Fusion proteins for CRISPR-based transcriptional repression

Abstract

Compositions for regulating nucleic acid expression and methods for using these compositions are provided. The compositions comprise fusion proteins that include one or both of the repression domains of SALL1 and SUDS3. In some embodiments, the compositions are Cas fusion proteins that can be used together with gRNA or other RNA. Additionally or alternatively, the compositions are RNA repression domain complexes.

Description

[Technical field]

[0001] INCORPORATION OF RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 146,419, filed February 5, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the field of CRISPR-based transcriptional repression. [Background technology]

[0003] The biotechnology industry is now familiar with the CRISPR-Cas9 system, which allows for the specific targeting and editing of genes, and which was originally discovered in archaea and bacteria but shows great promise for human applications. The basic CRISPR / Cas9 system has Cas9 protein and guide RNA (gRNA). A spacer sequence (also called target sequence) in the gRNA guides the Cas9 protein to a genomic target site based on the complementarity between the spacer sequence and the sequence of the target site. After the Cas9 protein is guided to the target site, the Cas9 protein cleaves the target DNA, leading to DNA editing. Meanwhile, inactive Cas9 (dCas9) can be used for sequence-specific targeting and other effectors with various functions.

[0004] Because the CRISPR / Cas9 system is effective at localizing and editing target sites, researchers have explored how to use this system to provide functions other than those provided by the active site of the naturally occurring Cas9 protein, and have also begun to explore the use of other Cas proteins that rely on the specificity of gRNAs to deliver the Cas9 protein to target sites.

[0005] Although researchers originally discovered the CRISPR-Cas9 system in low-level microorganisms, the system has been used very successfully for gene editing applications in mammalian cells. M.Jinek,etal.,“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”Science.337:p.816-821(2012). Researchers have also been able to abolish the nuclease activity of Cas9 proteins by introducing point mutations into catalytic residues (D10A and H840A in the commonly used Streptococcus pyogenes Cas9 protein), resulting in an inactive Cas9 that retains the ability to bind to target DNA when guided by sequence-specific guide RNA. When dCas9 is fused to transcriptional regulators and guided to gene promoter regions, it induces RNA-directed transcriptional regulation. CRISPR system technologies for transcriptional regulation include CRISPR interference (CRISPRi) for transcriptional repression and CRISPR activation to upregulate transcription (Qi, LS, et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell, 152(5): p.1173-83(2013); AW Cheng, et al., “Multiplexed activation of endogeneous genes by CRISPR-on, an RNA-guided transcriptional activator system,” Cell Res., 23(10): p.1163-71(2013)).

[0006] One known approach to CRISPR system for transcriptional repression is to use Kruppel-associated box domain (KRAB) from zinc finger protein 10 (KOX1) as a transcriptional repressor. LA Gilbert, et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell, 154(2): p. 442-51 (2013); LA Gilbert et al., “Genome-scale CRISPR-mediated control of gene repression and activation,” Cell, 159: p. 647-661 (2014). However, this approach has limitations. Researchers have shown that it does not provide sufficient repression in all applications, resulting in weaker repression of the target gene. L. Stojic et al., “Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis,” Nucleic Acids Research, 46(12): p. 5950-5966 (2018); Yeo, et al., “An enhanced CRISPR repressor for targeted mammalian gene regulation,” Nat. Methods, 15(8): p. 611-616 (2018). Although CRISPR-KRAB fusion proteins are the most commonly used fusion proteins for transcriptional repression, the diversity of reported functions indicates that further CRISPR-based approaches for transcriptional repression are needed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] M. Jinek, etal., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”Science.337:p.816-821(2012). [Non-Patent Document 2] Qi, LS, et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell, 152(5):p.1173-83 (2013). [Non-Patent Document 3] AWCheng, et al., “Multiplexed activation of endogeneous genes by CRISPR-on, an RNA-guided transcriptional activator system,” Cell Res., 23(10):p.1163-71 (2013). [Non-Patent Document 4] LA Gilbert, et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell, 154(2): p. 442-51 (2013). [Non-Patent Document 5] LA Gilbert et al., “Genome-scale CRISPR-mediated control of gene repression and activation,” Cell, 159: p. 647-661 (2014). [Non-Patent Document 6] L. Stojic et al., “Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis,” Nucleic Acids Research, 46(12): p. 5950-5966 (2018). [Non-Patent Document 7] Yeo, et al., “An enhanced CRISPR repressor for targeted mammalian gene regulation,” Nat. Methods, 15(8): p. 611-616 (2018). Summary of the Invention

[0008] The present invention provides novel fusion proteins, nucleic acid sequences encoding these fusion proteins, and methods of gene suppression using these fusion proteins and / or nucleic acids.Various embodiments of the present invention can be used to efficiently and effectively modulate gene expression. In a first embodiment, the invention provides a Cas fusion protein having a Cas9 protein and one or both of a SALl1 repression domain and a SUDS3 repression domain, in some embodiments, the Cas protein is inactivated, also referred to as dead or attenuated. In a second embodiment, the present invention provides a nucleic acid encoding a Cas fusion protein of the present invention. In a third embodiment, the present invention provides an RNA repression domain complex comprising: a) a gRNA molecule comprising 30 to 180 nucleotides; b) i) a ligand binding moiety that binds directly to the gRNA molecule or ii) a ligand binding moiety that binds to the gRNA molecule via a ligand binding moiety linker; c) a ligand that can reversibly bind to the ligand binding moiety; and d) a fusion protein comprising a SALL1 repression domain and a SUDS3 repression domain, wherein the fusion protein either i) binds directly to the ligand or ii) binds to the ligand via a linker.

[0009] In a fourth embodiment, the present invention provides a method for controlling expression of a target nucleus, comprising the step of introducing a Cas fusion protein or an RNA repression domain complex of the present invention or a nucleic acid of the present invention into a cell, such as a eukaryotic cell, or into a mammalian tissue, such as a human. In some embodiments, the introduction is performed in vitro, in vivo, or ex vivo. In a fifth embodiment, the invention provides a kit comprising a Cas fusion protein of the invention or a nucleic acid encoding a Cas fusion protein of the invention, and in an embodiment, the kit may further comprise a gRNA or a nucleic acid encoding a gRNA.

[0010] In a sixth embodiment, the invention provides a kit comprising a nucleic acid encoding an RNA repression domain complex or an RNA ligand binding domain and ligand repressor dyad of the invention. In a seventh embodiment, the present invention provides a protein having a sequence at least 80% similar to, essentially corresponding to, or consisting of, SEQ ID NO:10. [Brief description of the drawings]

[0011] [Figure 1] 1 depicts a Cas fusion protein of the present invention in association with a single guide RNA (sgRNA) and target DNA. [Diagram 2]1 shows an example of an sgRNA that can be used in various embodiments of the present invention. [Figure 3A] Graph showing gene knockdown in K562 cells nucleofected with either dCas9-KRAB or dCas9-SALL1-SUDS3 mRNA. [Figure 3B] Graph showing gene knockdown in Jurkat cells nucleofected with either dCas9-KRAB or dCas9-SALL1-SUDS3 mRNA. [Figure 3C] Graph showing gene knockdown in U2OS cells nucleofected with either dCas9-KRAB or dCas9-SALL1-SUDS3 mRNA. [Figure 4] Figure 1 shows the target gene silencing effect of dCas9-SALL1-SUDS3 eGFP mRNA versus dCas9-KRAB eGFP mRNA in HCT116 cells. All genes were targeted with a pool of three synthetic sgRNAs provided at 25 nM. Cells were transfected and stored for 24 hours in two categories: GFP negative (GFP Neg) and the top 10% expressing GFP (Top10%). After 24 hours of harvest, cells were analyzed for transcriptional silencing of the target genes. [Figure 5A] We compare the inhibitory effects of the dCas9-KRAB-containing system and the dCas9-SALL1-SUDS3-containing system in various cell lines: U2OS (Figure 5A); Jurkat (Figure 5B); and hiPS-stable hEF1α (Figure 5C). [Figure 5B] We compare the inhibitory effects of the dCas9-KRAB-containing system and the dCas9-SALL1-SUDS3-containing system in various cell lines: U2OS (Figure 5A); Jurkat (Figure 5B); and hiPS-stable hEF1α (Figure 5C). [Figure 5C]We compare the inhibitory effects of the dCas9-KRAB-containing system and the dCas9-SALL1-SUDS3-containing system in various cell lines: U2OS (Figure 5A); Jurkat (Figure 5B); and hiPS-stable hEF1α (Figure 5C). [Figure 6A] Figure 1 shows gene silencing effects of dCas9-KRAB and dCas9-SALL1-SUDS3 against BRCA1, PSMD7, SEL1L, and ST3GAL4 in K562 cells. [Figure 6B] Figure 1 shows gene silencing effects of dCas9-KRAB and dCas9-SALL1-SUDS3 against BRCA1, PSMD7, SEL1L, and ST3GAL4 in A375 cells. [Figure 7A] We compare the suppression of various gene targets by dCas9-KRAB with that by dCas9-SALL1-SUDS3 over six consecutive days in U2OS cells: BRCA1 (Figure 7A); CD46 (Figure 7B); HBP1 (Figure 7C); and SEL1L (Figure 7D). [Figure 7B] We compare the suppression of various gene targets by dCas9-KRAB with that by dCas9-SALL1-SUDS3 over six consecutive days in U2OS cells: BRCA1 (Figure 7A); CD46 (Figure 7B); HBP1 (Figure 7C); and SEL1L (Figure 7D). [Figure 7C] We compare the suppression of various gene targets by dCas9-KRAB with that by dCas9-SALL1-SUDS3 over six consecutive days in U2OS cells: BRCA1 (Figure 7A); CD46 (Figure 7B); HBP1 (Figure 7C); and SEL1L (Figure 7D). [Figure 7D] We compare the suppression of various gene targets by dCas9-KRAB with that by dCas9-SALL1-SUDS3 over six consecutive days in U2OS cells: BRCA1 (Figure 7A); CD46 (Figure 7B); HBP1 (Figure 7C); and SEL1L (Figure 7D). [Figure 8A]When introduced with the Cas fusion proteins of the present invention, we demonstrate suppression of PPIB, SEL1L, and RAB11A using individual sgRNAs, and suppression of these targets using pools of sgRNAs. [Figure 8B] When introduced with the Cas fusion proteins of the present invention, we demonstrate suppression of BRCA1, PSMD7, SEL1L, and ST3GAL4 using individual sgRNAs, and suppression of these targets using pools of sgRNAs. [Figure 9] Figure 1 shows the expression of PRIB, RAB11A and SEL1 genes in hiPSC cells in the presence of gRNA and dCas9-SALL1-SUDS3 when multiplexed with sgRNAs against multiple genes. [Figure 10] Functional phenotypes of PSMD3, PSMD8, and PSMD11 repression in U2OS-Ubi(G76V)-EGFP reporter cell lines in the presence of gRNA and dCas9 fused to KRAB or SALL1-SUDS3 at the N-terminal amino acid of dCas9 or the C-terminal amino acid of dCas9. [Figure 11] FIG. 13 is a graph showing the transcriptional repression effect in a system having a plasmid expressing a gRNA and a plasmid expressing a co-transfected fusion protein in A375 cells. [Figure 12] 1 is a graph showing the transcriptional repression effect in a system having a plasmid expressing a gRNA and a plasmid expressing a co-transfected fusion protein in U2OS cells. [Figure 13] Graph showing the effect of combining SALL1 or SUDS3 with an additional repression domain, respectively. [Figure 14A] 1 is a graph comparing the suppressive effect of dMAD7-SALL1-SUDS3 with the suppressive effect of dMAD7 in U2OS cells. [Figure 14B] This is a graph comparing the inhibitory effect of dCasPhi8-SALL1-SUDS3 with the inhibitory effect of dCasPhi8 in U2OS cells. [Figure 15A]FIG. 13 is a graph showing the effect of using sgRNAs with various crRNA target sizes together with non-inactivated Cas9 for simultaneous silencing and gene editing. [Figure 15B] 13 is a graph measuring the inhibitory effect of MRE11a during co-editing of the LBR. [Figure 15C] 13 is a graph measuring the inhibitory effect of MRE11a during co-editing of PPIB. [Figure 15D] 13 is a graph measuring the inhibitory effect of SEL1L during co-editing of the LBR. [Figure 15E] 13 is a graph measuring the inhibitory effect of SEL1L during co-editing of PPIB. [Figure 16] Graph showing the inhibitory effect of a system with a single reporter dCas9 fusion protein in U2OS-Ubi(G76V)-EGFP reporter cells. [Figure 17] Graph comparing transcriptional repression in U2OS cells stably expressing dCas9-KRAB, dCas9-KRAB MeCP2, or dCas9-SUDS3 transfected with synthetic guide RNA. [Figure 18A] 13 depicts the phenotypic effects of gene knockdown in U2OS Ubi "G76V"-EGFP reporter cells expressing either dCas9-KRAB or dCas9-SEL1L-SUDS3 and transfected with a synthetic guide target protease gene. [Figure 18B] The transcriptional repression effect on the corresponding target protease gene is shown. [Figure 19A] Figure 1 shows the transcriptional repression effect on PPIB and SEL1L in U2OS cells stably expressing either dCas9-SALL1-SUDS or guide RNA from a single lentiviral vector or from two separate vectors. [Figure 19B]Figure 1 shows the transcriptional repression effect on PPIB and SEL1L in HCT116 cells stably expressing either dCas9-SALL1-SUDS or guide RNA from a single lentiviral vector or from two separate vectors. [Figure 20A] 13 shows the transcriptional repression of BRCA1, PSMD7, SEL1L, and ST3GAL4 by either synthetic or plasmid sgRNA in U2OS cells stably expressing dCas9-SALL1-SUDS. [Figure 20B] Figure 1 shows the transcriptional repression of BRCA1, PSMD7, SEL1L, and ST3GAL4 by either synthetic or plasmid sgRNA in A375 cells stably expressing dCas9-SALL1-SUDS.

[0012] [Figure 21] 13 shows the transcriptional repression effect of either synthetic sgRNA or synthetic crRNA:tracrRNA complex on CD151, SEL1L, SETD3, and TFRC in U2OS cells stably expressing dCas9-SALL1-SUDS3. [Figure 22] This shows the transcriptional repression effect of synthetic sgRNAs with a 5' truncated 14-nt target region or a full-length 20-nt target region on LBR, MRE11a, XRCC4, and SEL1L in U2OS cells stably expressing dCas9-SALL1-SUDS3. [Figure 23A] Figure 1 shows the phenotypic effects of gene knockdown of PSMD7 and PSMD11 with synthetic sgRNAs bearing various combinations of two 2'-O methyl and phosphorothioate linkages (2xMS) and two locked nucleic acid (LNA) modifications at the 5' and 3' ends of the sgRNA in U2OS Ubi[G76V]-EGFP reporter cells expressing dCas9-SALL1-SUDS3. [Figure 23B]Figure 1 shows the phenotypic effects of gene knockdown of PSMD7 and PSMD11 in U2OS Ubi[G76V]-EGFP reporter cells expressing dCas9-SALL1-SUDS3, induced by synthetic sgRNAs end-stabilized with two 2'-O methyl and phosphorothioate linkages (2xMS) and various fixed nucleic acids (LNAs) at different positions in the target region. [Figure 24A] We show transcriptional repression of BRCA1, CD151, and SETD3 by synthetic crRNA:tracrRNA complexes, in which tracrRNA has MS2 stem-loops at different positions (at stem-loop 2 or at the 3' end of tracrRNA) and recruits MCP-SALL1-SUDS3 to dCas9. [Figure 24B] We show transcriptional repression of BRCA1, CD151, and SETD3 by synthetic crRNA:tracrRNA complexes, in which tracrRNA has a variety of MS2 stem-loop sequences and recruits MCP-SALL1-SUDS3 to dCas9. [Figure 25A] FIG. 13 shows transcriptional silencing and protein knockdown of CXCR3 in primary human CD4+ cells nucleofected with dCas9-SALL1-SUDS3 and either a synthetic non-targeting control or a pool of three guide RNAs targeting control genes after 1 and 3 days of nucleofection. [Figure 25B] FIG. 1 shows CXCR3 and CD4 protein expression in the above T cell populations. Detailed Description of the Invention

[0013] The present invention will now be described in detail with reference to various embodiments thereof, with specific examples being given in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, unless otherwise indicated or apparent from the context, the details are merely illustrative and should not be construed as limiting the scope of the present invention in any manner. Furthermore, features described in connection with various or specific embodiments should not be considered as being suitable for use in connection with other embodiments disclosed herein, unless otherwise expressly stated or apparent from the context. Additionally, the headings used herein are provided for the convenience of the reader and do not limit the scope of any embodiments disclosed herein.

