Crispr-based modular tool for specific introduction of ephenogenetic modification at a

Abstract

The present invention relates to a complex comprising i) a catalytically inactivated site-specific nuclease linked to ii) an array of 2-10, preferably 3-7 effector domains, each effector domain having a specific chromatin modification activity, the present invention relates to a nucleic acid activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, in which the effector domains are each separated by a linker that provides a sufficient distance between the domain and the nuclease, the present invention relates to novel complexes that do not substantially interfere with their specific chromatin modification activity, binding to site-specific nuclease enzymes, methods of using the complexes, and uses of the complexes.

Description

Technical Field

[0001] The present invention relates to a complex comprising i) a catalytically inactive site-specific nuclease, which is connected to ii) an array of 2-10, preferably 3-7 effector domains, each effector domain having a specific chromatin modification activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are each separated by a linker, which provides sufficient space between the domains and the nuclease so as not to substantially interfere with their specific chromatin modification activities and the binding of the site-specific nuclease, and also relates to various methods using the complex and the uses of the complex. Background Art

[0002] Coordinated regulation of transcription is essential for nearly all biological processes, from development to maintenance of homeostasis to disease. Therefore, understanding the nature, role, and environmental dependence of the molecular mechanisms that regulate gene expression is one of the core goals of modern biology.

[0003] The regulation of eukaryotic transcription is guided by a complex interplay between transcription factors (TFs), cis-regulatory elements, and epigenetic machinery. The epigenetic system is defined as a sequence-independent combination of regulatory molecules, heritable or otherwise, that influence chromatin structure, genome function, and transcriptional activity.

[0004] Most notably, the so-called epigenome is characterized by post-translational histone modifications and DNA methylation.

[0005] Loss of genes or functions involved in epigenetic regulation often results in embryonic lethality or morbidity (10), highlighting their essentiality for life. Furthermore, the dynamic nature of epigenetic modifications places them at the interface of genetic, developmental, and environmental interactions that ultimately produce phenotypes.

[0006] This has fueled a global initiative to map epigenetic modifications across developmental and disease contexts and to correlate them with the transcriptome, genome structure, and genetic variation (4, 11). For example, histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 acetylation (H3K27ac) are typically enriched at promoters of transcriptionally active genes in normal or diseased cell types. In contrast, H3K9me2, H3K27me3, H2AK119ub, and DNA methylation are typically associated with transcriptional repression, while H3K36me3 is enriched in the body of transcribed genes (12). These groundbreaking studies have provided unprecedented insights into genome function and revealed that chromatin modifications are important for controlling gene expression levels and normal cellular function.

[0007] In support of a critical role for chromatin modifications, manipulation of enzymes catalyzing H3K27me3 (trimethylation of histone 3 lysine 27), H2A119Kub, H3K4me3, H3K36me3, H3K9me3, and DNAme has resulted in widespread abnormal gene expression and embryonic lethality (1, 7–9). In addition, alterations in the epigenetic landscape have been directly implicated in a variety of diseases, including cancer and aging (3). However, clarifying direct and indirect effects remains difficult. Indeed, decoding the quantitative effects of chromatin marks themselves, as well as tracing their causal relationships, is plagued by the pleiotropic effects of global dysregulation and the non-histone substrates and non-catalytic functions of chromatin modifiers. For example, although H3K4me1 / H3K27ac is closely associated with active enhancers, recent evidence suggests that these marks may play a relatively minor role in enhancer function (10, 11). Therefore, the field must move beyond mapping changes in these marks to accurately defining their causal and context-dependent functions, which is important for understanding disease mechanisms. At the same time, there is a need to develop tools that can specifically "edit" chromatin modifications at specific loci in order to precisely reverse or manipulate abnormal gene activity in the diseases caused by them.

[0008] The advent of epigenetic editing technologies that allow for site-specific regulation of chromatin modifications can effectively address these challenges. One of the current tools fuses chromatin-modifying proteins to nuclease-inactive (d) Cas9, which is targeted to the locus via a guide (g) RNA (12).

[0009] Nakamura M et al. (in CRISPR technologies for precise epigenome editing. Nat Cell Biol. 2021Jan; 23(1): 11-22. doi: 10.1038 / s41556-020-00620-7. Epub 2021Jan 8. PMID: 33420494) disclosed that the epigenome involves a complex set of cellular processes that regulate genomic activity. Analyzing these complex mechanisms requires the development of tools that can specifically manipulate these processes. The redesign of the prokaryotic CRISPR system has enabled the development of a variety of technologies for epigenome engineering. They reviewed the current status of epigenetic manipulations that can be achieved and their relative applications. They concluded that with future optimization, CRISPR-based epigenome editing can serve as a powerful tool for understanding and controlling biological functions.

[0010] Goell JH and Hilton BI (in CRISPR / Cas-Based Epigenome Editing: Advances, Applications, and Clinical Utility. Trends Biotechnol. 2021 Jul; 39 (7): 678-691. doi: 10.1016 / j.tibtech.2020.10.012. Epub 2021 May 7. PMID: 33972106) disclosed that the epigenome dynamically regulates gene expression and guides cell differentiation during the life cycle of eukaryotic organisms. Recent advances in epigenome editing technology based on clustered regularly interspaced palindromic repeats (CRISPR) / Cas have enabled researchers to perform site-specific epigenetic modifications on endogenous DNA and histones and manipulate the structure of native chromatin. As a result, epigenome editing has helped reveal the relationship between epigenetic marks and gene expression. As epigenome editing tools continue to develop, researchers have applied them in new ways to explore the function of the epigenome in human health and disease. They discussed recent technical advances in CRISPR / Cas-based epigenome editing that have advanced clinical research and looked ahead to how these technologies might be improved for greater utility in the future.

[0011] Okada M et al. (in Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin. 2017 May 8; 10: 24. doi: 10.1186 / s13072-017-0129-1. PMID: 28503202; PMCID: PMC5422987) disclosed that epigenome editing is expected to manipulate gene transcription and cell fate, and revealed gene expression mechanisms in different cell types. For functional epigenome editing, it is necessary to evaluate the chromatin context-dependent activity of artificial epigenetic modifiers. They applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to mouse primary T cells, focusing on the plug box P3 (Foxp3) locus, a master transcription factor of regulatory T cells (Treg). The Foxp3 locus is regulated by combinatorial epigenetic modifications that determine Foxp3 expression. Foxp3 expression is unstable in transforming growth factor β (TGF-β)-induced Tregs (iTregs) but relatively stable in thymus-derived Tregs (tTregs). To stabilize Foxp3 expression in iTregs, the authors introduced dCas9-TET1CD (dCas9 fused to the catalytic domain of 10-11 translocation dioxygenase 1 (TET1), a methylcytosine dioxygenase) and dCas9-p300CD (dCas9 fused to the CD of p300 histone acetyltransferase), along with guide RNA (gRNA) targeting the Foxp3 locus. Although dCas9-TET1CD induced partial demethylation at an enhancer locus (called conserved noncoding DNA sequence 2, CNS2), robust Foxp3 stability was not observed. In contrast, dCas9-p300CD, which targeted a promoter site, partially maintained Foxp3 transcription in cultured primary T cells, even under inflammatory conditions in vitro. In addition, dCas9-p300CD promoted the expression of Treg signature genes and enhanced their suppressive activity in vitro. They concluded that artificial epigenome editing modified the epigenetic state and gene expression of the target locus and transformed cell function together with endogenous epigenetic modifications, indicating the effective use of these technologies to help analyze the relationship between chromatin state and gene expression.

[0012] Huang YH et al. (in DNA epigenome editing using CRISPR-Cas SunTag-directedDNMT3A. Genome Biol. 2017 Sep 18; 18(1): 176. doi: 10.1186 / s13059-017-1306-z. PMID: 28923089; PMCID: PMC5604343) disclosed that DNA methylation has a wide range of effects on gene expression during development. However, due to the low correlation between global DNA methylation patterns and gene expression, our ability to assign specific functions to specific regions of DNA methylation is still very limited. They used a nuclease-inactive Cas9 protein fused to a repetitive peptide epitope (SunTag) to recruit multiple copies of antibody-fused de novo synthesized DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate the target genomic site. They showed that dCas9-SunTag-DNMT3A significantly increased CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. In addition, with a single guide RNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5-kb genomic region and inhibit the expression of the HOXA5 gene. Reduced representative bisulfite sequencing and RNA-seq showed that dCas9-SunTag-DNMT3A had minimal effects on methylation of the target region, the global DNA methylation profile, and the transcriptome. They concluded that the efficient and precise tools discussed enable site-specific DNA methylation manipulation and can be used to address the relationship between DNA methylation and gene expression.

[0013] CN111748583A discloses an inducible DNA methylation editing system based on CRISPR / dCas9, characterized in that it comprises a guide element, an anchor element and an editing effector element that can act in sequence, the anchor element comprises a stimulus response protein A and an inactivated SpCas9, the editing effector element comprises a stimulus response protein B and a DNA methylation editing effector protein, the stimulus response protein A and the stimulus response protein B can bind to each other under the action of stimulation, and the binding is released after the stimulation disappears.

[0014] WO 2018 / 053037A1 discloses a composition and method for delivering enhanced demethylation activity to a target DNA sequence in mammalian cells. The composition and method can be used for regulating the activity of a target gene, or for constructing a gene regulatory network.

[0015] Vojta et al. (Repurposing the CRISPR-Cas9 system for targeted DNA methylation, Nucl Acid Res 2016 vol. 44, no. 12, doi: 10.1093 / nar / gkw159, ISSN 0305-1048, pages 5615–5628) developed a CRISPR-Cas9-based tool for specific DNA methylation, consisting of an inactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase DNMT3A, targeting any 20 bp DNA sequence followed by an NGG trinucleotide by co-expressing a guide RNA. They showed that the fusion protein can achieve targeted CpG methylation in a wide region of ~35 bp. They also showed that multiple guide RNAs can target the dCas9-DNMT3A construct to multiple adjacent sites, which allows methylation of most gene promoters. DNA methylation activity is specific to the targeted region and is heritable in mitosis. Finally, they showed that targeted DNA methylation of broader promoter regions of the target loci IL6ST and BACH2 reduced their expression.

[0016] Cas9 proteins (e.g., dCas9) containing mutations in the nuclease domain that are nuclease-deficient or nuclease-deficient still retain DNA binding activity when complexed with sgRNA. The dCas9 protein can be fused to and localize the effector domain or protein tag by fusion with the site matching the sgRNA, thereby forming an RNA-guided DNA binding enzyme. dCas9 can be fused to a transcriptional activation domain (e.g., VP64) or an inhibitory domain (e.g., KRAB), respectively, to activate or inhibit the target gene guided by sgRNA. dCas9 can also be fused to fluorescent proteins to achieve fluorescent labeling of chromosomal regions in living cells. Moreover, in the case where multi-copy protein tags or effector fusions are required to reach some biological thresholds or signal detection thresholds, the polymerization of effectors or protein tags by direct fusion with dCas9 proteins is technically limited by the difficulty of delivering large DNA encoding such fusions, or the difficulty of translating or transporting such large proteins to the nucleus due to the volume of the protein.

[0017] O'Geen H et al. (dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 2017 Sep 29; 45(17): 9901-9916. doi: 10.1093 / nar / gkx578. PMID: 28973434; PMCID: PMC5622328) reported that different epigenomic profiles of histone marks have been associated with gene expression, but questions about their causal relationship remain. They investigated the activity of a panel of genome-targeted epigenetic regulators (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A, and the first targetable version of Ezh2 and GATA-1 partner (FOG1)) that reflect epigenetic marks associated with repressive chromatin states. dCas9 fusions produced 0- to 10-fold repression of target genes that varied depending on locus and cell type. dCpf1 fusions failed to repress gene expression. The most persistent gene repression required the action of some effector domain; however, contrary to past reports, KRAB-dCas9 did not contribute to this persistence. The “direct ligation” strategy, which attached the Ezh2 methyltransferase enzyme to dCas9, and the “recruitment strategy,” which attached the N-terminal 45 residues of FOG1 to dCas9 to elicit an endogenous nucleosome remodeling and deacetylase complex, both successfully achieved targeted deposition of H3K27me3. However, surprisingly, repression was not associated with deposition of either H3K9me3 or H3K27me3. Their results suggest that so-called repressive histone modifications are not sufficient to achieve gene repression.

[0018] Chen et al. (Evelyn Chen, Enrique Lin-Shiao, Mohammad Saffari Doost, Jennifer A. Doudna Decorating chromatin for enhanced genome editing using CRISPR-Cas9 bioRxiv2022.03.15.484540; doi: https: / / doi.org / 10.1101 / 2022.03.15.484540) describe that CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. After CRISPR-Cas-induced double-stranded DNA breaks, homology-directed repair (HDR) in the presence of a donor template is the most common way to introduce precise editing today, but HDR efficiency is generally lower than that of the end-joining pathway that causes insertions and deletions (indels). They tested the hypothesis that HDR could be enhanced using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K4me3 and H3K36me3 that have been shown to mediate homologous recombination in human cells. The results showed that this fusion protein specifically contacts chromatin at the Cas9 cut site in DNA, doubling the observed HDR efficiency and increasing the HDR:indel ratio threefold compared to that caused by Cas9 alone. HDR enhancement occurred in multiple cell lines without an increase in off-target genome editing. These findings highlight the importance of chromatin structure in the selection of DNA repair pathways during CRISPR-Cas genome editing and provide new strategies to improve HDR efficiency.

