNF189, ZNF197, ZNF202, ZNF213, ZNF214, ZNF224 , ZNF221, ZNF222, ZNF224, ZNF225, ZNF226, ZNF250, ZNF254, ZNF257, ZNF264, ZNF273, ZNF274 , ZNF282, ZNF320, ZNF324, ZNF331, ZNF350, ZNF354A, ZNF37A, ZNF394, ZNF398, ZNF416, ZNF419 , ZNF436, ZNF490, ZNF528, ZNF543, ZNF547, ZNF554, ZNF557, ZNF566, ZNF582, ZNF595, ZNF596, ZNF610, ZNF669, ZNF675, ZNF677, ZNF680, ZNF729, ZNF764, ZNF785, ZNF8, and ZNF816.For additional information regarding human KRAB-ZFPs, see, e.g., Ecco et al., KRAB zinc finger proteins, Development 144(15):2719-29 (2017); Lupo et al., KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions, Curr Genomics 14(4):268-78 (2013); Urrutia, KRAB-containing zinc-finger repressor proteins, Genome Biol. 4(10):231 (2003); and Alerasool et al., An efficient KRAB domain for CRISPRi applications in human cells, Nat Methods 17(11):1093-96 (2020), the entire contents of which are incorporated herein by reference.

[0206] In some embodiments, the KRAB domain is a KOX1 KRAB domain. In some embodiments, the KRAB domain is a ZIM3 KRAB domain.

[0207] In some embodiments, the KRAB domain comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 155, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 155. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 155. In some embodiments, the KRAB domain comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 155.

[0208] In some embodiments, the fusion protein does not include any domain that recruits histone methyltransferases, hi some embodiments, the fusion protein does not include any KRAB domains.

[0209] histone methyltransferase In some embodiments, the fusion protein further comprises a histone methyltransferase or a homolog thereof. In some embodiments, the histone methyltransferase comprises an enhancer of zeste homolog 2 (EZH2) protein or a fragment thereof (e.g., having histone methyltransferase catalytic activity). In some embodiments, the histone methyltransferase comprises human EZH2 (see, e.g., Grzenda et al., "Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression," Epigenetics Chromatin 6(1):3 (2013)). In some embodiments, the histone methyltransferase comprises human EZH2 isoform alpha (see, e.g., NCBI Reference Sequence: NP_004447.2).

[0210] Nuclear localization signal (NLS) In some embodiments, the fusion proteins disclosed herein further comprise a nuclear localization signal (NLS).

[0211] In some embodiments, the NLS is a bipartite NLS. In some embodiments, the bipartite NLS comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 149, for example, at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the sequence set forth in SEQ ID NO: 149. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 149. In some embodiments, the bipartite NLS comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 149.

[0212] In some embodiments, the NLS is an SV40 NLS. In some embodiments, the SV40 NLS comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 151, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO: 151. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 151. In some embodiments, the SV40 NLS comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 151.

[0213] In some embodiments, the NLS comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:149 or SEQ ID NO:151, e.g., at least about 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the sequence set forth in SEQ ID NO:149 or SEQ ID NO:151. In some embodiments, the sequence has about 80-99.9%, 80-99.8%, 85-99.8%, 85-99.6%, 88-99.6%, 88-99.5%, 90-99.5%, 90-99.4%, 92-99.4%, 92-99.2%, 95-99.2%, 95-99%, or 98-99% sequence identity to the sequence set forth in SEQ ID NO: 149 or SEQ ID NO: 151. In some embodiments, the NLS comprises an amino acid sequence having 100% sequence identity to the sequence set forth in SEQ ID NO: 149 or SEQ ID NO: 151.

[0214] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a switchable nuclear localization sequence. In some embodiments, the switchable nuclear localization sequence allows for precise, user-defined control, e.g., temporal control, of nuclear localization. For additional information regarding switchable nuclear localization, see, e.g., Niopek et al., Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells, Nat Commun. 5:4404 (2014), Di Ventura & Kuhlman, Go in! Go out! Inducible control of nuclear localization, Curr Opin Chem Biol. 34:62-71 (2016), and Shin et al., Cytosolic microRNA-inducible nuclear translocation of Cas9 protein for disease-specific genome modification, Nucleic Acids Res. 50(10):5919-33 (2022), the entire contents of which are incorporated herein by reference. In some embodiments, the switchable nuclear localization sequence comprises a sequence encoding a sensitizing variant of the human estrogen receptor, ERT2, that can be activated by the tamoxifen metabolite trans-4-OH-tamoxifen (trans-4-OHT). See, e.g., Li et al., Multidimensional control of therapeutic human cell function with synthetic gene circuits, Science 378(6625):1227-34(2022), the entire contents of which are incorporated herein by reference.