[0014] (definition) Unless otherwise stated or apparent from the context, the following words and phrases have the meanings set forth below. "2'-modification" refers to a nucleotide unit having a sugar moiety modified at the 2'-position of the sugar. An example of a 2'-modification is a 2'-O-alkyl modification to form a 2'-O-alkyl-modified nucleotide, or a 2'-halogen modification to form a 2'-halogen-modified nucleotide. "2'-O-alkyl modified nucleotide" refers to a nucleotide unit having a sugar moiety, such as a deoxyribosyl or ribosyl moiety modified at the 2' position, in which an oxygen atom is attached to both the carbon atom at the 2' position of the sugar and to the alkyl group. In various embodiments, the alkyl group is a moiety consisting of carbon and hydrogen or consisting essentially of carbon and hydrogen. When the O-moiety and the alkyl group attached to the O-moiety are considered as a single functional group, such groups are referred to as O-alkyl groups, such as -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-ethyl-O-methyl (-OCH2CH2OCH3), and -O-ethyl-OH (-OCH2CH2OH). Additionally, the 2'-O-alkyl modified nucleotide may be substituted or unsubstituted.

[0015] "2' halogen modified nucleotide" refers to a nucleotide unit having a sugar moiety, such as a deoxyribosyl moiety modified at the 2' position, in which the 2' carbon is directly bonded to a halogen species such as Fl, Cl, or Br. "Complementarity" refers to the ability of a nucleic acid sequence to form one or more hydrogen bonds with another nucleic acid sequence, either by typical Watson-Crick base pairing or other non-typical base pairing. Percent complementarity refers to the percentage of nucleic acid residues in a nucleic acid molecule sequence that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 residues that can hydrogen bond out of 10 residues indicate 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). Here, "complete complementarity" means that all consecutive residues of a nucleic acid sequence form the same number of hydrogen bonds as the consecutive residues of the second nucleic acid sequence. As used herein, "substantial complementarity" refers to a degree of complementarity, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% complementarity. The region may be, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 contiguous nucleotides, or even more contiguous nucleotides. Alternatively, "substantial complementarity" refers to two nucleic acid sequences that hybridize under stringent conditions.

[0016] "Encode" refers to the ability of a nucleotide or amino acid sequence to provide descriptive information for the nucleotide or amino acid sequence of another sequence or molecule. Thus, a nucleotide sequence encodes a molecule that contains the same nucleotides as are in the nucleotide sequence that encodes it, including molecules that contain complementary nucleotides according to the Watson-Crick base pairing rules; molecules that contain the RNA equivalents of the nucleotides that code for it; molecules that contain the RNA equivalents of the nucleotide complements that code for it; molecules that contain an amino acid sequence generated based on consecutive codons in an amino acid sequence; and molecules that contain an amino acid sequence generated based on the complements of consecutive codons in a sequence. "gRNA" refers to a guide RNA. A gRNA comprises, consists essentially of, or consists of a CRISPR RNA (crRNA). In some embodiments, a gRNA may comprise a trans-activating CRISPR RNA (tracrRNA). A gRNA may be synthetically or enzymatically produced and may be in the form of a continuous nucleotide strand. In this case, the gRNA is a "sgRNA" and is formed by hybridization of a crRNA and a tracrRNA that are not covalently linked to each other in some embodiments to form a continuous nucleotide strand. Also, each gRNA (or its components, e.g., crRNA and tracrRNA, if present) is encoded separately by a plasmid, lentivirus or AAV (adeno-associated virus), retrovirus, adenovirus, coronavirus, Sendai virus, or other vector. The gRNA is specifically introduced into the CRISPR / Cas system. Its specificity is determined in part by base pairing between the target DNA and the sequence of the gRNA region, referred to as the spacer region or target region.

[0017] Another factor that influences the specificity of gRNA binding to a target DNA sequence is the presence of a PAM (protospacer adjacent motif) sequence (also called a PAM site) in the target sequence. Each target sequence and the corresponding PAM site / sequence are collectively referred to as a Cas target site. For example, the Streptococcus pyogenes class 2 CRISPR system uses a target site with N12-20NGG, where NGG represents the PAM site from Streptococcus pyogenes and N12-20 represents the 12-20 nucleotides directly 5' to the PAM site. PAM site sequences from other species of bacteria include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. See, for example, US 20140273233, WO 2013176772, Cong et al., Science 339 (6121): 819-823 (2012), Jinek et al., Science 337 (6096): p. 816-821 (2012), Mali et al., Science 339 (6121): p. 823-826 (2013), Gasiunas et al., Proc. Natl Acad. Sci. USA, 109 (39): p. E2579-E2586 (2012), Cho et al., Nature Biotechnology 31: p. 230-232 (2013), Hou et al., Proc. Natl Acad. Sci. US A. 110(39): p.15644-15649 (2013), Mojica et al., Microbiology 155 (Pt 3): p. 733-740 (2009), the contents of which are incorporated herein by reference in their entireties.

[0018] "Hybridization" and "hybridizing" refer to the process by which fully, substantially, or partially complementary nucleic acid strands come together under specific hybridization conditions to form a double helix structure or a region in which the two stranded components are joined by hydrogen bonds. Unless otherwise specified, the hybridization conditions may be naturally occurring or artificial. Furthermore, hydrogen bonds are typically formed between adenine and thymine or uracil (A and T or U) or cytidine and guanine (C and G), although other base pairs may also be formed (see, infra, Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0019] "Ligand binding site" refers to an aptamer, e.g., an oligonucleotide, peptide, or other compound that binds to a specific ligand and can bind reversibly or irreversibly to that ligand. Reversibly binds means that two molecules or complexes can remain bound to each other by non-covalent forces, e.g., hydrogen bonds, and can be separated from each other without the molecules or complexes losing their ability to bind to other molecules or complexes.

[0020] "Modified nucleotide" refers to a nucleotide with at least one modification in the chemical structure of the base, sugar and / or phosphate, including but not limited to the following: 5-position pyrimidine modification, 8-position purine modification, cytosine exocyclic amine modification, 5-bromouracil or 5-iodouracil substitution, and 2' modifications include but are not limited to the following: sugar-modified ribonucleotides in which the 2'-OH is replaced with a group such as H, OR, R, halogen, SH, SR, NH2, NHR, NR2 or CN. "Modified bases" refers to nucleotide bases, such as adenine, guanine, cytosine, thymine, uracil, xanthine, isocine, and quercein, that have been modified by substitution or addition of one or more atoms or groups. Examples of these types of modifications include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, used alone and in various combinations. More specifically, modified bases include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, and other nucleotides modified at the 5-position; 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, and deazanucleotides, such as 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine. Other thio bases include 2-thiouridine, 4-thiouridine, 2-thiocytidine; dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups; O- and N-alkylated purines and pyrimidines, such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one; phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene; modified cytosines that act as G-clamp nucleotides, such as 8-substituted adenines and guanines, 5-substituted uracils, thymines and azapyrimidines, carboxyhydroalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides.Additionally, modified nucleotides include nucleotides with modified sugar moieties and nucleotides having non-ribosyl sugars and sugar analogs, such as those based on mannose, arabinose, glucopyranose, galactopyranose, 4-thioribose, and other sugars, heterocycles, or carbocycles.

[0021] "Nucleotide" refers to ribonucleotides, deoxyribonucleotides, or modifications and analogs of these compounds. Nucleotides include purines, such as adenine, hypoxanthine, guanine, and derivatives and analogs of these compounds. Additionally, pyrimidines, such as cytosine, uracil, thymine, and derivatives and analogs thereof. Preferably, nucleotides include cytosine, uracil, thymine, adenine, or guanine moieties. Additionally, "nucleotides" include detectable labels, such as radioactive or fluorescently active moieties, or mass labels attached to the nucleotide. Additionally, "nucleotides" include commonly known bases known to those of skill in the art. For example, commonly known bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine. Nucleotide analogs include, for example, nucleotides containing bases such as inosine, queosine, xanthine, sugars such as 2'-methylribose, and non-natural phosphodiester internucleotide linkages, such as methylphosphonates, phosphorothioates, phosphoroacetates, and peptides.

[0022] "Repression domain" refers to an amino acid sequence that forms a domain of a repression molecule that inhibits gene expression. "Subject" and "patient" are used interchangeably herein and refer to a tissue, e.g., a vertebrate, more preferably a mammal, even more preferably a human. Mammals include, but are not limited to, monkeys, humans, farm animals, sport animals, and pets such as dogs and cats. Herein, tissues, cells, and the progeny of certain microorganisms, or other biological entities obtained in vivo or cultured in vitro, are also included within "subject" or "patient". In some embodiments, the subject may be an invertebrate, e.g., an insect or nematode, while in other cases the subject may be a plant or a fungus.

[0023] "Terminal amino acid" refers to the most terminal amino acid in a protein or in a fusion protein region. In a fusion protein, the terminal amino acid of a Cas protein is bound, for example, to another amino acid in the Cas protein region of the fusion protein, as well as to a repression domain or linker. Similarly, in a fusion protein, the terminal amino acid of a repression domain is bound, for example, to another amino acid in the repression domain, as well as to another repression domain, or to a Cas protein region or linker of the fusion protein. The terminal amino acid may be the C-terminal amino acid or the N-terminal amino acid.

[0024] As used herein, the terms "treatment", "treat", "alleviate" and "ameliorate" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to an improvement or effect in one or more diseases, disease states or conditions under treatment associated with the treatment. For a prophylactic effect, the complexes of the present invention are administered to a subject, or to cells or tissues of a subject, or administered exogenously to cells or tissues of another patient at risk of developing a particular disease, disease state or condition before re-administration, or administered to a disease reporting one or more physiological symptoms. Such administration can be performed even if the disease, disease state or condition is not yet evident.

[0025] "Vector" refers to a molecule or complex that transports another molecule, including but not limited to a nucleic acid molecule that can transport another nucleic acid molecule already bound to it or integrated into the sequence of the vector. Vectors are also called "expression vectors" because they are introduced into cells and organisms to express RNA transcripts, proteins and peptides. Examples of vectors include, but are not limited to, plasmids, lentiviruses, alphaviruses, adenoviruses, or adeno-associated viruses. The vector may be single-stranded, double-stranded, or may have at least one region that is single-stranded and at least one region that is double-stranded. The nucleic acid may be RNA or DNA, essentially composed of nucleic acid or DNA, or may consist of nucleic acid or DNA.

[0026] As disclosed herein, a number of ranges of values ​​are described. It is understood that each value between the upper and lower limits of the range is specifically disclosed, to one tenth of the unit of the lower limit unless the context clearly indicates otherwise. Subranges between a stated value or value falling within a stated range and another stated value or value falling within a stated range are encompassed within the invention. The upper and lower limits of the subranges may be independently included or excluded within the range. Each of these subranges, including either the upper or lower limit, or including neither limit, or including both limits, is encompassed within the invention, assuming the specifically excluded limit within the stated range. When the stated range includes one or both limits, ranges excluding either or both of the included limits are also encompassed within the invention. The term "about" generally means plus or minus 10% of the indicated number. For example, "about 10%" refers to a range of 9% to 11%, and "about 20" means 18 to 22. Alternative meanings of "about" are obvious from the context, and include rounding of values, for example, "about 1" may mean 0.5 to 1.4.

[0027] Various embodiments of the present invention are directed to fusion proteins and their uses. A fusion protein is a molecule that contains a portion or the complete amino acid sequence of each of two or more proteins. The components of the fusion protein are directly fused to each other, for example, by covalent bonds or via a linker as described below. Fusion proteins may also be linked to sites that do not contain amino acids, such as nucleotide sequences.

[0028] (Cas fusion proteins) In a first embodiment, the present invention is directed to a Cas fusion protein comprising, essentially comprising, or consisting of a Cas protein and one or both of a SALL1 repression domain and a SUDS3 repression domain, or comprising, essentially comprising, or consisting of a Cas protein and one or both of a sequence that is at least 80%, at least 85%, at least 90% or at least 95% identical to one of the repression domains. The Cas proteins may be, for example, CRISPR-associated proteins naturally occurring in archaea or bacteria, or modified, i.e., inactive, truncated, or derivative, CRISPR-associated proteins in archaea or bacteria. Amino acid and nucleic acid sequences for many Cas proteins are available from public sources, such as the National Institutes of Health, and are incorporated herein by reference in their entireties.

[0029] Examples of Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, Cas12j, Mad7, CasX, CasY, Cas 13a, Casl4, C2cl, C2c2, C2c3, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and analogs or modifications thereof. Unless otherwise noted or clear from the context, any listing of Cas proteins includes active and inactive forms, as well as analogs and modifications thereof. In one embodiment, the Cas protein is a type II Cas protein, such as Cas9, or a type IV Cas protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, Cas12j, and MAD7.

[0030] Modified Cas proteins that may be used in the present invention include, but are not limited to, catalytically inactive, such as dCas9 and dCas12, or modified to have reduced catalytic activity and nicking function, such as nickase Cas9. Nicking enzymes are enzymes that cleave one strand of double-stranded DNA at a specific recognized nucleotide sequence. These enzymes cleave only one strand of the DNA complex, generating a nicked rather than cleaved DNA molecule. Examples of amino acid sequences of Cas proteins that may be used in connection with the present invention include the following: Inactive Cas9:

[0031] Inactive MAD7: Inactive CasPhi8 (dCasPhi8): MVDGSGPAAKRVKLDSGGIKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIANIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEAAGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFE EIKAFDDKGYLLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPIGEPGYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKKYHKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICD QNGSCKLATVAVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLESSIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEKAQVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEFNKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENRWWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELNADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLREAVKRPAATKKAGQAKKKK (SEQ ID NO: 40) Cas proteins can be used with repression domains. The repression domain of SALL1 is: MSRRKQAKPQHFQSDPEVASLPRRDGDTEKGQPSRPTKSKDAHVCGRCCAEFFELSDLLLHKKNCTKNQLVLIVNENPASPPETFSPSPPPDNPDEQMNDTVNKTDQVDCSDLSEHNGLDREESMEVEAPVANKSGSGTSSGSHSSTAPSSSSSSSSSGGGGSSSTGTSAITTSLPQLGDLT (SEQ ID NO: 1). The repression domain of SUDS3 is: MSAAGLLAPAPAQAGAPPAPEYYPEEDEELESAEDDERSCRGRESDEDTEDASETDLAKHDEEDYVEMKEQMYQDKLASLKRQLQQLQEGTLQEYQKRMKKLDQQYKERIRNAELFLQLETEQVERNYIKEKKAAVKEFEDKKVELKENLIAELEEKKKMIENEKLTMELTGDSMEVKPIMTRKLRRRPNDPVPIPDKRRKPAPAQLNYLLTDEQIMEDLRTLNKLKSPKRPASPSSPEHLPATPAESPAQRFEARIEDGKLYYDKRWYHKSQAIYLESKDNQKLSCVISSVGANEIWVRKTSDSTKMRIYLGQLQRGLFVIRRRSAA (SEQ ID NO: 2).

[0032] In some embodiments, the Cas fusion protein comprises, comprises, or consists essentially of a Cas protein and a SALL1 repression domain, or comprises, comprises, or consists essentially of a Cas protein and a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:1. In some embodiments, the SALL1 repression domain, or a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:1, is bound to the N-terminal amino acid of the Cas protein. In some embodiments, the SALL1 repression domain, or a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:1, is bound to the C-terminal amino acid of the Cas protein. In some embodiments, the Cas fusion protein comprises, comprises, or consists essentially of a Cas protein and a SUDS3 repression domain, or comprises, comprises, or consists essentially of a Cas protein and a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:2. In some embodiments, the SUDS3 repression domain, or a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:2, is bound to the N-terminal amino acid of the Cas protein. In some embodiments, the SUDS3 repression domain, or a repression domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:2, is bound to the C-terminal amino acid of the Cas protein.

[0033] In some embodiments, the Cas fusion protein comprises, essentially comprises, or consists of a Cas protein and both a SALL1 repression domain and a SUDS3 repression domain. In some embodiments, the Cas fusion protein is constructed of one of the following (listed from N-terminus to C-terminus): [Cas protein]-[SALL1 repression domain]-[SUDS3 repression domain] [Cas protein]-[SUDS3 repression domain]-[SALL1 repression domain] [SALL1 repression domain]-[SUDS3 repression domain]-[Cas protein] [SUDS3 repression domain]-[SALL1 repression domain]-[Cas protein] [SALL1 repression domain]-[Cas protein]-[SUDS3 repression domain] [SUDS3 repression domain]-[Cas protein]-[SALL1 repression domain] In some embodiments, the Cas fusion protein comprises a SALL1 repression domain and a SUDS3 repression domain, wherein the SALL1 repression domain has, comprises primarily, or consists of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:1; and the SUDS3 repression domain has, comprises primarily, or consists of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:2. Also, in some embodiments, the Cas fusion protein comprises a SALL1 repression domain and a SUDS3 repression domain, wherein the SALL1 repression domain has, comprises primarily, or consists of the same sequence as SEQ ID NO:1; and the SUDS3 repression domain has, comprises primarily, or consists of the same sequence as SEQ ID NO:2.

