Recombinant gene effectors and compositions and methods of use thereof

Abstract

The present disclosure provides one or more recombinant gene effectors, as well as systems, compositions and methods of use thereof, wherein the one or more recombinant gene effectors of the present disclosure can be used to regulate a target gene in a cell (e.g., an endogenous target gene in a cell).The one or more recombinant gene effectors of the present disclosure can be operably linked to a heterologous endonuclease, such as a CRISPR / Cas protein.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 489,873, filed March 13, 2023; U.S. Provisional Patent Application No. 63 / 381,255, filed October 27, 2022; and U.S. Provisional Patent Application No. 63 / 323,248, filed March 24, 2022, all of which are incorporated by reference in their entireties.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application has been filed with an electronic Sequence Listing, which has been provided as a 132 kilobyte file with the file name 55176-727_601_SL.xml, created on March 10, 2023. The information set forth in this electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Various effectors (e.g., transcriptional regulators) can be used to regulate the expression or activity of target genes in cells. For example, heterologous gene effectors can be introduced (e.g., delivered or expressed) into cells, and the heterologous gene effectors alone or in combination with another agent can regulate the target gene. In some examples, the other agent can be a CRISPR (clustered regularly interspaced short palindromic repeats) / CRISPR-associated protein (Cas) that specifically binds to a target gene (e.g., a target deoxyribonucleic acid (DNA) sequence or a target ribonucleic acid (RNA) sequence (e.g., a foreign DNA sequence or a foreign RNA sequence) of the target gene), and the heterologous gene effector combined with CRISPR / Cas can regulate the expression amount or activity level of the target gene. Such gene effectors can be used, for example, as gene therapy for treating or alleviating a condition (e.g., a disease) in a subject. Summary of the Invention [Means for solving the problem]

[0004] The VP64-p65-Rta fusion polypeptide (VPR) is a standard gene effector that can activate target genes in cells. However, in some cases, VPR may not be optimal or function sufficiently to regulate all genes. In addition, or alternatively, the size of VPR (e.g., approximately 518 amino acid residues in length) may be too large to be packaged with at least one other agent (e.g., one or more guide RNAs, a transgene encoding a therapeutic polynucleotide or therapeutic protein, various types of Cas enzymes, etc.) in a particular delivery method (e.g., a viral vector, e.g., an adeno-associated virus (AAV) vector). Therefore, in various embodiments of the present disclosure, there are provided recombinant effectors that are not identical to VPR but are as effective as VPR in activating the expression or activity levels of one or more target genes, as well as compositions and methods for using the same.

[0005] Kruppel-associated box (KRAB) is a domain (e.g., a domain of about 75 amino acid residues or less) found in eukaryotic Kruppel-type C2H2 zinc finger proteins (ZFPs). KRAB is a standard gene effector that can repress target genes in cells. However, in some cases, KRAB may not be optimal or function sufficiently to regulate all genes. Thus, in various embodiments of the present disclosure, there are provided recombinant effectors that are not identical to KRAB but are as effective as KRAB in repressing the expression or activity levels of one or more target genes, as well as compositions and methods of use thereof.

[0006] Provided herein is a recombinant gene effector comprising a polypeptide, the polypeptide is heterologous to any member selected from the group consisting of VP16, VP64, p65, and Rta; the recombinant gene effector has a maximum size of about 500 amino acid residues in length; The recombinant gene effector is capable of activating the expression level of a target gene in a cell, and the expression level of the target gene activated via the recombinant gene effector is at least about 80% of the expression level of the target gene activated by a VP64-p65-Rta fusion polypeptide (VPR) in a control cell. Recombinant gene effectors are disclosed.

[0007] Provided herein is a recombinant gene effector comprising a polypeptide linked to an additional polypeptide, the polypeptide comprises an amino acid sequence having at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:1; the additional polypeptide comprises at least a portion of one or more members selected from the group consisting of VP16, VP64, p65, and Rta; the recombinant gene effector has a size of about 250 amino acid residues in length or less; Recombinant gene effectors are disclosed.

[0008] Provided herein is a recombinant gene effector comprising a polypeptide, the polypeptide comprises an amino acid sequence having at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:1; The recombinant gene effector is not identical to SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; Recombinant gene effectors are disclosed.