[0019] Morita S et al. (Targeted DNA demethylation in vivo using dCas9-peptiderepeat and scFv-TET1 catalytic domain fusions. (2016) Nat Biotechnol 34: 1060-1065) disclosed that, despite the importance of DNA methylation in health and disease, there is still a lack of technology that can conveniently manipulate specific sequence methylation for functional analysis and therapeutic purposes. They improved the previously described dCas9-SunTag for efficient targeted demethylation of specific DNA loci. The original SunTag consists of 10 copies of a GCN4 peptide separated by a 5-amino acid linker. To achieve efficient recruitment of an anti-GCN4 scFv fused to 10-11 (TET) 1 hydroxylase (inducing demethylation), they changed the linker length to 22 amino acids. The system achieved >50% demethylation efficiency at 7 of the 9 loci tested. 4 of these 7 loci showed >90% demethylation efficiency. They demonstrated targeted demethylation of CpGs in regulatory regions in cell culture (embryonic stem cells, cancer cell lines, primary neural progenitor cells) as well as in vivo in mouse embryos, and a demethylation-dependent 1.7- to 50-fold upregulation of associated genes.

[0020] WO 2018 / 035495A1 relates to a method for modifying DNA methylation by contacting a genomic DNA sequence with a catalytically inactive site-specific nuclease fused to an effector domain having methylation or demethylation activity, and one or more guide sequences.

[0021] To date, studies have applied epigenetic editing to a subset of specific loci, with causal biological mechanisms largely inferred by measuring bulk expression changes (13). This approach to dissecting the epigenome has also attracted interest for biomedical and preclinical applications, such as reversing phenotypes of epigenome-dependent diseases (14). However, to achieve quantitative principles for understanding (or manipulating) chromatin function, we must currently (i) improve the on-target activity of these systems, (ii) expand their targeting capabilities to multiple markers and combinations thereof, (iii) measure effects at single-cell resolution to capture the distribution of responses, and, perhaps most critically, (iv) maximize throughput to more than hundreds of loci to systematically dissect context-dependent responses. Systematic genome-wide association studies (GWAS) have proven to be a powerful tool to advance genomic research toward the goal of predicting genotype-to-phenotype relationships. These efforts have shown that sequence variation (SNPs, indels) located in promoters and cis-regulatory elements (cREs) are the major source of phenotypic variation, suggesting that complex human traits are often characterized by inherited differences in gene regulation rather than coding sequence (15,16). Therefore, how diverse promoters / cREs and their cis-variants interact with epigenetic machinery to regulate gene activity and ultimately phenotype is a frontier challenge for understanding genome function, disease susceptibility, and evolutionary processes. Indeed, there is a complex connection between DNA sequence variation and epigenetic systems; chromatin state affects sequence-dependent transcription factor (TF) occupancy, and DNA sequence in turn affects chromatin state (17,18). The urgent need to understand genome function and the molecular mechanisms underlying a variety of biological processes has been accelerated by genome-scale perturbation strategies such as panel CRISPR screens by many research groups (including the inventors) (19-21). These advances have enabled multiparametric outputs of CRISPR-based screens (22-25). In particular, targeted perturbation sequencing (TAP-seq) technology (26) can accurately quantify the expression of thousands of target genes in single cells carrying a specific perturbation using thousands of perturbations across a population. Using these high-resolution methods to reveal the regulatory logic that the chromatin system uses to overlap DNA sequence, cis-variants, and cellular features to obtain quantitative gene control will be a key milestone in dissecting genotype-to-phenotype relationships and chromatin functions.

[0022] Chromatin modification is considered one of the key regulatory mechanisms of transcriptional regulation in normal and disease states. However, dissecting the precise causal function of specific chromatin modifications remains challenging, i.e., the field is limited by the technical ability to accurately and quantitatively study the causal role of epigenetic modifications in different genomic features. Even less is known about how chromatin states interact with diverse DNA sequences or cis-variants to quantitatively affect transcription, and how the cellular environment affects them. Understanding the causal relationship between epigenetic marks and gene expression remains a core question in chromatin biology, especially as recent advances in epigenome editing technologies begin to provide new insights into these processes. The discovery of CRISPR-Cas interference that blocks transcription by targeting a catalytically inactive mutant of Streptococcus pyogenes SpCas9 (dCas9) provides a valuable tool for gene expression regulation. Subsequently, multiple strategies have fused dCas9 to repressors or activators with well-defined functions (e.g., KRAB and VP64) to regulate gene expression with enhanced silencing and activation capabilities. In addition, novel labeling methods have made it possible to more efficiently recruit multiple effectors to a single dCas9 anchor bound to a specific genomic site. This recruitment strategy has been combined with chemical induction methods to provide control over the temporal dimension of transcriptional regulation. Finally, current research has focused on regulatory DNA sequences, by recruiting dCas9 fused to histone acetyltransferase p300 or dCas9 fused to DNA demethylase Tet1 to activate enhancers (Braun, SMG, Kirkland, JG, Chory, EJet al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat Commun 8, 560 (2017). https: / / doi.org / 10.1038 / s41467-017-00644-y). Therefore, there is a clear need for large-scale, targeted perturbation strategies to resolve the regulatory functions of chromatin marks across endogenous environments, so as to ultimately apply this technology to clinical scenarios and therapies. Summary of the invention

[0023] The goal of the present invention is to provide other novel tools derived from the above and used for epigenetics, methylation, precise genome control, and even related disease treatment. Other objects and advantages will become clear when the current specification is further studied in conjunction with the accompanying examples.

[0024] In the first aspect of the present invention, the object of the present invention is achieved by providing a complex (particularly a protein complex), which comprises i) a catalytically inactive site-specific nuclease, which is connected to ii) an array of 2-10, preferably 3-7 effector domains, each of which has a specific chromatin modification activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are each separated by a linker, and the linker provides a sufficient distance between the domain and the nuclease so as not to substantially interfere with its specific chromatin modification activity and the binding of the site-specific nuclease.

[0025] Preferred is the complex of the present invention, wherein the protein complex comprises a fusion protein of a nuclease linked to a protein sequence containing 3-7 effector domain binding motifs, such as Streptococcus pyogenes dCas9 GCN (3-7) , wherein the effector domain binding motifs are each separated by a linker. The complex also comprises a plurality of effector domains, each non-covalently bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA).

[0026] More preferred is the complex of the present invention, wherein the chromatin modifying activity is histone methylation, such as histone methylation that promotes stable or reversible regulation of gene expression.

[0027] In its second aspect, the object of the present application is achieved by providing a nucleic acid set, each nucleic acid encoding at least one of the proteins in the complex of the present invention, and / or guide RNA (gRNA) and / or tagBFP.

[0028] In its third aspect, the object of the present invention is achieved by providing a set of gene constructs (e.g., expression vectors) comprising a nucleic acid set of the present invention, wherein preferably, an effector domain, a CD361 and / or a CD441 are encoded. scFV Each nucleic acid of comprises an inducible promoter, such as a tet-responsive promoter. More preferably, the construct of the invention is a viral construct, such as a viral construct derived from an adeno-associated virus (AAV), a lentivirus or a retrovirus.

[0029] In its fourth aspect, the object of the present invention is achieved by providing a recombinant cell, which comprises the nucleic acid set of the present invention, and / or the gene construct set of the present invention.

[0030] In its fifth aspect, the object of the present invention is achieved by providing a method for preparing the complex of the present invention, which comprises expressing the nucleic acid set of the present invention and / or the gene construct set of the present invention in the recombinant cell of the present invention, optionally comprising a step of inducing expression, for example using tetracycline.

[0031] In its sixth aspect, the object of the present invention is achieved by providing a method for epigenetically modifying chromatin specifically in a cell, tissue, cell nucleus and / or a sample containing chromatin, the method comprising introducing the complex of the present invention and one or more guide RNAs into the cell, tissue, cell nucleus and / or sample, thereby specifically epigenetically modifying chromatin in the cell, tissue, cell nucleus and / or sample. Preferably, the epigenetic modification includes histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multiple combinatorial epigenetic editing of histones.

[0032] In its seventh aspect, the object of the present invention is achieved by providing a method for regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, the method comprising: introducing the complex of the present invention and one or more guide RNA sequences specific to at least one target DNA sequence into cells, tissues, cell nuclei and / or samples, thereby specifically epigenetically regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples. Preferably, at least one target DNA sequence comprises a nucleic acid sequence specific for a symptom and / or disease state associated with epigenetically modified chromatin, such as a genetic disease, a proliferative disease (e.g., cancer), autoantibody-producing immune cells, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0033] In its eighth aspect, the object of the present invention is achieved by providing a method for detecting the biological effects of specific epigenetic modifications of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, and / or for regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, the method comprising performing the method of the present invention and detecting at least one biological effect in cells, tissues, cell nuclei and / or samples containing chromatin, wherein the biological effect is selected from changes in gene expression, changes in protein amounts, cis-genetic effects, changes in nucleic acid splicing, changes in the localization of gene loci in the cell nucleus, changes in TAD formation and destruction, changes in termination sites, activation of promoters, inhibition of promoters, changes in genetic-epigenetic interactions, the functional relationship between genetic variations in chromatin and epigenetic states, changes in genetic methylation and imprinting, and associating specific epigenetic changes with diseases or cell phenotypes.

[0034] In its ninth aspect, the object of the present invention is achieved by providing a cell having a specific epigenetically modified chromatin, which cell is prepared by performing the method of the present invention, and optionally isolating the cell, wherein preferably the cell is a stem cell, a neuron, a post-mitotic cell, or a fibroblast, and / or wherein the cell is an animal cell, such as a mammalian cell, preferably a human cell or a rodent cell.

[0035] In its tenth aspect, the object of the present invention is achieved by providing a method for identifying a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, the method comprising performing the method of the present invention in the presence and absence of a test drug, wherein if the regulation and / or biological effect in the presence of the test drug is different from the regulation and / or biological effect in the absence of the drug or from the control, the test drug is identified as a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples containing chromatin.

[0036] In its eleventh aspect, the object of the present invention is achieved by providing a method for preventing or treating a disease associated with epigenetically modified chromatin in a subject in need of prevention or treatment, the disease being, for example, a genetic disease, a proliferative disease (e.g., cancer), an immune cell producing autoantibodies, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome), the method comprising administering to a subject in need of the treatment an effective amount of a complex of the present invention and at least one of one or more suitable guide RNA sequences, a nucleic acid set of the present invention, a gene construct (e.g., an expression vector) set of the present invention, a cell of the present invention, and / or a drug identified by the present invention.

[0037] Another preferred aspect relates to the use of the complex of the present invention and at least one of one or more suitable guide RNA sequences, the nucleic acid set of the present invention, the gene construct (e.g., expression vector) set of the present invention, the cell of the present invention, and / or the drug identified by the present invention in a subject in need of prevention or treatment of a disease, or in the prevention and / or treatment of a disease associated with epigenetic modification of chromatin, such as a genetic disease, a proliferative disease (e.g., cancer), autoantibody-producing immune cells, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0038] In its twelfth aspect, the present invention aims to specifically epigenetically modify chromatin in cells, tissues, cell nuclei and / or samples containing chromatin according to the present invention, regulate the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin according to the present invention, detect the biological effects of specific epigenetic modified chromatin in cells, tissues, cell nuclei and / or samples according to the present invention, and / or identify the use of drugs according to the present invention by providing a complex of the present invention, a nucleic acid set of the present invention, a gene construct (e.g., expression vector) set of the present invention, and / or a cell of the present invention.

[0039] As mentioned above, in its first aspect, the objects of the present invention are achieved by a complex, in particular a protein complex, comprising:

[0040] i) a catalytically inactive site-specific nuclease linked to

[0041] ii) an array of 2-10, preferably 3-7, effector domains, each effector domain having a specific chromatin modifying activity,

[0042] The effector domains are each separated by a linker, and the linker provides a sufficient distance between the domains and the nuclease so as not to substantially interfere with their specific chromatin modification activity and the binding of the site-specific nuclease.

[0043] The specific chromatin modification activity is preferably selected from a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity.

[0044] The present invention provides an improved epigenome editing platform to systematically program specific combinatorial chromatin modifications across tens of thousands of scenarios in living cells. This resource can be used to capture multimodal functional responses from multiple lines at allelic, single-cell resolution. Precision perturbations of unprecedented scale will help reveal the regulatory logic of the interaction of different chromatin modifications with genomic features, sequence variants, and cellular features to shape quantitative gene expression patterns and further clarify the cis-acting and trans-structural mechanisms that implement epigenetic functions (especially chromatin marks). In addition, the present invention can be used to reverse or manipulate abnormal genomic or chromatin states in diseases.

[0045] What has been developed is a modular CRISPR-based toolkit that, in its current form, enables precise and inducible deposition of nine different epigenetic modifications at endogenous target loci. The ability to site-specifically deposit epigenetic marks such as methylation, including H3K27me3, H3K4me3, H3K79me3, H2AK119ub, and H3K36me3, presents a powerful gain-of-function perturbation strategy to unambiguously assess their causal role. In a preferred embodiment, the current tool provides a dCas9 (dCas9 GCN4 ), each motif is separated by a linker sequence, which is designed with an optimized space to accommodate large proteins without causing steric hindrance to their catalytic activity. GCN4 Can carry multiple (e.g., 5) "effector" proteins or domains directed against a specific locus, wherein the effector domains are complexed with a scFv domain specific for GCN4 (see Figure 1 A). The inventors have engineered and tested a comprehensive set of effector domains that preferably contain only the catalytic domains (collectively referred to as CD scFV), such as Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV .

[0046] The complexes of the present invention have a number of other advantages, which when integrated result in an epigenetic editing platform technology with unique features that enable exploration. These advantages include:

[0047] Efficient editing. For example, 5 copies of a particular CD scFV Recruitment to target loci greatly enhances on-target programming of chromatin modifications in terms of intensity and genome breadth. This ensures de novo deposition of histone methylation comparable to strong endogenous peaks, contributing to both negative and positive functional effects.

