CRISPR-based modular tools for the specific introduction of epigenetic modifications at target loci

Abstract

The present invention relates to a complex comprising: i) a catalytically inactive site-specific nuclease, and ii) the sequences of 2 to 10, preferably 3 to 7 effector domains, each having a specific chromatin modification activity, such as a specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are separated from each other by linkers and a sufficient distance is ensured between the domains and the nuclease so that the binding of the specific chromatin modification activity and the site-specific nuclease is not substantially hindered, as well as to each method related to the complex and to the use of the complex.

Description

Technical Field

[0001] The present invention relates to a complex comprising: i) a catalytically inactive site-specific nuclease; and ii) an array of 2 to 10, preferably 3 to 7 effector domains, each having a specific chromatin modification activity, such as a specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are separated from each other by linkers and a sufficient distance is ensured between the domains and the nuclease so that the specific chromatin modification activity and the binding of the site-specific nuclease are not substantially hindered, as well as to each method related to the complex and to the use of the complex.

Background Art

[0002] Coordinated control of transcription is essential for almost all biological processes, from development to homeostasis to disease. Therefore, understanding the nature, effects, and context-dependency of the molecular mechanisms that regulate gene expression is a central goal of modern biology.

[0003] The control of eukaryotic transcription is carried out by complex interactions between transcription factors (TFs), cis-regulatory elements, and epigenetic mechanisms. Such epigenetic systems are defined as an ensemble of regulatory molecules that affect chromatin structure, genomic function, and transcriptional activity and that are hereditary or otherwise sequence-independent.

[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 control usually leads to embryonic lethality or pathologies (Non-Patent Document 10), emphasizing their indispensability for life. Furthermore, it is located at the boundary of genetic, developmental, and environmental interactions that ultimately give rise to the phenotype.

[0006] Therefore, global efforts have been promoted to map epigenetic modifications throughout development and disease and to relate them to transcriptomes, genomic structures, and genetic mutations, etc. (Non-Patent Documents 4, 11). For example, histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 acetylation (H3K27ac) are typically specifically enriched in gene promoters where transcription is activated in normal or diseased cells. Conversely, H3K9me2, H3K27me3, H2AK119ub, and DNA methylation often correlate with transcriptional repression, while H3K36me3 is enriched on the transcribed gene body (Non-Patent Document 12). Such pioneering studies have provided unprecedented insights into genomic function and revealed that chromatin modifications are important for controlling gene expression levels and normal cellular functions.

[0007] Manipulation of enzymes that catalyze H3K27me3 (histone 3 lysine 27 trimethylation), H2A119Kub, H3K4me3, H3K36me3, H3K9me3, and DNAme causes widespread gene misexpression and embryonic lethality (Non-Patent Documents 1, 7-9), supporting the important role of chromatin modification. Furthermore, epigenetic environmental changes are also directly related to various diseases including cancer and aging (Non-Patent Document 3). However, it has been found that it is difficult to distinguish between direct and indirect effects. In fact, elucidating the quantitative effects of chromatin marks themselves and causal relationships is difficult due to the multifaceted effects of global regulatory abnormalities, not only non-histone substrates and non-catalytic functions of chromatin modifiers. For example, although there is a close relationship between H3K4mel / H3K27ac and active enhancers, recent evidence indicates that these marks may play a relatively minor role in enhancer function (Non-Patent Documents 10, 11). Therefore, in this field, it is necessary to shift from mapping the correlative changes of these marks to precisely defining context-dependent functions that are important for understanding disease mechanisms. Furthermore, it is necessary to develop tools that specifically "edit" chromatin modifications at specific loci and accurately reverse or manipulate abnormal gene activity in the diseases they cause.

[0008] The emergence of epigenetic editing technologies that enable site-specific regulation of chromatin modification may effectively address these issues. One of the current tools fuses chromatin-modifying proteins to nuclease-inactive (d)Cas9 and targets loci via guide (g)RNA (Non-Patent Document 12).

[0009] Non-Patent Document 12 (Nakamura M et al. (in: CRISPR technologies for precise epigenome editing. Nat Cell Biol. 2021 Jan;23(1): 11-22. doi:10.1038 / s41556-020-00620-7. Epub 2021 Jan 8. PMID: 33420494)) discloses that a complex series of cellular processes that control the activity of the genome are involved in the epigenome. To analyze this complexity, the development of tools that can specifically manipulate these processes is necessary. By reusing the prokaryotic CRISPR system, the development of various technologies for epigenome engineering has become possible. The authors review the current state of achievable epigenetic manipulations and their corresponding applications. The authors conclude that with future optimization, CRISPR-based epigenome editing will become a powerful set of tools for understanding and controlling biological functions.

[0010] Non-Patent Document 27 discloses that the epigenome dynamically controls gene expression and guides cell differentiation throughout the life of eukaryotes. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR) / Cas-based epigenome editing technologies have enabled researchers to site-specifically program epigenetic modifications to endogenous DNA and histones and manipulate the structure of native chromatin. As a result, epigenome editing has helped to clarify the causal relationship between epigenetic marks and gene expression. As epigenome editing tools continue to be developed, researchers have applied them in new ways to explore the functions of the epigenome in human health and disease. The authors discuss recent technical improvements in CRISPR / Cas-based epigenome editing that have advanced clinical research and examine how these technologies can be further improved for future practical use.

[0011] Non-patent Document 28 discloses that epigenome editing is expected to manipulate transcription and cell fate and elucidate the gene expression mechanism in various cell types. Functional epigenome editing requires the evaluation of the chromatin context-dependent activity of artificial epigenetic modifiers. The authors focused on the Forkhead box P3 (Foxp3) locus, which is the master transcription factor of regulatory T cells (Tregs), and applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to primary T cells of mice. The Foxp3 locus is controlled by combinatorial epigenetic modifications that determine Foxp3 expression. The expression of Foxp3 is unstable in transforming growth factor-beta (TGF-β)-induced Tregs (iTregs) but stable in thymus-derived Tregs (tTregs). To stabilize the expression of Foxp3 in iTregs, the authors introduced dCas9-TETICD (dCas9 fused to the catalytic domain (CD) of ten-eleven translocation dioxygenase 1 (TET1), a methylcytosine dioxygenase) and dCas9-p300CD (dCas9 fused to the CD of p300, a histone acetyltransferase) together with guide RNA (gRNA) targeting the Foxp3 locus. dCas9-TETICD induced partial demethylation in an enhancer region called conserved non-coding DNA sequence 2 (CSN2), but strong stabilization of Foxp3 was not observed. In contrast, dCas9-p300CD targeting the promoter locus partially maintained Foxp3 transcription in primary T cells cultured even under in vitro inflammatory conditions. Furthermore, dCas9-p300CD promoted the expression of Treg signature genes and enhanced the inhibitory activity in vitro. The authors concluded that artificial epigenome editing changed the epigenetic state and gene expression of the target locus and manipulated cell functions in conjunction with endogenous epigenetic modifications, suggesting an effective use of these technologies to elucidate the relationship between chromatin state and gene expression.

[0012] Non-patent Document 29 discloses that DNA methylation extensively affects gene expression during development. However, the ability to assign specific functions to regions of DNA methylation is limited because the overall pattern of DNA methylation has a low correlation with gene expression. The authors recruited multiple copies of an antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) that utilizes a nuclease-inactivated Cas9 protein fused to a repetitive peptide epitope (SunTag) to amplify local DNMT3A concentration and methylate the target genomic site. The authors demonstrated that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. Furthermore, dCas9-SunTag-DNMT3A can methylate a 4.5-kb genomic region and suppress the expression of the HOXA5 gene using a single guide RNA. Reduced representation bisulfite sequencing and RNA-seq revealed that dCas9-SunTag-DNMT3A methylates the region of interest while minimizing its impact on the overall DNA methylome and transcriptome. The authors concluded that the effective and accurate tool as discussed could enable site-specific manipulation of DNA methylation and potentially be used to address the relationship between DNA methylation and gene expression.

[0013] Patent Document 1 discloses a CRISPR / dCas9-based inducible DNA methylation editing system that includes a guide element, an anchor element, and an editing effector element that can act in sequence. The anchor element includes a stimulus-responsive protein A and inactivated SpCas9, and the editing effector element includes a stimulus-responsive protein B and a DNA methylation editing effector protein. The stimulus-responsive protein A and the stimulus-responsive protein B can be bound to each other under a stimulating effect, and the binding is released after the stimulating effect disappears.

[0014] Patent Document 2 discloses compositions and methods for delivering enhanced demethylation activity to a target DNA sequence in mammalian cells. These compositions and methods are useful for regulating the activity of target genes or creating gene regulatory networks.

[0015] In Non-Patent Document 30, a CRISPR-Cas9-based tool for specific DNA methylation, consisting of an inactivated Cas9 (dCas9) nuclease and the catalytic domain of DNA methyltransferase DNMT3A, targeted to any 20 bp DNA sequence following an NGG trinucleotide, was developed. The authors demonstrated CpG methylation targeted in a region approximately 35 bp wide by the fusion protein. The authors also showed that multiple guide RNAs can target the dCas9-DNMT3A construct to multiple adjacent sites, enabling methylation of a larger portion of the gene promoter. The DNA methylation activity was specific to the target region and heritable through mitosis. Finally, the authors demonstrated that directed DNA methylation of the broader promoter regions of the target gene loci IL6ST and BACH2 decreased their expression.

[0016] Nuclease-deficient or nuclease-lacking Cas9 proteins (such as dCas9) with mutations in the nuclease domain retain DNA-binding activity even when complexed with sgRNA. The dCas9 protein can be localized by tethering an effector domain or protein tag by protein fusion to the site matched by the sgRNA, constituting an RNA-guided DNA binding enzyme. dCas9 can be fused to a transcriptional activation domain (e.g., VP64) or a repressor domain (e.g., KRAB) and be induced by the sgRNA to activate or repress the target gene, respectively. dCas9 can also be fused to a fluorescent protein to achieve live-cell fluorescence labeling of chromosomal regions. Also, when multiple copies of a protein tag or effector fusion are required to achieve a certain biological threshold or signal detection threshold, multimerization of the effector or protein tag by direct fusion with the dCas9 protein is technically limited due to constraints such as difficulty in delivering large DNA encoding such fusions or difficulty in translating or translocating such large proteins into the nucleus due to the protein size.

[0017] Non-patent Document 31 discloses that although a clear epigenomic profile of histone marks is associated with gene expression, doubts remain about the causal relationship. The authors examined the activities of a broad collection of genome-targeted epigenetic regulators (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A, and the first targetable version of Ezh2 and Friend of GATA-1 (FOG1)) that can write epigenetic marks associated with a repressed chromatin state. The dCas fusions produced repression of target genes in a range of 0 to 10-fold different depending on the locus and cell type. The dCpf1 fusion was unable to repress gene expression. The action of several effector domains was required for the most persistent gene repression, but KRAB-dCas9 did not contribute to persistence, in contrast to previous reports. Both the "direct binding" strategy of binding the Ezh2 methyltransferase enzyme to dCas9 and the "recruit" strategy of binding the first 45 residues of the N-terminus of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex were successful in the targeted deposition of H3K27me3. However, surprisingly, the repression was not correlated with the deposition of H3K9me3 or H3K27me3. The authors' findings suggest that so-called repressive histone modifications are insufficient for gene repression.

[0018] Non-patent Document 32 describes that CRISPR-associated (Cas) enzymes have brought about a revolution in biology by enabling RNA-guided genome editing. Homologous recombination repair (HDR) in the presence of a donor template is currently the most versatile method for introducing accurate editing after CRISPR-Cas-induced double-stranded DNA breaks, but the efficiency of HDR is generally lower compared to the end-joining pathway that leads to insertions and deletions (indels). The authors tested the hypothesis that HDR can be increased by using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K4me3 and H3K36me3, which have been shown to mediate homologous recombination in human cells. The results show that the fusion protein specifically contacts chromatin at the Cas9 cleavage site in DNA, doubling the observed HDR efficiency and tripling the HDR:indel ratio compared to that induced by Cas9 alone. HDR enhancement occurred in multiple cell lines, but off-target genome editing did not increase. These findings highlight the importance of chromatin structure in the choice of DNA repair pathways during CRISPR-Cas genome editing and provide a new strategy for enhancing the efficiency of HDR.

[0019] Non-patent Document 33 discloses that, although DNA methylation is important in health and disease, there is a lack of technology that can easily manipulate the methylation of specific sequences for functional analysis and therapeutic purposes. The authors adapted the Cas9-SunTag described above for efficient and targeted demethylation of specific DNA loci. The original SunTag consists of 10 copies of the GCN4 peptide separated by 5-amino acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the 10-11(TET)1 hydroxylase that induces demethylation, the linker length was changed to 22 amino acids. This system achieved demethylation efficiency exceeding 50% at 7 out of the 9 loci tested. Demethylation exceeding 90% was observed at 4 of these 7 loci. The authors demonstrated targeted demethylation of CpGs in regulatory regions and 1.7- to 50-fold upregulation of related genes dependent on demethylation both in cell culture (embryonic stem cells, cancer cell lines, primary neural progenitor cells) and in vivo in mouse fetuses.

[0020] Patent Document 3 relates to a method of modifying DNA methylation by contacting a catalytically inactive site-specific nuclease fused to an effector domain having methylation or demethylation activity and one or more guide sequences with a genomic DNA sequence.