[0009] Provided herein is a recombinant gene effector comprising multiple polypeptide domains, Each polypeptide domain of the plurality of polypeptide domains comprises a polypeptide comprising an amino acid sequence that exhibits at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO: 1; Recombinant gene effectors are disclosed. Further disclosed is a system comprising a recombinant gene effector according to any one of the preceding claims.

[0010] Disclosed herein are one or more polynucleotides that encode the systems disclosed herein.

[0011] Disclosed herein are cells comprising the systems disclosed herein.

[0012] Disclosed herein is a method of regulating a target gene in a cell, the method comprising contacting the cell with a system disclosed herein.

[0013] Those skilled in the art will readily appreciate further aspects and advantages of the present disclosure from the following detailed description, which shows and describes only illustrative embodiments of the disclosure. It will also be readily apparent that the present disclosure is susceptible to other and various embodiments, the details of which can be modified in various obvious respects without departing from the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0014] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that an incorporated-by-reference publication, patent, or patent application conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter. [Brief explanation of the drawings]

[0015] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures").

[0016] [Figure 1]Schematic representation of various examples of recombinant effector structures.

[0017] [Figure 2] Schematic showing an example of the engineering of target gene regulation by a recombinant effector complexed with a heterologous endonuclease and a guide nucleic acid molecule.

[0018] [Figure 3] Correlation between two genes regulated by recombinant effector candidates is shown.

[0019] [Figure 4] Predicted active polypeptide sequences for generating recombinant effectors are shown.

[0020] [Figure 5] Schematic representation of various examples of recombinant effectors.

[0021] [Figure 6] Further examples of recombinant effectors containing flexible linkers are shown schematically.

[0022] [Figure 7] Further examples of recombinant effectors containing multiple active domains are shown schematically.

[0023] [Figure 8] Another example of a recombinant effector containing multiple active domains is shown schematically.

[0024] [Figure 9] Further examples of recombinant effectors of various lengths are shown schematically.

[0025] [Figure 10] Schematic representation of further examples of recombinant effectors of various lengths.

[0026] [Figure 11]Schematic representation of further examples of various recombinant effectors.

[0027] [Figure 12] Activation of a heterologous target gene by a recombinant effector.

[0028] [Figure 13] Activation of endogenous genes encoding IFNs by recombinant effectors.

[0029] [Figure 14] Activation of a heterologous target gene by a recombinant effector at various time points is shown.

[0030] [Figures 15A-15B] Activation of the endogenous target gene encoding CXCR4 by the recombinant effector at various time points is shown.

[0031] [Figure 16] Activation of the endogenous gene encoding CD2 by recombinant effectors.

[0032] [Figure 17] Activation of the endogenous gene encoding CD45 by recombinant effectors.

[0033] [Figure 18A] Activation of heterologous target genes by recombinant gene effectors, each containing an activation domain at a different position.

[0034] [Figure 18B] Activation of endogenous target genes by recombinant gene effectors, each containing an activation domain at a different position.

[0035] [Figure 19] 1 shows various levels of expression of heterologous target genes activated by recombinant gene effectors.

[0036] [Figure 20] Figure 1 shows the various levels of expression of endogenous genes encoding IFNs activated by recombinant gene effectors.

[0037] [Figure 21] Figure 1 shows the various expression levels of the endogenous gene encoding CXCR4 activated by recombinant gene effectors.

[0038] [Figure 22] The ability of recombinant gene effectors to regulate heterologous and endogenous genes compared to control gene effectors is shown.

[0039] [Figure 23] The ability of recombinant gene effectors to regulate endogenous genes compared to control gene effectors is shown.

[0040] [Figure 24] The expression level of CXCR4 activated by the recombinant gene effector is shown 3 days after transfection.

[0041] [Figure 25] The expression level of CXCR4 activated by the recombinant gene effector is shown 7 days after transfection.

[0042] [Figure 26] Activation of endogenous and heterologous target genes by recombinant gene effectors.

[0043] [Figure 27] The expression level of CXCR4 suppressed by the recombinant gene effector 28 days after transfection is shown.

[0044] [Figure 28] The expression level of CXCR4 suppressed by the recombinant gene effector 40 days after transfection is shown.

[0045] [Figure 29] This shows that gene expression induced by the recombinant gene effector was suppressed by JQ1 or GNE049.