[0048] Specificity of the catalytic domain. When using tailored effector domains (e.g., only the catalytic core of these), the complex avoids unwanted side effects of targeting the full-length chromatin modifying protein. Because the full-length protein may have many non-catalytic functions and / or recruit other protein complexes, it is preferred to use the catalytic domain so that the function of the targeted chromatin mark itself can be assessed (see below for details).

[0049] Combinatorial epigenetic editing. The complexes and systems of the present invention are modular and can thus simultaneously recruit (preferably up to 5) different CD scFV This enables multiplexed and controllable epigenetic editing, allowing the construction of de novo synthesized sets of different chromatin modification domains (e.g., bivalent or multiplex combinations).

[0050] Off-target effects are minimized. Due to the effector domain and CD scFV Rather than being directly fused to dCas9, CDs generally lack their endogenous DNA-binding domain and they exhibit minimal off-target activity.

[0051] In the gene construct provided in the context of the present invention, each effector domain and / or CD scFV Dynamically inducible via a DOX-responsive promoter and can carry a protein destabilization (D2) domain that promotes rapid degradation upon DOX withdrawal. This allows for the study of temporal responses and epigenetic memory (chromatin persistence).

[0052] Dynamic tracking. Preferably, some or all effector groups are linked to superfolded GFP (sfGFP), and some or all gRNAs are linked to tagBFP, so that the system can be tracked in real time and can be used for cell purification and testing of drug dose-dependent responses (e.g., comparing GFP 低 and GFP 高 cell).

[0053] As a further control, we also generated scFV The same effector domain but containing a single point mutation that abolishes its enzymatic activity (catalytic variant effector, mut-CD scFV ). These include: Dot1l-mut-CD scFV 、p300-mut-CD scFV 、Prdm9-mut-CD scFV 、Setd2-mut-CD scFV 、Ring1b-mut-CD scFV 、Ezh2-mut-CD scFV 、G9a-mut-CD scFV 、Kmt5C-mut-CD scFV 、Dnmt3a3L-mut-CD scFV These effector variants are unable to make chromatin modifications on histones and DNA. Therefore, they serve as important controls to ensure that, for example, changes in gene expression are entirely caused by the deposition of the chromatin modification itself and not by other indirect events such as the recruitment of other protein complexes (which are only possible when wild-type CD3 is recruited). scFV Rather than the variant mut-CD scFV In addition, an effector containing only GFP (GFP scFV ) to control large-volume dCas9 GCN4 Steric hindrance caused by protein recruitment to chromatin.

[0054] Taken together, these properties allow for carefully controlled assessment of the functional impact of introducing a specific chromatin modification at any given locus, while also ruling out confounding effects. They also allow for the ability to introduce or reverse specific chromatin modifications in living cells, thereby enabling reversal of pathological outcomes.

[0055] Policarpi C, Dabin J, and Hackett AJ (in Epigenetic editing: Dissecting chromatin function in context. Bioessays. 2021 May; 43 (5): e2000316. doi: 10.1002 / bies.202000316. Epub 2021 Mar 16. PMID: 33724509) reviewed the SpdCas9-SunTag technology, which consists of a SpdCas9 enzyme fused to multiple (5-20) tandem repeats of the GCN4 binding motif (SunTag), which recruits any protein that binds to the small chain variable fragment (scFV) domain. This system allows multiple copies of the same effector domain (or different effector domains) to be recruited to specific gene sites, thereby promoting high on-target editing efficiency and inducing increased diffusion of epigenetic states, while minimizing off-target effects. The same strategy will analyze how combinatorial chromatin modifications affect transcription and genomic regulation in the future. However, the described dCas9-SunTag system is a fusion of the dCas9 protein with a GCN4 peptide tail capable of recruiting up to 10 copies of scFV-VP64, which amplifies the activation signal (Tanenbaum ME, Gilbert L.A., Qi LS, Weissman JS, Vale RDA protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014; 159: 635–646). In addition, it is reported that spacers as short as 5 amino acids are disclosed as being sufficient to separate peptides so that antibodies can bind to adjacent peptides.

[0056] WO 2021 / 247570 discloses a fusion protein, composition and method for manipulating the genome of a living organism. The fusion comprises a DNA methyltransferase domain, a first XTEN linker, a nuclease-deficient RNA-guided endonuclease, a second XTEN linker, and a Krüppel-associated box domain from the N-terminus to the C-terminus.

[0057] WO 2014 / 197748 discloses compositions related to systems based on clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9 and methods of using the compositions related to systems based on CRISPR / Cas9 to change gene expression and genome engineering. Compositions for changing gene expression and genome engineering in muscles (e.g., skeletal muscle and cardiac muscle) and methods of using the compositions are also disclosed. These tools use a single fusion protein containing the necessary KRAB (Krüppel-associated box domain). The current system does not require KRAB and is more specific (making lower "off-target effects" and more "fine-tuning" possible).

[0058] Carlini V, Policarpi C and Hackett JA (in Epigenetic inheritance is gated by pluripotency and Dppa2.EMBO J.2022Apr 4;41(7):e108677.doi:10.15252 / embj.2021108677.Epub 2022Feb 24.PMID:35199868;PMCID:PMC8982627) developed a CRISPR-based epigenetic programming tool. They used a catalytically inactive (d)Cas9 (dCas9) fused to five GCN4 repeats with optimized spacing. GCN4 ). They serve as docking sites to recruit up to five “effectors” to specific genomic loci via their single-chain antibody (scFv) domains. This modular system amplifies the quantitative level and domain size of on-target epigenetic editing relative to dCas-effector fusions, while minimizing off-target effects. To target de novo heterochromatin, they generated KRAB GFP-scFV and DNMT3A / 3L GFP-scFV Effectors promote direct deposition of H3K9me3 and DNA methylation, respectively. Current systems have been significantly improved and now support many different types of chromatin editing (see below for details).

[0059] In the context of the present invention, "protein complex" refers to a complex of biomolecules, which at least mostly comprises a peptide chain (e.g., protein) composed of amino acids. The complex is formed by covalent and / or non-covalent bonds, preferably including molecules that are complexed by antigen / antigen binding (e.g., based on antibody recognition) activity. The complex may also contain other proteins (e.g., fluorescent tags), as well as nucleic acids (e.g., gRNA) and / or other chemical groups (e.g., linkers or coupling agents).

[0060] In another aspect of the methods and compounds of the invention, a variety of tags and / or labels are used. Labels can be used for nucleic acid molecules that form part of the assay, as well as protein components (e.g., nucleases and / or fusions). Tags and labels can be included in the assay components (particularly nucleic acids and / or proteins), as well as constituting groups that are covalently or non-covalently attached.

[0061] In principle, any suitable catalytically inactive site-specific nuclease can be used in the context of the protein complex of the present invention. As described above, a nuclease-deficient or nuclease-deficient Cas9 protein (e.g., dCas9) having a mutation in the nuclease domain retains DNA binding activity when compounded with sgRNA. The dCas protein can tether and locate the effector domain or protein tag to a site matching the sgRNA by protein fusion, thereby forming an RNA-guided DNA binding enzyme. dCas9 can be fused with a transcriptional activation domain (e.g., VP64) or an inhibitory domain (e.g., KRAB), respectively, to activate or inhibit the target gene through sgRNA guidance. dCas9 can also be fused with a fluorescent protein to achieve fluorescent labeling of live cells in a chromosomal region. However, in such systems, since the sgRNA:Cas9 pairing is exclusive, only one Cas9-effector fusion can be achieved. At the same time, in cases where multiple copies of protein tags or effector fusions are required to reach some biological threshold or signal detection threshold, multimerization of effector or protein tags by direct fusion with dCas9 protein is technically limited, for example, by the difficulty of delivering larger DNA encoding these fusions, or by the difficulty of translating or transporting these larger proteins into the nucleus due to protein size. Preferred are catalytically inactive site-specific nucleases selected from catalytically inactive (d) Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, Scel, and dCas9 / fCas9 fusions of Streptococcus pyogenes. Because some preferred delivery systems (e.g., AAV) have only limited payload capacity (i.e., can only accommodate a certain number and size of protein molecules), this has stimulated the development of smaller CRISPR / Cas9 systems, such as those based on Staphylococcus aureus saCas9, which is a preferred nuclease.

[0062] Preferred is the complex of the present invention, wherein the complex comprises a fusion protein in which a nuclease is fused or linked to a protein sequence containing 3-7 effector domain binding motifs, such as Streptococcus pyogenes dCas9GCN4 (3-7), the effector domain binding motifs are each separated by a second linker sequence, and the complex optionally further comprises a plurality of effector domains, each bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA).

[0063] In the context of the present invention, an "effector domain" is a polypeptide or protein having at least one specific chromatin modification activity. Preferred are effector domains having specific chromatin modification activities, such as specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or specific chromatin demethylation / deacetylation activity. The present invention also includes combined effector domains that exhibit their chromatin modification activities, so-called "combined effector domains". The present invention also preferably uses modified effectors, i.e., catalytic domains (CDs) comprising full-length chromatin modification polypeptides or proteins, and / or more preferably catalytic domains (CDs) fused to scFV domains specific to effector domain binding motifs (CDscFV). Again, in view of limited payload delivery vectors (such as AAV-based vectors), it has other advantages and has been shown to enhance the activity and specificity of the system. Other means of delivery systems include non-viral delivery modes, including physical methods (e.g., electroporation, microinjection, sonoporation, and hydrodynamic delivery) and chemical methods (e.g., lipid particles, polymeric nanoparticles, gold nanoparticles, and cell penetrating peptides (CPP)).

[0064] In the context of the present invention, the term effector domain will also include polypeptides that are at least 50%, preferably to 70%, more preferably at least 80%, more preferably at least 90%, more preferably to 95% identical to the polypeptide sequences disclosed herein and have the chromatin modifying activity disclosed herein.

[0065] More preferred in the context of the present invention is a complex of the present invention, wherein the effector domain comprises a chromatin modifying protein or polypeptide selected from Dot1L (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), OGT (GIcNAc), a catalytic domain (CD, if feasible), or a catalytic domain (CD) thereof fused to a scFV domain specific for the effector domain binding motif (CDscFV, if feasible).

[0066] Histones can be modified in a variety of ways (see above), including acetylation, methylation (lysine), methylation (arginine), phosphorylation, ubiquitination, paraubiquitination, ADP ribosylation, deimination, and proline isomerization. Preferred are complexes of the invention wherein the chromatin modifying activity is histone methylation, such as histone methylation that contributes to stable or reversible gene expression regulation.

[0067] In the context of the present invention, preferred effector domains for histone modification are selected from the group consisting of HAT1, CBP / P300, PCAF / GCN5, TIP60, HB01 (ScESA1, SpMST1), ScSAS3, ScSAS2 (SpMST2), ScRTT109, SirT2 (ScSir2), SUV39H1, SUV39H2, G9a, ESET / SETDB1, EuHMTase / GLP, CLL8, SpClr4, MLL1, MLL2, MLL3, MLL4, MLL5, SET1A, SET1B, ASH1, Sc / Sp SET1, SET2 (Sc / Sp SET2), NSD1, SYMD2, DOT1, Sc / Sp DOT1, Pr-SET 7 / 8, SUV4 20H1, SUV4 20H2, SpSet 9. EZH2, RIZ1, LSD1 / BHC110, JHDM1a, JHDM1b, JHDM2a, JHDM2b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC 1. JMJD2D, CARM1, PRMT4, PRMT5, Haspin, MSK1, MSK2, CKII, Mst1, Bmi / Ring1A, RNF20 / RNF40 and ScFPR4.

[0068] Preferably, the effector domain may be appropriately labeled or tagged, for example with a fluorescent marker, such as sfGFP (superfolded GFP). sfGFP surprisingly helps recombinant or artificial proteins (eg according to the present invention) to fold better, thereby enhancing the complex of the present invention.

[0069] More preferably, the protein complex of the present invention comprises 3-7 effector domains, especially 5 effector domains. The effector domains may be at least 2 or more, at least 3 or more, at least 4 or more, or at least 5 or more different effector domains.

[0070] Also preferred are protein complexes of the invention, wherein the effector domain binding motif consists of an epitope comprising a peptide sequence of 17-29 amino acids, preferably 17-21 amino acids, and having little or no structural folding under physiological conditions. In principle, any suitable binding motif can be used, with the GCN4 epitope motif sequence being preferred due to its specificity and stability. The GCN4 epitope motif sequence (or "GCN4 peptide") comprises 22 amino acids (LLPKNYHLENEVARLKKLVGER; SEQ ID NO: 1). Nevertheless, the effector domain array ("tail") can also be complexed or fused to the nuclease in different ways, for example with a chemical bond.

[0071] In the complex of the present invention, a linker is also provided, which provides sufficient distance between the domains and between the domains and the nuclease, respectively, so as not to substantially interfere with its specific chromatin modification activity and the binding of the site-specific nuclease. The linker is known to those skilled in the art, and preferably has a linker sequence length of 19-25 amino acids, preferably 22, and more preferably contains glycine (G) and serine (S). It has been found that the linker (5 amino acids) in dCas9-SunTag is insufficient to support the optimal catalytic activity of the enzyme. As long as the linker does not interfere with the desired activity, the sequence of the linker is not particularly important, and the length / distance is found to be more important. "Substantially" in the context of the interference described above means that the complex of the present invention exhibits sufficient specific chromatin modification activity and sufficient site-specific nuclease binding.

[0072] More preferred is the complex of the present invention, wherein the effector domain is bound via a scFV domain specific for the effector domain binding motif, in particular a scFV domain specific for GCN4, wherein the scFV is optionally connected to the effector domain via an effector linker group, such as a group comprising a fluorescent protein (such as GFP or sfGFP protein), and wherein the effector domain can also be fused to a protein destabilization domain (such as d2).