[0021] In previous studies, epigenetic editing has been applied to a subset of specific loci, and causal biological insights have been inferred by measuring changes in bulk expression, usually (Non-Patent Document 13). Such an approach to decoding the epigenome has also attracted interest in biomedical and preclinical applications, such as reversing epigenome-dependent disease phenotypes (Non-Patent Document 14). However, to enable an understanding (or desirable manipulation) of the quantitative principles of chromatin function, it is necessary to (i) enhance the on-target activity of these systems, (ii) expand the ability to target multiple marks and their combinations, (iii) measure effects at single-cell resolution and capture the distribution of responses, and perhaps most importantly, (iv) maximize throughput to hundreds or more loci and systematically analyze context-dependent responses. Systematic studies and genome-wide association studies (GWAS) have proven to be powerful tools for advancing genomic research towards the goal of predicting the relationship between genotype and phenotype. Such efforts have suggested that sequence variants (SNPs, indels) located in promoters and cis regulatory elements (cREs) are the main cause of phenotypic variation, and that complex human traits often appear through genetic differences in gene regulation (rather than in coding sequences) (Non-Patent Documents 15, 16). How diverse promoters / cREs and their cis variants interact with epigenetic mechanisms to regulate gene activity and ultimately phenotype is a cutting-edge issue for understanding genomic function, disease susceptibility, and evolutionary processes. In fact, DNA sequence variation and epigenetic systems are intricately related, and chromatin state can potentially affect the occupancy of sequence-dependent transcription factors (TFs), and conversely, DNA sequence can affect chromatin state (Non-Patent Documents 17, 18).The pressing need to understand genomic functions and the molecular mechanisms underlying numerous biological processes has been accelerated by genome-scale perturbation strategies such as pooled CRISPR screening by many groups, including the present inventors (Non-Patent Documents 19 to 21). This progress has facilitated multi-parameter readouts from CRISPR-based screening (Non-Patent Documents 22 to 25). In particular, in the targeted perturb-seq (TAP-seq) approach (Non-Patent Document 26), the expression of thousands of target genes can be accurately quantified in single cells with specific perturbations involving thousands of perturbations across the population. Applying such high-resolution approaches to elucidate the regulatory logic by which chromatin-based systems intersect DNA sequences, cis-variants, and cell identity to generate quantitative control of genes will be an important milestone for understanding genotype-phenotype interactions and chromatin function.

[0022] Chromatin modification is recognized as one of the important regulatory mechanisms of transcriptional control under normal and diseased conditions. Nevertheless, it has been found to be difficult to analyze the exact causal functions of specific chromatin modifications, that is, in this field, the technical ability to investigate the causal relationships of epigenetic modifications accurately, quantitatively, and within well-defined genomic features is limited. Little is known about how chromatin states interact with diverse DNA sequences and cis-variants to quantitatively affect transcription and how the cellular environment influences this. Understanding the causal relationship between epigenetic marks and gene expression is a central issue in chromatin biology, and particularly with the recent advances in epigenome editing technologies, new light has begun to be shed on these processes. The discovery of CRISPR-Cas9 interference has provided a valuable tool for controlling gene expression by targeting the catalytically inactive variant (dCas9) of Streptococcus pyogenes SpCas9 to inhibit transcription. Since then, in several strategies, dCas9 has been fused to well-characterized repressors or activators (e.g., KRAB, VP64, etc.) to enhance silencing and activation capabilities and regulate gene expression. Furthermore, new tagging approaches have enabled more efficient recruitment of multiple effectors to a single dCas9 anchor bound to a specific genomic locus. Recruitment strategies can also provide temporal control of transcriptional regulation when combined with chemically inducible approaches. Finally, recent studies have also focused on control DNA sequences that activate enhancers by recruiting dCas9 fused to histone acetyltransferase p300 or dCas9 fused to DNA demethylase Tet1 (Non-Patent Document 34). Therefore, ultimately, for the clinical scenario and treatment applications of this technology, large-scale, targeted perturbation strategies for analyzing the causal regulatory function of chromatin marks across the entire endogenous context are clearly necessary.

Prior Art Documents

Patent Documents

[0023]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0024]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non - Patent Document 19

Non - Patent Document 20

Non - Patent Document 21

Non - Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non-Patent Document 33

Non-Patent Document 34

Summary of the Invention

Problems to be Solved by the Invention

[0025] An object of the present invention is to provide additional tools derived from the above in the fields of epigenetics, methylation, precise genome control, and further treatment of related diseases. Other objects and advantages will become apparent by further examining this specification with reference to the attached examples.

Means for Solving the Problems

[0026] In its first aspect, the object of the present invention is solved in particular by providing a protein complex, which complex comprises: i) a catalytically inactive site-specific nuclease; and ii) an array of 2 to 10, preferably 3 to 7 effector domains, each having a specific chromatin modification activity, such as a specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity, wherein the effector domains are separated from each other by linkers and a sufficient distance is ensured between the domains and the nuclease so that the specific chromatin modification activity and the binding of the site-specific nuclease are not substantially hindered.

[0027] Preferably, the protein complex of the present invention is a fusion protein of a nuclease bound to a protein sequence comprising 3 to 7 effector domain binding motifs separated by a linker sequence, such as Streptococcus pyogenes dCas9 GCN4 (3-7) The complex may further comprise a number of effector domains, each non-covalently bound to the binding motif, and the complex may optionally further comprise at least one suitable guide RNA (gRNA).

[0028] In a further preferred complex according to the invention, the chromatin modification activity is histone methylation, such as histone methylation contributing to stable or reversible gene expression control.

[0029] In its second aspect, the object of the present invention is solved by providing a set of nucleic acids encoding at least one of the protein and / or guide RNA (gRNA) and / or tagBFP of the complex according to the present invention.

[0030] In that third aspect, the object of the present invention is solved by providing a set of gene constructs such as expression vectors, preferably effector domains, CD and / or CD, which contain a set of nucleic acids according to the present invention. scFV Each nucleic acid encoding scFV contains an inducible promoter such as a tet-responsive promoter. More preferably, the construct according to the present invention is a viral construct such as a viral construct derived from, for example, adeno-associated virus (AAV), lentivirus or retrovirus.

[0031] In that fourth aspect, the object of the present invention is solved by providing a recombinant cell containing a set of nucleic acids according to the present invention and / or a set of gene constructs according to the present invention.

[0032] In that fifth aspect, the object of the present invention is solved by providing a complex according to the present invention, which comprises expressing a set of nucleic acids according to the present invention and / or a set of gene constructs according to the present invention in a recombinant cell according to the present invention, and optionally inducing the expression using, for example, tetracycline.

[0033] In that sixth aspect, the object of the present invention is a method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample, comprising introducing a complex according to the present invention and one or more guide RNAs into the cell, tissue, cell nucleus, and / or sample, thereby specifically epigenetically modifying the 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, histone multiplex epigenetic editing, H3K9me2 / 3 + DNA methylation, H3K4me3 + H3K36me3, H3K4me3 + H3K79me2, H3K36me3 + H3K79me2, H3K9me2 / 3 + H4K20me3, histone bivalent epigenetic editing, and / or histone polycomb epigenetic editing.

[0034] In that seventh aspect, the object of the present invention is solved by providing a method for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising introducing into the cell, tissue, cell nucleus, and / or sample a complex according to the present invention and one or more guide RNA sequences specific to the at least one target DNA sequence, thereby specifically and epigenetically regulating the expression of the at least one target DNA sequence in the cell, tissue, cell nucleus, and / or sample. Preferably, the at least one target DNA sequence is, for example, a nucleic acid sequence specific to an epigenetic disease such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoal infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological 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 use disorder, and imprinting disorders such as Prader-Willi syndrome, and / or a disease state associated with epigenetically modified chromatin.

[0035] In its eighth aspect, the object of the present invention is to specifically epigenetically modify chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin, and / or to detect a biological effect by 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 according to the present invention, as well as detecting at least one biological effect in cells, tissues, cell nuclei, and / or samples containing chromatin, the biological effect being selected from the group consisting of changes in gene expression, protein amount, changes in cis-gene effects, changes in nucleic acid splicing, changes in the nuclear arrangement of loci, changes in the formation and disruption of TADs, termination sites, activation of promoters, changes in the suppression of promoters, genetic epigenetic interactions, changes in the functional relationship between gene mutations and the epigenetic state of chromatin, changes in genetic methylation and imprinting, and specific epigenetic changes associated with diseases or cell phenotypes.

[0036] In its ninth aspect, the object of the present invention is solved by providing a cell having specifically epigenetically modified chromatin produced by carrying out the method of the present invention, and optionally isolating said cell, preferably, said cell is a stem cell, neuron, postmitotic cell, or fibroblast, and / or said cell is an animal cell, such as a mammalian cell, preferably a human cell or a rodent cell.

[0037] In that tenth aspect, the object of the present invention is to provide a method for identifying a biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, a drug for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising performing the method according to the present invention in the presence and absence of a test agent, wherein the test agent is a cell, tissue, cell nucleus, and / or sample containing chromatin when the regulation and / or biological effect in the presence of the agent is different from the regulation and / or biological effect in the absence of the agent or in the control. A drug that specifically epigenetically modifies chromatin, a drug that regulates the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or a biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin is identified.

[0038] In the 11th aspect, the object of the present invention is to provide a method for preventing or treating diseases related to epigenetically modified chromatin in a subject in need of prevention or treatment, such as, for example, 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, shortening of biological 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 use disorder, epigenetic diseases including imprinting abnormalities such as Prader-Willi syndrome, etc., which comprises administering to the subject in need of such treatment an effective amount of at least one of the complex according to the present invention, and one or more appropriate guide RNA sequences, a set of nucleic acids according to the present invention, a set of gene constructs such as expression vectors according to the present invention, cells according to the present invention, and / or a drug identified according to the present invention.

[0039] In another preferred aspect, it relates to the complex according to the present invention, and one or more appropriate guide RNA sequences, a set of nucleic acids according to the present invention, a set of gene constructs such as expression vectors according to the present invention, cells according to the present invention, and / or at least one of the drugs identified according to the present invention for use in preventing and / or treating diseases in a subject in need of prevention or treatment, or for preventing and / or treating diseases related to epigenetically modified chromatin, such as, for example, 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, shortening of biological 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 use disorder, epigenetic diseases including imprinting abnormalities such as Prader-Willi syndrome, etc.

[0040] In the twelfth aspect, the object of the present invention is to specifically epigenetically modify chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin according to the present invention, to regulate the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin according to the present invention, to detect the biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample according to the present invention, and / or to identify a drug according to the present invention, a complex according to the present invention, a set of nucleic acids according to the present invention, a set of gene constructs such as an expression vector according to the present invention, and / or the use of a cell according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described in the following examples with reference to the accompanying drawings, but is not limited thereto. For the purpose of the present invention, all references cited herein are incorporated by reference in their entirety.

[0042] This disclosure includes a sequence listing containing SEQ ID NO: 1 as part of the description, and the whole of it is also incorporated by reference.

[0043]

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 2-1

Figure 2-2

Figure 2-3

Figure 2-4

Figure 2-5

Figure 3-1

Figure 3-2

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0044] As described above, in a first aspect of the present invention, the object of the present invention is solved by a complex, particularly a protein complex, comprising i) a catalytically inactive site-specific nuclease and ii) the sequences of 2 to 10, preferably 3 to 7 effector domains each having a specific chromatin modification activity, wherein the effector domains are separated from each other by linkers and a sufficient distance is ensured between the domains and the nuclease so that the specific chromatin modification activity and the binding of the site-specific nuclease are not substantially hindered.

[0045] The specific chromatin modification activity is preferably selected from specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or specific chromatin demethylation / deacetylation activity.

[0046] The present invention provides an improved epigenome editing platform for systematically programming specific chromatin modifications and combinatorial chromatin modifications across tens of thousands of contexts in living cells. This resource can be used to capture multimodal functional responses at the allelic and single-cell resolution from diverse lineages. Precision perturbation on a scale without precedent reveals the control logic by which different chromatin modifications interact with genomic features, sequence variants, and cell identity, forms quantitative gene expression patterns, and further helps to identify trans-acting and cis-structure mechanisms that implement the function of epigenetics, particularly chromatin marks. Furthermore, the present invention can be utilized to reverse or manipulate abnormal genomic or chromatin states in diseases.

[0047] Developed is a modular CRISPR-based toolkit that, in its current form, can accurately and inductively program nine different epigenetic modifications to endogenous target loci. The ability to site-specifically deposit epigenetic markings such as methylation, including H3K27me3, H3K4me3, H3K79me3, H2AK119ub, and H3K36me3, represents a powerful gain-of-function perturbation strategy for explicitly assessing their causal effects. In a preferred embodiment, the tool provides dCas9 (dCas9 GCN4 ) linked to the sequences of five GCN4 motifs, each motif separated by a linker sequence designed at an optimal interval to accommodate bulkier proteins without sterically inhibiting catalytic activity. This dCas9 GCN4 is capable of carrying multiple (e.g., five) "effector" proteins or domains to a specific locus, with the effector domains complexed via GCN4-specific scFV domains (see Figure 1A). The inventors designed and tested a comprehensive suite of effector domains (collectively referred to as CD scFV ) preferably containing only the catalytic domains of chromatin-modifying enzymes, such as Setd2-CD scFV for H3K36me3 and Prdm9-CD scFV for H3K4me3.

[0048] The complexes of the present invention incorporate a number of other advantages that, collectively, give rise to an epigenetic editing platform technology with excellent capabilities enabling discovery.

[0049] These advantages include the following:

[0050] Very active editing. For example, five copies of a specific CD scFVWhen recruited to the target locus, on-target programming of chromatin modification is greatly amplified both in amplitude and genome-wide. This ensures de novo histone methylation deposition comparable to strong endogenous peaks, promoting both negative and positive functional effects.

[0051] Catalytic domain specificity. When using only the customized effector domains, such as these catalytic cores, the complex avoids unwanted side effects of targeting full-length chromatin modification proteins. Since full-length proteins have major non-catalytic functions and / or may recruit complexes of other proteins, preferentially using the catalytic domain allows the evaluation of the function of the target chromatin mark itself (see also below).

[0052] Combinatorial epigenetic editing. Since the complexes and systems of the present invention are modular, different CDs (preferably up to 5) scFV can be recruited simultaneously. This enables multiplexed and even adjustable epigenetic editing, allowing the establishment of de novo domains of different chromatin modifications (e.g., bivalent or polycomb).

[0053] Minimized off-targeting. Since the effector domains and CDs used herein are not directly fused to dCas9 and the CDs generally do not have endogenous DNA binding domains, off-target activity is minimized. scFV The effector domains and CDs are not directly fused to dCas9 and the CDs generally do not have endogenous DNA binding domains, so off-target activity is minimized.

[0054] Temporal resolution. In the gene constructs provided in the context of the present invention, each effector domain and / or CD scFV may also have a protein destabilization (d2) domain that is dynamically induced via a DOX-responsive promoter and promotes rapid degradation upon DOX removal. This allows the investigation of temporal responses and epigenetic memory (chromatin persistence).