[0046] [Figure 30] We demonstrate persistent and sustainable gene regulation by recombinant gene effectors.

[0047] [Figure 31] Activation levels of CD45, IFNγ, and CXCR4 by recombinant gene effectors at 3 days post-transfection (3 dpt) are shown.

[0048] [Figure 32] We show persistent activation (e.g., durable activation) of CD45, IFNG, CXCR4, and CD81 by recombinant gene effectors at various time points (e.g., 9–18 days after transfection).

[0049] [Figure 33] The correlation between maximal activation by recombinant gene effectors (3 days after transfection) and subsequent activation (9-18 days after transfection) is shown.

[0050] [Figure 34] VPR, p300, Rta, p65, VP64 and recombinant gene effectors are shown to have different sizes.

[0051] [Figure 35] Activation of CD45 by recombinant gene effectors at 9, 12 and 18 days after transfection is shown.

[0052] [Figure 36] Persistent activation (e.g., durable activation) of CD45, CXCR4, IFN-γ, and CD81 by recombinant gene effectors is shown.

[0053] [Figure 37] The robustness and potency of activation of CD45, CXCR4, IFN-γ, and CD81 by recombinant gene effectors is shown.

[0054] [Figure 38] Activation of CD45 by recombinant gene effectors at various times after transfection is shown.

[0055] [Figure 39] Activation of IFNG by recombinant gene effectors at various times after transfection is shown.

[0056] [Figure 40] Activation of CXCR4 by recombinant gene effectors at various times after transfection is shown.

[0057] [Figure 41] Activation of CD81 by recombinant gene effectors at various times after transfection is shown.

[0058] [Figure 42-43] Changes in expression (MFI) of the recombinant gene effector (mCherry) and sgRNA (BFP) by the recombinant gene effector at various time points after transfection are shown.

[0059] [Figures 44A-44C] Activation of CD45 and IFNG by recombinant gene effectors is shown 3 days (FIG. 44A), 6 days (FIG. 44B), and 9 days (FIG. 44C) after transfection.

[0060] [Figure 45] The predicted structures of CC.2, CC.4 and CC.5 are shown.

[0061] [Figure 46] The predicted structure of XV1.48 aligned to CC.2 is shown.

[0062] [Figure 47] The predicted structure of CC.32 is shown.

[0063] [Figure 48] The predicted structure of the Cas protein linked to XV1.32 is shown.

[0064] [Figure 49] Conserved sequence motifs in various human protein families are shown.

[0065] [Figure 50] Conserved sequence motifs in various viral protein families are shown.

[0066] [Figure 51] This shows a method for screening amino acids using a regularized logistic regression model (ElasticNet).

[0067] [Figure 52] The feature importance of the 20 identified amino acids is shown.

[0068] [Figure 53] Motif enrichment at the peptide level by various properties (e.g., charge) is shown.

[0069] [Figure 54] The predicted structure of VPR is shown.

[0070] [Figure 55] The predicted structure of XV1.1 is shown.

[0071] [Figure 56] Volcano plots showing the results of screening recombinant gene effectors.

[0072] [Figure 57] The predicted structures of the recombinant gene effectors (XV1.32 and XV1.33) are shown.

[0073] [Figure 58] The barrel of the coiled-coil variant is shown.

[0074] [Figure 59] Activation of multiple target genes by recombinant gene effectors.

[0075] [Figure 60] IFNG activation as a function of coding length (bp) of recombinant gene effectors.

[0076] [Figure 61] Activation of CD45 by recombinant gene effectors at various time points is shown.

[0077] [Figure 62] 1 shows the potency and durability of CD45 activation by recombinant gene effectors.

[0078] [Figure 63] Activation of IFNG by recombinant gene effectors at various time points is shown.

[0079] [Figure 64] 1 shows the potency and durability of IFNG activation by recombinant gene effectors.

[0080] [Figure 65] Activation of CXCR4 by recombinant gene effectors at various time points is shown.

[0081] [Figure 66] 1 shows the potency and durability of CXCR4 activation by recombinant gene effectors.

[0082] [Figure 67] Activation of CD81 by recombinant gene effectors at various time points is shown.

[0083] [Figure 68] 1 shows the potency and durability of CD81 activation by recombinant gene effectors.