[0073] Greer, Eric L and Yang Shi (in "Histone methylation: a dynamic mark in health, disease and inheritance." Nature reviews. Genetics vol. 13, 5 343-57. 3Apr. 2012, doi: 10.1038 / nrg3173) disclosed that organisms need to have an appropriate balance between the stability and reversibility of gene expression programs to maintain cell characteristics or enable them to respond to stimuli; epigenetic regulation is indispensable for this dynamic regulation. Post-translational modification of histones by methylation is an important and widespread form of chromatin modification, which is known to affect biological processes in development and cellular responses. They provide a comprehensive review of how histone methylation regulates and causes biological structures to explore how histone methylation contributes to stable or reversible control. The importance of maintaining or reprogramming histone methylation is illustrated by its association with disease and aging or possibly the transgenerational transmission of traits. Histone methylation occurs at all basic residues: arginine 3, lysine 4 and histidine 5. Lysine can be mono(me1)4, di(me2)6, or tri(me3)7 methylated on its ε-amino group, arginine can be mono(me1)3, symmetrically dimethylated (me2), or asymmetrically dimethylated (me2a) on its guanidine group8, and histidine has been reported to be monomethylated8,9, although this methylation appears to be rare and has not been further characterized. The most extensively studied histone methylation sites include histone H3 lysine 4 (H3K4), H3K9, H3K27, H3K36, H3K79, and H4K20. Sites methylated by arginine include H3R2, H3R8, H3R17, H3R26, and H4R3. However, a number of other basic residues throughout histones H1, H2A, H2B, H3, and H4 have recently been identified as being methylated.

[0074] The main advantage of the complex of the present invention is the possibility to specifically "tune" chromatin modification activities, for example, specific DNA methylation activities, histone methylation activities, specific histone acetylation or ubiquitination activities, and / or specific chromatin demethylation / deacetylation activities, or combinations thereof, based on the specific selection and combination of effector domains used. Thus, the complex may comprise at least two, at least three, at least four, or at least five different effector domains, for example, Setd2-CD33 for H3K36me3, Setd2-CD43 for H3K36me3, Setd2-CD53 for H3K36me3, Setd2-CD63 for H3K36me3, Setd2-CD73 for H3K36me3, Setd2-CD83 for H3K36me3, Setd2-CD93 for H3K36me3, Setd2-CD10 ... scFV With Prdm9-CD for H3K4me3 scFVand preferably without KRAB and / or VPR effector domains. It is also preferred that at least one chromatin modifying protein or polypeptide is selected from Dot1L (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Kmt2b (H3K4me3), Set1a (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Setdb1 (H3K9me3), Suv39h1 (H3K9me3), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), Ogt (GIcNAc), Prmt5 (H4R3me2s), Hdac1 / 2 / 3 / 4 (histone deacetylase), Sirt1 / 2 / 3 / 6 (histone deacetylase), Kat2a (lysine acetyltransferase), Lsd1 (H3K4me demethylase), Kdm5a / b / c (H3K4 demethylase), Kdm2b (H3K4 and H3K79 demethylase), Tet1 / 2 / 3 (methylcytosine dioxygenase), Utx (H3K27 demethylase), JMJD3 (H3K27 demethylase), Kdm4a / b / c / d (H3K36 and H3K9 demethylase), their catalytic domains (CD), or their catalytic domains (CD) fused to a scFV domain (CDscFV) or a fragment antigen binding (Fab) domain specific for an effector domain binding motif.

[0075] Another aspect of the present invention relates to a set of nucleic acids, each encoding at least one of a protein of the complex of the present invention and / or a guide RNA (gRNA) and / or a tagBFP.

[0076] In the context of the present invention, the term nucleic acid also includes sequences that are at least 50%, preferably to 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% identical to the sequences disclosed herein and also encode peptides or polypeptides having substantially the same activity as the polypeptides disclosed herein. For example, preferred is a complex of the present invention, wherein the guide RNA molecule comprises a sequence that is at least 80%, preferably greater than 90%, more preferably greater than 95%, and most preferably 100% complementary to the target RNA.

[0077] Some parts of the nucleic acid molecules used in the method of the present invention are found and / or designed to specifically hybridize with complementary parts in other molecules. It is known to those skilled in the art that hybridization and washing conditions are very important for this. If the sequences are 100% complementary, high stringency hybridization can be performed. However, according to the present invention, the hybridization and / or specific hybridization parts are complementary to at least 80%, preferably complementary to more than 90%, more preferably to more than 95%, and most preferably to be 100% complementary. The stringency of hybridization is determined by the hybridization temperature and the salt concentration in the hybridization buffer, and high temperature and low salt are more stringent. A commonly used washing solution is SSC (sodium citrate saline, i.e., a mixture of sodium citrate and NaCl). Hybridization can be carried out in solution, or more commonly, at least one component can be on a solid support such as nitrocellulose paper. Commonly used experimental experiments use blocking agents, such as casein or bovine serum albumin in skim milk powder, usually mixed with denatured and fragmented salmon sperm DNA (or any other high complexity heterologous DNA) and detergents (such as SDS). Very high concentrations of SDS are often used as blocking agents. The temperature may range from 42°C to above 65°C, and the buffer may be 3X SSC, 25 mM HEPES (pH 7.0), 0.25% SDS (final).

[0078] Thus, the nucleic acid group of the present invention encodes at least two, preferably three, four, five or all of the protein components in the complex of the present invention. A preferred example is a first group of nucleic acids comprising a catalytically inactive site-specific nuclease encoding a linker structure ("tail", which can usually be fused to the nuclease gene), and / or a second group of nucleic acids comprising a nucleic acid encoding an effector domain polypeptide used. A further preferred group is a nucleic acid comprising 2-10, preferably 3-7 effector domains, each of which has a specific chromatin modification activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity. Other preferred groups include nucleic acids encoding at least one suitable guide RNA (gRNA). These groups can be further combined with nucleic acids encoding tagBFP for co-expression and labeling of gRNA.

[0079] Preferred groups also include nucleic acids encoding scFV domains specific for effector domain binding motifs, particularly scFV domains specific for GCN4, wherein the scFV is optionally linked to the effector domain via an effector linker group, such as a group comprising a fluorescent protein (e.g., GFP or sfGFP protein), wherein the effector domain may also be fused to a protein destabilization domain (e.g., d2). Also encoded is a tag for the effector, such as sfGFP (superfolded GFP), such that the construct preferably includes a fluorescent marker.

[0080] The encoded effector domain may comprise a protein selected from the group consisting of Dot1L (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Kmt2b (H3K4me3), Set1a (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Setdb1 (H3K9me3), Suv39h1 (H3K9me3), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), Ogt (GIcNAc), Prmt5 (H4R3me2s), Hdac1 / 2 / 3 / 4 (histone deacetylation enzymes), Sirt1 / 2 / 3 / 6 (histone deacetylases), Kat2a (lysine acetyltransferases), Lsd1 (H3K4me demethylases), Kdm5a / b / c (H3K4 demethylases), Kdm2b (H3K4 and H3K79 demethylases), Tet1 / 2 / 3 (methylcytosine dioxygenases), Utx (H3K27 demethylases), JMJD3 (H3K27 demethylases), Kdm4a / b / c / d (H3K36 and H3K9 demethylases), chromatin modifying polypeptides in their catalytic domains (CDs), or chromatin modifying polypeptides in their catalytic domains (CDs) fused to scFV domains (CDscFVs) specific for effector domain binding motifs and fragment antigen binding (Fab) domains thereof. Also included are effectors that have an effect when combined (combination effectors), which are typically co-expressed.

[0081] According to the present invention, the nucleic acid is selected from DNA, RNA, PNA or a combination thereof.

[0082] In a preferred embodiment, the gene construct set is present in the form of at least one vector or plasmid, such as an expression vector, comprising a nucleic acid set of the present invention. Preferably, each nucleic acid encoding an effector domain, a CD and / or a CDscFV comprises an inducible promoter, such as a tet-responsive promoter, such as a dox-responsive promoter. This allows fine-tuning of the expression of the effector, thereby achieving additional preferred functional control.

[0083] A key element of epigenetic editing as a human biomedical strategy is to deliver the complex and / or its components to the relevant cells. Issues that need to be considered are safety, efficiency, and targeting specificity. There are a variety of vectors for CRISPR / Cas9 in vivo delivery. Viral delivery is the most commonly used platform for (epi)genomic therapy delivery. Lentivirus systems are also widely used, but have problems with integration into the host genome. Instead, adenovirus-associated virus (AAV) constructs are used, which do not integrate into the genome. However, AAV has only a limited load capacity, which requires the use of a smaller CRISPR / Cas9 system, such as Staphylococcus aureus (sa) Cas9. Nanoparticles can also be used to provide efficient and site-specific in vivo delivery of CRISPR / Cas9 loads. They have high carrying capacity and good stability, as well as the potential to control the release of their loads in a timely manner. Particles combined with nanomaterials are preferred to improve safety, cellular uptake, and specificity for specified cell types or tissues. In addition, extracellular vesicles can be used as load carriers for targeted delivery of epigenetic genome editing molecules. Related examples are known to those skilled in the art and are recorded in the relevant literature. Thus, most preferred are the gene construct sets of the present invention, wherein the construct is a viral construct, for example derived from AAV, lentivirus or retrovirus.

[0084] Also included in the present invention are methods for introducing nucleic acids and / or gene constructs into cells or tissues, including appropriately transforming or transfecting cells or tissues with nucleic acids and / or gene constructs. Methods are known in the art and include, for example, infection or electroporation. Methods can be implemented in vitro or in vivo.

[0085] Another aspect of the invention relates to a recombinant cell or tissue comprising a nucleic acid set of the invention, and / or a gene construct set of the invention. Preferably, the recombinant cell or tissue is prepared as described herein. Depending on the purpose, the cell or tissue can be prokaryotic or eukaryotic, such as a bacterial or mammalian cell (e.g., a human cell). Further examples are cells for producing or amplifying the complex of the invention, or cells associated with a disease phenotype and thus epigenetically treated using the complex of the invention (see below for details).

[0086] Another aspect of the invention relates to a method for producing a complex of the invention, comprising expressing a nucleic acid set of the invention and / or a gene construct set of the invention in a recombinant cell of the invention, optionally comprising a step of inducing expression, for example using an antibiotic (e.g., a tetracycline, such as doxycycline).

[0087] Another aspect of the present invention relates to a method for epigenetically modifying chromatin, particularly specifically epigenetically modifying chromatin, in cells, tissues, cell nuclei and / or samples containing chromatin, comprising introducing the complex of the present invention and one or more guide RNAs into cells, tissues, cell nuclei and / or samples, thereby specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples. The method can be implemented in vitro or in vivo. Strategies for delivering the complex of the present application and / or one or more guide RNAs, as well as constructs for delivery, have been described above. Preferably, the method epigenetically modifies cells associated with the disease phenotype and thus epigenetically treatable using the complex of the present invention and one or more guide RNAs of the present invention (see below for details).

[0088] Preferred is the method of the present invention, wherein the epigenetic modification comprises histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multiple combinatorial epigenetic editing of histones.

[0089] Also preferred are methods of the invention, wherein the epigenetic modification comprises temporarily inducing the expression of the complex of the invention and optionally one or more guide RNAs, for example using an antibiotic (such as a tetracycline, for example doxycycline). To this end, each nucleic acid encoding an effector domain, a CD and / or a CDscFV comprises an inducible promoter, for example a tet-responsive promoter, for example a dox-responsive promoter. This allows for temporary induction of expression of the complex or parts thereof (particularly the effector), thereby achieving additional preferred functional control.

[0090] Preferred is the method of the present invention, wherein the complex comprises at least two, at least three, at least four, or at least five different effector domains, such as Setd2-CD34 for H3K36me3. scFV and Prdm9-CD for H3K4me3 scFV preferably without KRAB and / or VPR effector domains.

[0091] Another aspect of the present invention relates to a method for achieving specific epigenetic modification of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, comprising introducing the complex of the present invention and one or more guide RNAs into cells, tissues, cell nuclei and / or samples, thereby specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples.

[0092] As described above, a major advantage of the complexes of the present invention is the possibility to specifically "tune" chromatin modification activities, for example, specific DNA methylation activities, histone methylation activities, specific histone acetylation or ubiquitination activities, and / or specific chromatin demethylation / deacetylation activities, or combinations thereof, based on the specific selection and combination of effector domains used. Thus, chromatin modifications may include histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multiple combinatorial epigenetic editing of histones.

[0093] Preferred is the method of the invention, wherein the epigenetic modification or modulation comprises transiently inducing the expression of the complex of the invention and one or more guide RNAs.

[0094] Therefore, preferred is the method of the present invention, wherein the complex comprises at least two, at least three, at least four, or at least five different effector domains, for example, Setd2-CD34 for H3K36me3. scFV With Prdm9-CD for H3K4me3 scFV and preferably without KRAB and / or VPR effector domains.

[0095] Another aspect of the present invention relates to a method for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus and / or a sample containing chromatin, the method comprising: introducing the complex of the present invention and one or more guide RNAs specific to at least one target DNA sequence into the cell, tissue, cell nucleus and / or sample, thereby specifically epigenetically modifying the expression of at least one target DNA sequence in the cell, tissue, cell nucleus and / or sample.

[0096] In general, the expression of any desired target DNA sequence can be regulated, i.e., increased, decreased, or stabilized based on chromatin modification. Expression can be regulated directly, such as by regulating transcription via a promoter or its accessibility, or indirectly, such as by regulating the regulatory environment or "hierarchy", such as splicing, enhancers, or other regulatory elements. Unlike complexes in the art, the present invention can provide indirect regulation.

[0097] Preferred are methods of the invention, wherein at least one target DNA sequence comprises a nucleic acid sequence specific for a symptom and / or disease state associated with epigenetically modified chromatin, such as genetic diseases, proliferative diseases (e.g. cancer), immune cells producing autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g. Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0098] Also preferred is a method of the invention, wherein 2, 3, 4, 5, 6, 7, 8 or 9 target DNA sequences are modulated in the cell.