[0055] Dynamic tracking. Preferably, since some or all of the effector groups are bound to superfolder GFP (sfGFP) and some or all of the gRNAs are bound to tagBFP, this system can be tracked in real time, cells can be purified, and dose-dependent responses to drugs can be tested (e.g., comparison of GFP low cells and GFP high cells).

[0056] Control of indirect / cross effects. As an additional control, effector domains that are identical to CD scFV but contain a single-point mutation that inactivates enzyme activity were also generated (catalytic mutant effectors, 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 . The mutant effectors cannot write chromatin modifications to histones and DNA. Thus, they represent an important control to confirm that changes in gene expression are caused only by the deposition of chromatin modifications and not by indirect events such as the recruitment of other protein complexes (changes in gene expression are achieved only when wild-type CD scFV is recruited and mutant mut-CD scFV is not recruited). Additionally, GFP-only effectors (GFP GCN4 ) were also created to control for steric hindrance effects caused by the recruitment of the bulky dCas9 scFV protein to chromatin. Collectively, these features enable a carefully controlled assessment of the functional impact of introducing a specific chromatin modification at a specific locus while excluding confounding effects. They also enable the ability to introduce or reverse specific chromatin modifications in living cells to reverse pathological outcomes.

[0057] Non-Patent Document 13 reviews the SpdCas9-SunTag technology, which is constructed by fusing the SpdCas9 enzyme to several (5-20) tandem repeats of the GCN4 binding motif (SunTag). In this technology, any protein bound to the small-chain variable fragment (scFV) domain is recruited. This system allows multiple copies of the same effector domain (or different effector domains) to be recruited to a specific genomic site, minimizing off-target effects while promoting high on-target editing efficiency and the spread of induced epigenetic states. In the future, it may be possible to use the same strategy to analyze how combinatorial chromatin modifications affect transcription and genome control. Nevertheless, the described dCas9-SunTag system is a fusion of the dCas9 protein and the tail of the GCN4 peptide, which can recruit up to 10 copies of scFV-VP64 to amplify the activation signal (Tanenbaum M.E., Gilbert L. A., Qi L.S., Weissman J.S., Vale R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159:635-646). It is also disclosed that even a short spacer of about 5 amino acids can sufficiently separate the peptides to allow an antibody to bind to adjacent peptides.

[0058] WO 2021 / 247570 discloses a fusion protein, a composition, and a method of manipulating a biological genome. This fusion comprises, from the N-terminus to the C-terminus, a DNA methyltransferase domain, a first XTEN linker, a nuclease-deficient RNA-guided endonuclease enzyme, a second XTEN linker, and a Kruppel-associated box domain.

[0059] WO 2014 / 197748 discloses compositions related to the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9-based system and methods of using said CRISPR / Cas9-based system compositions for gene expression alteration and genome engineering. Also disclosed are compositions for gene expression alteration and genome engineering in muscles such as skeletal muscle and cardiac muscle, and methods of using said compositions. These tools use a single fusion protein containing the essential KRAB (Kruppel-associated box domain). This system does not require KRAB and is more specific (fewer "off-target effects" and allows for "fine-tuning").

[0060] Non-Patent Document 20 developed a CRISPR-based epigenetic programming tool. The authors used a fusion of catalytically dead (d)Cas9 and the sequence of five GCN4 repeats arranged at optimal intervals (dCas9 GCN4 ). These function as docking sites for recruiting up to five "effectors" to specific genomic loci via single-chain antibody (scFV) domains. This modular system amplified both the on-target epigenome editing quantitative level and the domain size compared to dCas9 effector fusions, and minimized off-target effects. The authors used KRAB GFP-scFV and DNMT3A / 3L GFP-scFV to promote direct deposition of H3K9me3 and DNA methylation, respectively, to target de novo heterochromatin.An effector was generated. The system was significantly improved, enabling various types of chromatin editing (see below).

[0061] In the context of the present invention, the "protein complex" shall mean a complex of biological molecules containing peptide chains composed at least mostly of amino acids such as proteins. The complex is formed by covalent and / or non-covalent bonds and preferably includes molecules complexed by antigen / antigen binding (e.g., based on antibody recognition) activity. Furthermore, the complex may include not only other proteins such as fluorescent labels, but also nucleic acids such as gRNA, and other chemical groups such as linkers and coupling agents.

[0062] In yet another aspect of the methods and compounds of the present invention, multiple labels and markers are used. Markers can be used for both nucleic acid molecules forming part of the assay and protein components (such as nucleases and fusions). Labels and markers can be included in the components of the assay (especially nucleic acids and / or proteins) and also include moieties attached by either covalent or non-covalent bonds.

[0063] In principle, any suitable catalytically inactive site-specific nuclease can be used in the protein complexes of the present invention. As described above, nuclease-deficient or nuclease-lacking Cas9 proteins (e.g., dCas9) with mutations in the nuclease domain retain DNA-binding activity even when complexed with sgRNA. The dCas9 protein can be localized by ligating an effector domain or protein tag to a site matching the sgRNA by protein fusion, constituting an RNA-guided DNA-binding enzyme. dCas9 can be fused to a transcriptional activation domain (e.g., VP64) or a repressor domain (e.g., KRAB) and is induced by sgRNA to activate or repress the target gene, respectively. dCas9 can also be fused to a fluorescent protein to achieve live-cell fluorescence labeling of chromosomal regions. However, in such a system, since the pairing of sgRNA:Cas9 is exclusive, only one Cas9 effector fusion can be made. Also, when multiple copies of a protein tag or effector fusion are required to achieve a certain biological threshold or signal detection threshold, multimerization of the effector or protein tag by direct fusion with the dCas9 protein is technically limited due to constraints such as difficulty in delivering large DNA encoding such a fusion, or difficulty in translating or translocating such a large protein to the nucleus due to the protein size. Preferably, it is a catalytically inactive site-specific nuclease selected from the group consisting of catalytically inactive (d)Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, SceI, and dCas9 / fCas9 fusions derived from Streptococcus pyogenes. Since some preferred delivery systems such as AAV have limited loading capacity (i.e., can only accommodate a specific number and size of protein molecules), the development of smaller CRISPR / Cas9 systems has been promoted, such as systems constructed around the preferred nuclease staphylococcusaureus saCas9.

[0064] In a preferred complex according to the present invention, the complex is a fusion protein of a nuclease fused or linked to a protein sequence comprising 3 to 7 effector domain binding motifs each separated by a second linker sequence, such as Streptococcus pyogenes dCas9 GCN4 (3-7) and the complex optionally further comprises a number of effector domains each bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA).

[0065] As used herein, an "effector domain" is a polypeptide or protein having at least one specific chromatin modification activity. Preferably, for example, an effector domain having a specific chromatin modification activity such as a 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 effector domains that exhibit chromatin modification activity in combination, so-called "combinatorial effectors". The present invention also advantageously uses effectors comprising a trimmed, i.e., catalytic domain (CD) of a full-length chromatin-modifying polypeptide or protein, and / or more preferably, its catalytic domain (CD scFV ) fused to an effector domain binding motif-specific scFV domain. Again, this has additional advantages from the perspective of delivery media with limited payloads, such as AAV-based media, and has also been found to enhance the activity and specificity of the system. Other methods of delivering the system include non-viral delivery modes including physical methods (e.g., electroporation, microinjection, sonoporation, hydrodynamic delivery) and chemical approaches (e.g., lipid particles, polymer nanoparticles, gold nanoparticles, and cell-penetrating peptides (CPPs)).

[0066] In the context of the present invention, the term effector domain includes polypeptides having at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity to the polypeptide sequences disclosed herein and having the chromatin modification activity disclosed herein.

[0067] Even more preferred in the context of the present invention is a complex according to the present invention, wherein the effector domain comprises a chromatin modification protein or polypeptide selected from the group consisting of Dot1l (H3K79me2), p300 (H3K27ac), Prdm9 (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), OGT (GlcNAC), and preferably comprises its catalytic domain (CD) if possible, and if possible, its catalytic domain (CD) is fused to an effector domain-binding motif-specific scFV domain (CD scFV )).

[0068] Histones can be modified in several ways, including acetylation, methylation (lysine), methylation (arginine), phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, deimination, and proline isomerization (see also above). Preferably, the complex according to the present invention has a chromatin modification activity that is histone methylation, for example, histone methylation that contributes to stable or reversible regulation of gene expression.

[0069] 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-SET7 / 8, SUV420H1, SUV420H2, SpSet 9, EZH2, RIZ1, LSD1 / BHC110, JHDM1a, JHDM1b, JHDM2a, JHDM2b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, CARM1, PRMT4, PRMT5, Haspin, MSK1, MSK2, CKII, Mst1, Bmi / RinglA, RNF20 / RNF40, and ScFPR4.

[0070] Preferably, the effector domain can be appropriately labeled or tagged with a fluorescent marker such as sfGFP (superfolder GFP). sfGFP surprisingly helps recombinant / artificial proteins (such as those according to the present invention) fold better, thereby improving the complexes of the present invention.

[0071] More preferably, the protein complex according to the present invention comprises 3 to 7 effector domains, particularly 5 effector domains. The domains are at least 2 or more, at least 3 or more, at least 4 or more, or at least 5 or more different effector domains.

[0072] More preferably, the protein complex of the present invention, wherein the effector domain binding motif consists of an epitope containing a peptide sequence of 17 to 29 amino acids, preferably 17 to 21 amino acids, and has little or no structural folding under physiological conditions. In principle, any suitable binding motif can be used, but for reasons of specificity and stability, the GCN4 epitope motif sequence is preferred. The GCN4 epitope motif sequence (or "GCN4 peptide") contains 22 amino acids (LLPKNYHLENEVARLKKLVGER; SEQ ID NO: 1). However, the sequence of the effector domain ("tail") may be complexed or fused with the nuclease in various ways such as chemical bonding.

[0073] In the complex according to the present invention, further, a linker is provided that provides sufficient distance between domains and between the domain and the nuclease so as not to substantially interfere with their specific chromatin modification activity and the binding of the site-specific nuclease, respectively. Linkers are known to those skilled in the art, and the length of the linker sequence is 25 to 19 amino acids, preferably 22, and more preferably contains glycine (G) and serine (S) amino acids. The linker (5 amino acids) in dCas9-SunTag was found to be insufficient for the optimal catalytic activity of the enzyme. It has been found that the sequence of the linker is not particularly important as long as the linker does not interfere with the desired activity, and the length / distance is more important. "Substantially" in the context of the interference described above means that the complex according to the present invention exhibits both sufficient specific chromatin modification activity and sufficient binding of the site-specific nuclease.

[0074] More preferably, in the complex according to the present invention, the effector domain is bound via an effector domain-binding motif-specific scFV domain, particularly a GCN4-specific scFV domain, and the scFV is optionally linked to the effector domain via an effector linker group, such as a group containing a fluorescent protein such as GFP or sfGFP protein, and the effector domain may be further fused to a protein destabilizing domain such as, for example, d2.

[0075] Greer, Eric L, and Yang Shi (in: "Histonemethylation: a dynamic mark in health, disease and inheritance." Naturereviews. Genetics vol. 13,5 343-57. 3 Apr. 2012, doi: 10.1038 / nrg3173) have shown that organisms require an appropriate balance of stability and reversibility in gene expression programs to maintain cell identity and respond to stimuli, and that epigenetic control is essential for this dynamic control. Post-translational modification of histones by methylation is an important and widespread type of chromatin modification known to affect biological processes in the context of development and cellular responses. These provide a broad overview of how histone methylation is regulated and leads to biological outcomes, and evaluate how histone methylation contributes to stable or reversible control.

[0076] The importance of properly maintaining or reprogramming histone methylation is indicated by its association with diseases, aging, or the transmission of traits across generations. Histone methylation occurs at all basic residues such as arginine 3, lysine 4, and histidine 5. Lysine can be mono-(me1)4, di-(me2)6, or tri-(me3)7 methylated on its amine group, arginine can be mono-(me1)3, symmetric dimethylated (me2s), or asymmetrically dimethylated (me2a) on its guanidinyl group8, and histidine has been reported8,9 to be monomethylated, although this methylation is rare and not further characterized. The most extensively studied histone methylation sites include histone H3 lysine 4 (H3K4), H3K9, H3K27, H3K36, H3K79, and H4K20. Arginine methylation sites include H3R2, H3R8, H3R17, H3R26, and H4R3. However, many other basic residues throughout the histone proteins H1, H2A, H2B, H3, and H4 have recently been confirmed to be methylated.

[0077] The main advantage of the complex according to the present invention is that, based on the specific selection of the effector domains used, and thus the combination of effector domains, for example, specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or specific chromatin demethylation / deacetylation activity, or combinations thereof, the chromatin modification activity can be specifically "regulated". Thus, the complex of the present invention has at least two or more, at least three or more, at least four or more, or at least five or more different effector domains, for example, Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFVmay include the combination of, and preferably does not include the KRAB and / or VPR effector domain. More preferably, at least one chromatin-modifying protein or polypeptide is 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 (GlcNAC), 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), these catalytic domains (CD), these catalytic domains (CD scFV ) or their antigen-binding fragment (Fab) domains fused to these catalytic domains (CD) are selected from the group consisting of.

[0078] Another aspect of the present invention relates to a set of nucleic acids encoding at least one of the protein and / or guide RNA (gRNA) and / or tagBFP of the complex according to the present invention.

[0079] In the context of the present invention, the term nucleic acid is intended to include sequences encoding peptides or polypeptides having at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity to the sequences disclosed herein and having substantially the same activity as the polypeptides disclosed herein. For example, preferably, in the complex of the present invention, the guide RNA molecule comprises a sequence that is at least 80%, preferably 90% or more, more preferably 95% or more, and most preferably 100% complementary to the target RNA.