[0215] Fusion proteins Does not contain the KRAB domain In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, or Cas12a), a ZFP, or a TALE); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain); c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or f) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, or a TALE), and a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain).

[0216] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain; c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or f) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0217] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or f) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0218] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domains; c) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or f) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0219] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP; or f) Comprising a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0220] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain; c) a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP; or f) Comprises a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0221] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) TALE, DNMT3 methyltransferase binding domain (e.g., Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE; or f) Comprising a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0222] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain; c) a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, TALE, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE; or f) A TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0223] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain); c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) A DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0224] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a Dnmt3L (e.g., Mus musculus or Apodemus silvaticus DNMT3L) C-terminal domain; c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) A Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aurus9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396).

[0225] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) A DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0226] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domains; c) a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0227] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0228] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain; c) a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) Comprising a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0229] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain); c) a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) Comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0230] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE; b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain; c) a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), or d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0231] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain).

[0232] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0233] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0234] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domains.

[0235] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP; or b) comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0236] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP; or b) comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0237] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE; or b) Comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0238] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE; or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0239] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0240] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0241] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0242] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0243] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP.

[0244] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP.

[0245] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE.

[0246] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE.

[0247] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain).

[0248] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminus.

[0249] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0250] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0251] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0252] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0253] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0254] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0255] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0256] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0257] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0258] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0259] In some embodiments, the fusion protein comprises, from N- to C-terminus, a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0260] In some embodiments, the fusion protein comprises, from N- to C-terminus, a ZFP, a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0261] In some embodiments, the fusion protein comprises, from N- to C-terminus, a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0262] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0263] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0264] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus sCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0265] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0266] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0267] In some embodiments, the fusion protein comprises, from N- to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0268] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0269] In some embodiments, the fusion protein comprises, from N- to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0270] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0271] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0272] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0273] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0274] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0275] In some embodiments, the fusion protein comprises, from N- to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP.

[0276] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP.

[0277] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE.

[0278] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE.

[0279] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0280] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0281] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0282] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0283] In some embodiments, the fusion protein comprises, from N- to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0284] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0285] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0286] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0287] Contains the KRAB domain In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, or Cas12a), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0288] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aurueus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0289] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) DNMT3 methyltransferase binding domains (e.g., Dnmt3L C-terminal domain, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and KRAB domains (e.g., KOX1 or ZIM3 KRAB domains); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0290] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), Dnmt3L (e.g., Mus musclus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), c) a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0291] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A ZFP comprising an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0292] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A ZFP comprising an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0293] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A TALE comprising an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0294] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); d) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); e) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or f) A TALE comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0295] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) A DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0296] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), c) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0297] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) A DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0298] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and KRAB domain (e.g., KOX1 or ZIM3 KRAB domain), c) a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0299] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0300] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) comprising a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0301] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0302] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain); c) a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or d) A Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0303] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFT or a TALE) and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0304] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenenes dCas9, S. aureus dCas9, dCas12a, or Cas12f), a ZFP, or a TALE), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0305] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0306] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0307] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0308] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0309] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0310] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), or b) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0311] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0312] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0313] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0314] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0315] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0316] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0317] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0318] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0319] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0320] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0321] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0322] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0323] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0324] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0325] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0326] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0327] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0328] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0329] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0330] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0331] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0332] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0333] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0334] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0335] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0336] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0337] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0338] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0339] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0340] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0341] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0342] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0343] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0344] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0345] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0346] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0347] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0348] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a ZFP, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0349] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0350] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a TALE, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0351] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0352] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0353] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0354] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0355] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0356] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0357] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0358] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain).