[0099] In general, any cell comprising the target DNA sequence can be used. Preferred is the method of the invention, wherein the cell is a stem cell, a cell in a tissue, a neuron, a post-mitotic cell, a cancer cell or a fibroblast, and / or the cell is an animal cell, such as a mammalian cell, preferably a human or rodent cell.

[0100] Another aspect of the present invention relates to a method for detecting the biological effects of specific epigenetic modifications of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, and / or for regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, comprising performing the method of the present invention and detecting at least one biological effect in cells, tissues, cell nuclei and / or samples containing chromatin, wherein the biological effect is selected from changes in gene expression, changes in protein amounts, cis-genetic effects, changes in nucleic acid splicing, changes in the localization of gene loci in the cell nucleus, changes in TAD formation and destruction, changes in termination sites, activation of promoters, repression of promoters, changes in genetic-epigenetic interactions, functional relationships between genetic variations in chromatin and epigenetic states, changes in inherited methylation and imprinting, and associating specific epigenetic changes with diseases or cell phenotypes.

[0101] In this regard, the complex of the present invention can be preferably used to test or verify the results in EWAS (epigenome association studies) showing that a specific epigenetic change is associated with a certain disease or phenotype. See, for example, Bhat B and Jones GT (Data Analysis of DNA Methylation Epigenome-Wide Association Studies (EWAS): A Guide to the Principles of Best Practice. Methods Mol Biol. 2022; 2458: 23-45. doi: 10.1007 / 978-1-0716-2140-0_2. PMID: 35103960) and the literature cited therein.

[0102] Preferred is the method of the invention, wherein the method is performed using a complex capable of tracking an effect in cells, tissues, cell nuclei and / or chromatin-containing samples, preferably in real time.

[0103] Also preferred is the method of the invention, wherein the method further comprises detecting a transcriptional activator (e.g. VPR or VPR scFV ) or transcriptional repressors (such as KRAB or KRAB scFV ), preferably as a control for guide RNA targeting efficiency, and / or for detecting fluorescent proteins (e.g., GFP, or sfGFP protein or tagBFP). The method of the present invention may also include epigenetic targeted perturbation sequencing for detecting at least one biological effect in cells, tissues, cell nuclei and / or samples containing chromatin.

[0104] Preferably, the method of the present invention can be at least partially automated and / or carried out in a high-throughput format, i.e. can be partially or completely automated, for example operated completely or partially by a robot. The method of the present invention can involve the use of computers and associated databases for executing and / or analyzing the results obtained.

[0105] In general, any biological effect, disease or phenotype associated with epigenetically modified chromatin can be detected, such as genetic diseases, proliferative diseases (such as cancer), immune cells that produce autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases such as Prader-Willi syndrome).

[0106] Another aspect of the present invention relates to a cell having a specific epigenetically modified chromatin, which is produced by implementing the method of the present invention and optionally isolating the cell, wherein preferably the cell is a stem cell, a neuron, a cancer cell, a cell in a tissue, a post-mitotic cell or a fibroblast, and / or wherein the cell is an animal cell, such as a mammalian cell, preferably a human or rodent cell.

[0107] Another aspect of the present invention relates to a method for identifying a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specific epigenetic modification of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, comprising performing the method of the present invention in the presence and absence of a test drug, wherein, if the regulation and / or biological effect in the presence of the drug is different from the regulation and / or biological effect in the absence of the drug or compared with a control, the test drug is identified as a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specific epigenetic modification of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin.

[0108] The method of the present invention is intended to identify pharmaceutically active compounds that participate in regulating epigenetic modifications of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin (preferably in human cells). Conveniently, this form of the method can be relatively flexible, requiring an appropriate combination of components, i.e., the complex of the present invention or its functional fragment, and at least one pharmaceutically active candidate compound, that is, the substance to be screened / identified for functional regulatory activity. The combination can be provided as a cell system (i.e., functioning in cells such as mammalian cell cultures), and the components can be partially or completely recombinantly provided, or the system can be in vitro, for example, as an in vitro translation system that can be conveniently adjusted (if necessary) according to the purpose of the present invention. Examples are recorded in the literature and are known to those skilled in the art.

[0109] Preferred are methods of the invention, wherein the method comprising at least one candidate compound is performed in vitro, in cell culture, or in vivo, preferably in a non-human mammal, or comprises bioinformatics molecular modeling, for example using a suitable computer program. Also preferred are methods of the invention, which further comprise a preselection step, comprising, for example using a computer program (e.g. SwissDock), molecular modeling of the binding of at least one candidate compound to a complex or epigenetic modification site (e.g. histone) or a target nucleic acid or a functional fragment thereof. That is, the invention herein comprises preselection based on binding properties based on bioinformatics modeling (e.g. based on all or part of the structural elements, alone or in the context of a complex) before subjecting the compound to a more complex and comprehensive in vivo and / or in vitro test.

[0110] The methods of the invention can be performed using automation (eg, robotics) and can be performed in a high-throughput format.

[0111] Candidate compounds and / or to be identified or screened in the context of the present invention may be any chemical substance or any combination thereof. Preferably, the compound is selected from chemical substances, substances in peptide libraries, small molecule organic compound libraries, combinatorial libraries, cell extracts (especially plant cell extracts), "small molecule drugs" (i.e., molecular weight less than about 500 Da), repurposable drugs, proteins and / or protein fragments, and antibodies or fragments thereof, and suitable derivatives thereof. Small molecule drugs are preferred.

[0112] Preferably, the method of the present invention further comprises testing the dose-dependent response of the drug. The relevant methods are known to those skilled in the art.

[0113] The selected or screened compound can be modified. This modification can be performed as an additional preferred step of the method of the present invention as described herein, wherein, for example, after analyzing the complex and / or epigenetic modification regulation, for example, in the presence and absence of the selected compound, the compound is further chemically modified as described in the following examples, and its effect on binding and / or regulation is analyzed again. This "modification round" can be performed once or multiple times in all methods to optimize the performance of the compound, such as to improve its specificity for elements (such as complexes or histones), and / or to enhance its specificity for epigenetic modifications to be affected. This strategy is also referred to as "directed evolution" because it involves multiple steps, including modification and selection, wherein the binding compound is selected during the "evolution" process of optimizing its specific characteristics (such as its binding activity, its ability to activate, inhibit or regulate activity, especially epigenetic modification activity).

[0114] The modification may also be simulated bioinformatically before further testing to confirm or validate the effect of the selected or screened compound for the modification from the first round of screening. Various software programs are known in the art and are readily available to the skilled artisan.

[0115] Modification can be achieved by various methods known in the art, including, but not limited to, the introduction of new side chains or the exchange of functional groups, such as the introduction of halogens (especially F, Cl or Br), the introduction of lower alkyl groups (preferably having one to five carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or isopentyl), lower alkenyl groups (preferably having two to five carbon atoms), lower alkynyl groups (preferably having two to five carbon atoms), or by the introduction of groups selected from, for example, NH2, NO2, OH, SH, NH, CN, aryl, heteroaryl, COH or COOH groups.

[0116] Therefore, it is preferred that the method of the present invention further comprises: c) chemically modifying the identified candidate compounds (see above) and repeating the method of the present invention to identify second generation candidate compounds with improved binding and / or regulatory properties, and optionally d) repeating step c) at least once, twice, three times or more.

[0117] The term "functional fragment" refers to fragments and / or regions of a peptide or polypeptide that ultimately participate in the regulation of epigenetic modifications. These regions are usually involved in the binding of compounds (modulators).

[0118] In the context of the present invention, unless otherwise stated, the term "about" means + / - 10% of the specified numerical value.

[0119] Also included in the present invention is a method for preparing a pharmaceutically acceptable preparation, comprising providing nucleic acid of the present invention and / or genetic construct and cell or tissue, and mixing or preparing nucleic acid of the present invention and / or genetic construct with at least one pharmaceutically acceptable diluent or carrier. Each method is known to those skilled in the art. Pharmaceutically acceptable carriers or excipients include diluents (fillers, expanders, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, crosslinked sodium carboxymethyl cellulose), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), glidants (such as colloidal SiO2), solvents / cosolvents (such as aqueous carriers, propylene glycol, glycerol), buffers (such as citrate, gluconate, lactate), preservatives (such as sodium benzoate, parabens (Me, Pr and Bu), BKC), antioxidants (such as BHT, BHA, ascorbic acid), wetting agents (such as polysorbates, sorbitan esters), thickeners (such as methylcellulose, or hydroxyethylcellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame), flavoring agents (such as mint, lemon oil, butterscotch, etc.), humectants (such as propylene glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients are described in particular in Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5 th Ed., Govi-Verlag Frankfurt (1997). Those skilled in the art are aware of suitable formulations, such as topical formulations, for each compound and will be able to conveniently select suitable pharmaceutically acceptable carriers or excipients based on, for example, the formulation and administration route of the pharmaceutical composition.

[0120] Another aspect of the present invention relates to a pharmaceutically acceptable formulation prepared, comprising the cells, nucleic acids and / or genetic constructs of the present invention, and a pharmaceutically acceptable adjuvant or carrier.

[0121] It should be understood that the current compound and / or pharmaceutical composition comprising the current compound is used to treat human patients. The term "administering" refers to the administration of a single therapeutic agent or a combination with another therapeutic agent. Therefore, it is envisioned that the pharmaceutical composition of the present invention can be used in a combination therapy method, i.e., co-administered with other drugs or medicaments and / or any other therapeutic agents that may be beneficial in the context of the present method. However, if desired, other drugs or medicaments and / or any other therapeutic agents can be administered separately from the selected or screened compound and / or the compound for use, as long as they act in conjunction with the currently selected and / or screened compound (i.e., directly and / or indirectly, preferably synergistically).

[0122] Another important aspect of the present invention relates to the application of the present invention in medicine (see, for example, Policarpi C, Dabin J, Hackett JA. "Epigenetic editing: Dissecting chromatin function in context". Bioessays. 2021 May; 43 (5): 2000316. doi: 10.1002 / bies. 202000316. Epub 2021 Mar 16. PMID: 33724509). In addition to its potential to reveal biological mechanisms, epigenetic editing is also an exciting prospect for the development of therapeutic agents. Epigenetic editing presents a precise and non-invasive strategy for dealing with specific disease states, and a variety of possible applications make it complementary to, and in some cases even preferred to, gene therapy or wild-type Cas9 genetic methods.

[0123] In principle, epigenetic editing has a better safety profile than genetic editing because it does not change the host DNA sequence and is reversible in nature. In addition, it relies on endogenous cis elements to regulate target gene expression, which is a more physiological method compared to gene (cDNA) delivery. For example, it promotes conversion to endogenous genes ON while retaining co-transcriptional and post-transcriptional signals for appropriate expression regulation.

[0124] Furthermore, due to the ability to target multiple genes simultaneously, multiplexed epigenetic editing can be used to (i) upregulate the expression of multiple genes simultaneously, or (ii) exploit the orthogonality between CRISPR-Cas9 systems to switch some genes ON and others OFF in parallel. CRISPR-based epigenetic therapies have shown great potential for application in a subset of human diseases.

[0125] Many types of diseases have become attractive targets for the development of epigenetic therapies ( Figure 5). For example, hundreds of human diseases are associated with haploinsufficient dosage, and selectively enhancing gene expression can compensate for defective gene products. This can be implemented by targeting Cas9 fused with a transcriptional activation module (e.g., VP64 and its derivatives) or a histone modification enzyme (e.g., p300, PRDM9, or DOTIL) herein. In a mouse model of a severe epileptic encephalopathy called Dravet syndrome, significant relief of the disease was shown.

[0126] Other diseases that could benefit from the in vivo development of CRISPR activation systems are X-linked diseases, which are caused by a defective allele of the active X chromosome, while the quiescent X chromosome contains a functional copy. Rett syndrome (caused by mutations in the MeCP2 gene) and Cdkl5 disease are typical examples of such diseases. Upregulating the expression of functional gene copies of the quiescent X chromosome in brain cells through programmable epigenetic editing can indeed complement the mutant allele. Epigenetic editing can potentially be applied to a variety of neurological diseases. For example, fragile X syndrome is the most prevalent form of intellectual disability in males, in which aberrant DNA methylation in neurons mediates epigenetic silencing of the FMR1 gene. Interestingly, the use of the dCas9-Tet1 system to specifically demethylate the FMR1 promoter reversed its heterochromatin state and restored FMRI expression in iPSC-derived neurons. This strategy can be extended to imprinting diseases caused by aberrant DNA methylation patterns. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are classic examples where similar approaches have been successfully used, as some forms of PWS are caused by abnormal methylation of the paternal allele, while 4% of AS arises from lack of methylation of the maternal allele. Therefore, allele-specific therapies based on targeting dCas9-Tet1 for the former, or dCas9-Dnmt3 for the latter, are promising.

[0127] In addition to targeted activation, programmed gene silencing can address disease phenotypes, especially those caused by dominant-acting genes. For example, dCas9-KRAB has been used in a mouse model of hypercholesterolemia to target the Pcsk9 gene, resulting in lower cholesterol levels in treated mice. Many inflammatory and pain-related diseases could benefit from this strategy by targeting (i) cytokines involved in relevant inflammatory pathways, such as hypothesized for the treatment of degenerative disc disease, or (ii) pain receptors located in the skin. Indeed, the skin constitutes an accessible and attractive organ for CRISPR-mediated gene silencing, as it is associated with many monogenic and autosomal dominant diseases such as Olmsted syndrome or familial localized cutaneous amyloidosis.

[0128] Importantly, to ensure that the functional gene copy remains active, allele-specific epigenetic silencing can be employed in dominant-acting diseases. This strategy has been applied to silence mutant HTT alleles in a diverse clinical population representing neurological Huntington's disease. Similarly, recent studies have shown that targeting the mutant DMD allele with the CRISPR / Cas9 system in a mouse model of Duchenne muscular dystrophy significantly improved the mice's muscle contraction ability.