[0080] Certain portions of the nucleic acid molecules used in the method of the present invention are found and / or designed to specifically hybridize with complementary portions of other molecules. As is known to those skilled in the art, hybridization conditions and washing conditions are important for this purpose. When the sequences are 100% complementary, high-stringency hybridization can be performed. However, according to the present invention, the hybridizing portion and / or the specifically hybridizing portion is at least 80% complementary, preferably 90% or more complementary, more preferably 95% or more complementary, and most preferably 100% complementary. The stringency of hybridization is determined by the hybridization temperature and the salt concentration in the hybridization buffer, and the higher the temperature and the lower the salt concentration, the higher the stringency. The commonly used washing solution is SSC (sodium citrate saline, a mixture of sodium citrate and sodium chloride). Hybridization can also be performed in solution, but more commonly, at least one component may be on a solid support such as nitrocellulose paper. In a commonly used protocol, blocking reagents such as casein derived from skim milk powder or bovine serum albumin are used in combination with a denatured fragmented salmon sperm DNA (or other highly complex heterologous DNA) and a surfactant such as SDS. In many cases, a very high concentration of SDS is used as a blocking agent. The temperature may be 42 to 65 °C or higher, and the buffer may be 3xSSC, 25 mM HEPES, pH 7.0, 0.25% SDS final.

[0081] Thus, the set according to the invention encodes at least two, preferably three, four, five, or all of the protein components of the complex according to the invention. Preferred examples include a first set comprising a nucleic acid encoding a catalytically inactive site-specific nuclease comprising a linker structure ("tail") that can be genetically fused to a nuclease enzyme, and / or a second set comprising a nucleic acid encoding an effector domain polypeptide to be used. A more preferred set is a set comprising nucleic acids encoding 2 to 10, preferably 3 to 7, effector domains each having a specific chromatin modification activity such as, for example, a specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or a specific chromatin demethylation / deacetylation activity. Other preferred sets include nucleic acids encoding at least one suitable guide RNA (gRNA). These sets are further combined with a nucleic acid encoding tagBFP for co-expression and then tagged to the gRNA.

[0082] Preferred sets further include nucleic acids encoding an effector domain-binding motif-specific scFV domain, particularly a GCN4-specific scFV domain, which scFV is optionally linked to the effector domain via an effector linker group such as a group comprising a fluorescent protein such as, for example, GFP or sfGFP protein, and the effector domain may be further fused to a protein destabilizing domain such as, for example, d2. Also, an effector tag such as, for example, sfGFP (superfolder GFP) is also encoded, and thus it is preferred that the construct contains a fluorescent marker.

[0083] The effector domains to be encoded are 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 (GlcNAC), Prmt5 (H4R3me2s), Hdac1 / 2 / 3 / 4 (histone deacetylases), Sirt1 / 2 / 3 / 6 (histone deacetylases), Kat2a (lysine acetyltransferase), Lsd1 (H3K4me demethylase), Kdm5a / b / c (H3K4 demethylases), Kdm2b (H3K4 and H3K79 demethylase), Tet1 / 2 / 3 (methylcytosine dioxygenases), Utx (H3K27 demethylase), JMJD3 (H3K27 demethylase), Kdm4a / b / c / d (H3K36 and H3K9 demethylases), these catalytic domains (CD), these catalytic domains (CD scFV ) or chromatin-modifying polypeptides selected from the group consisting of these catalytic domains (CD) fused to antigen-binding fragment (Fab) domains. Also included are effectors that exert an effect when combined (combination effectors), which are usually co-expressed.

[0084] According to the present invention, the nucleic acid is selected from DNA, RNA, PNA, or combinations thereof.

[0085] In one preferred embodiment, the set of gene constructs is present in the form of at least one vector or plasmid (e.g., an expression vector) comprising the set of nucleic acids according to the present invention. Preferably, the effector domain, CD and / or CD scFVEach of the nucleic acids encoding it includes an inducible promoter, such as a tet-responsive promoter, for example, a dox-responsive promoter. Thereby, the expression of the effector can be finely adjusted, and more preferable functional control becomes possible.

[0086] An important element of epigenetic editing as a biomedical strategy in humans is to deliver the complex and / or its components to the relevant cells. The issues to be considered are safety, efficiency, and target specificity. Several vehicles have been identified for in vivo delivery of CRISPR / Cas9. Viral delivery is the most common platform for (epi)genome therapeutic delivery. Lentiviral systems are widely used but have problems associated with integration into the host genome. Instead, adeno-associated virus (AAV) constructs that do not integrate into the genome are used. However, since AAV has limited cargo capacity, it is necessary to use small CRISPR / Cas9 systems such as staphylococcusaureus (sa)Cas9. Furthermore, nanoparticles can also be used to efficiently and site-specifically deliver CRISPR / Cas9 cargo in the body. They have high loading capacity, excellent stability, and the potential to timely control the release of the cargo. Particles conjugated to nanomaterials are preferred to improve safety, cellular uptake, and specificity for a designated cell type or tissue. Additionally, extracellular vesicles may be used as a cargo system for targeted delivery of epigenome editing molecules. Each example is known to those skilled in the art and is described in the literature. Therefore, most preferably, it is a set of gene constructs according to the present invention, wherein the constructs are viral constructs derived from, for example, AAV, lentivirus, or retrovirus.

[0087] The present invention further includes a method for introducing nucleic acids and / or gene constructs into cells or tissues, which method comprises appropriately transforming or transfecting the cells or tissues with the nucleic acids and / or gene constructs. These are methods known in the art, for example, infection or electroporation. These methods can be carried out in vitro or in vivo.

[0088] Yet another aspect of the present invention relates to a recombinant cell or tissue comprising a set of nucleic acids according to the present invention and / or a set of gene constructs according to the present invention. Preferably, the recombinant cell or tissue is produced as described herein. Depending on the purpose, the cell or tissue can be a prokaryote or a eukaryote such as a bacterial cell or a mammalian cell (e.g., a human cell). Further examples include cells used to produce or propagate the complex according to the present invention, or cells related to a disease phenotype (see below), and thus cells epigenetically treated using the complex according to the present invention.

[0089] Yet another aspect of the present invention relates to a method for producing a complex according to the present invention, which method comprises expressing a set of nucleic acids according to the present invention and / or a set of gene constructs of the present invention in a recombinant cell of the present invention, and optionally comprises the step of inducing expression using an antibiotic such as tetracycline, doxycycline.

[0090] Yet another aspect of the present invention relates to a method of epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, particularly a method of specifically epigenetically modifying chromatin, which comprises introducing a complex according to the present invention and one or more guide RNAs into a cell, tissue, cell nucleus, and / or sample, thereby specifically epigenetically modifying chromatin in the cell, tissue, cell nucleus, and / or sample. This method can be carried out in vitro or in vivo. Strategies for delivering the complex according to the present invention, as well as one or more guide RNAs and delivery constructs, are described above. Preferably, it is a method of epigenetically modifying cells associated with a disease phenotype (see below) and epigenetically treating using the complex according to the present invention and one or more guide RNAs.

[0091] Preferably, it is a method according to the present invention, 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 polycomb epigenetic editing of histones.

[0092] More preferably, it is a method according to the present invention, wherein the epigenetic modification includes transient induction of the expression of the complex according to the present invention and optionally one or more guide RNAs, including, for example, the use of antibiotics such as tetracycline and doxycycline. For this purpose, preferably, the effector domain, CD and / or CD scFVEach of the nucleic acids encoding it contains an inducible promoter, such as a tet-responsive promoter, for example, a dox-responsive promoter. Thereby, the expression of the complex or a part thereof, particularly the effector, can be temporarily induced, and more preferable functional control becomes possible.

[0093] Preferably, in the method according to the present invention, the complex comprises at least two or more, at least three or more, at least four or more, or at least five or more different effector domains, for example, Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV and preferably does not contain a KRAB and / or VPR effector domain.

[0094] Yet another aspect of the present invention relates to a method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, which comprises introducing the complex according to the present invention and one or more guide RNAs into the cell, tissue, cell nucleus, and / or sample, thereby specifically epigenetically modifying the chromatin in the cell, tissue, cell nucleus, and / or sample.

[0095] As described above, the main advantage of the complex according to the present invention is that the chromatin modification activity can be specifically "regulated" based on, for example, a specific selection and combination of effector domains used, such as specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or specific chromatin demethylation / deacetylation activity, or combinations thereof. Thus, chromatin modification may include histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, histone multiple epigenetic editing, H3K9me2 / 3 + DNA methylation, H3K4me3 + H3K36me3, H3K4me3 + H3K79me2, H3K36me3 + H3K79me2, H3K9me2 / 3 + H4K20me3, histone bivalent epigenetic editing, and / or histone polycomb epigenetic editing.

[0096] Preferably, in the method according to the present invention, the epigenetic modification or regulation includes a transient induction of the expression of the complex according to the present invention and one or more guide RNAs.

[0097] Thus, preferably, in the method according to the present invention, the complex of the present invention comprises at least two or more, at least three or more, at least four or more, or at least five or more different effector domains, for example, Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV and preferably does not include the KRAB and / or VPR effector domains.

[0098] Yet another aspect of the present invention relates to a method of regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample comprising chromatin, said method comprising introducing into the cell, tissue, cell nucleus, and / or sample a complex according to the present invention and one or more guide RNA sequences specific for the at least one target DNA sequence, thereby specifically epigenetically regulating the expression of the at least one target DNA sequence in the cell, tissue, cell nucleus, and / or sample.

[0099] Generally, based on chromatin modification, the expression of any target DNA sequence can be regulated, i.e., increased, decreased, or stabilized. Expression can be regulated directly, for example, by regulating transcription via a promoter or its accessibility, or indirectly by regulating the regulatory environment or "layer", such as splicing, enhancers, or other regulatory elements. In contrast to complexes in the art, the present invention can also provide this indirect regulation.

[0100] Preferably, in the method according to the present invention, the at least one target DNA sequence comprises a nucleic acid sequence specific for an epigenetically modified chromatin-related condition and / or disease state, such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological 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 use disorder, and imprinting disorders such as Prader-Willi syndrome.

[0101] More preferably, in the method of the present invention, 2, 3, 4, 5, 6, 7, 8, or 9 target DNA sequences are regulated intracellularly.

[0102] Generally, any cell containing the target DNA sequence can be used. Preferably, in the method of the present invention, the cell is a stem cell, a tissue cell, 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 cell or a rodent cell.

[0103] In another aspect of the present invention, a method for detecting a biological effect by specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising performing the method according to the present invention and detecting at least one biological effect in a cell, tissue, cell nucleus, and / or sample containing chromatin, wherein the biological effect is selected from the group consisting of a change in gene expression, a change in protein amount, a change in cis-gene effect, a change in nucleic acid splicing, a change in the nuclear arrangement of a locus, a change in the formation and disruption of TADs, a change in termination sites, a change in promoter activation, a change in promoter repression, a change in genetic epigenetic interaction, a change in the functional relationship between gene mutation and the epigenetic state of chromatin, a change in genetic methylation and imprinting, and a specific epigenetic change associated with a disease or cell phenotype.

[0104] In this aspect, the complex according to the present invention can preferably be used to test or verify hits from EWAS (epigenome wide association studies) indicating that specific epigenetic changes are associated with a specific disease or phenotype. For example, see Bhat B, and Jones GT (in: 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-l-0716-2140-0_2. PMID: 35103960) and the references cited therein.

[0105] Preferably, the method according to the present invention is performed using a complex that enables tracking, preferably in real time, of the effects in cells, tissues, cell nuclei, and / or samples containing chromatin.

[0106] More preferably, the method according to the present invention further includes, preferably as a control of the gRNA targeting efficiency, detecting the effect of an effector domain that is a transcriptional activator such as VPR or VPR scFV or a transcriptional repressor such as KRAB or KRAB scFV and / or detecting a fluorescent protein such as GFP or sfGFP protein and / or tagBFP. The method according to the present invention may further include epigenetic target perturbation sequencing to detect at least one biological effect in cells, tissues, cell nuclei, and / or samples containing chromatin.

[0107] Preferably, the method according to the invention is at least partially automated and / or can be performed in a high-throughput format, i.e., can be automated partially or completely, for example, can be performed completely or partially by a robot. The method according to the invention can involve the use of a computer and respective databases for the execution and / or analysis of the results obtained.

[0108] Generally, any biological effects and diseases or phenotypes associated with epigenetically modified chromatin can potentially be detected, such as 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, shortening of biological 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 use disorder, epigenetic diseases including imprinting disorders such as Prader-Willi syndrome.

[0109] Another aspect of the invention relates to cells having specifically epigenetically modified chromatin generated by performing the method of the invention, and optionally isolated said cells, preferably the cells are stem cells, neurons, cancer cells, cells in tissues, post-mitotic cells, or fibroblasts, and / or the cells are mammalian cells, preferably animal cells such as human cells or rodent cells.

[0110] Another aspect of the present invention relates to a method for identifying a pharmaceutically active compound involved in regulating epigenetic modification of chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, a drug for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or a biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising performing the method according to the present invention in the presence and absence of a test agent, wherein the test agent is a cell, tissue, cell nucleus, and / or sample containing chromatin when the regulation and / or biological effect in the presence of the agent is different from the regulation and / or biological effect in the absence or control of the agent. A drug that specifically epigenetically modifies chromatin, a drug that regulates the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or a biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin is identified as

[0111] The method according to the present invention aims to identify a pharmaceutically active compound involved in regulating epigenetic modification of chromatin in a cell (preferably a human cell), tissue, cell nucleus, and / or sample.

[0112] Conveniently, the format of the method of the present invention is very flexible. In the method of the present invention, a suitable combination of a constituent, namely a complex according to the present invention or a functional fragment thereof, and at least one pharmaceutically active candidate compound, i.e., a substance to be screened / identified for activity to regulate a function, is required. This combination can be provided as a cell system (i.e., one that functions intracellularly, such as mammalian cell culture), the constituent can be provided by partial or complete recombination, or this system can be in vitro, such as an in vitro translation system, which can be easily adjusted for the purposes of the present invention as required. Examples are described in the literature and are known to those skilled in the art.

[0113] Preferably, in the method according to the present invention, the method using at least one candidate compound is carried out in vitro, in cell culture, or in vivo, preferably in a non-human mammal, or includes in silico molecular modeling, for example, using a suitable computer program. More preferably, in the method according to the present invention, a pre-selection step is further included, which includes molecular modeling of the binding of at least one candidate compound to a complex or an epigenetic modification site (such as histone) or a target nucleic acid or a functional fragment thereof, for example, using a computer program such as SwissDock. That is, the present invention includes a pre-selection based on in silico modeled binding properties, for example, based on all or part of the structural elements isolated or in relation to the complex, before including the compound in a more complex complete in vivo and / or in vitro assay.