[0084] [Figure 69] Activation of CXCR4 mRNA levels by recombinant gene effectors at 40 days is shown.

[0085] [Figure 70] Activation of CD45 mRNA levels at day 27 by recombinant gene effectors is shown.

[0086] [Figure 71] Schematic representation of the epigenetic mechanism of persistent activation by recombinant gene effectors.

[0087] [Figure 72] Expression of CD45 by recombinant gene effectors on day 3 is shown.

[0088] [Figure 73] Expression of CD45 by recombinant gene effectors on day 9 is shown.

[0089] [Figure 74] Expression of CD45 by recombinant gene effectors 96 hours after administration of inhibitors is shown.

[0090] [Figure 75] 1 shows mitotic tolerance gene activation by recombinant gene effectors. DETAILED DESCRIPTION OF THE INVENTION

[0091] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will also recognize that various variations, changes, and substitutions are possible without departing from the invention. Various alternative aspects of the embodiments of the invention described herein may also be employed.

[0092] When the terms "at least," "greater than," or "greater than or equal to" appear before or after the first or last number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" always apply to each number in the series. For example, "1, 2, or 3 or more" is the same as "1 or more, 2 or more, or 3 or more."

[0093] When the term "within," "less than," or "less than or equal to" appears after the last number in a series of two or more numbers, the term "within," "less than," or "less than or equal to" always applies to each number in the series. For example, "less than or equal to 1, 2, or 3" means the same as "less than or equal to 1, 2, or 3."

[0094] The term "about" typically refers to a range within an acceptable error of a particular numerical value, as determined by one of ordinary skill in the art; this acceptable error range depends in part on how the numerical value is measured or determined, i.e., in part on the limitations of the measurement system. For example, "about" may mean within one standard deviation per measurement or more than one standard deviation. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a numerical value. Alternatively, particularly with respect to biological systems or processes, the term "about" may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a numerical value. Where specific numerical values ​​are described in this application and claims, unless otherwise specified, the term "about" is intended to mean within an acceptable error range of the particular numerical value.

[0095] The use of alternatives (e.g., those indicated by "or") means one, both, or a combination of the alternatives. The term "and / or" means one or both of the alternatives.

[0096] As used herein, the term "heterologous," when used with respect to a polypeptide or nucleic acid sequence, refers to (1) the polypeptide or nucleic acid sequence being located in a location where it is not normally found (e.g., a location where it is not normally found in nature) (e.g., an environment, such as a cell, a virus, or a fusion polypeptide or polynucleotide molecule); or (2) the polypeptide or nucleic acid sequence comprising two or more subsequences that exist in a relationship not normally found in nature. For example, a polypeptide may comprise a first polypeptide sequence and a second polypeptide sequence that are not found together in a single polypeptide in nature, and thus the first and second polypeptide sequences may be heterologous to each other. In another example, a polynucleotide may comprise a first polynucleotide sequence and a second polynucleotide sequence that are not found together in a single polynucleotide in nature, and thus the first and second polynucleotide sequences may be heterologous to each other.

[0097] The term "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may be from any organism having one or more cells. Some examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of single-celled eukaryotes, protozoan cells, cells obtained from plants (e.g., cells obtained from cereal plants, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, bryophytes, liverworts, or mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells obtained from mushrooms), animal cells, cells obtained from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells obtained from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells obtained from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. In some cases, the cells may not be derived from a naturally occurring organism (e.g., the cells may be synthetically produced, sometimes referred to as "artificial cells").

[0098] As used herein, the term "nucleotide" generally refers to a combination of a base, a sugar, and a phosphate. A nucleotide may be a synthetic nucleotide. A nucleotide may be a synthetic nucleotide analog. A nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). The term "nucleotide" may include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), and guanosine triphosphate (GTP), as well as deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, "nucleotide" may also refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Specific examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or may be detectably labeled by known techniques. Labeling may also be performed using quantum dots. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from PerkinElmer (Foster City, CA); and FluoroLink deoxynucleotides FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink available from Amersham (Arlington Heights, IL). Cy5-dUTP; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim (Indianapolis, IN); and Molecular Available chromosome-labeled nucleotides include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, all available from Probes, Inc. (Eugene, Oregon). Nucleotides can also be labeled by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some examples of biotinylated dNTPs include, but are not limited to, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0099] As used herein, the terms "polynucleotide," "oligonucleotide," or "nucleic acid" are used interchangeably and generally refer to a polymeric form of nucleotides of a certain length, which may be deoxyribonucleotides or ribonucleotides, or analogs thereof, and which may be single-stranded, double-stranded, or multi-stranded. A polynucleotide may be exogenous to a cell or endogenous to a cell. A polynucleotide may be in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If a modification is included in the nucleotide structure, the modification can be imparted before or after assembly of the nucleotide. Some examples of analogs include, but are not limited to, 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorescent dyes (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-labeled nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine.Examples of polynucleotides include, but are not limited to, coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from a sequence, RNA isolated from a sequence, cell-free polynucleotides such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. Non-nucleotide components can be inserted into the nucleotide sequence.