[0359] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, or Cas12a), a ZFP, or a TALE); b) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase-binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or d) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0360] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE); b) a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), c) a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or d) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0361] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f); b) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., Dnmt3L C-terminal domain), a KRAB domain (e.g., KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or d) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0362] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aurus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f); b) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, KRAB domain (e.g., KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a C-terminal domain of Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or d) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0363] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP; b) a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP; or d) A ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0364] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP; b) a ZFP, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a C-terminal domain of Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP; or d) A ZFP comprising an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0365] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE; b) TALE, DNMT3 methyltransferase binding domain (e.g., Dnmt3L C-terminal domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), an H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE; or d) A TALE comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0366] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE; b) a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87); c) the C-terminal domain of Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE; or d) A TALE comprising H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a C-terminal domain of Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L).

[0367] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or b) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0368] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), or b) A DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0369] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or b) comprising a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0370] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCaus9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), or b) Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0371] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP; or b) A ZFP comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0372] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP; or b) A ZFP comprising a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0373] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE; or b) A TALE comprising a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0374] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: a) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE; or b) A TALE comprising a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0375] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0376] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0377] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0378] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0379] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a ZFP.

[0380] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a ZFP.

[0381] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), and a TALE.

[0382] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, and a TALE.

[0383] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a or dCas12f), a ZFP, or a TALE), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0384] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0385] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0386] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0387] In some embodiments, the fusion protein comprises, from N- to C-terminus, a ZFP, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0388] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0389] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0390] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87).

[0391] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0392] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, e.g., the sequence set forth in SEQ ID NO: 81 or 87), and a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE).

[0393] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0394] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, e.g., the sequence set forth in SEQ ID NO: 81 or 87), and a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f).

[0395] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP.

[0396] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a ZFP.

[0397] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain), a KRAB domain (e.g., a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain) H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE.

[0398] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Dnmt3L (e.g., Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain, a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), and a TALE.

[0399] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0400] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNA-binding domain (e.g., a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), a ZFP, or a TALE), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a C-terminal domain of Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L).

[0401] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0402] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Cas (e.g., Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, S. aureus dCas9, dCas12a, or dCas12f), H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396 of a sequence such as that set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a C-terminal domain of Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L).

[0403] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0404] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a ZFP, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0405] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a DNMT3 methyltransferase binding domain (e.g., a Dnmt3L C-terminal domain).

[0406] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a TALE, H3K4me0 (e.g., a 12-aa or 30-aa N-terminal fragment of SEQ ID NO: 393, 394, 395, or 396, such as the sequence set forth in SEQ ID NO: 81 or 87), a KRAB domain (e.g., a KOX1 or ZIM3 KRAB domain), and a Dnmt3L (e.g., a Mus musculus or Apodemus sylvaticus DNMT3L) C-terminal domain.

[0407] In some embodiments, the fusion protein comprises, from N- to C-terminus, H3K4me0, a DNMT3 methyltransferase binding domain, and a DNA binding domain (e.g., dCas9, ZFP, or TALE). In some embodiments, the fusion protein comprises, from N- to C-terminus, H3K4me0 (e.g., SEQ ID NO: 87), a DNMT3 methyltransferase binding domain, a DNA binding domain (e.g., dCas9), and a KRAB domain. In some embodiments, the fusion protein comprises, from N- to C-terminus, H3K4me0 (e.g., SEQ ID NO: 87), a KRAB domain, a DNMT3 methyltransferase binding domain, and a DNA binding domain (e.g., dCas9).

[0408] In some embodiments, the fusion proteins disclosed herein comprise, from N- to C-terminus, an H3K4me0, a DNA binding domain, and a DNMT3 methyltransferase binding domain. In some embodiments, the fusion proteins disclosed herein comprise, from N- to C-terminus, an H3K4me0, a DNA binding domain, a DNMT3 methyltransferase binding domain, and a KRAB domain.

[0409] In some embodiments, the fusion protein comprises: a) an H3K4me0 having at least about 80% sequence identity to at least one of the sequences set forth in SEQ ID NOs: 81 and 87; b) a Dnmt3L C-terminal domain having at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NOs: 31 to 50 and 71 to 75; or c) a dCas9 having at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or any combination of the foregoing.

[0410] In some embodiments, the fusion protein comprises: a) H3K4me0 having 100% sequence identity to the sequence set forth in SEQ ID NO: 81 or 87; b) a Dnmt3L C-terminal domain having 100% sequence identity with the sequences set forth in SEQ ID NOs: 31 to 50 and 71 to 75, or c) dCas9 having 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or any combination of the foregoing.