[0114] The method according to the present invention can be carried out using automation (robotics) and can be executed in a high-throughput format.

[0115] Candidate compounds and / or candidate compounds identified or screened in the context of the present invention can be any chemical substance or mixture thereof. Preferably, said compounds are selected from substances selected from chemical substances, peptide libraries, libraries of small organic molecules, combinatorial libraries, cell extracts, particularly plant cell extracts, "small molecule drugs" (i.e., those with a molecular weight of 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.

[0116] The method according to the present invention preferably further comprises testing the dose-dependent response of the drug. Each method is known to those skilled in the art.

[0117] The selected or screened compound can then be modified. Said modification can be carried out in an additional preferred step of the method of the present invention described herein, for example, in the presence and absence of said selected compound, for example, after analyzing the binding to a complex and / or the regulation of epigenetic modification, the compound is further chemically modified as described, for example, in the following examples, and its effect on binding and / or regulation is analyzed again. Said series of "modifications" can be carried out one or more times in all methods in order to optimize the effect of the compound, for example, to improve the specificity for the binding element (e.g., complex or histone), and / or to improve the specificity for the epigenetic modification affected. This strategy is also called "directed evolution" because it involves a number of steps including modification and selection, and the binding compound is selected in an "evolutionary" process that optimizes its ability with respect to specific properties (e.g., its binding activity, ability to activate, inhibit, or modulate activity (particularly epigenetic modification activity)).

[0118] For modifications, prior to performing additional tests, simulations can also be carried out in silico, which are done to confirm or verify the effects of modified selections or screened compounds in the first screening round. Each software program is known in the art and readily available to those skilled in the art.

[0119] Modifications can be further carried out by various methods known in the art, including the introduction of a new side chain, or the exchange of a functional group, such as the introduction of a halogen, especially F, Cl or Br, a lower alkyl group, preferably a lower alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or isopentyl group, preferably a lower alkenyl group having 2 to 5 carbon atoms, preferably a lower alkynyl group having 2 to 5 carbon atoms, or the introduction of a group selected from the group consisting of, for example, NH2, NO2, OH, SH, NH, CN, aryl, heteroaryl, COH or COOH groups, but not limited thereto.

[0120] Thus, preferably, the method according to the invention comprises c) chemically modifying the identified candidate compound (see above), and repeating the method according to the invention to identify a second generation of candidate compounds with improved binding properties and / or regulatory properties, and optionally d) repeating step c) at least once, twice, three or more times.

[0121] The term "functional fragment" means the region and / or part of a peptide or polypeptide that is ultimately involved in the regulation of epigenetic modification. These regions are generally involved in the binding of a compound (modulator).

[0122] In the context of the present invention, unless otherwise specified, the term "about" shall mean + / - 10% of a given value.

[0123] The present invention provides nucleic acids and / or gene constructs and cells or tissues according to the present invention, and further methods for producing pharmaceutically acceptable preparations, including mixing or formulating the nucleic acids and / or gene constructs according to the present invention with at least one pharmaceutically acceptable diluent or carrier. Each method is known in the art. Pharmaceutically acceptable carriers or excipients include diluents (fillers, extenders, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, croscarmellose sodium), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), glidants (such as colloidal SiO2), solvents / co-solvents (such as aqueous media, 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 polysorbate, sorbitan esters), thickeners (such as methyl cellulose, or hydroxyethyl cellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame), flavors (such as peppermint, 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, 15th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5th Ed., Govi-Verlag Frankfurt (1997). Those skilled in the art know the appropriate formulation for each compound, for example for topical use, and will be able to easily select the appropriate pharmaceutically acceptable carrier or excipient depending on, for example, the formulation and route of administration of the pharmaceutical composition.

[0124] Another aspect of the present invention relates to a manufactured pharmaceutically acceptable preparation comprising a cell, nucleic acid and / or gene construct according to the present invention, together with a pharmaceutically acceptable adjuvant or carrier.

[0125] It is understood that the compounds of the present invention and / or pharmaceutical compositions comprising the compounds of the present invention are used for administration to human patients. The term "administration" means the administration of a single therapeutic agent or the co-administration with other therapeutic agents. Thus, the pharmaceutical compositions of the present invention are contemplated for use in combination therapy approaches, i.e., co-administration with other agents or drugs and / or other therapeutic agents that may be beneficial in the context of the methods of the present invention. However, other agents or drugs and / or other therapeutic agents can be administered separately, if desired, as long as they act in combination with (i.e., directly and / or indirectly, preferably synergistically) the selected and / or screened compounds of the present invention.

[0126] Another important aspect of the present invention relates to its use in medicaments (see, for example, Policarpi C, Dabin J, Hackett JA. Epigenetic editing: Dissecting chromatin function in context. Bioessays. 2021 May;43(5):e2000316. doi: 10.1002 / bies.202000316. Epub 2021 Mar 16. PMID: 33724509). Epigenetic editing holds great promise not only for elucidating biological mechanisms but also in the development of therapeutic approaches. Epigenetic editing is an accurate and non-invasive strategy for combating specific disease states, is complementary due to its various applications, and in some cases is preferred over gene therapy and wild-type Cas9 gene approaches.

[0127] In principle, epigenetic editing has a better safety profile than gene editing because it does not alter the host DNA sequence and is essentially reversible. Furthermore, since it relies on endogenous cis - elements to control the expression of target genes, it represents a more physiological approach than gene (cDNA) delivery. For example, it facilitates the switching - on of endogenous genes while retaining the co - transcriptional and post - transcriptional signals for appropriate expression control.

[0128] Moreover, since multiple genes can be targeted simultaneously, multiplex epigenetic editing can be used to (i) co - upregulate the expression of multiple genes simultaneously or (ii) utilize the orthogonality between CRISPR - Cas9 systems to perform in parallel actions such as switching some genes on and others off. CRISPR - based epigenetic therapies hold great promise for a subset of human diseases.

[0129] Several disease classes are attractive targets for the development of epigenetic therapies (Figure 5). For example, hundreds of human diseases are associated with haploinsufficiency, and selectively enhancing gene expression may be able to compensate for the defective gene product. This can be achieved by targeting transcriptional activation modules such as VP64 and its derivatives, or dCas9 fused to histone - modifying enzymes such as p300, PRDM9, DOT1L, as described herein. A significant reduction in symptoms was shown in a mouse model of severe epileptic encephalopathy called Dravet syndrome.

[0130] Other medical conditions that could benefit from the in vivo development of CRISPR activation systems are X-linked disorders, which result from the expression of a defective allele from the active X chromosome and the presence of a functional copy on the inactive X chromosome. Rett syndrome and Cdkl5 disorder, caused by mutations in the MeCP2 gene, are good examples of such diseases. Using programmable epigenetic editing to upregulate the expression of a functional gene copy from the inactive X chromosome in brain cells complements the mutant allele. Epigenetic editing may be applicable to several neurological diseases. For example, Fragile X syndrome, the most common intellectual disability found in males, is mediated by abnormal DNA methylation that epigenetically silences the FMR1 gene in neurons. Interestingly, using the dCas9-Tet1 system to specifically demethylate the FMR1 promoter reversed the heterochromatic state and restored FMR1 expression in iPSC-derived neurons. This strategy may also be applicable to imprinting disorders resulting from abnormal DNA methylation patterns. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are good examples that have successfully utilized a similar approach, as some forms of PWS are caused by abnormal methylation of the paternal allele, while 4% of AS cases are caused by a lack of methylation of the maternal allele. Therefore, allele-specific therapies targeting dCas9-Tet1 for the former and dCas9-Dnmt3 for the latter are promising.

[0131] In addition to targeted activation, programmed gene silencing can address disease phenotypes arising from dominant genes in particular. For example, dCas9-KRAB has been used to target the PCSK9 gene in a mouse model of hypercholesterolemia, resulting in reduced cholesterol levels in treated mice. This strategy may benefit many inflammatory and pain-related diseases by (i) targeting cytokines involved in inflammatory pathways for the treatment of degenerative disc disease or (ii) targeting pain receptors in the skin. Indeed, the skin is an accessible and attractive organ for CRISPR-mediated gene silencing as it is associated with numerous single-gene and autosomal dominant diseases such as Olmsted syndrome and familial primary localised cutaneous amyloidosis.

[0132] Importantly, allele-specific epigenetic silencing may be exploitable in dominant gene disorders to ensure that functional gene copies remain active. Such a strategy emerged to silence mutant HTT alleles in different clinical populations presenting with neurological Huntington's disease. Similarly, recent studies have shown that targeting mutant DMD alleles in a mouse model of Duchenne muscular dystrophy with the CRISPR / Cas9 system significantly improved muscle contractility in the mice.

[0133] Finally, complex diseases such as cancer may benefit from multiplex epigenetic editing not only by the strategies described above, but also by activating tumour suppressor genes and simultaneously inhibiting the expression of oncogenes. Indeed, simultaneous activation and suppression of different genes in the same cell has been achieved in vitro by conjugating dCas9 to chemical and photoinducible effector domains and designing scaffold gRNA molecules that can recruit transcriptional regulators. Furthermore, proof-of-concept for simultaneous gene activation has been demonstrated in vivo in mice.

[0134] Histone Methylation and Aging Proteins that modify histone methylation have also been shown to play roles in controlling the lifespan of organisms and the aging of tissues. When the appropriate balance of stable and dynamic methyl marks in adult stem cells is lost, individual tissue functions may decline with aging 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 use disorder (see Basavarajappa BS, Subbanna S. Histone Methylation Regulation in Neurodegenerative Disorders. Int J Mol Sci. 2021 Apr 28;22(9):4654. doi:10.3390 / ijms22094654. PMID: 33925016; PMCID: PMC8125694).

[0135] The object of the present invention is to provide a method for preventing or treating epigenetic diseases related to chromatin modified epigenetically, such as genetic diseases, proliferative diseases such as cancer, immune cells producing autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular diseases, shortening of biological 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 use disorder, and imprinting disorders such as Prader-Willi syndrome, which is solved by administering to a subject in need of prevention or treatment an effective amount of at least one of the complex according to the present invention, one or more appropriate guide RNA sequences, a set of nucleic acids according to the present invention, a set of gene constructs such as an expression vector according to the present invention, a cell according to the present invention, and / or a drug identified according to the present invention.

[0136] In this aspect, the nucleic acid encoding the complex of the present invention or 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 performed by any suitable method, for example, as a pharmaceutical composition comprising an isolated component of at least one complex (polypeptide and / or nucleic acid) according to the present invention together with a suitable stabilizer or carrier. In another embodiment, it is to provide the complex encoded on at least one nucleic acid vector to a patient, cell, tissue, sample, or nucleus. These pharmaceutical compositions and their use constitute preferred embodiments of the present invention. This aspect also includes the step of monitoring the treatment.

[0137] This aspect combines the diagnostic approach of the present invention with a separate "usual" medical treatment and also includes use for monitoring the treatment. This method includes subjecting the cell, tissue, or organism to an appropriate treatment, particularly a specific medical treatment, performing the method according to the present invention as described above, and modifying the treatment of the disease or condition based on chromatin modifications and disease modifications detected when compared, for example, to healthy controls (e.g., controls based on a group of healthy samples or samples of the disease).

[0138] "Treatment" or "treating" means any treatment for a disease or disorder in a mammal, including preventing or protecting against the disease or disorder, i.e., preventing the occurrence of clinical symptoms of the disease, inhibiting the disease, i.e., preventing or suppressing the occurrence of clinical symptoms, and / or alleviating the disease, i.e., causing regression of clinical symptoms. "Improvement" means preventing, alleviating, or mitigating a condition, or improving the condition of a subject, and improvement of stress is canceling out the negative aspects of stress. Improvement includes, but does not require, complete recovery or complete prevention of stress.

[0139] The object of the present invention is for use in the prevention and / or treatment of diseases in a subject in need of prevention or treatment of a disease, or for example, hereditary diseases, proliferative diseases such as cancer, immune cells that produce autoantibodies, bacterial or viral infections, protozoal infections, fragile X syndrome, muscular dystrophy, kidney damage, cardiovascular diseases, shortening of biological 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 use disorder, epigenetic diseases including imprinting disorders such as Prader-Willi syndrome, etc. It is solved by at least one of the complex according to the present invention, and one or more appropriate guide RNA sequences, a set of nucleic acids according to the present invention, a set of gene constructs such as an expression vector according to the present invention, a cell according to the present invention, and / or a drug identified according to the present invention.

[0140] Another aspect of the present invention relates to the use of the complex according to the present invention, a set of nucleic acids according to the present invention, a set of gene constructs such as an expression vector according to the present invention, and / or a cell according to the present invention for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin according to the present invention, for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin according to the present invention, for detecting the biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample according to the present invention, and / or for identifying a drug according to the present invention. All of these aspects are further described above.

[0141] In the context of the present invention, the subject can be a mammal, preferably a mouse or a human.

[0142] In another preferred embodiment of the present invention, the object of the present invention is solved by providing a kit containing materials for carrying out the method of the present invention as a detection system, for example as part of a diagnostic kit. This kit can also be used in a therapeutic kit containing isolated components of at least one complex according to the present invention (polypeptide and / or nucleic acid encoding it, and / or virus particles containing them) together with a suitable stabilizer or carrier, or in a pharmaceutical composition. In another embodiment, it contains additional materials for supplying the complex encoded on at least one nucleic acid vector to a subject, cell, tissue, sample or nucleus.

[0143] Preferably, the kit is provided in one or more containers and contains suitable enzymes, buffers, excipients, and instructions for use. Its components can be (at least partially) immobilized on a substrate, and the substrate can be exposed to the cells, tissues, and / or samples.

[0144] In the context of the present invention, unless explicitly stated otherwise, the term "about" shall mean a value indicated in the range of + / - 10%. The method according to the present invention can be carried out in vivo or in vitro, for example in a living being, cell, tissue, and / or part thereof such as a nucleus, or in an in vitro assay such as a diagnostic assay.