[0034] In some embodiments, the Cas fusion protein comprises, comprises essentially, or consists of a Cas protein and two or more copies of both a SALL1 repression domain and a SUDS3 repression domain, In some embodiments, the Cas fusion protein is constructed as one of the following: [SALL1 repression domain]-[SUDS3 repression domain]-[Cas protein]-[SALL1 repression domain]-[SUDS3 repression domain] [SALL1 repression domain]-[SUDS3 repression domain]-[Cas protein]-[SUDS3 repression domain]-[SALL1 repression domain] [SUDS3 repression domain]-[SALL1 repression domain]-[Cas protein]-[SALL1 repression domain]-[SUDS3 repression domain] [SUDS3 repression domain]-[SALL1 repression domain]-[Cas protein]-[SUDS3 repression domain]-[SALL1 repression domain] Also, in some embodiments, the Cas fusion protein has multiple SALL1 repression domains and multiple SUDS3 repression domains, where each SALL1 repression domain has, primarily comprises, or consists of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:1. Furthermore, each SUDS3 repression domain has, primarily comprises, or consists of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:2. Also, in some embodiments, the Cas fusion protein has multiple SALL1 repression domains and multiple SUDS3 repression domains, where each SALL1 repression domain has, primarily comprises, or consists of the same sequence as SEQ ID NO:1. Furthermore, each SUDS3 repression domain has, primarily comprises, or consists of the same sequence as SEQ ID NO:2. In some embodiments, the Cas fusion protein has a domain of another inhibitory protein, "R." In some embodiments, "R" is selected from the following group: NIPP1 inhibitory domain, KRAB inhibitory domain, DNMT3A inhibitory domain, BCL6 inhibitory domain, CbpA inhibitory domain, H-NS inhibitory domain, MBD3 inhibitory domain, and KRAB-Me-CP2 inhibitory domain.

[0035] The NIPP1 repression domain is represented as follows: MVQTAVVPVKKKRVEGPGSLGLEESGSRRMQNFAFSGGLYGGLPPTHSEAGSQPHGIHGTALIGGLPMPYPNLAPDVDLTPVVPSAVNMNPAPNPAVYNPEAVNEPKKKKYAKEAWPGKKPTPSLLI (SEQ ID NO: 34) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:34. The KRAB repression domain is represented as follows: MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLV (SEQ ID NO: 35) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:35. The BCL6 repression domain is represented as follows: MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEM (SEQ ID NO: 173) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:173. The CbpA repression domain is represented as follows: MELKDYYAIMGVKPTDDLKTIKTAYRRLARKYHPDVSKEPDAEARFKEVAEAWEVLSDEQRRAEYDQMWQHRNDPQFNRQFHHGDGQSFNAEDFDDIFSSIFGQHARQSRQRPATRGHDIEIEVAVFLEETLTEHKRTISYNLPVYNAFGMIE QEIPKTLNVKIPAGVGNGQRIRLKGQGTPGENGGPNGDLWLVIHIAPHPLFDIVGQDLEIVVPVSPWEAALGAKVTVPTLKESILLTIPPGSQAGQRLRVKGKGLVSKKQTGDLYAVLKIVMPPKPDENTAALWQQLADAQSSFDPRKDWGKA (SEQ ID NO: 174) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:174. The H-NS repression domain is represented as follows: MSEALKILNNIRTLRAQARECTLETLEEMLEKLEVVVNERREEESAAAAEVEERTRKLQQYREMLIADGIDPNELLNSLAAVKSGTKAKRAQRPAKYSYVDENGETKTWTGQGRTPAVIKKAMDEQGKSLDDFLIKQ (SEQ ID NO: 175) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:175. The MBD3 repression domain is represented as follows: MERKRWECPALPQGWEREEVPRRSGLSAGHRDVFYYSPSGKKFRSKPQLARYLGGSMDLSTFDFRTGKMLMSKMNKSRQRVRYDSSNQVKGKPDLNTALPVRQTASIFKQPVTKITNHPSNKVKSDPQKAVDQPRQLFWEKKLSGLNAFDIAEELVKTMDLPKGLQGVGPGCTDETLLSAIASALHTSTMPITGQLSAAVEKNPGVWLNTTQPLCKAFMVTDEDIRKQEELVQQVRKRLEEALMADMLAHVEELARDGEAPLDKACAEDDDEEDEEEEEEEPDPDPEMEHV (SEQ ID NO: 176) or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO:176. The KRAB-MeCP2 repression domain is represented as follows: MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVSGGGSGGSGSSPKKKRKVEASVQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKE (SEQ ID NO: 177) Or a sequence which is at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO: 177. Examples of constructs of these sequences are depicted below: [Cas protein]-[SALL1 repression domain]-[R] [Cas protein]-[SUDS3 repression domain]-[R] [R]-[SUDS3 repression domain]-[Cas protein] [R]-[SALL1 repression domain]-[Cas protein] [Cas protein]-[R]-[SUDS3 repression domain] [Cas protein]-[R]-[SALL1 repression domain] [SALL1 repression domain]-[R]-[Cas protein] [SUDS3 repression domain]-[R]-[Cas protein] [R]-[Cas protein]-[SUDS3 repression domain] [R]-[Cas protein]-[SALL1 repression domain] [SALL1 repression domain]-[Cas protein]-[R] [SUDS3 repression domain]-[Cas protein]-[R]

[0036] Also, in one embodiment, the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and each of a SALL1 repression domain, a SUDS3 repression domain, and an "R" repression domain. When all three repression domains are present, the domains are all present on the C-terminal amino acid of the Cas protein, all present on the N-terminal amino acid of the Cas protein, two present on the C-terminal amino acid of the Cas protein and one present on the N-terminal amino acid of the Cas protein, or two present on the N-terminal amino acid of the Cas protein and one present on the C-terminal amino acid of the Cas protein. Examples of construction of these sequences are provided below. [Cas protein]-[SALL1 repression domain]-[R]-[SUDS3 repression domain] [Cas protein]-[SALL1 repression domain]-[SUDS3 repression domain]-[R] [Cas protein]-[SUDS3 repression domain]-[SALL1 repression domain]-[R] [Cas protein]-[SUDS3 repression domain]-[R]-[SALL1 repression domain] [Cas protein]-[R]-[SUDS3 repression domain]-[SALL1 repression domain] [Cas protein]-[R]-[SALL1 repression domain]-[SUDS3 repression domain] [SALL1 repression domain]-[R]-[SUDS3 repression domain]-[Cas protein] [SALL1 repression domain]-[SUDS3 repression domain]-[R]-[Cas protein] [SUDS3 repression domain]-[SALL1 repression domain]-[R]-[Cas protein] [SUDS3 repression domain]-[R]-[SALL1 repression domain]-[Cas protein] [R]-[SUDS3 repression domain]-[SALL1 repression domain]-[Cas protein] [R]-[SALL1 repression domain]-[SUDS3 repression domain]-[Cas protein] [SALL1 repression domain]-[Cas protein]-[R]-[SUDS3 repression domain] [SALL1 repression domain]-[Cas protein]-[SUDS3 repression domain]-[R] [SUDS3 repression domain]-[Cas protein]-[SALL1 repression domain]-[R] [SUDS3 repression domain]-[Cas protein]-[R]-[SALL1 repression domain] [R]-[Cas protein]-[SUDS3 repression domain]-[SALL1 repression domain] [R]-[Cas protein]-[SALL1 repression domain]-[SUDS3 repression domain] [R]-[SUDS3 repression domain]-[Cas protein]-[SALL1 repression domain] [SUDS3 repression domain]-[R]-[Cas protein]-[SALL1 repression domain] [SALL1 repression domain]-[R]-[Cas protein]-[SUDS3 repression domain] [R]-[SALL1 repression domain]-[Cas protein]-[SUDS3 repression domain] [SUDS3 repression domain]-[SALL1 repression domain]-[Cas protein]-[R] [SALL1 repression domain]-[SUDS3 repression domain]-[Cas protein]-[R] As a non-limiting example, in one embodiment, in the Cas fusion protein, the Cas protein is dCas9 or dCas12, such as dCas12a, and the Cas fusion protein comprises, comprises essentially, or consists of both a SALL1 repression domain and a SUDS3 repression domain.

[0037] Examples of amino acid sequences of fusion proteins of the present invention include, but are not limited to, the following: MCP-SALL1-SUDS3 amino acid sequence: MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSMSRRKQAKPQ HFQSDPEVASLPRRDGDTEKGQPSRPTKSKDAHVCGRCCAEFFELSDLLLHKKNCTKNQLVLIVNENPASPPETFSPSPPPDNPDEQMNDTVNKTDQVDCCSDLSEHNGLDREESMEVEAPVANKSGSGTSSGSHSSTAPSSSSSSSSSSGGGGSSSTGTSAITTSLPQLGD LTGSGGGSGGSGSMSAAGLLAPAPAQAGAPPAPEYYPEEDEELESAEDDERSCRGRESDEDTEDASETDLAKHDEEDYVEMKEQMYQDKLASLKRQLQQLQEGTLQEYQKRMKKLDQQYKERIRNAELFLQLETEQVERNYIKEKKAAVKEFEDKKVELKENLIAELEEKKKMIENEKLTMELTGDSMEVKPIMTRKLRRRPNDPVPIPDKRRKPAPAQLNYLLTDEQIMEDLRTLNKLKSPKRPASPSSPEHLPATPAESPAQRFEARIEDGKLYYDKRWYHKSQAIYLESKDNQKLSCVISSVGANEIWVRKTSDSTKMRIYLGQLQRGLFVIRRRSAA (SEQ ID NO: 41) SEQ ID NO:41, for example, is encoded by a nucleic acid having, essentially comprising, or consisting of SEQ ID NO:170. SUDS3-SALL1-Active Cas9 amino acid sequence:

[0038] SEQ ID NO:171 is, for example, encoded by a nucleic acid having, essentially comprising or consisting of SEQ ID NO:172.

[0039] Protein and polypeptide sequences that are fragments of SEQ ID NOs: 41 and 171, and derivatives of these sequences having substantially the same function, are within the scope of the present invention. In certain embodiments, the protein and polypeptide sequences are at least 80%, at least 85%, at least 90% or at least 95% similar to either SEQ ID NOs: 41 or 171. Additionally, nucleic acid sequences having, comprising essentially or consisting of SEQ ID NOs: 170 or 172, or their complements, or sequences that are at least 80%, at least 85%, at least 90% or at least 95% similar to either SEQ ID NOs: 170 and 172, are within the scope of the present invention.

[0040] (Linker) When the repression domain is fused to a Cas protein, the fusion is a direct bond (e.g., a covalent bond) between the N-terminal amino acid of the repression protein and the C-terminal amino acid of the Cas protein, or between the C-terminal amino acid of the repression domain and the N-terminal amino acid of the Cas protein. However, instead of connecting or forming a direct bond between two components (i.e., two or more repression domains, or a repression domain and a Cas protein), a linker can be used. In some embodiments, the linker has, consists essentially of, or consists of an amino acid sequence, e.g., 1 to 100 amino acids in length, 3 to 90 amino acids in length, or 10 to 50 amino acids in length. Also, in some embodiments, the linker has, consists essentially of, or consists of a sequence that is not an amino acid sequence.

[0041] When the linker is sandwiched between the Cas protein and the repression domain, the linker is referred to as a Cas linker. In one embodiment, the Cas protein has a C-terminal amino acid, and the Cas fusion protein has a Cas linker. In this case, the Cas linker is covalently bound to the C-terminal amino acid of the Cas protein. In another embodiment, the Cas protein has an N-terminal amino acid, and the Cas fusion protein has a Cas linker. In this case, the Cas linker is covalently bound to the N-terminal amino acid of the Cas protein. In another embodiment, the Cas protein has a C-terminal amino acid and an N-terminal amino acid, and the Cas fusion protein has two Cas linkers. In this case, the first Cas linker is covalently bound to the C-terminal amino acid of the Cas protein, and the second Cas linker is covalently bound to the N-terminal amino acid of the Cas protein. Here, when a first Cas linker and a second Cas linker are present, the first Cas linker can bind to the first repression domain, and the second Cas linker can bind to the second repression domain.

[0042] In some embodiments, there is one Cas linker, and the Cas linker has, comprises, or consists of a sequence at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO: 7: GSGGGSGGSGS. Also, in some embodiments, the Cas linker has, comprises, or consists of a sequence of SEQ ID NO: 7. In some embodiments, there are two Cas linkers, and each Cas linker has, comprises, or consists of a sequence at least 80%, at least 85%, at least 90% or at least 95% similar to SEQ ID NO: 7: GSGGGSGGSGS. In some embodiments, each of the two Cas linkers has, comprises, or consists of a sequence of SEQ ID NO: 2. Moreover, in some embodiments, the Cas linker is covalently linked to a Cas protein and to a repression domain having, comprising, or consisting essentially of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 1. In some embodiments, the Cas linker is covalently linked to a Cas protein and to a repression domain having, comprising, or consisting essentially of the sequence of SEQ ID NO:1. In some embodiments, the Cas linker is covalently linked to a Cas protein and to a repression domain having, comprising, or consisting essentially of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 2. Also, in some embodiments, the Cas linker is covalently linked to a Cas protein and to a repression domain having, comprising, or consisting essentially of the sequence of SEQ ID NO:2.

[0043] When a Cas fusion protein has two or more repression domains, the two or more repression domains are located on the same side of the Cas protein, i.e., either on the N-terminal side or on the C-terminal side, and each pair of two repression domains can be directly, e.g., covalently, linked to each other, or can be linked via a linker. The linker linking the two repression domains can also be referred to as a repression linker. The inhibitory linker may be the same as or different from the Cas linker. In some embodiments, the inhibitory linker has, comprises, or consists of a sequence at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 7: GSGGGSGGSGS. In some embodiments, the inhibitory linker has, comprises, or consists of a sequence of SEQ ID NO: 7. Also, as a non-limiting example, in an embodiment, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has a C-terminal amino acid, and the Cas fusion protein further has a Cas linker and an inhibitory linker. The Cas linker is covalently linked to the C-terminal amino acid of the Cas protein and the N-terminal amino acid of the SALL1 inhibitory domain. The inhibitory linker is flanked by the SALL1 inhibitory domain and the SUDS3 inhibitory domain.

[0044] Also, as another non-limiting example, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has a C-terminal amino acid, and the Cas fusion protein further has a Cas linker and an inhibitory linker. The Cas linker is covalently linked to the C-terminal amino acid of the Cas protein and the SUDS3 inhibitory domain. The inhibitory linker binds to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain. In yet another non-limiting example, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has an N-terminal amino acid, and the Cas fusion protein further has a Cas linker and an inhibitory linker. The Cas linker is covalently linked to the N-terminal amino acid of the Cas protein and the SUDS3 inhibitory domain. The inhibitory linker binds to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain. In yet another non-limiting example, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has an N-terminal amino acid, and the Cas fusion protein further has a Cas linker and an inhibitory linker. The Cas linker is covalently linked to the N-terminal amino acid of the Cas protein and the SALL1 inhibitory domain. The inhibitory linker binds to both the SALL1 inhibitory domain and the inhibitory SUDS3 domain. In addition, as another non-limiting example, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has an N-terminal amino acid and a C-terminal amino acid, and the Cas fusion protein further has a first Cas linker and a second Cas linker. The first Cas linker is covalently linked to the N-terminal amino acid of the Cas protein and the SUDS3 repression domain. The second Cas linker is linked to the C-terminal amino acid of the Cas protein and the SALL1 repression domain. In addition, as another non-limiting example, in the Cas fusion protein of the present invention, the Cas protein is dCas9 or dCas12, such as dCas12a protein. Here, the Cas protein has an N-terminal amino acid and a C-terminal amino acid, and the Cas fusion protein further has a first Cas linker and a second Cas linker. The first Cas linker is covalently linked to the C-terminal amino acid of the Cas protein and the SUDS3 repression domain. The first Cas linker is linked to the C-terminal amino acid of the Cas protein and the SUDS3 repression domain. The second Cas linker is linked to the N-terminal amino acid of the Cas protein and the SALL1 repression domain.