[0100] The term "sequence identity" typically refers to a perfect nucleotide-to-nucleotide correspondence between two polynucleotide sequences or a perfect amino acid-to-amino acid correspondence between two polypeptide sequences. Techniques for determining sequence identity generally involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded by that nucleotide sequence and then comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid sequences) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is determined by dividing the number of perfectly matched residues between the two aligned sequences by the length of the longer sequence and multiplying by 100. For example, percent identity may be determined by comparing sequence information using the advanced BLAST computer program (e.g., version 2.2.9) available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), which is discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). The BLAST program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are set to optimize searches with short query sequences, for example, using the blastp program.The BLAST program can further employ a SEG filter to mask specific segments of the query sequence as determined by the SEG program described in Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired sequence identity range is between about 50% and 100%, including integer values ​​within that range. Typically, the present disclosure includes sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the sequences provided herein.

[0101] The term "gene" generally refers to a nucleic acid (e.g., DNA, such as genomic DNA or cDNA) and the corresponding nucleotide sequence involved in encoding an RNA transcript. As used herein with respect to genomic DNA, the term also includes intervening non-coding and regulatory regions, including the 5' and 3' ends. In some uses, the term "gene" encompasses transcribed sequences, such as 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some uses of the term "gene," the term "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") required to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as, for example, a ribosomal RNA gene (rRNA) or a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, such as enhancers and promoters. "Gene" may refer to an "endogenous gene" or a native gene that is present in its natural location in the genome of an organism. "Gene" may also refer to a "foreign gene" or a non-native gene. A "non-native gene" may refer to a gene that is not normally found in the host organism, but that has been introduced into the host organism by gene transfer. Furthermore, a "non-native gene" may refer to a gene that is not present in its natural location in the genome of an organism. A "non-native gene" may refer to a naturally occurring nucleic acid sequence or polypeptide sequence that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0102] The term "expression" typically refers to one or more processes by which a polynucleotide (e.g., mRNA or other RNA transcript) is transcribed from a DNA template and / or the process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. A transcript and the encoded polypeptide can be collectively referred to as a "gene product." When a polynucleotide is derived from genomic DNA, "expression" can also include splicing of mRNA in eukaryotic cells. "Up-regulation," with respect to expression, typically refers to an increase in the expression level of a polynucleotide sequence (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the wild-type expression level, while "down-regulation" typically refers to a decrease in the expression level of a polynucleotide sequence (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the wild-type expression level. Expression of a transfected gene can occur transiently or stably in a cell. In "transient expression," the transfected gene is not transferred to daughter cells during cell division. In transient expression, expression occurs only in transfected cells, and gene expression is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selectable advantage to the transfected cells. Such a selectable advantage may be resistance to a particular toxin to which the cells are exposed.