[0145] In this specification, the inventors provide an epigenome editing platform for systematically programming chromatin modifications in combination with specific chromatin modifications across hundreds of contexts in living cells. This resource is used to capture collective functional responses from diverse lineages at the allelic, single-cell resolution. Through perturbation at an unprecedented scale, the control logic by which different chromatin modifications interact with genomic features, sequence variants, and cellular identity to form quantitative gene expression patterns is revealed. The present invention further identifies trans-acting and cis-structure mechanisms that implement the function of chromatin marks by integrating datasets and conducting gene screening.

[0146] Precise epigenetic editing strategies are used to define the transcriptional function and memory of heterochromatin epialleles at endogenous loci. A method of multiplexedly recruiting multiple "effector" modules using dCas9 GCN4 facilitates the programming of major (over 10 kb) heterochromatin domains sufficient to promote potent epigenetic silencing. These de novo domains are composed of H3K9me3, H4K20me3, and DNA methylation, and the accompanying loss of H3K4me3, and their modification levels are equivalent to or higher than those of endogenous heterochromatin regions and are thought to self-propagate by "read-write" enhancement (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 discovered that naive pluripotent cells act to fundamentally prevent the inheritance of heterochromatin domains that occur outside the normal genomic context, even when providing selective advantages such as silencing of p53.

[0147] As one preferred embodiment, the inventors developed a modular CRISPR-based toolkit that can accurately and inductively program nine different epigenetic modifications (in this case) to an endogenous target locus (see Figures 1B - H). Among these, the H3K27ac and H3K4me3 marks are usually associated with active transcription, while DNA methylation, H2AK119ub, and trimethylation of lysine 9 or 27 of histone H3 (H3K9me3 and H3K27me3 respectively) are associated with repressed chromatin regions (Berger SL et al. 2007). However, whether the observed correlations indicate causal relationships remains unclear, especially considering that removing some of these marks from the genome of mouse embryonic stem cells (mESCs) results in only minor transcriptional changes (Tsumura A et al. 2006; Chamberlain SJ et al. 2008; Sze CC et al. 2017; Zhang T et al. 2020). Similarly, the H3K36me3 mark has been found to peak in active genes in vivo, but has been shown to function as a transcriptional repressor in both yeast and mammals (Carrozza MJ et al. 2005; Neri F et al. 2017). Genetic approaches (including genetic manipulation of the DNA sequence underlying the mark, gene deletion of upstream chromatin-modifying enzymes, or mutation of specific histone residues) are widely used to investigate the functional relevance of epigenetic marks (Stricker SH et al 2017; Grosswendt S et al. 2020). These approaches have generally been successful in causing widespread changes in gene expression, but have not clearly established the causal contribution of the chromatin marks themselves to the observed phenotypes because it is impossible to distinguish direct effects from indirect effects.

[0148] Therefore, the ability to site-specifically deposit marks such as H3K27me3, H3K4me3, H2AK119ub, and H3K36me3 represents a powerful gain-of-function perturbation strategy for explicitly evaluating their causal effects. Our system or toolkit utilizes dCas9 linked to the tail-array of five GCN4 motifs (dCas9 GCN4 ), with each motif separated by a linker designed at optimal intervals to accommodate proteins bulky enough not to sterically interfere with catalytic activity. This embodiment of dCas9 GCN4 links up to five "effector" proteins to a specific locus via a GCN4-specific scFV domain (Figure 1A). We designed and tested a comprehensive suite of effectors (collectively referred to as CD scFV ) that contain only the catalytic domains of chromatin-modifying enzymes, for example, Setd2-CD scFV for H3K36me3 and Prdm9-CD scFV for H3K4me3.

[0149] The above considerations are also mutatis mutandis applicable to other chromatin-modifying proteins or polypeptides used as effectors in the present invention, and the chromatin-modifying protein or polypeptide is 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 (GlcNAC), 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), these catalytic domains (CD), these catalytic domains (CD scFV ) or may be selected from the group consisting of these catalytic domains (CD) fused to an effector domain binding motif-specific scFV domain or an antigen-binding fragment (Fab) domain.

[0150] Epigenetic modifications may include histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, histone multiplex epigenetic editing, H3K9me2 / 3 + DNA methylation, H3K4me3 + H3K36me3, H3K4me3 + H3K79me2, H3K36me3 + H3K79me2, H3K9me2 / 3 + H4K20me3, histone bivalent epigenetic editing, and / or histone polycomb epigenetic editing.

[0151] The system of the present invention incorporates a plurality of other advancements that together result in an epigenetic editing platform technology with excellent functions enabling discoveries. Its attributes include the following:

[0152] Very active editing. In this case, when five copies of a specific CD scFV are recruited to the target locus, on-target programming of chromatin modification is significantly amplified both in terms of amplitude and genomic width. This ensures de novo histone modification deposition comparable to strong endogenous peaks, facilitating both negative and positive functional conclusions.

[0153] Catalytic domain specificity. By separating only the catalytic core, the confounding effects targeting full-length chromatin modification proteins can be eliminated. Since full-length proteins have major non-catalytic functions and / or may recruit complexes of other proteins, preferentially using the catalytic domain allows the evaluation of the function of the target chromatin mark itself.

[0154] Combinatorial epigenetic editing. Since the system of the present invention is modular, for example, five different CDs scFV can be recruited simultaneously. This enables multiplex epigenetic editing to establish de novo domains or combinations of different chromatin modifications (e.g., bivalent or polycomb).

[0155] Minimized off-targeting. The CDs scFV used herein generally lack endogenous DNA-binding domains and are not directly fused to dCas9, thus minimizing off-target activity.

[0156] Temporal resolution. Here, each CD scFVIt is dynamically induced via a DOX-responsive promoter, also has a protein destabilization (d2) domain, and promotes rapid degradation upon DOX removal. This enables the investigation of temporal responses and epigenetic memory (chromatin persistence).

[0157] Dynamic tracking. In this embodiment, since all effectors are bound to superfolder GFP (sfGFP) and all gRNAs are bound to tagBFP, the system can be tracked in real time, cells can be purified, and dose-dependent responses (e.g., comparison between GFP low cells and GFP high cells) can be tested.

[0158] The inventors created a comprehensive set of controls. First, the inventors designed point mutants (mut-CD scFV ) for every CD scFV , which specifically inhibited catalytic activity and enabled direct comparison with active CD scFV . Next, as additional negative controls, the inventors employed GFP scFV alone recruitment, non-induced (-DOX) cells, and scrambled gRNA. Finally, as positive controls for gRNA targeting efficiency, the inventors utilized the recruitment of well-characterized transcriptional activators (VPR scFV ) and transcriptional repressors (KRAB scFV ).

[0159] Combining these features enables a carefully controlled use of introducing specific chromatin modifications at specific loci while excluding confounding effects.

[0160] The present invention circumvents the central limitations of existing epigenome perturbation approaches by separating the functional relationship between the genome and epigenome while eliminating pleiotropy and redundancy. A further outcome is a deeper understanding of how specific chromatin states instruct or reflect gene regulation. This serves to inform the design of strategies towards precision medicine and provides guidance for attributing functional significance to epigenomic profiles in health and disease.

[0161] Whether defined chromatin modifications at specific loci directly impact gene expression in specific cell types remains essentially unknown at the quantitative level. The studies conducted herein provide a systematic strategy to answer this question and will yield fundamental insights into genome biology that address a major challenge in the field. The unprecedented scale of precise perturbation also provides a resource for predicting the functional activity of chromatin marks within defined contexts. Such modeling underpins several important advancements. First, it allows for greater confidence in attributing functional relevance to the changes observed across the vast amount of developmental and disease-related epigenomic profiles generated by the community. In other words, it contributes to untangling cause and effect.

[0162] Second, the knowledge obtained herein will generate rules that will form the basis for designing and optimizing future epigenetic editing strategies for precision medicine applications. For example, when deriving design principles for inducing desirable changes in the magnitude, penetrance, or persistence of target gene transcription to modulate disease. Proof-of-principle has been demonstrated in fragile X, muscular dystrophy, and kidney injury, and additional knowledge is critically important to maximize treatment potential. Third, the output provides a means to analyze the functional relationship between genetic mutations and chromatin states. This provides a basis for mechanistically understanding how eQTLs associated with human genetic traits are regulated and exert their effects across specific tissues, diseases, and evolution as a whole. Beyond our framework of expected impacts, high-content readouts and unbiased screening provide ample opportunity for new discoveries of interactions and mechanisms, and thus unexpected research trajectories. There is also great scope to expand the experimental strategies herein towards understanding the mechanisms of genetically diverse human cells, designing desirable cell characteristics, and in vivo applications.

[0163] As described herein, the present invention particularly relates to the following items.

[0164] Item 1. A complex comprising: i) a catalytically inactive site-specific nuclease; and ii) the sequences of 2 to 10, preferably 3 to 7, effector domains each having a specific chromatin modification activity, such as a specific DNA methylation activity, histone methylation activity, 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 a sufficient distance is ensured between the domain and the nuclease so that the specific chromatin modification activity and the binding of the site-specific nuclease are not substantially hindered. Item 2. The catalytically inactive site-specific nuclease is the complex according to item 1, selected from the group consisting of catalytically inactive (d)Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, SceI, and dCas9 / fCas9 fusions derived from Streptococcus pyogenes. Item 3. The complex comprises a fusion protein of a nuclease bound to a protein sequence comprising 3 to 7 effector domain binding motifs separated by a linker sequence, e.g., Streptococcus pyogenes dCas9 GCN4 (3-7) respectively, and the complex optionally further comprises a number of effector domains each bound to a binding motif, and the complex optionally further comprises at least one suitable guide RNA (gRNA), the complex according to item 1 or 2. Item 4. The complex according to any one of items 1 to 3, wherein the protein complex comprises 4 to 6 effector domains, preferably 5 effector domains. Item 5. The effector domain binding motif consists of an epitope comprising a peptide sequence of 17 to 29 amino acids, preferably 17 to 21 amino acids, e.g., the GCN4 epitope motif sequence, and has little or no structural folding under physiological conditions, the complex according to any one of items 1 to 4. Item 6. The length of the linker sequence is 25 to 19 amino acids, preferably 22 amino acids, and more preferably, the linker sequence comprises glycine (G) and serine (S) amino acids, the complex according to any one of items 1 to 5. Item 7. The effector domain is one that is bound via an effector domain-binding motif-specific scFV domain, particularly a GCN4-specific scFV domain, and the scFV is optionally linked to the effector domain via an effector linker group, such as a group containing a fluorescent protein such as a GFP or sfGFP protein, and the effector domain may be further fused to a protein destabilizing domain such as d2, the complex according to any one of Items 1 to 6. Item 8. The effector domain is DotL (H3K79me3), p300 (H3K27ac), Prdm9 (H3K4me3), Setd2 (H3K36me3), Ring1b (H2AK119ub), Ezh2 (H3K27me3), G9a (H3K9me2), Kmt5C (H4K20me3), Dnmt3a3L (DNAme), OGT (GlcNAC), these catalytic domains (CD), these catalytic domains (CD scFV ) fused to an effector domain-binding motif-specific scFV domain, the complex according to any one of Items 1 to 7, comprising a chromatin-modifying polypeptide selected from the group consisting of. Item 9. The chromatin-modifying activity is histone methylation activity, for example, histone methylation activity that contributes to stable or reversible gene expression control, the complex according to any one of Items 1 to 8. Item 10. The complex comprises at least two or more, at least three or more, at least four or more, or at least five or more different effector domains, for example, Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV in combination, preferably not containing the KRAB and / or VPR effector domains, the complex according to any one of Items 1 to 9. Item 11. A set of nucleic acids encoding at least one of the protein and / or guide RNA (gRNA) and / or tagBFP of the complex according to any one of Items 1 to 10. Item 12. Each nucleic acid encoding preferably an effector domain, CD and / or CD scFV is included in a set of gene constructs such as expression vectors, which includes the set of nucleic acids according to item 11 and contains an inducible promoter such as a tet-responsive promoter, for example a dox-responsive promoter. Item 13. The set of gene constructs according to item 12, wherein the gene constructs are viral constructs such as viral constructs derived from, for example, AAV, lentivirus or retrovirus. Item 14. A recombinant cell comprising the set of nucleic acids according to item 11 and / or the set of gene constructs according to item 12 or 13. Item 15. A method for producing the complex according to any one of items 1 to 10, which includes expressing the set of nucleic acids according to item 11 and / or the set of gene constructs according to item 12 or 13 in the recombinant cell according to item 14, and optionally inducing the expression using, for example, tetracycline. Item 16. A method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, which includes introducing the complex according to any one of items 1 to 10 and one or more guide RNAs into the cell, tissue, cell nucleus, and / or sample, thereby specifically epigenetically modifying the chromatin in the cell, tissue, cell nucleus, and / or sample. Item 17. The method according to item 16, wherein the epigenetic modification includes histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, histone multiplex epigenetic editing, H3K9me2 / 3 + DNA methylation, H3K4me3 + H3K36me3, H3K4me3 + H3K79me2, H3K36me3 + H3K79me2, H3K9me2 / 3 + H4K20me3, histone bivalent epigenetic editing, and / or histone polycomb epigenetic editing. Item 18. An epigenetic modification, the method according to item 16 or 17, comprising transient induction of the expression of the complex according to any one of items 1 to 10 and one or more guide RNAs. Item 19. The method according to any one of items 16 to 18, wherein the complex comprises a combination of at least two, at least three, at least four, or at least five different effector domains, such as Setd2-CD for H3K36me3 scFV and Prdm9-CD for H3K4me3 scFV and preferably does not contain a KRAB and / or VPR effector domain. Item 20. A method of regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising introducing into the cell, tissue, cell nucleus, and / or sample the complex according to 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 and epigenetically regulating the expression of at least one target DNA sequence in the cell, tissue, cell nucleus, and / or sample. Item 21. The method according to item 20, wherein the at least one target DNA sequence comprises a nucleic acid sequence specific to an epigenetic modification-related condition and / or disease state, such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoal infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological 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 use disorder, and imprinting disorders such as Prader-Willi syndrome. Item 22. The method according to item 20 or 21, wherein the expression of 2, 3, 4, 5, 6, 7, 8, or 9 target DNA sequences is regulated in the cell. Item 23. The method according to any one of items 16 to 22, wherein the cell is a stem cell, neuron, post-mitotic cell, or fibroblast, and / or the cell is an animal cell, such as a mammalian cell, preferably a human cell or a rodent cell. Item 24. A method for detecting a biological effect by specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or by regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, the method comprising performing the method according to any one of items 16 to 23, and detecting at least one biological effect in a cell, tissue, cell nucleus, and / or sample containing chromatin, wherein the biological effect is selected from the group consisting of changes in gene expression, changes in protein amount, changes in cis-gene effects, changes in nucleic acid splicing, changes in the nuclear arrangement of loci, changes in the formation and disruption of TADs, changes in termination sites, changes in promoter activation, changes in promoter suppression, changes in genetic epigenetic interactions, changes in the functional relationship between gene mutations and the epigenetic state of chromatin, changes in genetic methylation and imprinting, and specific epigenetic changes associated with diseases or cell phenotypes. Item 25. The method according to item 24, wherein the method is performed using a complex that enables tracking of the effect in a cell, tissue, cell nucleus, and / or sample containing chromatin, preferably in real time. Item 26. The method preferably includes detecting the effect of an effector domain that is a transcriptional activator such as VPR or VPR scFV as a control for gRNA targeting efficiency, or a transcriptional repressor such as KRAB or KRAB scFV and / or detecting a fluorescent protein such as GFP or sfGFP protein and / or tagBFP. The method according to item 24 or 25. Item 27. The method according to any one of items 24 to 26, further comprising epigenetic target perturbation sequencing to detect at least one biological effect in a cell, tissue, cell nucleus, and / or sample containing chromatin. Item 28. The method according to any one of items 16 to 27, which is at least partially automated and / or performed in a high-throughput format. Item 29. The disease or phenotype is related to epigenetically modified chromatin, such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological lifespan, tissue aging, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), drug abuse including alcohol use disorder, epigenetic diseases including imprinting disorders such as Prader-Willi syndrome, etc. The method according to any one of items 16 to 28. Item 30. A cell having specifically epigenetically modified chromatin, optionally an isolated cell, produced by performing the method according to any one of items 16 to 19, preferably, the cell is a stem cell, neuron, post-mitotic cell, or fibroblast, and / or the cell is an animal cell, such as a mammalian cell, preferably a human cell or a rodent cell. Item 31. A method for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, a method for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or a method for identifying a biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, which method comprises performing the method according to any one of Items 16 to 29 in the presence and absence of a test agent, wherein the test agent is a drug that specifically epigenetically modifies chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin, a drug that regulates the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin, and / or a drug whose biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin is identified when the regulation and / or biological effect in the presence of the drug is different from the regulation and / or biological effect in the absence or control of the drug. Item 32. The method according to Item 30, wherein the test agent is selected from the group consisting of a chemical molecule, a molecule selected from a library of small organic molecules, a molecule selected from a combinatorial library, a cell extract, particularly a plant cell extract, a small molecule drug, a protein, a protein fragment, a molecule selected from a peptide library, and an antibody or a fragment thereof. Item 33. The method according to Item 31 or 32, further comprising testing a dose-dependent response of the drug. Item 34. A method for preventing or treating a disease related to epigenetically modified chromatin in a subject in need of prevention or treatment, such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological 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 use disorder, epigenetic diseases including imprinting abnormalities such as Prader-Willi syndrome, etc., comprising administering to a subject in need of such treatment an effective amount of at least one of the complex according to any one of Items 1 to 10, and one or more appropriate guide RNA sequences, the set of nucleic acids according to Item 11, the set of gene constructs such as the expression vector according to Item 12 or 13, the cell according to Item 30, and / or the agent identified by the method according to any one of Items 31 to 33. Item 35. For use in preventing and / or treating a disease in a subject in need of prevention or treatment of a disease, or for preventing and / or treating a disease related to epigenetically modified chromatin, such as a genetic disease, a proliferative disease such as cancer, immune cells producing autoantibodies, bacterial or viral infection, protozoan infection, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortening of biological 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 use disorder, epigenetic diseases including imprinting abnormalities such as Prader-Willi syndrome, etc., at least one of the complex according to any one of Items 1 to 10, and one or more appropriate guide RNA sequences, the set of nucleic acids according to Item 11, the set of gene constructs such as the expression vector according to Item 12 or 13, the cell according to Item 30, and / or the agent identified by the method according to any one of Items 31 to 33. Item 36. A complex according to any one of Items 1 to 10, a set of nucleic acids according to Item 11, a set of gene constructs such as an expression vector according to Item 12 or 13, and / or the use of a cell according to Item 30, for specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample containing chromatin according to any one of Items 16 to 19 and 29, for regulating the expression of at least one target DNA sequence in a cell, tissue, cell nucleus, and / or sample containing chromatin according to any one of Items 20 to 23 and 28, for detecting the biological effect of specifically epigenetically modifying chromatin in a cell, tissue, cell nucleus, and / or sample according to any one of Items 24 to 27, and / or for identifying a drug by the method according to any one of Items 30 to 32.