[0045] Furthermore, as another example, in certain embodiments, the Cas fusion protein has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:10. . In one embodiment, the Cas fusion protein has a sequence identical to SEQ ID NO:10. In one embodiment, the Cas fusion protein is represented as follows: [dCas9]-[Cas linker]-[SALL1 repression domain]-[repression linker]-[SUDS3 repression domain] Also, in one embodiment, the Cas fusion protein is as follows: [dCas9]-[Cas linker]-[SUDS3 repression domain]-[repression linker]-[SALL1 repression domain] In one embodiment, the Cas fusion protein is: [dMAD7]-[Cas linker]-[SALL1 repression domain]-[repression linker]-[SUDS3 repression domain] In one embodiment, the Cas fusion protein is: [dMAD7]-[Cas linker]-[SUDS3 repression domain]-[repression linker]-[SALL1 repression domain] In one embodiment, the Cas fusion protein is: [SALL1 repression domain]-[repression linker]-[SUDS3 repression domain]-[Cas linker]-[dCas9] In one embodiment, the Cas fusion protein is: [SUDS3 repression domain]-[repression linker]-[SALL1 repression domain]-[Cas linker]-[dCas9] In one embodiment, the Cas fusion protein is: [SALL1 repression domain]-[repression linker]-[SUDS3 repression domain]-[Cas linker]-[dMAD7] In one embodiment, the Cas fusion protein is: [SUDS3 repression domain]-[repression linker]-[SALL1 repression domain]-[Cas linker]-[dMAD7]

[0046] (gRNA) The Cas fusion proteins of the present invention can be used with gRNA. In some embodiments, the gRNA comprises 30 to 180 nucleotides, 45 to 135 nucleotides, or 60 to 120 nucleotides. The gRNA can be chemically synthesized or enzymatically synthesized. If enzymatically synthesized, the synthesis can be performed in vitro, in vivo, or ex vivo. The nucleotides of the gRNA can be all modified ribonucleotides, all unmodified ribonucleotides, or a combination of modified and unmodified ribonucleotides. In some embodiments, the gRNA contains one or more modifications, such as a 2' modification, e.g., a 2'-O-alkyl, such as 2'-O-methyl or 2'-O-ethyl, or a 2'-halogen modification, such as 2'fluoro. In some embodiments, the gRNA contains one or more modified internucleotide linkages, such as a phosphorothioate linkage. In one embodiment, the gRNA has the following modifications: 2'-O-methyl modifications on the first and second most terminal 5' nucleotides; a 2'-O-methyl modification on the penultimate 3' nucleotide (the second most terminal 3' nucleotide) and the third penultimate 3' nucleotide (the third most terminal 3' nucleotide); All other nucleotides are unmodified at the 2' position. phosphothioate bonds between the first and second most 5' nucleotides, between the second and third most 5' nucleotides, between the penultimate 3' nucleotide and the penultimate 3' nucleotide, and between the penultimate 3' nucleotide and the most terminal 3' nucleotide; · The bonds between all other nucleotides are phosphodiester bonds.

[0047] In one embodiment, the gRNA has the following modifications: 2'O-methyl modifications on the first and second most terminal 5' nucleotides and All other nucleotides are unmodified at the 2' position phosphothioate bonds between the first and second most 5' nucleotides and between the second and third most 5' nucleotides; · The bonds between all other nucleotides are phosphodiester bonds.

[0048] In some embodiments, the gRNA comprises, primarily comprises, or consists of the crRNA. In some embodiments, the gRNA comprises, primarily comprises, or consists of the crRNA and tracrRNA sequences. When the gRNA comprises, primarily comprises, or consists of the crRNA and tracrRNA sequences, the crRNA and tracrRNA are part of the sgRNA or each is present on a separate single strand of nucleotides to form the crRNA and tracrRNA molecules, each being a polynucleotide. Also, when the two sequences are two separate nucleotides, one of the tracrRNA and crRNA molecules is referred to as the first RNA molecule, and the other of the tracrRNA and crRNA molecules is referred to as the second RNA molecule. When the tracrRNA and crRNA molecules are present separately, the total number of nucleotides of the two molecules can be combined to be the same as, for example, the sgRNA described in various embodiments of the present invention. Also, chemical modifications to the sgRNA can be present on one or both of the tracrRNA and crRNA molecules. Furthermore, multiple internucleotide modifications between sgRNAs can be present in either or both of the tracrRNA and crRNA molecules, and modifications described as being present at the 5' or 3' end of the gRNA, in the case of an sgRNA, can be present at the 5' or 3' end of the sgRNA, and in the case of separate tracrRNA and crRNA molecules, each having a 5' or 3' end, can be present at the 5' or 3' end of the tracrRNA or crRNA molecule. The crRNA comprises, consists essentially of, or consists of a Cas-associated region and a spacer region (also referred to as a target region). The target region is sufficiently complementary to and capable of hybridizing with a predetermined target site of interest. In various embodiments, the target-specific element of the guide sequence has from about 10 nucleotides to about 25 nucleotides, but can have up to, for example, 36 nucleotides. In some embodiments, the base-paired region between the guide sequence and its corresponding target site sequence has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides. In some embodiments, the target region has a length of 12 to 30 nucleotides, or 14 to 25 nucleotides, or about 17 to 20 nucleotides, or about 14 nucleotides, or about 20 nucleotides. The target region has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementarity to a region of the target dsDNA of at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, or between 14 and 20 contiguous nucleotides.

[0049] When used with an active Cas protein whose target region is about 20 nucleotides long and capable of cleaving both DNA strands, a double-stranded break occurs in the target DNA, resulting in an insertion / deletion (indel) in the genome. If suppression is desired without creating indels, an inactive Cas9 protein, such as an inactive Cas9 protein, may be used. On the other hand, when using an active Cas9 protein that is capable of cleaving two strands of DNA or a Cas nickase mutant capable of cleaving one strand of the target DNA, a gRNA with a relatively short target region, such as about 14 nucleotides long, may be used for gene suppression. Guide RNAs with a 20 nucleotide long target region guide the active Cas9 suppressor to another genomic site for DNA cleavage and subsequent editing. The Cas-associated region is, for example, a portion of the crRNA having a length of about 18-36 nucleotides. The crRNA enables the crRNA (and thus the gRNA) to remain bound to the Cas protein. In one embodiment, binding to the Cas protein is possible in the absence of the tracrRNA. In another embodiment, said binding requires the presence of the tracrRNA.

[0050] When the crRNA requires the presence of the tracrRNA to bind to the Cas protein, the Cas-associated region hybridizes to an anti-repeat region in the tracrRNA, including a terminal region 3' to the anti-repeat region that is not complementary to any region of the crRNA. When there is hybridization between the Cas-associated regions, also called the repeat and anti-repeat regions, the repeat:anti-repeat region of the gRNA scaffold is divided into three parts: the stem, the expansion, and the superstem. The stem is six base pairs in length and forms both Watson-Crick base pairs and no Watson-Crick base pairs. This is followed by a six nucleotide expansion structure. Finally, there is a superstem that consists of a four base pair structure. When the gRNA is sgRNA, in one embodiment, the single strand comprises a complementary region. When the complementary region hybridizes, the region in the single strand can bind to Cas proteins, such as type II Cas enzymes, including but not limited to Cas9 in active or inactive form, and type V Cas enzymes, such as Cas12c, Cas12d, Cas12e, and Cas12f in active or inactive form. In another embodiment, when the gRNA is sgRNA, there is no complementary sequence, but the sgRNA can bind to Cas enzymes. The Cas enzymes are type V Cas enzymes, such as Cas12a, MAD7 (a genetically engineered mutant of ErCas12a), Cas12h, Cas12i, and Cas12j (CasΦ), in active or inactive form.

[0051] A non-limiting example of an sgRNA is shown in Figure 2. The sgRNA in Figure 2 has the following sequence (SEQ ID NO: 11): 5'mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U3 * indicates a phosphorothioate bond, and N represents any of A, C, G, or U. As shown in Figure 2, the crRNA and tracrRNA regions are connected by a tetraloop, and the tracrRNA has three stem-loop regions. This example sgRNA is 100 nucleotides. In an embodiment, the sgRNA is 60 to 120 nucleotides or 90 to 110 nucleotides long. The depicted N region is 20 nucleotides in length. In certain embodiments, the N region is 10 to 36 nucleotides in length, 14 to 26 nucleotides in length, or 18 to 22 nucleotides in length.

[0052] Various tracrRNA sequences are known in the art, and are exemplified by SEQ ID NOs: 27-34 and active portions of these sequences. GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:28); UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 29); AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 30); CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 31); UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO: 32); UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:33); and UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:34). As used herein, an active portion of a tracrRNA maintains the ability to form a complex with a Cas protein, such as Cas9, dCas9 or nCas9.

[0053] Also, by way of non-limiting example, the gRNA may comprise a programmable gRNA in which the crRNA described above is fused to a tracrRNA to mimic the native crRNA:tracrRNA duplex. An example of this hybrid includes crRNA:tracrRNA, gRNA sequence:5'-(20 nucleotide long guide). GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3' (SEQ ID NO:27). Methods for generating crRNA:tracrRNA hybrid RNAs (also known as sgRNAs) are known in the art. In some embodiments, the crRNA and tracrRNA are provided as sgRNAs, with the two components linked to each other via a tetra-stem loop. In some embodiments, the repeat-anti-repeat region is extended, for example, by 2, 3, 4, 5, 6, 7 or more than 7 bases on either side of the repeat-anti-repeat. In other embodiments, the repeat-anti-repeat region extends 7 nucleotides on either side of the stem. This 7-base extension on each side results in a region that is 14 base pairs long. In some embodiments, the extension can be more than 7 bases. See, for example, WO2014099750, US20140179006, and US20140273226 for additional disclosure of tracrRNA, the contents of which are incorporated herein by reference in their entirety.

[0054] In some embodiments, the tracrRNA is of or derived from Streptococcus pyogenes. In some embodiments, the target site is present in DNA. The target nucleic acid strand in the DNA can be either of two strands, for example, in the genomic DNA of the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stabilized plasmids. However, it will be understood that the current method can also be performed with other dsDNA present in the host cell, such as unstable plasmid DNA, viral DNA, and plasmid DNA, so long as there is a Cas target site. In an embodiment, rather than using a fusion protein together with a gRNA, a fusion protein of the invention can be used together with a scoutRNA and an applicable crRNA. For example, a fusion protein of the invention can be used as part of a system or complex having: (a) a crRNA, wherein the crRNA is 30 to 60 nucleotides long, said crRNA having a Cas-associated region and a target region, said Cas-associated region being 15 to 30 nucleotides long and said target region being 15 to 30 nucleotides long; (b) a scoutRNA, wherein said scoutRNA is 20 to 100 nucleotides long, said scoutRNA having an anti-repeat region, wherein said anti-repeat region is 3 to 10 nucleotides long and said anti-repeat region is complementary to at least three consecutive nucleotides in the Cas-associated region. Furthermore, said anti-repeat region is hybridizable to said at least three consecutive nucleotides in the Cas-associated region to form a hybridized region. Furthermore, the crRNA and the scoutRNA can maintain binding with the RNA-binding domain of V-type Cas protein.

[0055] (RNA repression domain complex) In one embodiment, the invention is directed to the use of an RNA repression domain complex that comprises, comprises essentially, or consists of a gRNA, such as a gRNA as described above, or a scoutRNA and / or a crRNA capable of binding to a scoutRNA as described above, a ligand binding site, a ligand, and one or more domains. The RNA repression domain complex can be used to bind to a Cas fusion protein of the invention or to another Cas protein that is not a fusion protein. The gRNA, scoutRNA, or crRNA capable of binding to the scoutRNA can be fused directly to the ligand binding site or can be linked to the ligand binding site via a ligand binding site linker. The linker is linked to the repression domain directly or via a ligand linker. The repression domain can be any effector. When one or both of the ligand binding site linker and the ligand linker are present, each of the ligand binding site linker and the ligand linker comprises, primarily comprises, or consists of nucleotides, amino acids, and other organic and inorganic moieties, or combinations thereof.

[0056] Non-limiting examples of ligand binding site-ligand pairs for use in various embodiments of the present invention are shown in Table 1. Both unmodified and chemically modified ligand binding sites and ligands are within the scope of the present invention.

[0057] [Table 1]

[0058] 1. Telomerase Ku binding site / Ku heterodimer a) Ku-binding hairpin 5'- UUCUUGUCGUACUUAUAGAUCGCUACGUUAUUUCAAUUUUGAAAAUCUGAGUCCUGGGAGUGCGGA-3' (SEQ ID NO: 12) b) Heterodimer MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPFDMSIQCIQSVYISKIISSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELDQFKGQQGQKRFQDMMGHGSDYSLSEVLWVCAN LFSDVQFKMSHKRIMLFTNEDNPHGNDSAKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDIISIAEDEDLRVHFEESSKLEDLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKALKPPPIKLYRETNEPVKTKTRTFN TSTGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFDDPGLMLMGFKPLVLLKKHHYLRPSLFVYPEESLVIGSSTLFSALLIKCLEKEVAALCRYTPRRNIPPYFVALVPQEEELDDQKIQVTPPGFQLVFLPFADDKRKMPFTEKIMATPEQVGKMKAIVEKLRFTYRSDSFENPVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRLGSLVDEFKELVYPPDYNPEGKVTKRKHDNEGSGSKRPKVEYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKSGLKKQELLEALTKHFQD (SEQ ID NO: 13) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFGTDGTDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQHE TIGKKFEKRHIEIFTDLSRFSKSQLDIIIHSLKKCDISERHSIHWPCRLTIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDDDETEVLKEDIIQGFRYGSDIVP FSKVDEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVLKVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLVYVQLPFMEDLRQYMFSSLKNSKKYAPTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAK (SEQ ID NO: 14) 2. Telomerase Sm7 binding site / Sm7 homoheptamer c) Sm consensus site (single stranded) 5'-AAUUUUUGGA-3' (SEQ ID NO: 15) d) Monomeric Sm-like proteins (Archaea) GSVIDVSSQRVNVQRPLDALGNSLNSPVIIKLKGDREFRGVLKSFDLHMNLVLNDAELEDGEVTRRLGTVLIRGDNIVYISP (SEQ ID NO: 16)

[0059] 3. MS2 phage operator stem loop / MS2 coat protein a) MS2 phage operator stem loop 5'- GCACAUGAGGAUCACCCAUGUGC-3' (SEQ ID NO: 17) b) MS2 coat protein MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 18) 4. PP7 phage operator stem loop / PP7 coat protein a) PP7 phage operator stem loop 5'-AUAAGGAGUUUAUAUGGAAACCCUUA-3' (SEQ ID NO: 19) b) PP7 coat protein (PCP) (SEQ ID NO: 20) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR. 5. SfMu Com stem-loop / SfMu Com binding protein a) SfMu Com stem loop 5'-CUGAAUGCCUGCGAGCAUC-3' (SEQ ID NO: 21) b) SfMu Com-binding protein MKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRY (SEQ ID NO:22)

[0060] 6.BoxB Aptamer / Lambda N22 Plus e) BoxB aptamer 5'- GCCCUGAAGAAGGGC-3' (SEQ ID NO: 23) f) Lambda N22 Plus Protein MNARTRRRERRAEKQAQWKAAN (SEQ ID NO:24) 7. Cys4-binding stem-loop / Cys4[H29A] a)Cys4 binding site 5'-CUGCCGUAUAGGCAGC-3' (SEQ ID NO: 25) b) Cys4[H29A] MDHYLDIRLRPDPEFPPAQLMSVLFGKLAQALVAQGGDRIGVSFPDLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF (SEQ ID NO: 26) 8. Q beta-binding stem loop [Q65H] a) Q beta phage operator stem loop 5'- ATGCTGTCTAAGACAGCAT -3' (SEQ ID NO: 180) b) Q beta coat protein [Q65H] MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVHVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO: 181)

[0061] For each of the above sequences, for example, the same sequence or sequences with one or more insertions, deletions or substitutions in one or both sequences of the binding pair can be used. As non-limiting examples, sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the above sequences for one or both of the binding pairs can be used. In some embodiments, a complex is formed that comprises, primarily comprises, or consists of a Cas fusion protein of the invention and an RNA repression domain complex of the invention. Thus, if the Cas fusion protein comprises a Cas protein fused to a SUDS3 repression domain, the ligand can be fused to a SALL1 repression domain, or another unknown or known repression domain. Similarly, if the Cas fusion protein comprises a Cas protein fused to a SALL1 repression domain, the ligand can be fused to a SUDS3 repression domain, or another unknown or known repression domain. Also, in some embodiments, the Cas fusion protein comprises, primarily comprises, or consists of a Cas protein, a SALL1 repression domain, and a SUDS3 repression domain. Furthermore, the RNA repression domain complex comprises, primarily comprises, or consists of a gRNA, a ligand binding site, a ligand, and one or more repression domains other than SALl1 or SUDS3. As a non-limiting example, the one or more repression domains can be selected from the group consisting of NIPP1, KRAB, and DNMT3A.

[0062] Alternatively, an RNA repression domain complex can be used with a Cas enzyme that is not part of a Cas fusion protein complex. For example, the RNA repression domain complex has a gRNA, a ligand binding site, and one or both of the SUDS3 repression domain and the SALL1 repression domain as defined above. The repression linker as defined above is between the SUDS3 repression domain and the SALL1 repression domain, and the ligand can bind to the SALL1 repression domain and one of the SUDS3 repression domain directly or via a linker.

[0063] (Nucleic acid encoding a Cas fusion protein) In one embodiment, the invention provides a nucleic acid encoding a fusion protein of the invention, the nucleic acid may be single-stranded, double-stranded, or have at least one region that is single-stranded and at least one region that is double-stranded, and the nucleic acid may comprise, consist essentially of, or consist of RNA or DNA. In some embodiments, the nucleic acid encoding only the fusion protein has only the nucleotides for the fusion protein and any linker present therein, while in other embodiments, the nucleic acid encoding the fusion protein is part of a larger nucleic acid or vector. In some embodiments, the present invention is also directed to a vector having a nucleic acid encoding the fusion protein of the present invention. In some embodiments, the vector is a plasmid or a viral vector. When the vector is a viral vector, in some embodiments, the viral vector is a lentiviral vector. In some embodiments, rather than a vector having a polynucleotide sequence encoding a Cas fusion protein, the present invention is directed to an mRNA encoding the Cas fusion protein of the present invention.