[0103] The term "expression profile" generally refers to the quantitative (e.g., abundance) and qualitative expression of one or more genes in a sample (e.g., cells). The one or more genes may be expressed and identified in the form of nucleic acid molecules (e.g., mRNA or other RNA transcripts). Additionally or alternatively, the one or more genes may be expressed and identified in the form of polypeptides (e.g., proteins as measured by Western blot). The expression profile of a gene may be defined as the amount of expression of the gene over a certain time frame (e.g., a time frame of at least about 1 hour or less, at least about 2 hours or less, at least about 3 hours or less, at least about 4 hours or less, at least about 5 hours or less, at least about 6 hours or less, at least about 7 hours or less, at least about 8 hours or less, at least about 9 hours or less, at least about 10 hours or less, at least about 11 hours or less, at least about 12 hours or less, 2 hours or less, at least about or about 16 hours or less, at least about or about 18 hours or less, at least about or about 24 hours or less, at least about or about 36 hours or less, at least about or about 48 hours or less, at least up to about 3 days, at least up to about 4 days, at least up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, at least up to about 14 days, etc.).Alternatively, a gene expression profile may be defined as the amount of expression of a gene at a time point of interest (e.g., at least about 1 hour or at most about 1 hour, at least about 2 hours or at most about 2 hours, at least about 3 hours or at most about 3 hours, at least about 4 hours or at most about 4 hours, at least about 5 hours or at most about 5 hours, at least about 6 hours or at most about 6 hours, at least about 7 hours or at most about 7 hours, at least about 8 hours or at most about 8 hours, at least about 9 hours or at most about 9 hours, at least about 10 hours or at most about 10 hours, at least about 11 hours or at most about 11 hours, The expression level of the gene measured may be at least about 12 hours or at most about 12 hours, at least about 16 hours or at most about 16 hours, at least about 18 hours or at most about 18 hours, at least about 24 hours or at most about 24 hours, at least about 36 hours or at most about 36 hours, at least about 48 hours or at most about 48 hours, at least at most about 3 days, at least at most about 4 days, at least at most about 5 days, at least at most about 6 days, at least at most about 7 days, at least at most about 8 days, at least at most about 9 days, at least at most about 10 days, at least at most about 11 days, at least at most about 12 days, at least at most about 13 days, or at least at most about 14 days).

[0104] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and generally refer to a polymer of at least two amino acid residues linked by a peptide bond. These terms do not imply a specific length of the polymer, nor do they refer to or distinguish between peptides produced using recombinant technology, chemical synthesis, enzymatic synthesis, or natural origin. These terms also apply to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, non-amino acids can be inserted into these polymers. These terms also include amino acid chains of any length, including full-length proteins, proteins with secondary and / or tertiary structure (e.g., domains), or proteins lacking secondary and / or tertiary structure. These terms also include modified amino acid polymers, such as those modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, or other manipulations, such as conjugation with a labeling component. As used herein, the term "amino acid" generally refers to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids include natural and unnatural amino acids that have been chemically modified to contain groups or chemical moieties not found in naturally occurring amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0105] As used herein, the terms "derivative," "variant," or "fragment" when used with respect to a polypeptide generally refer to a polypeptide that is related to a wild-type polypeptide, e.g., a polypeptide that is related in amino acid sequence, structure (e.g., secondary and / or tertiary structure), activity (e.g., enzymatic activity), and / or function to the wild-type polypeptide. Polypeptide derivatives, variants, and fragments may include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

[0106] As used herein, the terms "recombined," "chimeric," or "recombinant" with respect to a polypeptide molecule (e.g., a protein) generally refer to a polypeptide molecule having a heterologous or modified amino acid sequence, as well as a cell or organism expressing the polypeptide molecule, resulting from genetic engineering of the nucleic acid encoding the polypeptide molecule. As used herein, the terms "recombined" or "recombinant" with respect to a polynucleotide molecule (e.g., a DNA molecule or an RNA molecule) generally refer to a polynucleotide molecule having a heterologous or modified nucleic acid sequence, resulting from genetic engineering. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, a recombined or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered with a gene editing moiety. For example, the heterologous endonuclease (e.g., a recombinant Cas protein) disclosed herein is not a naturally occurring nuclease (e.g., is not a naturally occurring Cas protein). In another example, the recombinant gene effector disclosed herein is not a naturally occurring gene effector.

[0107] As used herein, the terms "recombinant" and "modified" are used interchangeably. As used herein, the terms "recombining" and "modifying" are used interchangeably. As used herein, the terms "recombinant cell" and "modified cell" are used interchangeably. As used herein, the terms "recombinant characteristic" and "modified characteristic" are used interchangeably.

[0108] The terms "enhanced expression," "increased expression," or "upregulated expression" generally refer to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) at an expression level that exceeds the normal expression level of the moiety of interest in a host strain (e.g., a host cell). The normal expression level may be substantially zero (or null) or may be an expression level greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. The moiety of interest may comprise a heterologous gene or heterologous polypeptide construct introduced into the host strain. For example, to enhance expression of a polypeptide of interest in a host strain, a heterologous gene encoding the polypeptide of interest can be knocked in (KI) into the genome of the host strain.