[0411] In some embodiments, the fusion protein comprises: a) H3K4me0 having 80% sequence identity with the sequence set forth in SEQ ID NO: 81; b) a Dnmt3L C-terminal domain having 80% sequence identity to the sequence set forth in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 38; or c) dCas9 having 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or any combination of the foregoing.

[0412] In some embodiments, the fusion protein comprises: a) H3K4me0 having 100% sequence identity with the sequence set forth in SEQ ID NO: 81; b) a Dnmt3L C-terminal domain having 100% sequence identity to the sequence set forth in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 38; or c) dCas9 having 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or any combination of the foregoing.

[0413] In some embodiments, the fusion protein comprises: a) an H3K4me0 having at least about 80% sequence identity to the sequence set forth in SEQ ID NO: 87; b) a Dnmt3L C-terminal domain having at least about 80% sequence identity to the sequence set forth in SEQ ID NO: 32; or c) a dCas9 having at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or any combination of the foregoing.

[0414] In some embodiments, the fusion protein comprises: a) H3K4me0 having 100% sequence identity to the sequence set forth in SEQ ID NO: 87; b) a Dnmt3L C-terminal domain having 100% sequence identity with the sequence set forth in SEQ ID NO: 32, or c) dCas9 having 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; d) or any combination of the foregoing.

[0415] In some embodiments, the fusion protein comprises: a) H3K4me0 having at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NO: 87, SEQ ID NO: 81, and SEQ ID NOs: 393 to 396; b) a DNMT3 methyltransferase binding domain having at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NOs: 31 to 50 and 71 to 75; c) a DNA-binding domain having at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 489, SEQ ID NOs: 3 to 15, and SEQ ID NOs: 454 to 469; d) a KRAB domain having at least 80% ...

Claims

1. A fusion protein comprising a DNA-binding domain, a DNMT3 methyltransferase-binding domain, and H3K4me0.

2. The fusion protein of claim 1, wherein the fusion protein further comprises a Kruppel-associated box (KRAB) domain, and optionally the KRAB domain is a KOX1 KRAB domain or a ZIM3 KRAB domain.

3. a) the fusion protein lacks nuclease activity; b) the fusion protein lacks a DNA methyltransferase catalytic domain; c) the DNMT3 methyltransferase binding domain binds to the catalytic domain of DNMT3A, and optionally the DNMT3 methyltransferase binding domain comprises a DNA methyltransferase 3-like protein (Dnmt3L) or a C-terminal fragment of Dnmt3L; 3. The fusion protein of claim 1 or 2, wherein the fusion protein is any combination of the foregoing.

4. 4. The fusion protein of any one of claims 1 to 3, wherein the DNA-binding domain comprises a DNA-binding domain of a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a tetracycline-controlled repressor (tetR), a meganuclease, a homing (HO) endonuclease, a eukaryotic programmable RNA-guided endonuclease, or any combination of the foregoing.

5. the DNA binding domain comprises a Cas, and optionally the Cas comprises: a) Streptococcus pyogenes dCas9, b) Staphylococcus aureus dCas9, c)S. aureus dCas9, d) dCas12a, or e) The fusion protein of any one of claims 1 to 4, which is dCas12f.

6. The fusion protein of any one of claims 1 to 4, wherein the DNA-binding domain comprises a zinc finger protein (ZFP) or a transcription activator-like effector (TALE).

7. 5. The fusion protein of claim 1, wherein the DNA-binding domain comprises a leucine zipper domain, a winged-helix domain, a helix-turn-helix domain, a helix-loop-helix domain, a chromatin-associated high mobility group (HMG) box domain, a white-opaque regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB)-fold domain, an immunoglobulin domain, or a B3 DNA-binding domain.

8. From the N-terminus to the C-terminus, a) H3K4me0, the DNMT3 methyltransferase binding domain, and the DNA binding domain; or b) the H3K4me0, the DNA binding domain, and the DNMT3 methyltransferase binding domain;

9. From the N-terminus to the C-terminus, a) the H3K4me0, the DNMT3 methyltransferase binding domain, the DNA binding domain, and the KRAB domain; b) the H3K4me0, the KRAB domain, the DNMT3 methyltransferase binding domain, and the DNA binding domain; c) the H3K4me0, the DNA binding domain, the DNMT3 methyltransferase binding domain, and the KRAB domain; d) the H3K4me0, the KRAB domain, the DNA binding domain, and the DNMT3 methyltransferase binding domain; or The fusion protein according to any one of claims 2 to 8, comprising: a) the H3K4me0, the DNA binding domain, the KRAB domain, and the DNMT3 methyltransferase binding domain.