[0064] In one embodiment, the nucleic acid has sequences encoding a Cas protein and at least one repression domain, such as SUDS3 or SALL1, and in one embodiment, the nucleic acid has sequences encoding a Cas protein and at least two repression domains, such as SUDS3 and SALL1, and in one embodiment, the nucleic acid has sequences encoding a Cas protein and at least three repression domains, such as SUDS3 and SALL1, and one or more of NIPP1, KRAB, and DNMT3A. In one embodiment, the nucleic acid has, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:4, which encodes the SALL1 repression domain. ATG AGT AGG AGA AAA CAA GCA AAA CCA CAG CAC TTT CAA AGT GAT CCT GAG GTA GCA AGC CTT CCA CGG GAC GGT GAC ACG GAG AAG GGT CAA CCA AGT CGA CCC ACG AAA AGC AAA GAT GCT CAT GTA TGT GGA CGC TGT TGC GCA GAA TTT TTT GAA TTG TCC GAT CTT CTT CTT CAC AAA AAG AAC TGC ACG AAG AAT CAG TTG GTT TTG ATA GTA AAC GAA AAT CCA GCT TCA CCC CCA GAA ACT TTT TCC CCG TCA CCT CCT CCA GAT AAT CCT GAT GAA CAA ATG AAT GAC ACC GTA AAT AAA ACC GAC CAA GTA GAC TGT TCT GAT TTG AGC GAA CAC AAC GGT TTG GAT CGA GAA GAG TCA ATG GAA GTA GAG GCC CCA GTT GCC AAT AAG TCA GGC AGC GGT ACT TCT TCC GGC TCC CAC AGT TCA ACA GCT CCA TCC TCA AGT AGT TCA AGC TCT TCT AGT TCA GGA GGC GGG GGG AGT AGC TCT ACC GGC ACT TCT GCC ATC ACA ACC TCA CTT CCT CAG CTT GGA GAC TTG ACA. In one embodiment, the nucleic acid sequence has, essentially comprises, or consists of the sequence of SEQ ID NO:4. In one embodiment, the nucleic acid sequence has, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:5, which encodes a SUDS3 repression domain. ATG TCT GCA GCT GGC CTT TTG GCT CCT GCC CCC GCA CAA GCG GGA GCT CCT CCC GCA CCG GAG TAC TAT CCA GAA GAG GAT GAG GAA CTG GAA TCT GCC GAA GAC GAC GAG CGC AGT TGC CGG GGG AGG GAA TCT GAC GAG GAT ACT GAG GAT GCT TCT GAG ACC GAC CTC GCG AAA CAT GAT GAG GAA GAC TAC GTT GAA ATG AAA GAG CAG ATG TAC CAA GAC AAA CTT GCT AGC CTC AAG AGA CAG TTG CAG CAA CTG CAA GAA GGC ACG CTC CAG GAG TAC CAG AAG AGA ATG AAA AAA CTC GAC CAG CAG TAC AAG GAA CGA ATT AGA AAC GCA GAG CTC TTT CTT CAG CTG GAG ACT GAA CAG GTT GAG CGC AAT TAT ATT AAG GAA AAA AAA GCC GCT GTG AAG GAG TTC GAA GAC AAG AAA GTG GAA CTT AAA GAA AAC CTC ATC GCC GAA CTG GAG GAG AAG AAG AAG ATG ATA GAG AAC GAA AAA CTC ACA ATG GAA CTG ACG GGT GAT TCC ATG GAG GTA AAA CCG ATT ATG ACC CGA AAG CTC CGC CGA CGC CCA AAC GAT CCG GTA CCG ATC CCT GAT AAG CGG CGC AAG CCC GCA CCG GCT CAG CTC AAT TAC CTG CTG ACC GAC GAA CAA ATA ATG GAG GAC CTG CGG ACT CTT AAT AAG CTG AAG AGT CCT AAA CGG CCA GCT TCC CCC AGT TCC CCC GAA CAC CTG CCC GCT ACT CCC GCG GAG AGC CCT GCTCAG CGC TTT GAG GCC CGA ATC GAG GAC GGA AAA TTG TAC TAT GAC AAA CGC TGG TAT CAT AAG AGC CAG GCT ATA TAC CTG GAG TCA AAA GAT AAC CAA AAG TTG TCA TGT GTA ATC TCC TCA GTC GGG GCT AAC GAA ATA TGG GTG CGG AAG ACC TCT GAT AGT ACG AAG ATG CGC ATA TAT CTG GGA CAA TTG CAA AGA GGA CTT TTT GTT ATA AGA CGG AGA AGC GCT GCT.

[0065] In one embodiment, the nucleic acid sequence has, essentially comprises, or consists of the sequence of SEQ ID NO:5. In one embodiment, the nucleic acid sequence has, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:6, which encodes a NIPPI repression domain. ATGGTGCAAACTGCAGTGGTCCCAGTCAAGAAGAAGCGTGTGGAGGGCCCTGGCTCCCTGGGCCTGGAGGAATCAGGGAGCAGGCGCATGCAGAACTTTGCCTTCAGCGGAGGACTCTACGGGGGCCTGCCCCCCACACACAGTGAAGCAGGCTCCCAGCCACATGGCATCCATGGGACAGCACTCATCGG TGGCTTGCCCATGCCATACCCAAACCTTGCCCCTGATGTGGACTTGACTCCTGTTGTGCCGTCAGCAGTGAACATGAACCCTGCACCAAACCCTGCAGTCTATAACCCTGAAGCTGTAAATGAACCCAAGAAGAAGAAATATGCAAAAGAGGCTTGGCCAGGCAAGAAGCCCACACCTTCCTTGCTGATT. In another embodiment, the nucleic acid sequence has, essentially comprises, or consists of the sequence of SEQ ID NO:6. In one embodiment, the nucleic acid sequence has, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:37, which encodes the KRAB repression domain. ATGGACGCGAAATCACTTACGGCATGGTCGAGAACACTGGTTACGTTCAAGGACGTGTTTGTGGACTTTACACGTGAGGAGTGGAAATTGCTGGATACTGCGCAACAAATTGTGTATCGAAATGTCATGCTTGAGAATTACAAGAACCTCGTCAGTCTCGGATACCAGTTGACGAAACCGGATGTGATCCTTAGGCTCGAAAAGGGGGAAGAACCTTGGCTGGTA. In another embodiment, the nucleic acid sequence has, essentially comprises, or consists of the sequence of SEQ ID NO:37. In one embodiment, the nucleic acid sequence has, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:38, which encodes the DUNMT3A repression domain. CCCTCCCGGCTCCAGATGTTCTTCGCTAATAACCACGACCAGGAATTTGACCCTCCAAAGGTTTACCCACCTGTCCCAGCTGAGAAGAGGAAGCCCATCCGGGTGCTGTCTCTCTTTGATGGAATCGCTACAGGGCTCCTGGTGCTGAAGGACTTGGGCATTCAGGTGGACCGCTACATTGCCTCGGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGGGAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCCCATTCGATCTGGTGATTGGGGGCAGTCCCTGCAATGACCTCTCCATCGTCAACCCTGCTCGCAAGGGCCTCTACGAGGGCACTGGCCGGCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTCGAGTCCAACCCTGTGATGATTGATGCCAAAGAAGTGTCAGCTGCACACAGGGCCCGCTACTTCTGGGGTAACCTTCCCGGTATGAACAGGCCGTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAGGAGTGTCTGGAGCATGGCAGGATAGCCAAGTTCAGCAAAGTGAGGACCATTACTACGAGGTCAAACTCCATAAAGCAGGGCAAAGACCAGCATTTTCCTGTCTTCATGAATGAGAAAGAGGACATCTTATGGTGCACTGAAATGGAAAGGGTATTTGGTTTCCCAGTCCACTATACTGACGTCTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCATGGAGCGTGCCAGTCATCCGCCACCTCTTCGCTCCGCTGAAGGAGTATTTTGCGTGTGTG。

[0066] In one embodiment, the nucleic acid sequence has, essentially comprises, or consists of the sequence of SEQ ID NO:38. Further, in one embodiment, the nucleic acid sequence encodes at least one linker sequence and has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO:8. GGATCCGGTGGGGGATCTGGGGGATCTGGCTCG In one embodiment, the nucleic acid sequence has, consists essentially of, or comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to or complementary to SEQ ID NO: 184, which encodes both the SALL1 and SUDS3 repression domains.

[0067] Additionally or alternatively, in some embodiments, the nucleic acid sequence has, essentially comprises, or consists of the same sequence as SEQ ID NO: 183, which encodes an inactive Cas9 (dCas9).

[0068] Additionally or alternatively, in some embodiments, the nucleic acid sequence has, essentially comprises, or consists of a sequence identical to SEQ ID NO: 178, which encodes inactive MAD7 (dMAD7). In one embodiment, the nucleic acid sequence has, consists essentially of, or consists of a sequence identical to, or at least 80%, at least 85%, at least 90%, or at least 95% complementary to, SEQ ID NO:178.

[0069] Additionally or alternatively, in some embodiments, the nucleic acid sequence has, essentially comprises, or consists of the same sequence as SEQ ID NO: 179, which encodes an inactive CasPhi8 (dCasPhi8). In one embodiment, the nucleic acid sequence has, consists essentially of, or comprises a sequence identical to, or at least 80%, at least 85%, at least 90%, or at least 95% complementary to, SEQ ID NO:179.

[0070] In some embodiments, the fusion protein of the present invention is linked to a nuclear localization signal (NLS), an epitope tag, or a reporter gene sequence. Examples of nuclear localization signals include, but are not limited to, the nuclear localization signals of SV40 large T antigen, nucleoplasmin, EGL-13, and TUS proteins. Examples of epitope tags include, but are not limited to, FLAG tags, V5 tags, histidine (His) tags, and influenza hemagglutinin (HA) tags. Examples of reporter genes include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), small ubiquinone-like modifiers (SUMO), ubiquinone, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), and luciferase. In some embodiments, the nucleic acid or vector encoding the fusion protein of the present invention can also encode various regulatory elements or selection markers. Regulatory elements include, but are not limited to, promoters such as cytomegalovirus (CMV) promoter or human EF1α promoter, transcriptional promoters such as woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or HIV-1Rev response element (RRE), polyadenylation signals, self-cleaving peptides such as T2A, and internal ribosome entry sites (IRES). Examples of selection markers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), puromycin N-acetyltransferase (PAC) that confers resistance to puromycin, hygromycin resistance gene, and blasticidin-S deaminase (BSD).

[0071] (Method of Regulating Expression) In one embodiment, the present invention is directed to a method of regulating expression of a target nucleic acid in a eukaryotic cell. The method includes providing a cell with a gRNA and a Cas fusion protein comprised in any embodiment of the present invention. When bound to the gRNA as shown in FIG. 1, a Cas protein (shown here as dCas protein) 110 fused to SALL1 120, which is fused to SUDS3 130, acts on a target region of genomic DNA 150. In an embodiment, the method includes introducing multiple gRNAs with a Cas protein. The multiple gRNAs can be two or more, for example, 2-10 or 4-8 gRNAs. Two or more or all of the gRNAs can target the same gene or the same locus within a gene. When two or more gRNAs target the same locus, the gRNAs can have the same or overlapping spacer sequences, or non-overlapping sequences. In some embodiments, two or more or all of the gRNAs can target different genes or multiple different loci within a gene. Alternatively, one or more gRNAs can be provided to the cell by introducing a nucleic acid encoding the gRNA into the cell. Also, the Cas fusion protein can be provided to the cell by introducing a nucleic acid encoding the Cas fusion protein into the cell. The cell can be under conditions to express the gRNA and the Cas fusion protein.

[0072] Also, in one embodiment, the invention is directed to a method of modulating expression of a target nucleic acid in a eukaryotic cell by introducing a Cas fusion protein and an RNA repression domain complex. In one embodiment, the invention is directed to a method of modulating expression of a target nucleic acid in a eukaryotic cell by introducing a Cas protein and an RNA repression domain complex that is not a fusion protein. In some embodiments, the eukaryotic cell is a yeast cell, a plant cell, or a mammalian cell, such as a human or mouse cell, hi some embodiments, the cell is a cell line, such as, for example, HEK293, K562, Jurkat, or US2OS. When introducing a fusion protein, the fusion protein can be synthesized extracellularly or extramicrobially. Alternatively, an mRNA encoding the fusion protein can be introduced. In one embodiment, a gRNA is synthesized extracellularly and a nucleic acid encoding a Cas fusion protein is introduced into the cell, thereby providing the Cas fusion protein to the cell. The Cas fusion protein, RNA repression domain complex, and / or gRNA can be delivered to a target cell or microorganism by a variety of other methods and in a variety of formats (DNA, RNA, or protein), or a combination of these various approaches. For example, the various components can be delivered in the following formats: a) a DNA polynucleotide encoding a sequence for the Cas fusion protein or gRNA; b) an RNA (mRNA) encoding a sequence for the Cas fusion protein and a synthetic gRNA; c) a purified protein for the Cas fusion protein; d) an RNA encoding the gRNA; and e) a purified RNA repression domain complex.

[0073] When delivering Cas fusion proteins in protein form, the Cas fusion proteins are assembled together with a compatible gRNA to form a ribonucleoprotein complex (RNP) for delivery to a target cell, microorganism or specimen. For example, the assembled components or complexes ([Cas fusion protein]-[gRNA]) can be delivered together or separately by electroporation, nucleofection, transfection, via nanoparticles, virus-mediated RNA delivery, non-virus-mediated delivery, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., genetically modified yeast). In addition, other methods that can encapsulate molecules can deliver the molecules to target living cells without changing the genomic morphology. Other methods include, but are not limited to, transiently introducing DNA polynucleotides with sequences related to protein recruitment without integration to transcribe the desired molecules into the desired RNA molecules. These methods include, but are not limited to, using DNA alone vehicles (e.g., plasmids, MiniCircles, MiniVectors, MiniStrings, protelomerase-generated DNA molecules (e.g., Doggybones), artificial chromosomes (e.g., HACs), and cosmids), via DNA vehicles such as nanoparticles and extracellular vesicles (such as exosomes and microvesicles), via eukaryotic cell transfer (such as genetically modified yeast), transient viral transfer via AAV, non-integrating viral particles (such as lentivirus and retrovirus-based systems), cell-penetrating peptides, and other techniques that allow for the introduction of DNA into cells without direct integration into a genomic form.

[0074] Another method for introducing an RNA moiety includes the use of integrative gene transfer techniques to stably introduce machinery for RNA transcription into the genome of a target cell. Such methods can be controlled by constitutive or promoter-inducible systems to attenuate RNA expression. Furthermore, these methods can be designed to allow the system to be removed after use (e.g., introduction of the Cre-Lox recombination system). Such stable gene transfer techniques include, but are not limited to, methods that incorporate viral particles (e.g., lentivirus, adenovirus, and retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and Piggybac), DNA cleavage (e.g., using CRISPR and TALEN) techniques, and the use of non-homologous repair processes introduced by alternative DNA molecules, and other techniques to integrate target DNA into insect cells. The various components of the complex of the present invention, if not enzymatically synthesized in a cell or solution, can be chemically synthesized. Alternatively, if naturally occurring, they can be isolated and purified from naturally occurring sources. Methods for chemically and enzymatically synthesizing the various embodiments of the present invention are well known to those skilled in the art. Similarly, methods for linking or introducing covalent bonds between the components of the present invention are well known to those skilled in the art.

[0075] (kit) In one embodiment, the invention is directed to a kit comprising, consisting essentially of, or consisting of a Cas fusion protein of the invention, or a polynucleotide having a nucleic acid sequence encoding a protein of the invention. In one embodiment, the kit further comprises a gRNA or a nucleic acid encoding a gRNA, a gRNA library, and optionally a transfection reagent and / or other delivery reagent to a cell or subject. In one embodiment, the kit comprises a nucleic acid capable of expressing both a gRNA and a Cas fusion protein of the invention. Also, in one embodiment, the kit comprises a cell line engineered to express a Cas fusion protein of the invention, and optionally further comprises a gRNA or a nucleic acid encoding a gRNA. In one embodiment, the invention is directed to a kit comprising, essentially comprising, or consisting of an RNA repression domain complex of the invention, which in one embodiment further comprises a Cas protein or a Cas fusion protein, or a nucleic acid encoding a Cas protein or a Cas fusion protein, and optionally includes reagents for transfection and / or other delivery to a cell or subject. In one embodiment, the invention provides a kit, characterized in that the kit comprises: (1) a first lentiviral particle, the first lentiviral particle comprising a first polynucleotide, such as dCas9-SALL1-SUDS3, encoding a Cas fusion protein of the invention, and (2) a second lentiviral particle, the second lentiviral particle comprising a second polypeptide, the second polynucleotide encoding an sgRNA. In another embodiment, the invention provides a kit comprising: (1) a lentiviral particle comprising a first polynucleotide, such as dCas9-SALL1-SUDS3, encoding a Cas fusion protein of the invention; and (2) a second polynucleotide, wherein the second polynucleotide is a plasmid, the plasmid comprising the second polynucleotide, and the second polynucleotide is an sgRNA. In yet another embodiment, the present invention provides a kit comprising: (1) a first polynucleotide, the first polynucleotide being an mRNA, such as dCas9-SALL1-SUDS3, encoding a Cas fusion protein of the invention; and (2) a second polynucleotide, the second polynucleotide being an sgRNA.