[0129] Finally, complex diseases such as cancer could potentially benefit from the strategies discussed above, as well as multiplexed epigenetic editing by simultaneously activating tumor suppressor genes and inhibiting oncogene expression. Indeed, simultaneous activation and repression of different genes in the same cell has been achieved in vitro by combining dCas9 with chemical- and light-inducible effector domains or by engineering scaffold gRNA molecules that recruit transcriptional regulators. Furthermore, proof-of-concept for simultaneous gene activation has been shown in mice in vivo.

[0130] It has been shown that histone methylation and aging histone methylation-modified proteins play a role in regulating the life span of organisms and tissue aging. Loss of the appropriate balance between stable and dynamic methyl marks in adult stem cells may contribute to the degeneration of individual tissue function with age (see Basavarajappa BS, Subbanna S. "Histone Methylation Regulation in Neurodegenerative Disorders". Int J Mol Sci. 2021Apr28; 22 (9): 4654. doi: 10.3390 / ijms22094654. PMID: 33925016; PMCID: PMC8125694), such as in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and drug abuse (including alcohol abuse disorder).

[0131] The above-mentioned object of the present invention is achieved by a method for preventing or treating a disease associated with epigenetic modification of chromatin in a subject in need of prevention or treatment, the disease including genetic diseases, proliferative diseases (such as cancer), immune cells producing autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular diseases, shortened lifespan, tissue aging, neurodegenerative diseases (such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome) , The method comprises administering to a subject in need of the treatment an effective amount of the complex of the present invention and one or more suitable guide RNA sequences, the nucleic acid set of the present invention, the genetic construct set of the present invention (such as expression vector), the cell of the present invention, and / or at least one of the agents identified in the present invention.

[0132] In this aspect, the complex or the nucleic acid encoding all or part thereof is used as the actual active ingredient in prevention and / or treatment. Delivery of the complex to a patient, cell or sample can be carried out in any suitable manner, for example, as a pharmaceutical composition comprising the separated components (polypeptides and / or nucleic acids) of at least one complex of the present invention and a suitable stabilizer or carrier. Another embodiment is to provide a complex encoded on at least one nucleic acid vector to a patient, cell, tissue, sample or cell nucleus. These pharmaceutical compositions and their uses constitute preferred embodiments of the present invention. This aspect also includes the step of monitoring treatment.

[0133] This aspect combines the diagnostic method of the invention with an otherwise "conventional" medical treatment and also includes applications for monitoring treatment. The method comprises providing the cell, tissue or organism with an appropriate treatment (particularly a specific medical treatment), performing the method of the invention described above, and modifying the treatment of the disease or medical condition based on the detected chromatin modifications and disease changes, for example, when compared to healthy controls (e.g., controls based on a set of healthy or disease samples).

[0134] "Treatment" refers to any treatment of a disease or condition in a mammal, including: preventing or protecting against a disease or condition, i.e., so that clinical symptoms of the disease do not develop; inhibiting the disease, i.e., preventing or inhibiting the development of clinical symptoms; and / or relieving the disease, i.e., causing regression of clinical symptoms. "Amelioration" refers to preventing, alleviating, or relieving a condition, or improving the condition of a subject; relieving stress refers to counteracting the negative effects of stress. Amelioration includes, but does not require complete recovery or complete prevention of stress.

[0135] The above-mentioned object of the present invention is achieved by the use of at least one of the complex of the present invention and one or more suitable guide RNA sequences, the nucleic acid set of the present invention, the genetic construct set of the present invention (e.g., expression vector), the cell of the present invention, and / or the agent identified in the present invention in a subject in need of prevention and treatment for preventing and / or treating a disease, or preventing and / or treating a disease associated with epigenetically modified chromatin, for example, a genetic disease, a proliferative disease (e.g., cancer), an immune cell producing autoantibodies, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0136] Another aspect of the invention relates to the use of the complex of the invention, the set of nucleic acids of the invention, the set of genetic constructs of the invention (e.g., expression vectors), and / or the cell of the invention in specifically epigenetically modifying chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin according to the invention, regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei, and / or samples containing chromatin according to the invention, detecting the biological effects of specific epigenetic modifications of chromatin in cells, tissues, cell nuclei, and / or samples according to the invention, and / or identifying drugs according to the invention. All these aspects have been further described above.

[0137] In the context of the present invention, the subject may be a mammal, preferably a mouse or a human.

[0138] In another preferred aspect, the object of the present invention is achieved by providing a kit comprising materials for performing the method of the present invention as a detection system, for example as part of a diagnostic kit. The kit can also be used in a therapeutic kit, or a pharmaceutical composition comprising isolated components of at least one complex of the present invention (polypeptides and / or nucleic acids encoding them, and / or viral particles comprising these) and a suitable stabilizer or carrier. Another embodiment comprises additional materials for providing the complex encoded on at least one nucleic acid vector to a subject, cell, tissue, sample or cell nucleus.

[0139] Preferably, the kit is provided in one or more containers and comprises suitable enzymes, buffers and excipients, and instructions for use.The components may be (at least partially) immobilized on a matrix, wherein the matrix may be exposed to the cell, tissue and / or sample.

[0140] In the context of the present invention, unless expressly stated otherwise, the term "about" refers to + / - 10% of a given value.

[0141] The methods of the invention may be performed in vivo or in vitro, for example in an organism, a cell, a tissue and / or a part thereof (eg a cell nucleus), or in an in vitro assay (eg a diagnostic assay).

[0142] Here, the inventors provide an epigenome editing platform to systematically program specific combinations of chromatin modifications across tens of thousands of scenarios in living cells. This resource can be used to capture multimodal functional responses from multiple lines at allelic, single-cell resolution. Precision perturbations at an unprecedented scale will reveal the regulatory logic by which different chromatin modifications interact with genomic features, sequence variants, and cellular characteristics to shape quantitative gene expression patterns. By integrating datasets and applying genetic screening, the present invention will also identify trans-acting and cis-structural mechanisms that implement chromatin mark functionality.

[0143] Precise epigenetic editing strategies can be used to define the transcriptional function and memory of heterochromatin epialleles at endogenous loci. The method of multiple "effector" molecules recruited using dCas9GCN4 multiplexes promotes the reprogramming of major (>10kb) heterochromatin regions, which is sufficient to drive robust epigenetic silencing. These de novo generated domains include H3K9me3, H4K20me3, and DNA methylation, and are accompanied by H3K4me3 loss, and their modification levels are comparable to or even higher than endogenous heterochromatin regions, which are considered to be self-maintained by "read-write" reinforcement (see also Reinberg D, Vales LD. "Chromatin domains rich in inheritance." Science. 2018 Jul 6; 361 (6397): 33-34. doi: 10.1126 / science.aat7871. PMID: 29976815). The inventors have discovered that naive pluripotent stem cell function is a major barrier to inheritance of heterochromatin domains occurring outside of the normal genomic context, even when afforded a selective advantage such as silencing p53.

[0144] As a preferred embodiment, the inventors have developed a modular CRISPR-based toolkit that can precisely and inducibly program nine (in this case) different epigenetic modifications to endogenous target loci (see Figure 1BH). Of these, H3K27ac and H3K4me3 marks are generally associated with active transcription, while DNA methylation, H2AK119ub, and trimethylation of lysine 9 or 27 on histone H3 (H3K9me3 and H3K27me3, respectively) are associated with repressed chromatin regions (see Berger SL et al. 2007). However, whether these observed correlations indicate causality remains unclear, especially considering that removal of these marks from the genome of mouse embryonic stem cells (mESCs) only causes mild transcriptional changes (see Tsumura A et al. 2006; Chamberlain SJ et al. 2008; Sze C et al. 2017; Zhang T et al. 2020). Similarly, the H3K36me3 mark was found to be highest within active gene bodies, but it has still been shown to act as a transcriptional repressor in yeast and mammals (Carrozza MJ et al. 2005; Neri F et al. 2017). Genetic approaches, which include gene editing of the DNA sequence underlying the mark, genetic deletion of upstream chromatin modifying enzymes, or mutation of specific histone residues, have been widely used to explore the functional relevance of epigenetic marks (Stricker SH et al. 2017; Grosswendt S et al. 2020). Although these approaches are often successful in triggering widespread changes in gene expression, they have not yet clearly established the causal role of the chromatin mark itself on the observed phenotype due to the inability to distinguish between direct and indirect effects.

[0145] Therefore, the ability to site-specifically deposit marks (H3K27me3, H3K4me3, H2AK119ub, and H3K36me3) presents a powerful gain-of-function perturbation strategy to unambiguously assess their causal impact. GCN4 ), each separated by a linker designed with optimized spacing to accommodate bulky proteins without steric hindrance to their catalytic activity. GCN4 In one embodiment, up to five "effector" proteins are anchored to specific loci ( Figure 1 A). The inventors have generated and tested a panel of effectors that consist only of the catalytic domains of chromatin-modifying enzymes (collectively referred to as CD scFV ), such as Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV .

[0146] The above considerations also apply mutatis mutandis to other chromatin modifying proteins or polypeptides that can be used as effectors of the present invention, which can be selected from Dot1L (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Kmt2b (H3K4me3), Set1a (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Setdb1 (H3K9me3), Suv39h1 (H3K9me3), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), Ogt (GIcNAc), Prmt5 (H4R3me3), H3K27me3. dac1 / 2 / 3 / 4 (histone deacetylase), Sirt1 / 2 / 3 / 6 (histone deacetylase), Kat2a (lysine acetyltransferase), Lsd1 (H3K4me demethylase), Kdm5a / b / c (H3K4 demethylase), Kdm2b (H3K4 and H3K79 demethylase), Tet1 / 2 / 3 (methylcytosine dioxygenase), Utx (H3K27 demethylase), JMJD3 (H3K27 demethylase), Kdm4a / b / c / d (H3K36 and H3K9 demethylase), their catalytic domains (CD), or their catalytic domains (CD) fused to a scFV domain (CDscFV) or a fragment antigen binding (Fab) domain specific for an effector domain binding motif.

[0147] Epigenetic modifications can include histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multi-combinatorial epigenetic editing of histones.

[0148] The system of the present invention has a number of other advantages, which collectively result in an epigenetic editing platform technology with unique features that enable exploration. These advantages include:

[0149] Efficient editing. 5 copies (in this case) of a particular CD scFV Recruitment to target loci greatly enhances on-target programming of chromatin modifications in terms of intensity and genome breadth. This ensures de novo deposition of histone methylation comparable to strong endogenous peaks, contributing to both negative and positive functional outcomes.

[0150] Specificity of the catalytic domain. By isolating only the catalytic core, the confounding effects of targeting full-length chromatin modifying proteins can be avoided. Because full-length proteins may have extensive non-catalytic functions and / or recruit other protein complexes, it is preferred to use the catalytic domain so that the function of the targeted chromatin mark itself can be assessed.

[0151] Combinatorial epigenetic editing. The system of the present invention is modular, so that for example, 5 different CD4+ T cells can be recruited simultaneously. scFV This enables multiplexing and allows the construction of de novo domains of different chromatin modifications (e.g., bivalent or multiplex combinations) and combinations thereof for epigenetic editing.

[0152] Off-target effects are minimized. scFV Lacking their endogenous DNA-binding domain, and because they are not directly fused to dCas9, they exhibit minimal off-target activity.

[0153] Here, each CD scFV Dynamically inducible via a DOX-responsive promoter and can carry a protein destabilization (D2) domain that promotes rapid degradation upon DOX withdrawal. This allows for the study of temporal responses and epigenetic memory (chromatin persistence).

[0154] Dynamic tracking. In this embodiment, all effectors are linked to superfolded GFP (sfGFP), and all gRNAs are linked to tagBFP, so that the system can be tracked in real time and can be used for cell purification and testing of drug dose-dependent responses (e.g., comparing GFP 低 and GFP 高 cell).

[0155] The inventors have generated a comprehensive control group. First, the inventors scFV A point mutant (mut-CD scFV ), which specifically eliminates the catalytic activity, allowing the active CD scFV Second, as another negative control, the inventors used a GFP-only scFV Finally, as a positive control for gRNA targeting efficiency, the inventors used a well-characterized transcriptional activator (VPR scFV ) and inhibitors (KRAB scFV ) recruitment.

[0156] Taken together, these properties allow for the carefully controlled introduction of a specific chromatin modification per se at any particular locus, while excluding confounding effects.

[0157] The present invention circumvents the core limitations of existing epigenomic perturbation methods by eliminating pleiotropy and redundancy and isolating functional genome × epigenome relationships. An additional result is a deeper understanding of how specific chromatin states guide - or reflect - gene regulation. This will help design strategies for precision medicine and provide guiding principles for attributing functional significance in health and disease to epigenomic maps.

[0158] Whether defined chromatin modifications at specific loci affect any gene expression in a given cell type is still largely unknown at a quantitative level. The research explored here will provide a systematic strategy to answer this question and thereby generate fundamental insights into genome biology that will address key challenges in the field. Precise perturbations at an unprecedented scale will provide a source for predicting the functional activity of chromatin marks in a specific context. Such modeling lays the foundation for several important advances. First, it allows for greater confidence in attributing functional relevance to changes observed across developmental- and disease-related epigenomic profiles generated by populations. In other words, this helps to tease out causes from results. Second, the knowledge gained here will generate rational rules to design and optimize future epigenetic editing strategies for precision medicine applications. For example, guiding design principles about directing changes in the intensity, penetration, or persistence of desired target gene transcription to regulate disease. Its proof of principle has been shown in fragile X, muscular dystrophy, and kidney injury, and further research is essential to maximize its full therapeutic potential. Third, the current output will provide tools to resolve the functional relationship between genetic variants and chromatin states. This provides a foundation for a mechanistic understanding of how human trait-associated eQTLs are regulated to exert their effects in specific tissues, disease settings, and during evolution. High-throughput data readout and unbiased screening provide ample opportunities for discovery of interactions and mechanisms, as well as unexpected research paths, outside the framework of our expected effects. There is enormous scope to extend our experimental strategy to mechanistic understanding of genetically diverse human cells, tailoring of desired cellular properties, and in vivo applications.