10. a) the H3K4me0 has at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NO: 87, SEQ ID NO: 81, and SEQ ID NOs: 393-396; b) the DNMT3 methyltransferase binding domain has at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NOs: 31-50 and 71-75; c) the DNA-binding domain has at least 80% sequence identity with at least one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-15, and SEQ ID NOs:454-469; or or any combination of the foregoing.

11. a) the H3K4me0 has 100% sequence identity with the sequences set forth in SEQ ID NO: 87, SEQ ID NO: 81, and SEQ ID NOs: 393-396; b) the DNMT3 methyltransferase binding domain has 100% sequence identity with the sequences set forth in SEQ ID NOs: 31-50 and 71-75; c) the DNA-binding domain has 100% sequence identity with at least one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-15, and SEQ ID NOs:454-469; or or any combination of the foregoing.

12. a) the H3K4me0 has at least 80% sequence identity to SEQ ID NO:87, SEQ ID NO:81, or both; b) the DNMT3 methyltransferase binding domain has at least 80% sequence identity to SEQ ID NO: 31, SEQ ID NO: 32, or both; c) the DNA-binding domain has at least 80% sequence identity with the sequences set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-9, and SEQ ID NOs:454-461; or any combination of the foregoing.

13. a) the H3K4me0 has 100% sequence identity with SEQ ID NO: 87 or SEQ ID NO: 81; b) the DNMT3 methyltransferase binding domain has 100% sequence identity to SEQ ID NO: 31 or SEQ ID NO: 32; c) the DNA-binding domain has 100% sequence identity with the sequences set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-9, and SEQ ID NOs:454-461; or any combination of the foregoing.

14. The fusion protein of any one of claims 2 to 13, wherein the KRAB domain has at least 80% sequence identity with SEQ ID NO:

155.

15. The fusion protein of any one of claims 2 to 14, wherein the KRAB domain has 100% sequence identity with SEQ ID NO:

155.

16. 16. The fusion protein of any one of claims 1 to 15, wherein the H3K4me0 is connected to the DNMT3 methyltransferase binding domain by a linker of about 30 to 50 amino acids in length.

17. The fusion protein of any one of claims 2 to 15, wherein the H3K4me0 is connected to the KRAB domain by a linker of about 30 to 50 amino acids in length.

18. 17. The fusion protein of claim 16, wherein the linker has 100% sequence identity with the sequences set forth in SEQ ID NOs:89-100, 113-128, 145, 147, and 506.

19. 19. The fusion protein of claim 17 or 18, wherein the linker has 100% sequence identity with the sequences set forth in SEQ ID NOs:89-100, 113-128, 145, 147, and 506.

20. 20. The fusion protein of claim 19, wherein the linker has 100% sequence identity with SEQ ID NO:96 or SEQ ID NO:

506.

21. The fusion protein of any one of claims 1 to 20, wherein the fusion protein has at least 80% sequence identity with at least one sequence set forth in SEQ ID NOs: 157-169, 397, 398, and 470-488.

22. A polynucleotide encoding the fusion protein of any one of claims 1 to 21.

23. 23. The polynucleotide of claim 22, wherein the polynucleotide is about 6 kilobases (kb) or less in length.

24. 24. The polynucleotide of claim 22 or 23, wherein the polynucleotide is about 4.7 kb or less in length.

25. The polynucleotide according to any one of claims 22 to 24, wherein the polynucleotide is a single-stranded linear DNA.

26. A vector comprising the polynucleotide of any one of claims 22 to 25.

27. 27. The vector of claim 26, wherein the vector is about 6 kb or less in length.

28. 28. The vector of claim 26 or 27, wherein the vector is about 4.7 kb or less in length.

29. A gene delivery system comprising the polynucleotide of any one of claims 22 to 25 or the vector of any one of claims 26 to 28.

30. 30. The gene delivery system of claim 29, comprising a viral gene delivery system.

31. the viral gene delivery system comprises an adeno-associated virus vector, an adenovirus vector, a herpes simplex virus vector, or a retrovirus vector, and optionally a) the adeno-associated viral vector comprises an AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAV10 viral vector, or a variant thereof; b) the adenoviral vector comprises AD100; c) the herpes simplex virus vector comprises HSV-2; or d) The gene delivery system of claim 30, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

32. 32. The gene delivery system of claim 30 or 31, wherein the viral gene delivery system comprises an adeno-associated virus (AAV).