[0076] The sgRNA of the kit can be designed to bind to a Cas fusion protein encoded by the polynucleotide described above, and optionally, the kit also includes one or more of the following: target cells, one or more selection chemicals and / or media (blasticidin, puromycin).

[0077] (application) In another embodiment, the invention provides methods for simultaneously suppressing multiple genes. In some of these methods, different gRNAs targeting different gene promoters or different transcription start sites of the same gene can be delivered to the same dCas9-repressing Cas fusion protein. In another embodiment, the invention provides methods for simultaneously suppressing and editing genes. In these methods, a standard gRNA (20 nucleotide target region) is delivered to a Cas-repressing Cas fusion protein to perform gene editing, and a truncated gRNA (14 nucleotide target region) to perform gene suppression. This method can be used to suppress inflammatory responses such as myeloid differentiation primary response 88 (My88) while performing gene editing. Alternatively, different genes involved in non-homologous end joining can be suppressed, resulting in an increased likelihood of homology-directed DNA repair (HDR), or host genes involved in regulating the repair of double-stranded DNA breaks, leading to different outcomes. These methods can be used to affect synthetic lethality, resulting in editing a gene target and suppressing a secondary gene target to suppress a cytotoxic response that is not present in cells with only one genomic perturbation.

[0078] Figure 15B-E shows the effect of using different sizes of crRNA regions with active Cas9 fused to SALL1 and SUDS3. When a 14-mer crRNA target region is used, transcriptional repression of the target occurs (left y-axis of the figure). In contrast, when a 20-mer crRNA target region is used, gene editing of the target occurs (right y-axis of the figure). Various embodiments of the present invention are used for sequence screening applications. For example, a library of sequenced gRNAs can be used for systematic loss-of-function studies. In one embodiment, 2-5 synthetic guide RNAs can be pooled for sequence screening applications. Various embodiments of the present invention can also be used for pooled lentivirus screening applications. For example, a pooled library of lentiviral sgRNA constructs can be used to target a set of gene targets or the whole genome for systematic loss-of-function studies. Such gRNAs can be delivered into cells expressing the Cas fusion construct of the present invention. Alternatively, such gRNAs can be delivered via a lentiviral construct expressing both Cas fusion protein and gRNA.

[0079] Additionally, in another embodiment, different CRISPR Cas systems can be combined with different effectors in the same cell, allowing one Cas system to cause transcriptional repression and another Cas system to cause a different effect (activation, gene editing, base editing, or epigenetic modification). Such methods provide for specific gene silencing in immune cells selected from, for example, T cells (including primary T cells), natural killer cells (NK cells), B cells, or CD34+ hematopoietic stem and progenitor cells (HSPCs). The immune cells may be engineered immune cells, such as T cells with chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs). Here, these methods can be applied to cells already modified to contain a CAR and / or TCR that are useful for therapy, so that gene expression can be further controlled. In another example, primary immune cells, either naturally occurring in a host animal or patient, or derived from stem cells or induced pluripotent stem cells (iPSCs), can be used for specific gene silencing using the methods and complexes described herein. Suitable stem cells include, but are not limited to, mammalian stem cells, such as human stem cells, including, but not limited to, hematopoietic stem cells, neural stem cells, embryonic stem cells, iPSCs, mesenchymal stem cells, mesodermal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells. Other stem cells include, but are not limited to: mammalian stem cells, such as mouse stem cells, such as mouse embryonic stem cells. Furthermore, methods provided herein are methods for genomic engineering (e.g., altering or manipulating the expression of one or more genes or one or more gene products) in prokaryotic or eukaryotic cells in vitro, in vivo, or ex vivo. In particular, the methods described herein are useful for modulating expression of target genes in mammalian cells, including primary human T cells, NK cells, CD34+ HSPCs, such as HSPCs isolated from cord blood or bone marrow and cells differentiated from cord blood or bone marrow.

[0080] Also provided herein are genetically engineered cells derived from hematopoietic stem cells, such as T cells, modified by the methods described herein. Here, by way of non-limiting example, various embodiments of the present invention can be used for the following applications: base editing, genome editing, genome screening, therapeutic cell generation, genome labeling, epigenome editing, karyotype manipulation, chromatin imaging, transcriptome and metabolic pathway manipulation, genetic circuit manipulation, cell signal sensing, intracellular event recording, genealogy reconstruction, gene drive, DNA genotyping, miRNA quantification, in vivo cloning, site-directed mutagenesis, genome diversification, and in situ proteomics analysis. In one embodiment, a cell or a cell population is exposed to the fusion protein of the present invention, and the cell or cell population is introduced into a subject by infusion. The aforementioned applications also include cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnostics, pathogen screening, microbiota reconstitution, stem cell reprogramming, immunogenomic engineering, vaccine development, and antibody production. In some embodiments, one or more molecules or complexes described herein are introduced into a subject.These molecules and complexes include Cas fusion proteins, fusion proteins Cas proteins, gRNAs, and nucleic acids that code the above substances that are introduced into a subject.For example, the method of introduction can be in the form of a drug.

[0081] (Example) In the examples below, protocols were used that were adapted from the methods and materials described below. (Generation of stable cell lines) U2OS Ubi[G76V]-EGFP (BioImage, discontinued), U2OS (ATCC, catalog no. HTB-96), A375 (ATCC, catalog no. CRL-161, K-562 (ATCC, catalog no. CCL-243), Jurkat (ATCC, catalog no. TIB-152), HCT 116 (ATCC, catalog no. CCL-247), and WTC-11 hiPS (Coriell Institute, U2OS cells stably expressing dCas9 were then transduced with lentiviral particles co-expressing the blasticidin resistance gene and a Cas system effector protein designed for CRISPRi at a multiplicity of infection (MOI) of 0.3. The cells were then cultured in cell line specific medium containing 5-10 μg / mL blasticidin for at least 10 days to select for a population of cells that stably expressed the CRISPRi effector protein. U2OS cells stably expressing dCas9 were then transduced with lentiviral particles co-expressing MCP-SALL1-SUDS3 and a hygromycin resistance gene at an MOI of 0.3. The cells were then cultured in medium containing 200 μg / mL hygromycin for 14 days to select for a population that stably expressed both MCP-SALL1-SUDS3 and dCas9.

[0082] (Guide RNA synthesis) All sgRNAs, crRNAs and tracrRNAs were synthesized at Horizon Discovery (formerly Dharmacon). sgRNAs and tracrRNA molecules were designed based on the CRISPRi2.1 version (v2.1) guide RNA prediction algorithm developed in 2016. MA Horlbeck et al., “Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation,” eLife.5, e19760 (2016). Unless otherwise stated, experiments using modified sgRNAs were delivered as an equimolar pool of the top three sgRNAs ranked by the algorithm, labeled g1-g3 in Table 2. The same target sequence was used for sgRNA, crRNA and sgRNA, except that the first base in the expressed sgRNA was always G.

[0083] (Lipid transfection using synthetic guide RNA) One day before transfection, U2OS or A375 cells were seeded in clear 96-well plates at 10,000 or 20,000 cells per well, respectively; one day before transfection, HCT116 cells were seeded in clear 6-well plates at 200,000 cells per well. They were transfected with synthetic guide RNAs targeting specific genes at a final concentration of 25 nM. Synthetic guide RNAs were mixed with DharmaFECT4 Transfection Reagent (Horizon Discovery, Cat. No. T-2005-01) in serum-free medium (GE Healthcare HyClone, Cat. No. SH30564.01) in each vessel for 20 minutes. The medium surrounding the cells was removed and replaced with the transfection mixture. Cells were cultured at 37°C, 5% CO2 for 24-144 hours until assays were performed.

[0084] (Cotransfection with dCas9 mRNA and synthetic sgRNA) One day before transfection, U2OS cells were seeded in clear 96-well plates at 10,000 cells per well; one day before transfection, HCT116 cells were seeded in clear 6-well plates at 200,000 cells per well. U2OS cells were co-transfected with 0.2 μg / well of dCas9-SALL1-SUDS3 or dCas9-KRAB mRNA and 25 nM of synthetic sgRNA; HCT116 cells were co-transfected with 2.5 μg / well of dCas9-SALL1-SUDS3 or dCas9-KRAB mRNA and 25 nM of synthetic sgRNA. dCas9 mRNA and sgRNA were mixed with DharmaFECT Duo Transfection Reagent (Horizon Discovery, Catalog No. T-2010) in serum-free (GE Healthcare HyClone, Catalog No. SH30564.01) for 20 minutes. The medium surrounding the cells was removed and replaced with the transfection mixture, and the cells were cultured at 37°C and 5% CO2.

[0085] (Nucleofection) K562 cells, Jurkat cells, WTC-11 human induced pluripotent stem cells (hiPS cells), and primary CD4+ T cells were electroporated per well using the Amaxa 96-well Shuttle System. 200,000 K562 cells per replicate were resuspended in SF buffer (Lonza, catalog number V4SC-2096) and nucleofected using the FF-120 program; 200,000 Jurkat cells were resuspended in SE buffer (Lonza, catalog number V4SC-1960) and nucleofected using the Cl-120 program. 80,000 hiPS cells were resuspended in P3 buffer (Lonza, catalog number V4SP-3096) and nucleofected using the DC-100 program. 250,000 primary human CD4+ T cells were resuspended in P3 buffer and nucleofected using the E0-115 program. Synthetic guide RNAs were delivered at cell line-dependent final concentrations between 2.5 and 9 μM. If cells did not stably express the dCas9 CRISPRi construct, dCas9-SALL1-SUDS3 or dCas9-KRAB mRNA was delivered at cell line-dependent concentrations ranging from 1-2.5 μg per nucleofection.

[0086] (Transfection with plasmid sgRNA) One day before transfection with CRISPRi sgRNA plasmids, U2OS and A375 cells were seeded in 96-well plates at 10,000 or 20,000 cells per well; plasmids were mixed with DharmaFECT kb Transfection Reagent (Horizon Discovery, Cat. No. T-2006) in serum-free medium (GE Healthcare HyClone, Cat. No. SH30564.01) for 10 minutes. The medium around the cells was removed and replaced with the transfection mixture. Cells were cultured at 37°C, 5% CO2 for 72 hours before assay. (Lentiviral transformation) U2OS and HCT116 cells were seeded at 10,000 cells per well and transduced with CRISPRi sgRNA lentiviral particles at a multiplicity of infection (MOI) of 0.3 to obtain cells with single integrants, which were then selected with 2.5 μg / mL puromycin for 7 days, passaged every 3-4 days before RT-qPCR analysis.

[0087] (RT-qPCR) Total RNA was isolated and reverse transcribed with dsDNase (ThermoFisher Scientific, Cat. No. K1672) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR, and assessed by qPCR using TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays. Relative expression of each gene was determined using the ΔΔCq method with GAPDH or ACTB as housekeeping genes and normalized to the non-targeting control (NTC). (Proteasome Assay - Functional Reporter Assay for Proteasome Gene Inhibition) The proteasome assay uses a recombinant U2OS cell line stably expressing a mutant ubiquitin fused to enhanced green fluorescent protein (Ubi[G76V]-EGFP). At the end of the experiment, the cell medium was replaced with Dulbecco's phosphate-buffered saline (Cytivia, catalogue no. SH30028.02) and EGFP fluorescence was measured on an EnVision® plate reader. Fluorescence values ​​of cell populations transfected with guide RNAs targeting key proteasome genes were normalized to those of untreated cell populations.

[0088] (Gene editing analysis by Sanger sequencing) Cells were lysed in 100 μL of buffer containing Protease K (Thermo Scientific, Cat. No. FEREO049), RNase A (Thermo Scientific, Cat. No. FEREN0531), and Phusion HF Buffer (Thermo Scientific, Cat. No. F-518L) at 56°C for 30 min, followed by heat inactivation at 95°C for 5 min. This cell solution was used to generate PCR amplicons of 400-600 nucleotides spanning the region containing the gene editing site. The unpurified PCR amplicons were subjected to Sanger sequencing. Gene editing efficiency was calculated from the ABI files using TIDE analysis. Brinkman et al., “Easy quantitative assessment of genome editing by sekuense trace decomposition,” Nucleic acids research, 42(22)(2014). TIDE quantified the frequency and type of small insertions and deletions (indels) at the target locus. Here, quantitative sequence trace data from the target sample normalized to the sequence trace data of the control sample was used.

[0089] (FACS analysis) 24 and 72 hours after nucleofection, functional knockdown of CXCR3 was assessed as the percentage of cells expressing the target gene by FACS analysis. Cells were resuspended in a 1:50 solution of FACS block (BD Biosciences, Cat. No. 564220) and stained for CD4 as an expression positive control using Alexa Flour 488-conjugated antibody (Biolegend, Cat. No. 50166932) and for CXCR3 using an APC-conjugated primary antibody (Biolegend, Cat. No. 353707). Unstained cells were used to sort CD4 and CXCR3 positive cells. The percentage of CXCR3 positive cells in the target population was normalized to that of the control population to determine functional knockdown. Example 1

[0090] Comparison of dCas-KRAB silencing with dCas9-SALL1-SUDS3 silencing delivered as mRNA. A comparison of dCas-KRAB silencing with dCas9-SALL1-SUDS3 silencing was performed for each of the following targets: BRCA1, PPIB, CD46, PSMD7, SEL1L, and ST3GAL4. K562 cells were nucleofected with either dCas-KRAB or dCas9-SALL1-SUDS3 mRNA, followed by gene knockdown. In both cases, cells were fed a 5 μM mixture of three pooled synthetic sgRNAs targeting each gene. Forty-eight hours after nucleofection, gene expression was measured compared to non-targeted controls, and the results are shown in Figure 3A. As the bar graphs show, in every case, silencing of gene expression by dCas9-SALL1-SUDS3 was comparable, if not better, than silencing by dCas-KRAB. Figure 3B shows the results of a similar study, except that the target cells were Jurkat cells and measurements were taken 72 hours after nucleofection, in which it can again be seen that in each case silencing of gene expression by dCas9-SALL1-SUDS3 was comparable, if not better, to silencing by dCas-KRAB. Figure 3C shows the results of another similar study, except that the target cells were U2OS cells, the reagents were delivered via lipid transfection with a 25 μM mixture of three pools of synthetic sgRNAs targeting each gene, and expression was measured 72 hours after transfection. In Figure 3C, it can again be seen that in each case, silencing of gene expression by dCas9-SALL1-SUDS3 was comparable, if not better, to silencing by dCas-KRAB. Example 2

[0091] (Comparison of the inhibitory effect of Cas fusion protein with that of dCas-KRAB) HCT116 cells were plated at 400,000 cells per well. After 24 hours, dCas9-SALL1-SUDS3 eGFP mRNA or dCas-KRAB eGFP mRNA and a 25 nM mixture of a pool of three synthetic sgRNAs targeting each of the following genes, as well as a non-targeting control (NTC), were co-transfected using DharmaFECT® Duo transfection reagent. The genes were PPIB, PSMD7, and SEL1L. After 24 hours of transfection, cells were trypsinized and FACS was performed. Cells were pooled into two groups, negative and top 10%, and then plated in 6-well dishes for harvesting. After 24 hours of harvesting (48 hours in total), total RNA was isolated and relative gene expression was measured using RT-qPCR. Relative gene expression of each gene was determined by the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. As shown in Figure 4, dCas9-SALL1-SUDS3 eGFP mRNA was used for FACS enrichment and resulted in greater suppression of the target gene than dCas-KRAB eGFP mRNA in both selected and unselected populations. Example 3

[0092] (Comparison of suppression between dCas-KRAB and dCas9-SALL1-SUDS3 in different cell lines) U2OS, Jurkat and hiPS cells stably expressing dCas9-SALL1-SUDS3 or dCas-KRAB were transfected or nucleofected with a pool of three synthetic sgRNAs targeting the listed genes. NTCs were also performed. After 72 hours, cells were cultured. In each cell line, dCas-KRAB or dCas9-SALL1-SUDS3 was under the control of hEF1α. Total RNA was isolated and relative gene expression was measured using RT-qPCR. Relative gene expression of each gene was determined by the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. As shown in Figure 5A, in the U2OS stable hEF1α cell line, dCas9-SALL1-SUDS3 showed greater gene suppression for BRCA1, PSMD7, SELIL, and ST3GAL4. As shown in Figure 5B, in the Jurkat stable hEF1α cell line, dCas9-SALL1-SUDS3 showed greater gene suppression for BRCA1, PSMD7, SELIL, and ST3GAL4. As shown in Figure 5C, in the U2OS stable hEF1α cell line, dCas9-SALL1-SUDS3 showed greater gene suppression for RAB11A, PPB, and SELIL. As shown in Figure 6A, in the K562 stable hEF1α cell line, dCas9-SALL1-SUDS3 showed greater gene suppression for BRCA1, PSMD7, SELIL, and ST3GAL4. As shown in Figure 6B, in the A375 stable hEF1α cell line, dCas9-SALL1-SUDS3 showed greater or similar gene repression for BRCA1, PSMD7, SELIL, and ST3GAL4. Example 4