Example

[0165] dCas9 GCN4 and all CDs scFV and FL scFV Epigenetic editing tools containing effectors were cloned into the PiggyBac recipient plasmid by homologous arm recombination using In-fusion HD-Cloning (Takara #639650) according to the manufacturer's instructions. Streptococcus pyogenes dCas9 GCN4 was PCR amplified from the PlatTET-gRNA2 plasmid (Morita et al., 2016; Addgene #82559) and cloned under the control of the TRE3G promoter in a PiggyBac backbone vector also containing a hygromycin resistance gene driven by the TET-ON3G transactivator and the EF-1a promoter.

[0166] For all effector plasmids, the scFV domain and sfGFP coding sequence were amplified from the PlatTET-gRNA2 plasmid (Addgene #82559) and fused in-frame with the catalytic domain (CD) or full-length version (FL) of mouse Prdm9, p300, Dot1L, G9a, Kmt5c, Setd2, Ezh2, and Ring1b amplified from cDNA samples. Alternatively, the 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 a PiggyBac plasmid under the control of the TRE3G promoter. These vectors also carry the constitutive expression of the neomycin resistance gene. Control GFP scFV Effectors were cloned as described above but lacked the chromatin modification domain. Finally, the catalytic mutant (mut-CD scFV ) effectors were also cloned as described above. Specific mutations that abolish catalytic activity were introduced during PCR amplification of the cDNA / plasmid template by oligonucleotide primers designed to mismatch nucleotides.

[0167] Guide RNA plasmids with the enhanced gRNA scaffold were amplified from Addgene plasmid #60955 and cloned into a PiggyBac recipient vector that constitutively expresses the puromycin resistance gene and TagBFP.

[0168] For the design of all gRNAs targeting the epigenetic editing system, the GPP Web portal (Broad Institute) was used. The gRNA forward and reverse strands with appropriate overhangs (final concentration 10 μM) were annealed in an annealing buffer containing 10 mM Tris, pH 7.5 - 8.0, 60 mM NaCl, and 1 mM EDTA at 95 °C for 3 minutes and cooled at room temperature for over 30 minutes. The annealed gRNAs were ligated to a PiggyBac recipient vector pre-digested with BlpI (NEB #R0585S) and BstXI (NEB #R0113S) restriction enzymes at 37 °C for 1 hour using T4-DNA ligase (NEB #M0202S). The final plasmid was amplified by bacterial transformation and purified by an endotoxin-free midiprep (ZymoResearch #D4200). The correct assembly and sequence were confirmed by Sanger sequencing (Azenta).

[0169] Results

[0170] In the context of the present invention, the inventors confirmed that the system enables specific and highly efficient on-target epigenetic editing at endogenous loci.

[0171] B - H in Figure 1 show the quantitative enrichment of seven chromatin modifications targeting Hbby by DOX induction. Importantly, the levels are comparable to the endogenous positive control (high on-target activity), while the off-target loci are hardly affected (low off-target activity). This is further shown in Figure 1I for H3K4me3 and H2AK119ub after targeting with a single gRNA. scFV

[0172] The inventors further investigated the chromatin function situation using a reporter system knocked into two specific genomic positions. The inventors, for each CD scFV ​We found that the histone modifications predicted by [mut-CD] were strongly and specifically enriched, but no enrichment was seen when targeting [mut-CD] scFV scFV or GFP scFV scFV . By using reporter activity, we were able to reproducibly detect the quantitative functional response to epigenetic editing at single-cell resolution (Figure 2). Programming H2AK119ub at promoters with Ring1b-CD scFV scFV (PRC1) strongly directed transcriptional repression in most cells, unlike its catalytic mutant. On the other hand, targeting H3K27me3 (PRC2) with Ezh2-FL scFV scFV showed a partially penetrant transcriptional response, and many cells did not show repression, indicating that H3K27me3 has weak instructive power at this locus.

[0173] Multi-programming of H2AK119ub and H3K27me3 produced a synergistic effect, and penetrant silencing was quantitatively improved compared to using either mark individually (Figure 3A). Examination of additional chromatin modifications such as H3K4me3 and H3K9me2 / 3 revealed different functions at the reporter locus, but other modifications such as H4K20me3 and H3K36me3 did not induce a transcriptional response. Collectively, these data support the accurate and highly efficient ability to program a panel of chromatin modifications and reveal single-cell transcriptional responses at a single test locus in ESCs.

[0174] The inventors further investigated the genetic epigenetic interactions in this synthetic system. For example, it was found that when programming H3K27ac, the suppressed reporter was activated, while inserting a short 10-nt MYC motif (E-box) into the same reporter experimentally weakened the effect (Figure 3B). Conversely, introducing a 9-nt OTX2 motif amplified the effect of H3K27ac transcription. The inventors discovered that inserting a short CTCF motif switched the behavior of H3K36me3 targeting the promoter from neutral (non-functional) to strong transcriptional repression (Figure 3C).

[0175] These data demonstrate a strong quantitative interaction between gene motifs and the underlying epigenomic functions. More generally, such results using a reductionist reporter strategy highlight the power of precise epigenetic perturbations for detecting quantitative responses and cis-genetic effects.

[0176] To further elucidate the context-dependent principle of chromatin function, the inventors propose the development of an epigenetic targeted perturbation sequencing: epiTAP-seq. This strategy enables the multi-dimensional evaluation of the functions of multiple chromatin states. This principle is based on TAP-seq (Non-Patent Document 26) and derives quantitative expression changes in single cells in response to induced perturbations (Figure 4). By intersecting this approach with our suite of chromatin perturbations and focusing on direct targets rather than network responses, the transcriptional outcomes of endogenous genes can be measured on an unprecedented scale. By programming 12 epigenetic marks and their combinations into a wide range of genes and further leveraging cis-gene mutations and multiple cell types, tens of thousands of contexts for analyzing causal relationships are generated. epi genetic ta rgeted p erturbation-sequencing:epiTAP-seq), which will enable the multi-dimensional evaluation of the functions of multiple chromatin states. This principle is based on TAP-seq (Non-Patent Document 26) and derives quantitative expression changes in single cells in response to induced perturbations (Figure 4). By intersecting this approach with our suite of chromatin perturbations and focusing on direct targets rather than network responses, the transcriptional outcomes of endogenous genes can be measured on an unprecedented scale. By programming 12 epigenetic marks and their combinations into a wide range of genes and further leveraging cis-gene mutations and multiple cell types, tens of thousands of contexts for analyzing causal relationships are generated.

[0177] This enables interrogation of a number of important questions. For example, the precise nature (quantification) of the transcriptional response to de novo chromatin marks, the functional penetrance (robustness) of modifications between single cells and between different genes, the quantitative consequences (context-dependence) of diverse genetic-epigenetic interactions at the level of genomic features or cis-variants, the influence of the cellular environment on regulatory responses (cell type specificity), and / or the degree of epigenetic and transcriptional persistence of chromatin states (memory). Overall, by implementing epiTAP-seq in the systems and methods of the present invention and integrating the data with a complementary series of unbiased screening and epigenome profiling approaches, a unique position is established from which complex genomic regulatory mechanisms can be analyzed in a controlled and comprehensive manner, leveraging the opportunities provided by precise epigenetic editing.