[0159] As described herein, the present invention particularly relates to the following items.

[0160] Item 1. A complex comprising i) a catalytically inactive site-specific nuclease connected to ii) an array of 2-10, preferably 3-7 effector domains, each effector domain having a specific chromatin modification activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are each separated by a linker, and the linker provides sufficient distance between the domain and the nuclease so as not to substantially interfere with its specific chromatin modification activity and the binding of the site-specific nuclease.

[0161] Item 2. A complex as described in Item 1, wherein the catalytically inactive site-specific nuclease is selected from catalytically inactive (d) Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, Scel, and dCas9 / fCas9 fusion of Streptococcus pyogenes.

[0162] Item 3. A complex as described in Item 1 or 2, wherein the complex comprises a fusion protein of a nuclease linked to a protein sequence containing 3-7 effector domain binding motifs, such as Streptococcus pyogenes dCas9GCN4 (3-7) , each binding motif is separated by a linker sequence, the complex optionally further comprises a plurality of effector domains, each effector domain is bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA).

[0163] Item 4. The complex as described in any one of Items 1 to 3, wherein the protein complex comprises 4-6 effector domains, preferably 5 effector domains.

[0164] Item 5. A complex as described in any one of Items 1 to 4, wherein the effector domain binding motif consists of an epitope comprising a peptide sequence of 17-29 amino acids (preferably 17-21 amino acids) and having little or no structural folding under physiological conditions, such as a GCN4 epitope motif sequence.

[0165] Item 6. A complex as described in any one of Items 1 to 5, wherein the length of the linker sequence is 19-25 amino acids, preferably 22, and more preferably contains glycine (G) and serine (S).

[0166] Item 7. A complex as described in any one of Items 1 to 6, wherein the effector domain is bound via a scFV domain specific to the effector domain binding motif (in particular, a scFV domain specific to GCN4), wherein the scFV is optionally connected to the effector domain via an effector linker group (for example, a group comprising a fluorescent protein (such as GFP or sfGFP protein)), and wherein the effector domain can also be fused to a protein destabilization domain (such as d2).

[0167] Item 8. A complex as described in any one of Items 1 to 7, wherein the effector domain comprises a chromatin modifying polypeptide selected from DotL (H3K79me3), p300 (H3K27ac), Prdm9 (H3K4me3), Setd2 (H3K36me3), Ringlb (HAK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), KmtSC (H4K20me3), Dnmt3a3L (DNAme), OGT (GIcNAC), its catalytic domain (CD), and its catalytic domain (CDscFV) fused to a scFV domain specific for the effector domain binding motif.

[0168] Item 9. The complex of any one of Items 1 to 8, wherein the chromatin modifying activity is histone methylation, for example, histone methylation that contributes to stable or reversible gene expression control.

[0169] Item 10. A complex as described in any one of Items 1 to 9, wherein the complex comprises at least two, at least three, at least four, or at least five different effector domains, such as Setd2-CD33 for H3K36me3. scFV With Prdm9-CD for H3K4me3 scFV and preferably without KRAB and / or VPR effector domains.

[0170] Item 11. A nucleic acid group, each nucleic acid encoding at least one of the proteins and / or guide RNA (gRNA) and / or tagBFP of the complex described in any one of Items 1 to 10.

[0171] Item 12. A gene construct set, the gene construct being, for example, an expression vector, comprising the nucleic acid set described in Item 11, wherein preferably each nucleic acid encoding an effector domain, CD and / or CDscFV comprises an inducible promoter, for example, a tet-responsive promoter, for example, a dox-responsive promoter.

[0172] Item 13. The gene construct set as described in Item 12, wherein the construct is a viral construct, such as a viral construct derived from AAV, lentivirus or retrovirus.

[0173] Item 14. A recombinant cell comprising the nucleic acid set described in Item 11 and / or the gene construct set described in Item 12 or 13.

[0174] Item 15. A method for preparing a complex as described in any one of Items 1 to 10, comprising expressing the nucleic acid group described in Item 11 and / or the gene construct group described in Item 12 or 13 in the recombinant cell described in Item 14, and optionally comprising a step of inducing expression, for example using tetracycline.

[0175] Item 16. A method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus and / or a sample containing chromatin, comprising introducing into the cell, tissue, cell nucleus and / or sample a complex described in any one of Items 1 to 10 and one or more guide RNAs, thereby specifically epigenetically modifying chromatin in the cell, tissue, cell nucleus and / or sample.

[0176] Item 17. A method as described in Item 16, wherein the epigenetic modification includes histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multiple combinatorial epigenetic editing of histones.

[0177] Item 18. A method as described in Item 16 or 17, wherein the epigenetic modification comprises temporarily inducing the expression of any complex described in Items 1 to 10 and one or more guide RNAs.

[0178] Item 19. A method as described in any one of Items 16 to 18, wherein the complex comprises at least two, at least three, at least four, or at least five different effector domains, such as Setd2-CD33 for H3K36me3. scFV With Prdm9-CD for H3K4me3 scFV and preferably without KRAB and / or VPR effector domains.

[0179] Item 20. A method for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus and / or a sample containing chromatin, the method comprising: introducing into the cell, tissue, cell nucleus and / or sample a complex described in any one of Items 1 to 10 and one or more guide RNA sequences specific to the at least one target DNA sequence, thereby specifically epigenetically modifying the expression of the at least one target DNA sequence in the cell, tissue, cell nucleus and / or sample.

[0180] Item 21. A method as described in Item 20, wherein at least one target DNA sequence comprises a nucleic acid sequence specific for symptoms and / or disease states associated with epigenetically modified chromatin, the symptoms and / or disease states being, for example, genetic diseases, proliferative diseases (e.g., cancer), immune cells that produce autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0181] Item 22. A method as described in Item 20 or 21, wherein 2, 3, 4, 5, 6, 7, 8 or 9 target DNA sequences are regulated in the cell.

[0182] Item 23. A method as described in any one of Items 16 to 22, wherein the cell is a stem cell, a neuron, a post-mitotic cell, or a fibroblast, and / or the cell is an animal cell, such as a mammalian cell, preferably a human or rodent cell.

[0183] Item 24. A method for detecting the biological effects of specific epigenetic modifications of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, and / or regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, comprising performing any of the methods described in Items 16 to 23 and detecting at least one biological effect in cells, tissues, cell nuclei and / or samples containing chromatin, wherein the biological effect is selected from changes in gene expression, changes in protein levels, cis-genetic effects, changes in nucleic acid splicing, changes in the localization of a locus in the cell nucleus, changes in TAD formation and destruction, changes in termination sites, activation of promoters, repression of promoters, changes in genetic-epigenetic interactions, functional relationships between genetic variants and epigenetic states of chromatin, changes in inherited methylation and imprinting, and associating specific epigenetic changes with diseases or cellular phenotypes.

[0184] Item 25. A method as described in Item 24, wherein the method is implemented using a complex that allows tracking of effects in cells, tissues, cell nuclei and / or chromatin-containing samples, preferably in real time.

[0185] Item 26. A method as described in Item 24 or 25, wherein the method further comprises detecting a transcriptional activator (e.g., VPR or VPR scFV ) or transcriptional repressors (such as KRAB or KRAB scFV)'s effector domain, preferably as a control for gRNA targeting efficiency, and / or for detecting fluorescent proteins (e.g., GFP, or sfGFP protein or tagBFP).

[0186] Item 27. A method as described in any one of Items 24 to 26, wherein the method also includes epigenetic targeted perturbation sequencing to detect at least one biological effect in cells, tissues, cell nuclei and / or chromatin-containing samples.

[0187] Item 28. A method as described in any one of Items 16 to 27, wherein the method is at least partially automated and / or implemented in a high-throughput format.

[0188] Item 29. A method as described in any one of Items 16 to 28, wherein the disease or phenotype is associated with epigenetically modified chromatin, such as a genetic disease, a proliferative disease (such as cancer), immune cells that produce autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0189] Item 30. A cell having specifically epigenetically modified chromatin, prepared by performing the method described in any one of Items 16 to 19, and optionally isolating the cell, wherein the cell is preferably a stem cell, a neuron, a post-mitotic cell or a fibroblast, and / or the cell is an animal cell, such as a mammalian cell, preferably a human or rodent cell.

[0190] Item 31. A method for identifying a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specific epigenetic modification of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, comprising performing the method described in any one of Items 16 to 29 in the presence and absence of a test drug, wherein if the regulation and / or biological effect in the presence of the drug is different from the regulation and / or biological effect in the absence of the drug or from a control, the test drug is identified as a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that regulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of specific epigenetic modification of chromatin in cells, tissues, cell nuclei and / or samples containing chromatin.

[0191] Item 32. A method as described in Item 30, wherein the test drug is selected from chemical molecules, molecules in a small molecule organic compound library, molecules in a combinatorial library, cell extracts (especially plant cell extracts), small molecule drugs, proteins, protein fragments, molecules in a peptide library, and antibodies or their fragments.

[0192] Item 33. The method of Item 31 or 32, further comprising testing the dose-dependent response of the drug.

[0193] Item 34. A method for preventing or treating a disease associated with epigenetically modified chromatin in a subject in need of prevention or treatment, the disease being, for example, a genetic disease, a proliferative disease (e.g., cancer), an immune cell producing autoantibodies, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome), comprising administering to a subject in need of such treatment an effective amount of a complex as described in any one of Items 1 to 10 and one or more suitable guide RNA sequences, a nucleic acid set as described in Item 11, a gene construct (e.g., an expression vector) set as described in Item 12 or 13, a cell as described in Item 30, and / or at least one of the drugs identified according to any one of Items 31 to 33.

[0194] Item 35. Use of the complex described in any one of Items 1 to 10 and one or more suitable guide RNA sequences, the nucleic acid set described in Item 11, the gene construct (e.g., expression vector) set described in Item 12 or 13, the cell described in Item 30, and / or at least one of the drugs identified according to any one of Items 31 to 33, in a subject in need of prevention or treatment of a disease, or the prevention and / or treatment of a disease associated with epigenetic modification of chromatin, wherein the disease is, for example, a genetic disease, a proliferative disease (e.g., cancer), an immune cell producing autoantibody, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)), drug abuse (including alcohol abuse disorder), epigenetic diseases (including imprinting diseases, such as Prader-Willi syndrome).

[0195] Item 36. A complex as described in any one of items 1 to 10 and one or more suitable guide RNA sequences, a nucleic acid group as described in item 11, a gene construct (e.g., expression vector) group as described in item 12 or 13, and / or a cell as described in item 30, for specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples containing chromatin according to any one of items 16-19 and 29, for regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin according to any one of items 20-23 and 28, for detecting the biological effects of specific epigenetically modified chromatin in cells, tissues, cell nuclei and / or samples containing chromatin according to any one of items 24-27, and / or for identifying drugs according to any one of items 30-32.

[0196] The present invention will be further described in the following examples with reference to the accompanying drawings, but the present invention is not intended to be limited thereto. For the purpose of the present invention, all references cited herein are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0197] The present disclosure includes a Sequence Listing containing SEQ ID No: 1 as part of the specification, which is also incorporated by reference in its entirety.

[0198] Figure 1 A is a schematic diagram showing a preferred embodiment of the composite of the present invention. Figure 1 BH shows that DOX-induced CD scFV Quantitative enrichment of seven chromatin modifications targeting Hbby was observed. Importantly, levels were comparable to endogenous positive controls (high on-target activity), while non-targeted loci were largely unaffected (low off-target activity). Figure 1 I further shows H3K4me3 and H2AK119ub after being targeted by a single gRNA.

[0199] Figure 2 Showing functional responses to epigenetic editing at single-cell resolution. scFV (PRC1), but not its catalytic mutant, programs H2AK119ub to the promoter of an active reporter gene ( Figure 2 H), robustly caused transcriptional repression in most cells. On the other hand, using Ezh2-FL scFV H3K27me3 targeting (PRC2, Figure 2 G) shows a partially penetrant transcriptional response, with many cells failing to show repression, suggesting that H3K27me3 has a weak instructive role at this locus.

[0200] Figure 3 AC shows that A) combined epigenetic editing of H3K27me3 and H2AK119ub shows a synergistic effect on suppressing reporter genes in single cells. B, C) Gene-epigenetic interaction. Introducing short MYC, OTX (3B) or CTCF (3C) motifs in the reporter gene, respectively, has little effect on the starting state, but specifically regulates the effects of de novo generation of H3K27ac and H3K36me3.

[0201] Figure 4 A schematic diagram showing the epiTAP-seq process according to the present invention.

[0202] Figure 5 Potential therapeutic applications of epigenome editing are shown. Different types of diseases may benefit from the development of different CRISPR / Cas9-driven epigenome editing strategies. Examples of how to manipulate mutant or wild-type alleles in specific disease contexts are shown here. The molecular etiology underlying each disease is shown, as well as dCas9-based repair strategies. Figure taken from Policarpi et al. 2021. DETAILED DESCRIPTION

[0203] Example

[0204] Will contain dCas9 GCN4 and all CDs scFV and FL scFVThe epigenetic editing tool for effectors was cloned into the PiggyBac recipient plasmid by homology arm recombination using seamless cloning technology (Takara #639650) according to the manufacturer's instructions. Streptococcus pyogenes dCas9 was PCR amplified from the PlatTET-gRNA2 plasmid (Morita et al, 2016; Addgene #82559) GCN4 , and cloned into a PiggyBac backbone vector under the control of the TRE3G promoter, which also contains the TET-ON3G transcriptional activator and a hygromycin resistance gene driven by the EF-1α promoter.