[0093] (dCas-KRAB vs. dCas9-SALL1-SUDS3 over a 6-day course) U2OS cells stably expressing dCas9-SALL1-SUDS3 or dCas-KRAB under the control of the hEF1α promoter were transfected with a pool of three synthetic sgRNAs targeting each of the following genes: BRCA1, CD46, HBP1, and SELIL. Suppression was measured for 6 days using samples cultured every 24 hours after transfection. Total RNA was isolated and relative gene expression was assessed using RT-qPCR. Relative gene expression of each gene was determined using the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. FIG. 7A shows that dCas9-SALL1-SUDS3 caused greater repression of BRCA1 at all time points compared to dCas-KRAB. FIG. 7B shows that dCas9-SALL1-SUDS3 caused greater repression of BRCA1 at all time points compared to dCas-KRAB. FIG. 7C shows that dCas9-SALL1-SUDS3 caused greater repression of HBP1 at all time points compared to dCas-KRAB. FIG. 7D shows that dCas9-SALL1-SUDS3 caused greater repression of SELIL at all time points compared to dCas-KRAB. However, in each case, dCas9-SALL1-SUDS3-mediated repression occurred more quickly than repression mediated by dCas-KRAB. Furthermore, compared with dCas-KRAB-mediated repression, dCas9-SALL1-SUDS3-mediated repression maintained near-maximal levels for a longer period of time. Example 5

[0094] (sgRNA pool) WTC-11 hiPS cells stably expressing dCas9-SALL1-SUDS3 and U2OS cells stably expressing dCas9-SALL1-SUDS3 were nucleofected or transfected with 3 μL of each sgRNA delivered by lipid transfection, targeting PPIB (3 μL), SELIL (3 μL), RAB11A (3 μL)-electroporated sgRNA, BRCA1 (25 nM), PSDM7 (25 nM), SELIL (25 nM) and ST3GAL4 (25 nM), individual sgRNAs or a pool of three synthetic sgRNAs. Cells were cultured for 72 h. Total RNA was isolated and relative gene expression was assessed using RT-qPCR. Relative gene expression of each gene was determined by the ΔΔCq method with GAPDH as the housekeeping gene and normalized by the non-targeting control group. As shown in Figure 8A, the pool performed as well as or better than each individual sgRNA in WTC-11 hiPS cells, and as shown in Figure 8B, the pool performed as well as or better than each individual sgRNA in U2OS hEF1α dCas9-SALL1-SUDS3. Example 6

[0095] (Combination of gRNAs to simultaneously suppress multiple genes) Human iPS cells stably expressing dCas9-SALL1-SUDS3 were nucleofected with individual sgRNAs targeting unique genes and pools of up to six sgRNAs. After 72 hours, cells were cultured. Total RNA was isolated and relative gene expression was measured using RT-qPCR. Relative gene expression of each gene was determined using the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. As shown in FIG. 9, when up to six genes were simultaneously suppressed in human iPS cells, the level of suppression of the target genes was similar to that when only one of the genes was targeted. Example 7

[0096] (Fusion with N-terminal and C-terminal amino acids of Cas protein) The structures of the three Cas proteins are shown at the bottom of Figure 10: dCas-KRAB, dCas9-SALL1-SUDS3, and SUDS3-SALL1-dCas. These Cas fusion proteins were expressed under human EF1α regulation. Along with a cell line stably expressing dCas-KRAB, U2OS Ubi[G76V]-EGFP cell lines were generated that stably express the various bipartite Cas fusion proteins. Cells were transfected with 25 nM synthetic sgRNA targeting genes known to be important for proteasome fusion. Non-targeting controls were also transfected. Fluorescence in each transfection condition was determined 72 hours after transfection using an EnVision® plate reader and values ​​were normalized to those of the untreated cell line. U2OS cells stably expressed a ubiquitin mutant fused to enhanced green fluorescent protein (Ubi[G76V]-EGFP). In untreated cells, the expressed ubiquitin-EGFP protein was constitutively downregulated, leaving only background fluorescence. In contrast, cells in which proteasome function was inhibited showed accumulation of EGFP. Thus, silencing of the target gene resulted in an increase in fluorescence intensity. As shown in Figure 10, Cas fusion proteins including dCas9, SUDS3, and SALL1 exhibited substantially stronger repression (greater mean GFP expression correlated with stronger repression) compared to dCas-KRAB, regardless of whether the fusion occurred at the N- or C-terminal amino acid of the Cas protein. Example 8

[0097] (Plasmid: Plasmid cotransfection in A375 and U2OS cells) Plasmid suppressors (1) hEF1α-dCas9KRAB or (2) dCas-SALL1-SUDS3 were co-transfected with guide (total amount = 100 ng) using 0.6 μL / well of DharmaFECT® kb. Gene expression was measured using RT-qPCR 3 days post-plasmid co-transfection of suppressor and target gene in A375 cells, as shown in FIG. 11. Gene expression was also measured using RT-qPCR 3 days post-plasmid co-transfection of suppressor and target gene in U2OS cells, as shown in FIG. 12. Both figures consistently show greater suppression in the system containing the plasmid for dCas9-SALL1-SUDS3. Example 9

[0098] (Additional Inhibitors) U2OS Ubi[G76V]-EGFP cell lines stably expressing various bipartite Cas fusion proteins were generated along with a cell line stably expressing dCas-KRAB. Cells were transfected with 25 nM synthetic sgRNA targeting genes known to be important for proteasome fusion. A non-targeting control was also generated. Fluorescence in each transfection condition was determined 72 hours post-transfection using an EnVision® plate reader and values ​​were normalized to those of the untreated cell line. As shown in Figure 13, (i) dCas9, (ii) either SUDS3 or SALL1, and (iii) Cas fusion proteins having KRAB or NIPP1 show the same or better inhibitory effect as dCas9. (The higher the bar, the stronger the inhibitory effect. This system was designed in the same manner as the system in Example 8.) Example 10

[0099] (V-type Cas protein-SALL1-SUDS3 fusion construct) Inactive MAD7 (engineered Cas12a protein)-SALL1-SUDS3 fusion construct (dMAD7-SALL1-SUDS3) was cloned and U2OS cells were generated that stably expressed the construct under the control of the minimal CMV (mCMV) promoter. Inactive CasPhi8 (Cas12J protein)-SALL1-SUDS3 fusion construct (dCasPhi8-SALL1-SUDS3) was also cloned and U2OS cells were generated that stably expressed the construct under the control of the mCMV promoter. These cells, together with U2OS cells stably expressing dMAD7 or dCasPhi8, were lipid transfected with synthetic guides designed for each Cas protein. In each case, they were delivered at 25 nM. The transcriptional repression was evaluated 48 hours after transfection. Figure 14A shows CRISPRi-induced transcriptional repression in U2OS cells stably expressing either dMAD7 or dMAD7-SALL1-SUDS3 and in NTCs for two individual synthetic guide RNAs for either BRCA1 or PPIB, as well as for a pool of synthetic guide RNAs. The figure shows significantly greater repression by dMAD7-SALL1-SUDS3 compared to dMAD7. Figure 14B shows CRISPRi-induced transcriptional repression in U2OS cells stably expressing either dCasPhi8 or dCasPhi8-SALL1-SUDS3, and in basal BRCA1 expression in untreated U2OS cells, for three repeats of individual synthetic guide RNAs targeting the same site in BRCA1. The figure shows significantly greater repression by dCasPhi8-SALL1-SUDS3 compared to dCasPhi8. The figures above demonstrate that SALL1 and SUDS3 can be fused to various V-type Cas proteins and programmed with synthetic guide RNAs to silence important target genes. Example 11

[0100] Simultaneous editing and suppression using active Cas fusion proteins U2OS cells stably expressing SUDS3-SALL1-WtCas9 under the control of the hEF1α promoter were transfected with a 25 nM pool of guide RNAs designed for both CRISPRi and CRISPR editing. Guides designed for CRISPRi had a truncated 14-nt target region. Guides designed for CRISPR editing contained the full-length 20-nt target region. Cells were cultured for 72 hours post-transfection. Total RNA was isolated and relative gene expression was measured using RT-qPCR. Relative gene expression was determined using the ΔΔCq method with GAPDH as housekeeping and normalized to a non-targeted control. Genomic DNA was isolated and the target region was amplified using PCR and Sanger analysis. Indel formation was analyzed using TIDE. FIG. 15B shows that MRE11A can be repressed while LBR is co-edited. FIG. 15C shows that MRE11A can be repressed while PPIB is co-edited. FIG. 15D shows that SELIL can be repressed while LBR is co-edited. FIG. 15E shows that SEL1L can be repressed while PPIB is co-edited. Example 12

[0101] Comparison of single repression domains as dCas9 fusion proteins U2OS Ubi[G76V]-EGFP cell lines stably expressing various single inhibitory dCas9 fusion proteins (BCL6, CbpA, H-NS, MBD3, NIPP1, SALL1, and SUDS3) were generated under the control of the human EF1α promoter, along with a cell line stably expressing dCas-KRAB. Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be important for proteasome fusion and non-targeting controls. Fluorescence for each transfection condition was determined 72 hours after transfection with an EnVision® plate reader, and values ​​were normalized to those of the untreated cell line. The U2OS cell line stably expressed a ubiquitin mutant fused to enhanced green fluorescent protein (U2OS Ubi[G76V]-EGFP). In untreated cells, the expressed ubiquitin EGFP was constitutively downregulated, leaving only background fluorescence. On the other hand, cells with inhibited proteasome function showed accumulation of EGFP. Thus, suppression of the target gene results in enhanced fluorescence. As shown in Figure 16, dCas9 fusion proteins with NIPP1, SALL1, and SUDS3 showed substantially stronger suppression effects than dCas-KRAB. (Higher mean GFP expression correlates with stronger suppression effects.) Example 13

[0102] Comparison of dCas9-SUDS suppression with dCas9-KRAB and dCas9-KRAB-MeCP2 systems We generated U2OS Ubi[G76V]-EGFP cell lines stably expressing either dCas9-KRAB, dCas9-KRAB MeCP2, or dCas9-SUDS3 under the control of human EF1α. Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be important for proteasome fusion, as well as non-targeting controls. Cells were cultured for 72 hours after transfection. Total RNA was isolated and gene expression was measured using RT-qPCR. Relative gene expression was determined by the ΔΔCq method with GAPDH as a housekeeping gene and normalized to the non-targeted control. Figure 17 shows that for each genetic target, dCas9-SUDS has substantially stronger transcriptional repression effects than either dCas9-KRAB or dCas9-KRAB-MeCP2 alone. Example 14

[0103] (Proteasome functional reporter assay and transcriptional repression) U2OS Ubi[G76V]-EGFP cell lines were generated that stably express either dCas9-KRAB or dCas9-SALL1-SUDS3 under the control of human EF1α. Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be important for proteasome fusion, as well as an untreated control. Fluorescence for each transfection condition was determined 72 hours post-transfection on an EnVision® plate reader, and values ​​were normalized to those of the untreated cell line. U2OS cell lines stably express a ubiquitin mutant fused to enhanced green fluorescent protein (U2OS Ubi[G76V]-EGFP). In untreated cells, expressed ubiquitin EGFP was constitutively downregulated, leaving only background fluorescence. In contrast, cells with inhibited proteasome function show accumulation of EGFP. Thus, suppression of the target gene results in increased fluorescence. Total RNA was isolated and expression of the target genes was measured using RT-qPCR. Relative gene expression was determined by the ΔΔCq method using GAPDH as a housekeeping gene and normalized to the non-targeted control. Figure 18A shows that dCas9-SALL1-SUDS3 knocks down the phenotype substantially more potently than dCas9-KRAB (higher mean GFP expression correlates with stronger suppression). Figure 18B also shows that the more pronounced phenotype observed with dCas9-SALL1-SUDS3 correlates with stronger transcriptional suppression of the target proteasome genes. Example 15

[0104] Lentiviral delivery Figure 19A shows the transcriptional repression of PPIB and SEL1L in US2OS cells stably expressing dCas9-SALL1-SUDS3 and guide RNA from either a single lentiviral vector or two separate vectors. Figure 19B shows the transcriptional repression of PPIB and SEL1L in HCT116 cells stably expressing dCas9-SALL1-SUDS3 and guide RNA from either a single lentiviral vector or two separate vectors. Lentiviral vectors were used to generate US2OS and HCT116 cells stably expressing dCas9-SALL1-SUDS3 under the control of the human EF1α promoter or the mouse CMV promoter (mCMV), respectively. These cells were subsequently transduced with lentiviral particles carrying vectors expressing individual guide RNAs from the human U6 promoter and targeting PPIB, SEL1L, or containing a non-targeting control sequence. Parental U2OS and HCT116 cells were transduced with lentiviral particles containing a single vector expressing dCas9-SALL1-SUDS3 under the control of the hEF1α (U2OS) or (HCT116) promoter, and individual guide RNAs from the human U6 promoter. These single vector systems also targeted PPIB and SEL1L, or had a non-targeting control sequence. 24 hours after transfection, transduced cells were enriched by adding medium containing 2.5 μg / mL puromycin. Cells were cultured in this medium for 7 days and subcultured every 3 to 4 days. Eight days after transduction, cells were harvested, total RNA was isolated, and the relative expression levels of the target genes were measured using RT-qPCR. Relative gene expression was determined using the ΔΔCq method with GAPDH as a housekeeping gene and normalized to the non-targeting control. Figure 19A shows that dCas9-SALL1-SUDS3 and guide RNA can be expressed using either a single lentiviral vector system or a dual lentiviral vector system to strongly suppress target genes in U2OS cells. Figure 19B shows that dCas9-SALL1-SUDS3 and guide RNA can be expressed using either a single lentiviral vector system or a dual lentiviral vector system to strongly suppress target genes in HCT116 cells. Example 16

[0105] (Plasmid sgRNA vs. synthetic sgRNA) U2OS cells and A375 cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1α promoter were transfected with individual sgRNAs, 25 nM synthetic sgRNA combinations, or 100 ng of plasmid sgRNA targeting BRCA1, PSDM7, SEL1L, and ST3GAL4. Cells were cultured for 72 hours post-transfection. Total RNA was isolated and the relative expression of each target gene was measured using RT-qPCR. Relative gene expression was determined using the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. Figure 20A shows that dCas9-SALL1-SUDS3 mediates substantially stronger expression of target genes when delivered with synthetic sgRNA compared to plasmid sgRNA in U2OS cells. FIG. 20B shows that dCas9-SALL1-SUDS3 mediates substantially more targeted gene expression when delivered with synthetic sgRNA compared to plasmid sgRNA in A375 cells. Example 17

[0106] (Synthetic sgRNA vs. crRNA: tracrRNA) U2OS cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1α promoter were transfected with a pool of 25 nM synthetic sgRNAs or synthetic crRNA:tracrRNA complex. Cells were cultured for 72 hours post-transfection. Total RNA was isolated and the relative expression of each target gene was measured using RT-qPCR. Relative gene expression was determined using the ΔΔCq method with GAPDH as a housekeeping gene and normalized to a non-targeting control. Figure 21 shows that a significant silencing effect was clearly observed when using the synthetic sgRNA pool, while both synthetic sgRNA and crRNA:tracrRNA complexes delivered with dCas9-SALL1-SUDS3 caused target gene silencing. Example 18

[0107] (5' truncated spacer) U2OS cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1α promoter were transfected with 25 nM synthetic sgRNA pools containing either the truncated 14-mer target region or the full 20-mer target region. Cells were cultured for 72 hours after transfection. Total RNA was isolated and the relative expression of each target gene was measured using RT-qPCR. Relative gene expression was determined using the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control group. Figure 22 shows that the target region of the guide gRNA can be shortened by at least 6 mers at the 5' end and still have a transcriptional repression effect when delivered with dCas9-SALL1-SUDS3. Example 19