33. 30. The gene delivery system of claim 29, comprising a non-viral gene delivery system.

34. 34. The gene delivery system of any one of claims 29 to 33, further comprising a guide RNA, optionally pre-complexed with the fusion protein.

35. 35. A composition comprising the fusion protein of any one of claims 1 to 21, the polynucleotide of any one of claims 22 to 25, the vector of any one of claims 26 to 28, or the gene delivery system of any one of claims 29 to 34, or any combination of the foregoing.

36. 36. A pharmaceutical composition comprising the composition of claim 35 and a pharmaceutically acceptable carrier.

37. A kit comprising a container and optionally instructions for use, said container comprising: a) a fusion protein according to any one of claims 1 to 21, b) a polynucleotide according to any one of claims 22 to 25, c) a vector according to any one of claims 26 to 28, d) a gene delivery system according to any one of claims 29 to 34; e) a composition according to claim 35, or f) the pharmaceutical composition of claim 36; or any combination of the foregoing.

38. The guide RNA is a) single guide RNA (sgRNA), or 38. The gene delivery system of claim 34, the composition of claim 35, the pharmaceutical composition of claim 36, or the kit of claim 37, comprising b) a CRISPR RNA (crRNA) and a trans-activated CRISPR RNA (tracrRNA) (cr:tracrRNA).

39. 38. A cell comprising the fusion protein of any one of claims 1 to 21, the polynucleotide of any one of claims 22 to 25, the vector of any one of claims 26 to 28, or the gene delivery system of any one of claims 29 to 34 and 38, or any combination of the foregoing.

40. A progeny cell derived from the cell of claim 39.

41. 41. The cell of claim 39 or 40, wherein the cell is in vitro or ex vivo.

42. 41. The cell of claim 39 or 40, wherein the cell is in vivo.

43. 29. A method of epigenetically modifying a genomic locus in a cell, the method comprising delivering to said cell a fusion protein of any one of claims 1 to 21, a polynucleotide of any one of claims 22 to 25, or a vector of any one of claims 26 to 28.

44. The method of claim 43, comprising delivering to said cell a polynucleotide according to any one of claims 22 to 25.

45. 45. The method of claim 43 or 44, wherein delivering the polynucleotide comprises transfection, viral infection, or delivery by lipid nanoparticles.

46. 44. The method of claim 43, comprising delivering to said cell a fusion protein according to any one of claims 1 to 21.

47. 47. The method of any one of claims 43-46, further comprising delivering to the cell an sgRNA or cr:tracrRNA that targets the genomic locus.

48. 48. The method of any one of claims 43 to 47, wherein the genomic locus comprises a cytosine-guanine dinucleotide (CpG).

49. 48. The method of any one of claims 43 to 47, wherein the genomic locus lacks cytosine-guanine dinucleotides (CpG).

50. 50. The method of any one of claims 43 to 49, wherein the cell expresses DNMT3A methyltransferase and the fusion protein recruits DNMT3A to the genomic locus in the cell.

51. 51. The method of any one of claims 43 to 50, wherein epigenetically modifying the genomic locus comprises methylating DNA at or near the genomic locus.

52. 52. The method of any one of claims 43 to 51, wherein the genomic locus comprises a target gene.

53. 53. The method of any one of claims 43 to 52, wherein epigenetically modifying the genomic locus reduces or silences expression of the target gene.

54. 54. The method of any one of claims 43 to 53, wherein expression of the fusion protein is self-silencing.

55. 55. An epigenetically modified cell or a progeny thereof produced by the method of any one of claims 43 to 54.

56. 42. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a fusion protein according to any one of claims 1 to 21, a polynucleotide according to any one of claims 22 to 25, a vector according to any one of claims 26 to 28, a gene delivery system according to any one of claims 29 to 34 and 38, a composition according to claim 35 or 38, a pharmaceutical composition according to claim 36 or 38, or a cell according to any one of claims 39 to 42.

57. 57. The method of claim 56, wherein the disease is a genetic disorder, an infectious disease, or a neurodegenerative disease.

58. 58. The method of claim 56 or 57, wherein the disease is a prion disease.

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