[0205] For all effector plasmids, the scFv domain and sfGFP coding sequences were amplified from the PlatTET-gRNA2 plasmid (Addgene #82559) and fused in frame to the catalytic domain (CD) or full-length version (FL) of mouse Prdm9, P300, Dot1L, G9a, Kmt5c, Setd2, Ezh2, and Ring1b, all cloned from cDNA samples. Dnmt3a CD, and the C-terminal portion of mouse Dnmt3L (3a3L) were amplified from pET28-Dnmt3a3L-sc27 (Addgene #71827). The resulting constructs were cloned into the PiggyBac plasmid under the control of the TRE3G promoter. These vectors also carry constitutive expression of the neomycin resistance gene. The control GFPscFv effector was cloned as described above, but lacked the chromatin modification domain. Finally, the catalytic variant (mut-CD scFV ) effector, also cloned as described above. During PCR of the cDNA / plasmid template, specific mutations that abolished the catalytic activity were introduced by designing oligonucleotide primers containing mismatched nucleotides.

[0206] The guide RNA plasmid, carrying the enhanced gRNA scaffold, was amplified from Addgene plasmid #60955 and cloned into a PiggyBac acceptor vector that also constitutively expresses the puromycin resistance gene and TagBFP.

[0207] For the design of all gRNAs for targeted epigenetic editing systems, the GPP portal (Broad Institute) was used. Forward and reverse gRNA chains (final concentration 10 μM) carrying appropriate end protrusions were annealed at 95°C for 3 minutes in an annealing buffer containing 10mM Tris (pH 7.5-8.0), 60mM NaCl, and 1mM EDTA, followed by cooling at RT for >30 minutes. The annealed gRNA was ligated to a PiggyBac receptor vector previously digested with BIpI (NEB#R0585S) and BstXI (NEB#R0113S) restriction endonucleases using T4-DNA ligase (NEB#M0202S) at 37°C for 1 hour. The final plasmid was amplified by bacterial transformation and purified by endotoxin-free medium preparation (ZymoResearch#D4200). Correct assembly and sequence were verified by Sanger sequencing (Azenta).

[0208] result

[0209] In the context of the present invention, the inventors have demonstrated that the current system is capable of on-target epigenetic editing at endogenous loci specifically and efficiently.

[0210] Figure 1 BH shows that DOX-induced CD scFV Quantitative enrichment of seven chromatin modifications targeting Hbby was observed. Importantly, levels were comparable to endogenous positive controls (high on-target activity), while non-targeted loci were largely unaffected (low off-target activity). Figure 1 I further shows H3K4me3 and H2AK119ub after being targeted by a single gRNA.

[0211] The inventors also tested various aspects of chromatin function using a reporter gene system knocked into two specific genomic locations. scFV Strong and specific enrichment of the expected histone modifications of scFV GFP scFV By exploring reporter gene activity, they were able to reproducibly detect quantitative functional responses to epigenetic editing at single-cell resolution ( Figure 2 ). For example, through Ring1b-CD scFV (but not its catalytic mutant) programs HAK119ub to the promoter (PRC1), robustly causing transcriptional repression in most cells. scFVThe H3K27me3 target (PRC2) exhibited a partially penetrant transcriptional response, with many cells failing to show repression, suggesting that H3K27me3 has a weak instructive power at this locus.

[0212] Multiple programming of H2AK119ub and H3K27me3 results in a synergistic effect, with penetrant silencing exceeding the effect of either mark alone. Figure 3 A). Detection of other chromatin modifications, such as H3K4me3 and H3K9me2 / 3, revealed differential functionality at the reporter gene locus, whereas others such as H4K20me3 and H3K36me3 did not elicit a transcriptional response.

[0213] Taken together, these data support the ability to precisely and efficiently program a set of chromatin modifications toward a target and reveal single-cell transcriptional responses of individual tested loci in ESCs.

[0214] The inventors also studied gene-epigenetic interactions in our synthetic system. For example, we found that programming H3K27ac can activate a repressed reporter gene, and experimentally inserting a short 10nt MYC motif (E-box) into the same reporter gene attenuated this effect ( Figure 3 B). In contrast, the introduction of a 9nt OTX2 motif amplifies the transcriptional effect of H3K27ac. The inventors also found that the insertion of a short CTCF motif caused a behavioral shift of H3K36me3 in the targeted promoter from neutral (non-functional) to strong transcriptional repression ( Figure 3 C).

[0215] These data demonstrate robust and quantitative interactions between genetic motifs and causal epigenomic functionality. More generally, these results using a reductionist reporter strategy highlight the power of precise epigenetic perturbations to detect quantitative responses and cis-genetic effects.

[0216] To further explore the context-dependent principle of chromatin function, the inventors proposed to develop an epigenetic targeted perturbation sequencing (epiTAP-seq). This strategy will enable multi-dimensional evaluation of the functions of various chromatin states. This principle is based on TAP-seq (Schraivogel, D., et al. Targeted Perturb-seq enables genome-scale genetic screens in single cells. 17: 629-635. Nat Methods. (2020)), which obtains quantitative expression changes in single cells in response to guided perturbations ( Figure 4). Combining this approach with our chromatin perturbations and focusing on direct targeting rather than network responses, we can measure transcriptional consequences of endogenous genes at an unprecedented scale. The complexity of reprogramming a wide range of genes with 12 epigenetic marks and combinations thereof, as well as utilizing cis-genetic variants and multiple cell types, generates tens of thousands of scenarios for dissecting causal relationships.

[0217] This allows exploration of a wide range of critical questions. For example, what is the transcriptional response to de novo generated chromatin marks (quantification), functional penetration between single cells or between different genes (robustness), the quantitative consequences of diverse genetic-epigenetic interactions at the level of genomic features or cis-variants (context dependence), the impact of cellular context on regulatory responses (cell type specificity), and / or the precise nature of the epigenetic and transcriptional persistence of chromatin states (memory). Overall, by implementing epiTAP-seq into the systems and methods of the present invention and combining the data with a complementary range of unbiased screening and epigenome mapping methods, we are uniquely positioned to investigate the opportunities presented by precise epigenetic editing to dissect complex genomic regulatory mechanisms in a controlled and comprehensive manner.

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[0248] Chamberlain,S.J.,Yee,D.,and Magnuson,T.(2008).Polycomb repressivecomplex 2is dispensable for maintenance of embryonic stem cellpluripotency.Stem Cells 26,1496–1505.

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Claims

1. A complex comprising i) a catalytically inactive site-specific nuclease, preferably a catalytically inactive site-specific nuclease selected from Streptococcus pyogenes catalytically inactive (d) Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, Scel, and dCas9 / fCas9 fusion, which is linked to ii) an array of 2-10, preferably 3-7 effector domains, each effector domain having a specific chromatin modification activity, such as a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are each separated by a linker that provides a sufficient distance between the domain and the nuclease so as not to substantially interfere with its specific chromatin modification activity and the binding of the site-specific nuclease.

2. The complex of claim 1, wherein the complex comprises a fusion protein of the nuclease linked to a protein sequence comprising 3-7 effector domain binding motifs, such as Streptococcus pyogenes dCas9GCN4 (3-7) , each effector binding domain binding motif is separated by a linker sequence, the complex optionally further comprises a plurality of effector domains, each effector domain is bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA), preferably wherein the protein complex comprises 4-6 effector domains, preferably 5 effector domains.

3. The complex as described in claim 1 or 2, wherein the length of the linker sequence is 19-25 amino acids, preferably 22, and further preferably contains glycine (G) and serine (S).

4. A complex as described in any of claims 1-3, wherein the effector domain is bound via a scFV domain specific for the effector domain binding motif, in particular a scFV domain specific for GCN4, wherein the scFV is optionally linked to the effector domain via an effector linker group, such as a group comprising a fluorescent protein such as GFP or sfGFP protein, and wherein the effector domain is also fused to a protein destabilization domain such as d2.

5. The complex of any one of claims 1 to 4, wherein the effector domain comprises a chromatin modifying polypeptide, wherein the chromatin modifying polypeptide is selected from Dot1L (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Kmt2b (H3K4me3), Set1a (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Setdb1 (H3K9me3), Suv39h1 (H3K9me3), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), Ogt (GIcNAc), Prmt5 (H4R3me2 s), Hdac1 / 2 / 3 / 4 (histone deacetylase), Sirt1 / 2 / 3 / 6 (histone deacetylase), Kat2a (lysine acetyltransferase), Lsd1 (H3K4me demethylase), Kdm5a / b / c (H3K4 demethylase), Kdm2b (H3K4 and H3K79 demethylase), Tet1 / 2 / 3 (methylcytosine dioxygenase), Utx (H3K27 demethylase), JMJD3 (H3K27 demethylase), Kdm4a / b / c / d (H3K36 and H3K9 demethylase), its catalytic domain (CD), its catalytic domain (CD) fused to a scFV domain specific for an effector domain binding motif (CDscFV) and its fragment antigen binding (Fab) domain.

6. A nucleic acid group, each nucleic acid encoding at least one of the proteins and / or guide RNA (gRNA) and / or tagBFP in the complex according to any one of claims 1 to 5.

7. A gene construct set, the gene construct is, for example, an expression vector, which comprises the nucleic acid set described in claim 6, wherein preferably each nucleic acid encoding an effector domain, CD and / or CDscFV comprises an inducible promoter, such as a tet-responsive promoter, such as a dox-responsive promoter, wherein preferably the construct is a viral construct, such as a viral construct derived from AAV, a lentivirus or a retrovirus. 8 . A recombinant cell comprising the nucleic acid set according to claim 6 and / or the gene construct set according to claim 7 .

9. A method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus and / or a sample containing chromatin, comprising introducing into the cell, tissue, cell nucleus and / or sample the complex described in any one of claims 1-5 and one or more guide RNAs, thereby specifically epigenetically modifying chromatin in the cell, tissue, cell nucleus and / or sample.

10. The method of claim 9, wherein the epigenetic modification comprises histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, multiple epigenetic editing of histones, H3K9me2 / 3+DNA methylation, H3K4me3+H3K36me3, H3K4me3+H3K79me2, H3K36me3+H3K79me2, H3K9me2 / 3+H4K20me3, bivalent epigenetic editing of histones, and / or multiple combinatorial epigenetic editing of histones, and / or wherein the epigenetic modification comprises temporarily inducing the expression of the complex described in any one of claims 1-5, and one or more guide RNAs.

11. A method for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus and / or a sample containing chromatin, the method comprising: Introducing the complex of any one of claims 1 to 5 and one or more guide RNA sequences specific to the at least one target DNA sequence into a cell, tissue, cell nucleus and / or sample, thereby specifically epigenetically modifying the expression of at least one target DNA sequence in the cell, tissue, cell nucleus and / or sample, wherein preferably the at least one target DNA sequence comprises a nucleic acid sequence specific to a disorder and / or disease state associated with epigenetic modification of chromatin, the disorder and / or disease state being, for example, a genetic disease, a proliferative disease such as cancer, an immune cell producing autoantibodies, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened life span, tissue aging, neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), drug abuse including alcohol abuse disorder, epigenetic diseases including imprinting diseases such as Prader-Willi syndrome, wherein more preferably 2, 3, 4, 5, 6, 7, 8 or 9 target DNA sequences in the cell are regulated.

12. A method for detecting biological effects of specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, and / or regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, comprising implementing the method of any one of claims 9-11 and detecting at least one biological effect in cells, tissues, cell nuclei and / or samples containing chromatin, wherein the biological effect is selected from changes in gene expression, changes in protein levels, cis-genetic effects, changes in nucleic acid splicing, changes in the localization of loci in the cell nucleus, changes in TAD formation and destruction, changes in termination sites, activation of promoters, inhibition of promoters, changes in genetic-epigenetic interactions, functional relationships between genetic variants and epigenetic states of chromatin, changes in inherited methylation and imprinting, and associating specific epigenetic changes with diseases or cell phenotypes, wherein preferably the method is implemented with a complex capable of tracking effects in cells, tissues, cell nuclei and / or samples containing chromatin, preferably in real time.

13. A cell having specifically epigenetically modified chromatin, prepared by implementing the method of claim 9 and optionally isolating the cell, wherein the cell is preferably a stem cell, a neuron, a post-mitotic cell or a fibroblast, and / or wherein the cell is an animal cell, such as a mammalian cell, preferably a human or rodent cell.

14. A method for identifying a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that modulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin, and / or a biological effect of a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, comprising carrying out the method of any one of claims 9 to 12 in the presence and absence of a test drug, wherein: If the regulation and / or biological effect in the presence of the drug is different from the regulation and / or biological effect in the absence of the drug or compared to the control, the test drug is identified as a drug that specifically epigenetically modifies chromatin in cells, tissues, cell nuclei and / or samples containing chromatin, a drug that modulates the expression of at least one target DNA sequence in cells, tissues, cell nuclei and / or samples containing chromatin and / or the biological effect of specifically epigenetically modifying chromatin in cells, tissues, cell nuclei and / or samples containing chromatin.

15. Use of the complex of any one of claims 1-5 and one or more suitable guide RNA sequences, the set of nucleic acids of claim 6, the set of gene constructs such as expression vectors of claim 7, the cell of claim 12, and / or at least one of the drugs identified in claim 13 for preventing and / or treating a disease in a subject in need of prevention or treatment, or preventing and / or treating a disease associated with epigenetic modification of chromatin, the disease being, for example, a genetic disease, a proliferative disease such as cancer, an immune cell producing autoantibody, a bacterial or viral infection, a protozoan infection, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), drug abuse including alcohol abuse disorder, epigenetic diseases including imprinting diseases such as Prader-Willi syndrome.

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