[0108] (LNA modified sgRNA) U2OS Ubi[G76V]-EGFP cells stably expressing dCas9-SALL1-SUDS3 under the control of the human EF1α promoter were transfected with 25 nM synthetic sgRNA targeting two genes known to be important for proteasome function and a non-targeting control. The guides contain various combinations of 2'-O-methyl and phosphorothioate linkages and anchored nucleic acids at both ends of the sgRNA molecule. Additionally, the guides contain bases 1 through 20 from the 5' end of the 20-base long target region. Fluorescence in each transfection condition was determined 144 hours after transfection using an EnVision® plate reader and normalized to that of the untreated cell line. U2OS cells stably expressed a ubiquitin mutant fused to enhanced green fluorescent protein (Ubi[G76V]-EGFP). In untreated cells, the expressed ubiquitin-EGFP was constitutively downregulated, leaving only background fluorescence, whereas cells in which proteasome function was inhibited showed accumulation of EGFP. Thus, suppression of a targeted gene results in increased fluorescence. FIG. 23A shows the effect of sgRNA molecules with various chemical end modifications on dCas9-SALL1-SUDS3-mediated knockdown of function. Incorporation of fixed nucleic acids at the 5' and 3' ends of the sgRNA molecule can be used to stabilize the sgRNA. Incorporation of two fixed nucleic acids at the 3' end of sgRNAs targeting PSDM7 and PSDM11 further improves target gene suppression. (Higher GFP expression correlates with greater suppression.) FIG. 23B also shows the impact of incorporation of fixed nucleic acids at fixed positions in the sgRNA target region on dCas9-SALL1-SUDS3-mediated knockdown of function. Incorporation of fixed nucleic acids at certain positions in the sgRNA target region can improve target gene suppression. Example 20

[0109] (Recruitment of RNA suppressor complexes) U2OS cells stably expressing SALL1-SUDS3 fused with dCas9 and MS2 coat protein ligand (MCP-SALL1-SUDS3), each under the control of the human EF1α promoter, were generated by sequence conversion of the respective lentiviral vectors. Cells were then transfected with 25 nM synthetic crRNA:tracrRNA complexes targeting BRCA1, CD151, and SETD3, together with NTC. Designs of tracrRNAs containing various sequences and positions of the MS2 ligand binding site were tested for each target gene and compared with tracrRNA without the MS2 ligand binding site and complexes with labeled crRNA:tracrRNA with and without MS2. Cells were cultured for 72 hours post-transfection. Total RNA was isolated and the relative expression of each target gene was measured using RT-qPCR. Relative gene expression was determined by the ΔΔCq method with GAPDH as the housekeeping gene and normalized to the non-targeting control. Figure 24A shows that MCP-SALL1-SUDS3 is recruited to dCas9 via the C-5 MS2 sequence located either in the sgRNA stem loop 2 or at the 3' end of the tracrRNA molecule. Recruitment of MCP-SALL1-SUDS3 can enhance the inhibitory effect of dCas9 binding, represented herein as MS2-containing / non-containing crRNA:tracrRNA. Figure 24B shows that MCP-SALL1-SUDS3 is recruited to dCas9 via both the C-5 MS2 sequence and the F-5MS2 sequence containing 2dAP chemical modification, significantly improving the inhibitory effect of dCas9 binding. Example 21

[0110] (Knockdown in T cells) Primary human CD4+ T cells were nucleofected with dCas9-SALL1-SUDS3 mRNA and pooled synthetic sgRNA via the Lonza 96-well Shuttle system. 24 and 72 hours after nucleofection, functional knockdown of CXCR3 was assessed as the percentage of cells expressing the target gene by FACS analysis. Cells were stained for CD4 using Alexa Flour488-conjugated antibodies as a positive expression control and compared to CXCR3 using APC-conjugated primary antibodies. Total RNA was isolated at each time point and CXCR3 mRNA expression was assessed using RT-qPCR. Relative gene expression of CXCR3 was determined using the ΔΔCq method with GAPDH as a housekeeping gene and normalized to the non-targeting control. Figure 25A is a graph showing transcriptional repression and protein level knockdown of CXCR3 in primary human CD4+ T cells nucleofected with dCas9-SALL1-SUDS3 and either a synthetic non-targeting control or a pool of three guides targeted to the gene of interest at 1 and 3 days after nucleofection. Figure 25B also shows that the onset of knockdown using dCas9-SALL1-SUDS3 was rapid and sustained for several days in clinically relevant primary cell morphology, as assessed by protein expression in the non-transfected control line at day 1, protein expression in the non-transfected control line at day 3, protein expression in the CXCR3 pool line at day 1, and protein expression in the CXCR3 pool line at day 3.

[0111] [Table 2] JPEG2024507451000004.jpg245170JPEG2024507451000005.jpg237170JPEG2024507451000006.jpg245170JPEG2024507451000007. jpg245170JPEG2024507451000008.jpg245170JPEG2024507451000009.jpg245170JPEG2024507451000010.jpg36170 Table 2. Synthetic sgRNAs (Sp Cas9) The targeted region is shown in bold and the chemical modifications are shown in italics.

[0112] [Table 3] JPEG2024507451000012.jpg137169Table 3. 5' truncated 14-base target region synthetic sgRNAs (Sp Cas9) The target region is shown in bold and the chemical modifications are shown in italics.

[0113] [Table 4] JPEG2024507451000014.jpg245167JPEG2024507451000015.jpg9170 Table 4. LNA modified synthetic sgRNAs (Sp Cas9) The target region is shown in bold and the sequence is shown in italics.

[0114] [Table 5] Table 5. Synthetic crRNAs (Sp Cas9) The target region is shown in bold and the chemical modifications are shown in italics.

[0115] [Table 6] Table 6. Synthetic tracrRNAs (Sp Cas9) The MS2 aptamer region is shown in bold and the chemical modifications are shown in italics.

[0116] [Table 7] Table 7. Synthetic V-type Cas crRNAs The target region is shown in bold and the chemical modifications are shown in italics.

[0117] [Table 8] JPEG2024507451000020.jpg25170Table 8. Lentiviral guide RNAs (Sp Cas9) delivered via particles or plasma The target region is shown in bold.

Claims

1. A Cas fusion protein having a Cas protein and one or both of a SALL1 repression domain and a SUDS3 repression domain.

2. The Cas fusion protein of claim 1, characterized in that the Cas fusion protein has the SALL1 repression domain.

3. The Cas fusion protein of claim 1, characterized in that the Cas fusion protein has the SUDS3 repression domain.

4. The Cas fusion protein of claim 1, characterized in that the Cas fusion protein has both the SALL1 repression domain and the SUDS3 repression domain.

5. The Cas fusion protein of any one of claims 2 to 4, further comprising an additional repression domain, said additional repression domain being a repression domain other than said SALL1 repression domain or said SUDS3 repression domain.

6. The Cas fusion protein of claim 5, wherein the additional repression domain is a NIPP1 repression domain.

7. The Cas fusion protein of any one of claims 1 to 4, characterized in that the Cas protein is catalytically inactive.

8. The Cas fusion protein of any one of claims 1 to 4, wherein the Cas protein is a nickase.

9. The Cas fusion protein according to any one of claims 1 to 4, characterized in that the Cas protein is in an active or inactive form.

10. The Cas fusion protein according to any one of claims 1 to 4, characterized in that the Cas protein is a type V Cas protein or a type II Cas protein.

11. 11. The Cas fusion protein of claim 10, wherein the Cas protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i and Cas12j.

12. The Cas fusion protein of claim 11, wherein the Cas protein is Cas12a.

13. The Cas fusion protein according to any one of claims 1 to 4, further comprising a Cas linker.

14. The Cas fusion protein of claim 13, wherein the Cas linker is an amino acid sequence having at least 80% similarity to SEQ ID NO:

7.

15. The Cas fusion protein of claim 14, wherein the Cas linker has SEQ ID NO:

7.

16. The Cas fusion protein of claim 15, wherein the Cas linker is SEQ ID NO:

7.

17. The Cas fusion protein of claim 13, characterized in that the Cas protein has an N-terminal amino acid, the Cas fusion protein has the SUDS3 repression domain, and the Cas linker binds to both the SUDS3 repression domain and the N-terminal amino acid of the Cas protein.

18. The Cas fusion protein of claim 13, wherein the Cas protein has an N-terminal amino acid, the Cas fusion protein has the SALL1 repression domain, and the Cas linker binds both the SALL1 repression domain and the N-terminal amino acid of the Cas protein.

19. The Cas fusion protein of claim 13, characterized in that the Cas protein has a C-terminal amino acid, the Cas fusion protein has the SUDS3 repression domain, and the Cas linker binds to both the SUDS3 repression domain and the C-terminal amino acid of the Cas protein.

20. The Cas fusion protein of claim 13, characterized in that the Cas protein has a C-terminal amino acid, the Cas fusion protein has the SALL1 repression domain, and the Cas linker binds to both the SALL1 repression domain and the C-terminal amino acid of the Cas protein.

21. The Cas fusion protein of claim 2 or claim 4, characterized in that the SALL1 repression domain has a sequence that is at least 80% similar to SEQ ID NO:

1.

22. The Cas fusion protein of claim 2 or claim 4, characterized in that the SALL1 repression domain has SEQ ID NO:

1.

23. The Cas fusion protein of claim 3 or claim 4, characterized in that the SUDS3 repression domain has a sequence that is at least 80% similar to SEQ ID NO:

2.

24. The Cas fusion protein of claim 3 or claim 4, characterized in that the SUDS3 repression domain has sequence number 2.

25. The Cas fusion protein of claim 4, wherein the SALL1 repression domain has a sequence at least 80% similar to SEQ ID NO:1, and the SUDS3 repression domain has a sequence at least 80% similar to SEQ ID NO:

2.

26. 26. The Cas fusion protein of claim 25, wherein the SALL1 repression domain has SEQ ID NO:1 and the SUDS3 repression domain has SEQ ID NO:

2.

27. 26. The Cas fusion protein of claim 25, wherein the SALL1 repression domain is SEQ ID NO: 1 and the SUDS3 repression domain is SEQ ID NO:

2.

28. The Cas fusion protein of claim 4, wherein the Cas protein is a dCas9 protein, the dCas9 protein has a C-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the C-terminal amino acid of the dCas9 protein and the N-terminal amino acid of the SALL1 repression domain, and the inhibitory linker is bound to both the C-terminal amino acid of the SALL1 repression domain and the N-terminal amino acid of the SUDS3 repression domain.

29. The Cas fusion protein of claim 4, wherein the Cas protein is a dCas9 protein, the dCas9 protein has a C-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the C-terminal amino acid of the dCas9 protein and the SUDS3 inhibitory domain, and the inhibitory linker is bound to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain.

30. The Cas fusion protein of claim 4, wherein the Cas protein is a dCas9 protein, the dCas9 protein has an N-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the N-terminal amino acid of the dCas9 protein and the SALL1 inhibitory domain, and the inhibitory linker is bound to both the SALL1 inhibitory domain and the SUDS3 inhibitory domain.

31. The Cas fusion protein of claim 4, wherein the Cas protein is a dCas9 protein, the dCas9 protein has an N-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the N-terminal amino acid of the dCas9 protein and the SUDS3 inhibitory domain, and the inhibitory linker is bound to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain.

32. The Cas fusion protein of any one of claims 28 to 31, wherein each of the Cas linker and the inhibitory linker has an amino acid sequence of 3 to 90 amino acids in length.

33. 33. The Cas fusion protein of claim 32, wherein the Cas linker and the inhibitory linker each have SEQ ID NO:

7.

34. The Cas fusion protein of claim 33, wherein each of the Cas linker and the inhibitory linker is SEQ ID NO:

7.

35. The Cas fusion protein of claim 4, wherein the Cas protein is a Cas12a protein, the Cas12a protein has a C-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently linked to the C-terminal amino acid of the Cas12a protein and the SALL1 inhibitory domain, and the inhibitory linker is linked to both the SALL1 inhibitory domain and the SUDS3 inhibitory domain.

36. The Cas fusion protein of claim 4, wherein the Cas protein is a Cas12a protein, the Cas12a protein has a C-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently linked to both the C-terminal amino acid of the Cas12a protein and the SUDS3 inhibitory domain, and the inhibitory linker is linked to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain.

37. The Cas fusion protein of claim 4, wherein the Cas protein is a Cas12a protein, the Cas12a protein has an N-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the N-terminal amino acid of the Cas12a protein and the SALL1 inhibitory domain, and the inhibitory linker is bound to both the SALL1 inhibitory domain and the SUDS3 inhibitory domain.

38. The Cas fusion protein of claim 4, characterized in that the Cas protein is a Cas12a protein, the Cas12a protein has an N-terminal amino acid, the Cas fusion protein further has a Cas linker and an inhibitory linker, the Cas linker is covalently bound to both the N-terminal amino acid of the Cas12a protein and the SUDS3 inhibitory domain, and the inhibitory linker is bound to both the SUDS3 inhibitory domain and the SALL1 inhibitory domain.

39. The Cas fusion protein of any one of claims 35 to 38, wherein each of the Cas linker and the inhibitory linker has SEQ ID NO:

7.

40. The Cas fusion protein of claim 39, wherein each of the Cas linker and the inhibitory linker is SEQ ID NO:

7.

41. The Cas fusion protein of claim 40, wherein the Cas linker and the inhibitory linker each have SEQ ID NO:

7.

42. The Cas fusion protein of claim 41, wherein each of the Cas linker and the inhibitory linker is SEQ ID NO:

7.

43. The Cas fusion protein of claim 4, wherein the Cas fusion protein has a sequence that is at least 80% similar to SEQ ID NO:

10.

44. The Cas fusion protein of claim 43, wherein the Cas fusion protein is SEQ ID NO:

10.

45. A nucleic acid encoding the Cas fusion protein of any one of claims 1 to 44.

46. 46. ​​The nucleic acid of claim 45, wherein the nucleic acid is present in a vector.

47. 47. The method of claim 46, wherein the vector is a plasmid or a viral vector. of nucleic acid.

48. 48. The nucleic acid of claim 47, wherein the nucleic acid is present in a viral vector selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a coronavirus, and a Sendai virus.

49. 45. A method for regulating expression of a target nucleic acid in a eukaryotic cell, comprising providing a gRNA and a Cas fusion protein of any one of claims 1 to 44 to the eukaryotic cell.

50. 50. The method of claim 49, wherein the gRNA is synthetically produced outside the cell and the Cas fusion protein is provided to the cell by introducing an mRNA encoding the Cas fusion protein into the cell.

51. 51. The method of claim 50, wherein the eukaryotic cell is a yeast cell, a plant cell, or a mammalian cell.

52. 52. The method of claim 51 , wherein the eukaryotic cell is a mammalian cell.

53. 53. The method of claim 52, wherein the mammalian cell is a human cell.

54. 50. The method of claim 49, wherein the providing step comprises obtaining the gRNA and the Cas fusion protein from a vector, the vector encoding the gRNA and the Cas fusion protein.

55. 50. The method of claim 49, further comprising synthesizing the gRNA outside of the cell and synthesizing the Cas fusion protein outside of the cell.

56. 11. A method for modulating expression of a target nucleic acid in a eukaryotic cell, the method comprising providing to said cell a Cas fusion protein and an RNA repression domain complex according to any one of claims 1 to 44, the RNA repression domain complex comprising: (a) gRNA molecule, (b) a ligand binding site; (c) a ligand, (d) a repression domain; the gRNA molecule comprises 30 to 180 nucleotides; the ligand binding site is either (i) directly attached to the gRNA molecule or (ii) attached to the gRNA molecule via a ligand binding site linker; the ligand is capable of reversibly binding to the ligand-binding site; The inhibition domain either (i) directly binds to the ligand, or (ii) binds to the ligand via a ligand linker. A method comprising:

57. 57. The method of claim 56, wherein the eukaryotic cell is a human cell.

58. 57. The method of claim 49 or claim 56, characterized in that the method is carried out in vitro or ex vivo.

59. A kit comprising a Cas fusion protein and an RNA repression domain complex according to any one of claims 1 to 44, wherein the RNA repression domain complex comprises: (a) gRNA molecule, (b) a ligand binding site; (c) a ligand, (d) Repression domain having the gRNA molecule comprises 30 to 180 nucleotides; the ligand binding site is either (i) directly attached to the gRNA molecule or (ii) attached to the gRNA molecule via a ligand binding site linker; the ligand is capable of reversibly binding to the ligand-binding site; The inhibition domain either (i) directly binds to the ligand, or (ii) binds to the ligand via a ligand linker. A kit characterized by:

60. Use of a Cas fusion protein according to any one of claims 1 to 44 for treating a subject, said subject being affected by a disease treatment requiring said transcriptional repression of a gene.

61. The use of claim 60, further comprising administering a gRNA.

62. The use of claim 61, further comprising the step of administering an mRNA encoding the gRNA.

63. A method for modulating expression of a target nucleic acid in a cell, comprising providing the cell with the sgRNA and Cas fusion protein of claim 1.

64. 64. The method of claim 63, wherein the sgRNA is chemically modified.

65. 65. The method of claim 63 or claim 64, wherein the sgRNA has a spacer region of 12 to 30 nucleotides in length.

66. A method for modulating expression of a target nucleic acid in a cell, comprising providing to the cell a crRNA molecule, a tracrRNA molecule and a Cas fusion protein of claim 1.

67. 67. The method of claim 66, wherein one or both of the crRNA molecule and the tracrRNA molecule are chemically modified.

68. 68. The method of claim 66 or claim 67, wherein the crRNA molecule has a spacer region of 12 to 30 nucleotides in length.

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