[0178] Cited References 1 Grosswendt, S., Kretzmer, H., Smith, Z. D., Kumar,A. S., Hetzel, S., Wittier, L., Klages, S., Timmermann, B., Mukherji, S. &Meissner, A. Epigenetic regulator function through mouse gastrulation. 584,102-108. Nature (2020). 2 De Santa, F., Totaro, M. G., Prosperini, E.,Notarbartolo, S., Testa, G. & Natoli, G. The histone H3 lysine-27demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated genesilencing. 130, 1083-1094. Cell (2007). 3 Cavalli, G. & Heard, E. Advances in epigenetics link genetics to the environment and disease. 571, 489-499. Nature (2019). 4 Roadmap Epigenomics, C. et al. Integrative analysis of 111 reference human epigenomes. 518, 317-330. Nature (2015). 5 Moore, J. E., Purcaro, M. J., Pratt, H. E., Epstein, C. B., Shoresh, N., Adrian, J., Kawli, T., Davis, C. A., Dobin, A. & Kaul, R. Expanded encyclopaedias of DNA elements in the human and mouse genomes. 583, 699-710. Nature (2020). 6 Stunnenberg, H. G., International Human Epigenome, C. & Hirst, M. The International Human Epigenome Consortium: A Blueprint for Scientific Collaboration and Discovery. 167, 1897. Cell (2016). 7 Allis, C. D. & Jenuwein, T. The molecular hallmarks of epigenetic control. 17, 487-500. Nat Rev Genet (2016). 8 Schuettengruber, B., Bourbon, H. M., Di Croce, L. & Cavalli, G. Genome Regulation by Polycomb and Trithorax: 70 Years and Counting. 171, 34-57. Cell (2017). 9 Greenberg, M. V. C. & Bourc'his, D. The diverse roles of DNA methylation in mammalian development and disease. 20, 590 - 607. Nat Rev Mol Cell Biol (2019). 10 Dorighi, K. M., Swigut, T., Henriques, T., Bhanu, N. V., Scruggs, B. S., Nady, N., Still, C. D., 2nd, Garcia, B. A., Adelman, K. & Wysocka, J. M113 and M114 Facilitate Enhancer RNA Synthesis and Transcription from Promoters Independently of H3K4 Monomethylation. 66, 568 - 576 e564. Molecular cell (2017). 11 Zhang, T., Zhang, Z., Dong, Q., Xiong, J. & Zhu, B. Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells. 21, 45. Genome Biol (2020). 12 Nakamura, M., Gao, Y., Dominguez, A. A. and Qi, L. S. CRISPR technologies for precise epigenome editing. 23, 11 - 22. Nat Cell Biol (2021). 13 Policarpi, C., Dabin, J. and Hackett, J. A. Epigenetic editing: Dissecting chromatin function in context. 43, e2000316. Bioessays (2021). 14 Liu, X. S., Wu, H., Krzisch, M., Wu, X., Graef, J., Muffat, J., Hnisz, D., Li, C. H., Yuan, B., Xu, C., Li, Y., Vershkov, D., Cacace, A., Young, R. A. & Jaenisch, R. Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene. Cell (2018). 15 Consortium, G. T. The GTEx Consortium atlas of genetic regulatory effects across human tissues. 369, 1318 - 1330. Science (2020). 16 Maurano, M. T. et al. Systematic localization of common disease - associated variation in regulatory DNA. 337, 1190 - 1195. Science (2012). 17 Izzo, F. et al. DNA methylation disruption reshapes the hematopoietic differentiation landscape. Nat Genet (2020). 18 Lambert, S. A., Jolma, A., Campitelli, L. F., Das, P. K., Yin, Y., Albu, M., Chen, X., Taipale, J., Hughes, T. R. & Weirauch, M. T. The Human Transcription Factors. 172, 650 - 665. Cell (2018). 19 Hanna, R. E., Hegde, M., Fagre, C. R., DeWeirdt, P.C., Sangree, A. K., Szegletes, Z., Griffith, A., Feeley, M. N., Sanson, K. R.,Baidi, Y., Koblan, L. W., Liu, D. R., Neal, J. T. & Doench, J. G. Massivelyparallel assessment of human variants with base editor screens. 184, 1064-1080 el020.Cell (2021). 20 Carlini, V., Policarpi, C. & Hackett, J. A.Epigenetic inheritance is gated by naive pluripotency and Dppa2, el08677. EMBOJ (2022). 21 Przybyla, L. & Gilbert, L. A. A new era infunctional genomics screens. Nat Rev Genet (2021). 22 Adamson, B. et al. A Multiplexed Single-Cell CRISPRScreening Platform Enables Systematic Dissection ofthe Unfolded ProteinResponse. 167, 1867-1882 el821. Cell (2016). 23 Dixit, A., Pamas, O., Li, B., Chen, J., Fulco, C.P., Jerby-Arnon, L., Marjanovic, N. D., Dionne, D., Burks, T., Raychowdhury,R., Adamson, B., Norman, T. M., Lander, E. S., Weissman, J. S., Friedman, N.& Regev, A. Perturb-Seq: Dissecting Molecular Circuits with ScalableSingle-Cell RNA Profiling of Pooled Genetic Screens. 167, 1853-1866 el817. Cell(2016). 24 Datlinger, P., Rendeiro, A. F., Schmidl, C.,Krausgruber, T., Traxler, P., Klughammer, J., Schuster, L. C., Kuchler, A.,Alpar, D. and Bock, C. Pooled CRISPR screening with single-cell transcriptomereadout. 14, 297-301. Nat Methods (2017). 25 Gasperini, M., Hill, A. J., McFaline-Figueroa, J.L., Martin, B., Kim, S., Zhang, M. D., Jackson, D., Leith, A., Schreiber, J.,Noble, W. S., Trapnell, C., Ahituv, N. & Shendure, J. A Genome-wideFramework for Mapping Gene Regulation via Cellular Genetic Screens. 176, 1516.Cell (2019). 26 Schraivogel, D., Gschwind, A. R., Milbank, J. H.,Leonce, D. R., Jakob, P., Mathur, L., Korbel, J. O., Merten, C. A., Velten, L.and Steinmetz, L. M. Targeted Perturb-seq enables genome-scale genetic screensin single cells. 17, 629-635. Nat Methods (2020).

[0179] Additional references Berger, S. The complex language of chromatin regulation during transcription.Nature 447, 407-412 (2007). Tsumura A, Hayakawa T, Kumaki Y, Takebayashi S, Sakaue M, Matsuoka C, ShimotohnoK, Ishikawa F, Li E, Ueda HR, Nakayama J, Okano M. Maintenance of selfrenewalability of mouse embryonic stem cells in the absence of DNA methyltransferasesDnmtl, Dnmt3a and Dnmt3b. Genes Cells. 2006 Jul;l l(7):805-14. Chamberlain, S.J., Yee, D., and Magnuson, T. (2008). Polycomb repressivecomplex 2 is dispensable for maintenance of embryonic stem cell pluripotency.Stem Cells 26, 1496-1505. Sze CC, Cao K, Collings CK, et al. Histone H3K4 methylation-dependent and-independent functions of SetlA / COMPASS in embryonic stem cell self-renewal and differentiation. Genes Dev. 2017;31(17): 1732-1737. Zhang, T., Zhang, Z., Dong, Q. et al. Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells. Genome Biol 21, 45 (2020). Carrozza MJ, Li B, Florens L, Suganuma T, Swanson SK, Lee KK, Shia WJ, Anderson S, Yates J, Washbum MP, Workman JL. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell. 2005 Nov 18; 123(4):581-92. Neri F, Rapelli S, Krepelova A, Incarnato D, Parlato C, Basile G, Maldotti M, Anselmi F, Oliviero S. Intragenic DNA methylation prevents spurious transcription initiation. Nature. 2017 Mar 2;543(7643):72-77. Stricker SH, Kbferle A, Beck S. From profiles to function in epigenomics. NatRev Genet.2017 Jan;18(l):51-66. Grosswendt S, Kretzmer H, Smith ZD, Kumar AS, Hetzel S, Wittier L, Klages S,Timmermann B, Mukherji S, Meissner A. Epigenetic regulator function throughmouse gastrulation. Nature. 2020 Aug;584(7819):102-108.

[0180] Terms in the drawings Figure 1 Effector GCN4-tail Catalytic domain ‘effectors’ enrichment norm. targeted untargeted Positive Negative DNA methylation Figure 2 Permissive locus reporter active Non-permissive locus reporter repressed targeted knock-in upstream coding sensor ~3kb baseline context Depleted of TF motifs, TE & histone mods Single-cell expression Permissive Non-permissive enrichment enrichment Figure 3 Single-cell expression Multiplexed programming motif Figure 4 gRNA library gRNA library Epigenetic editing F1 hybrid Differentiation Multi-lineage identity Direct function Memory function DOX withdrawal Quantitative expression of 100s target genes in single cells. Identify target gene for epigenetic editing in each cell Programmed chromatin mark Expression n=500 genes x 8 marks Cluster cells by identity Quantitative expression of epi-edited target gene per cell e.g. Programme H3K27me3 (or 7 x other) Endoderm Mesoderm Neural Gene n=500 genes x 8 marks x 6 cell types Figure 5 Class of disease Disease molecular aetiology Rescue Examples Haploinsufficiency X-linked diseases Activator Davet Syndrome Rett Syndrome Cdkl5 disorder Dominant acting Gain of function Repressor Hypercholesterolemia Olmsted Syndrome Muscular Dystrophy Imprinted or Fragile X Prader-Willis Angelman Complex disorders and Cancers Neurological disorders Wild-type allele Defective allele Target gene Active Inactive Methylated CpGs Unmethylated CpGs

Claims

1. i) A catalytically inactive site-specific nuclease, preferably selected from the group consisting of catalytically inactive (d)Cas9, asCas12, saCas9, miniCas9, dCas9, fCas9, SceI, and dCas9 / fCas9 fusions derived from Streptococcus pyogenes, and ii) A sequence of 2 to 10, preferably 3 to 7, effector domains, each having a specific chromatin modification activity, such as specific DNA methylation activity, histone methylation activity, specific histone acetylation or ubiquitination activity, and / or specific chromatin demethylation / deacetylation activity. A complex containing a combination of the following: The effector domains are each separated by a linker, and a sufficient distance is maintained between the domains and the nuclease, so that specific chromatin modification activity and site-specific nuclease binding are not substantially hindered in the complex.

2. The complex consists of a linker sequence, for example, Streptococcus pyogenes dCas9. ＧＣＮ４ （３－７） The complex according to claim 1, comprising a nuclease fusion protein bound to a protein sequence containing 3 to 7 effector domain binding motifs, each separated by, and the complex optionally further comprising a number of effector domains, each bound to a binding motif, and the complex optionally further comprising at least one suitable guide RNA (gRNA), preferably the protein complex comprising 5 effector domains.

3. The complex according to claim 1 or 2, wherein the linker sequence has a length of 25 to 19 amino acids, preferably 22 amino acids, and more preferably the linker sequence comprises glycine (G) and serine (S) amino acids.

4. The complex according to claim 1 or 2, wherein the effector domain is bound via an effector domain-binding motif-specific scFV domain, particularly a GCN4-specific scFV domain, the scFV is optionally linked to the effector domain via an effector linker group, such as a group containing a fluorescent protein such as GFP or sfGFP protein, and the effector domain may be further fused to a protein destabilization domain such as d2.

5. The effector domains are 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 (GlcNAC), 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 Demethylases), Tet1 / 2 / 3 (methylcytosine dioxygenase), Utx (H3K27 demethylase), JMJD3 (H3K27 demethylase), Kdm4a / b / c / d (H3K36 and H3K9 demethylases), their catalytic domains (CD), these catalytic domains (CD) fused to effector domain-binding motif-specific scFV domains ｓｃＦＶ The complex according to claim 1 or 2, comprising a chromatin-modified polypeptide selected from the group consisting of these catalytic domains (CD) fused to an antigen-binding fragment (Fab) domain.

6. A set of nucleic acids each encoding at least one of the proteins and / or guide RNA (gRNA) and / or tagBFP of the complex according to claim 1 or 2.

7. A set of gene constructs, such as an expression vector, comprising the set of nucleic acids described in claim 6, preferably an effector domain, CD and / or CD ｓｃＦＶ Each nucleic acid encoding a gene comprises an inducible promoter such as a tet-responsive promoter or a dox-responsive promoter, and preferably the gene construct is a set of gene constructs, such as a viral construct derived from AAV, lentivirus, or retrovirus.

8. Recombinant cells comprising the set of nucleic acids described in Claim 6.

9. A method for specifically epigenetically modifying chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin, comprising introducing the complex according to claim 1 or 2 and one or more guide RNAs into cells, tissues, cell nuclei, and / or samples to specifically epigenetically modify chromatin in cells, tissues, cell nuclei, and / or samples.

10. The method according to claim 9, wherein the epigenetic modification includes histone methylation, DNA methylation, histone acetylation, histone ubiquitination, DNA demethylation, histone deacetylation, histone multiple epigenetic editing, H3K9me2 / 3 + DNA methylation, H3K4me3 + H3K36me3, H3K4me3 + H3K79me2, H3K36me3 + H3K79me2, H3K9me2 / 3 + H4K20me3, histone bivalent epigenetic editing, and / or histone polycomb epigenetic editing, and / or the epigenetic modification includes transient induction of the expression of the complex described in claim 1 or 2 and one or more guide RNAs.

11. 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 described in claim 1 or 2 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 includes nucleic acid sequences specific to conditions and / or disease states related to epigenetically modified chromatin, such as hereditary diseases, proliferative disorders such as cancer, immune cells that produce autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), drug abuse including alcohol abuse disorder, and epigenetic disorders including imprinting disorders such as Prader-Willi syndrome. More preferably, a method in which the expression of 2, 3, 4, 5, 6, 7, 8, or 9 target DNA sequences is regulated within a cell.

12. A method for detecting a biological effect by specifically epigenetically modifying chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin, and / or a method for detecting a biological effect by regulating the expression of at least one target DNA sequence in cells, tissues, cell nuclei, and / or samples containing chromatin, comprising performing the method according to claim 9, and a method comprising detecting at least one biological effect in cells, tissues, cell nuclei, and / or samples containing chromatin, wherein the biological effect is selected from the group consisting of changes in gene expression, changes in protein quantity, changes in cis-gene effects, changes in nucleic acid splicing, changes in the nuclear arrangement of gene loci, changes in TAD formation and disruption, changes in termination sites, changes in promoter activation, changes in promoter repression, changes in genetic epigenetic interactions, changes in the functional relationship between gene mutations and the epigenetic state of chromatin, changes in genetic methylation and imprinting, and specific epigenetic changes associated with disease or cellular phenotype, Preferably, the method is carried out using a complex that allows tracking, preferably in real time, the effects on cells, tissues, cell nuclei, and / or samples containing chromatin.

13. Cells having specifically epigenetically modified chromatin produced by the method of claim 9, and optionally the cells are isolated cells, preferably stem cells, neurons, postmittal cells, or fibroblasts, and / or the cells are animal cells, such as mammalian cells, preferably human cells or rodent cells.

14. The method of claim 9 is carried out in the presence and absence of the test agent, Drugs that specifically epigenetically modify 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 Biological effects that specifically epigenetically modify chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin. A method for identifying, If the regulatory and / or biological effects in the presence of the test agent differ from those in the absence of the agent or in the control, Drugs that specifically epigenetically modify 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 Biological effects that specifically epigenetically modify chromatin in cells, tissues, cell nuclei, and / or samples containing chromatin. A method identified as such.

15. In subjects requiring prevention or treatment, For use in the prevention or treatment of disease, or For example, for use in the prevention or treatment of diseases related to epigenetically modified chromatin, such as hereditary disorders, proliferative disorders like cancer, immune cells that produce autoantibodies, bacterial or viral infections, protozoan infections, fragile X syndrome, muscular dystrophy, kidney injury, cardiovascular disease, shortened lifespan, tissue aging, neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), drug abuse including alcohol abuse disorder, and epigenetic disorders including imprinting abnormalities such as Prader-Willi syndrome. A pharmaceutical product comprising the complex described in claim 1 or 2.

Images