[0109] The terms "enhanced activity," "increased activity," or "upregulated activity" generally refer to the activity of a moiety of interest (e.g., a polynucleotide or polypeptide) that has been modified to exceed the normal activity level of the moiety of interest in a host strain (e.g., a host cell). The normal activity level may be substantially zero (or null) or an activity level greater than zero. The moiety of interest may include a polypeptide construct of the host strain. The moiety of interest may include a heterologous polypeptide construct introduced into the host strain. For example, to enhance the activity of a polypeptide of interest in a host strain, a heterologous gene encoding the polypeptide of interest can be knocked in (KI) into the genome of the host strain.

[0110] The terms "suppression of expression," "reduced expression," or "downregulated expression" generally refer to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) at an expression level that is below the normal expression level of the moiety of interest in a host strain (e.g., a host cell). Normal expression is an expression level that is greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked out or knocked down in the host strain. In some instances, suppression of expression of the moiety of interest may include complete inhibition of such expression in the host strain.

[0111] The terms "suppressed activity," "reduced activity," or "downregulated activity" generally refer to the activity of a moiety of interest (e.g., a polynucleotide or polypeptide) that has been altered to be below the normal activity level of the moiety of interest in a host strain (e.g., a host cell). A normal activity level is an activity that is greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked out or knocked down in the host strain. In some instances, reducing the activity of the moiety of interest may include complete inhibition of such activity in the host strain.

[0112] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and generally refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological matter obtained in vivo or in vitro culture.

[0113] The terms "treatment" or "treating" generally refer to an approach for obtaining a beneficial or desired result, such as, but not limited to, a therapeutic benefit and / or a prophylactic benefit. For example, "treatment" can include administering a system or cell population disclosed herein. A "therapeutic benefit" refers to a treatment-related improvement in, or an effect on, one or more diseases, conditions, or symptoms being treated. To obtain a prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or a subject experiencing one or more physiological symptoms of a disease, even before the disease, condition, or symptom appears.

[0114] The term "effective amount" or "therapeutically effective amount" generally refers to the amount of a composition sufficient to achieve a desired activity when administered to a subject in need thereof, such as a composition comprising a heterologous polypeptide, a heterologous polynucleotide, and / or a recombinant cell (e.g., a modified stem cell). In the context of the present disclosure, the term "therapeutically effective" generally refers to the amount of a composition sufficient to delay the onset of, arrest the progression of, or alleviate or relieve at least one symptom of, a disease treated by the methods of the present disclosure.

[0115] overview

[0116] In various embodiments, the present disclosure provides recombinant effectors (or recombinant gene effectors; herein, the terms "recombinant effector" and "recombinant gene effector" are used interchangeably) that can modulate (e.g., activate or suppress) the expression level or activity level of a target gene in a cell (e.g., an endogenous target gene or a heterologous target gene), as well as compositions and methods of use thereof. Such recombinant effectors can, for example, regulate the expression level or activity level of a target gene in a cell by acting in concert with a heterologous endonuclease (e.g., a recombinant CRISPR / Cas nuclease or an inactive variant thereof), thereby treating or alleviating a condition (e.g., a disease) in a subject. Gene expression underpins various physiological and pathological effects in cells and tissues, thereby contributing to numerous diseases and conditions. Therefore, compositions and methods utilizing the recombinant gene effectors of the present disclosure offer therapeutic advantages by allowing the expression of specific genes to be modulated in a desired manner.

[0117] Recombinant gene effectors and compositions and methods thereof

[0118] In some embodiments, the disclosure provides a recombinant gene effector (e.g., a recombinant gene activator, e.g., a transcription activator) that is not identical to VP16 (e.g., comprising the polypeptide sequence of SEQ ID NO: 5), VP64 (e.g., comprising the polypeptide sequence of SEQ ID NO: 6), p65 (e.g., comprising the polypeptide sequence of SEQ ID NO: 7), Rta (e.g., comprising the polypeptide sequence of SEQ ID NO: 8), and VP64-p65-Rta fusion polypeptide (VPR) (e.g., comprising the polypeptide sequence of SEQ ID NO: 9). In some embodiments, the disclosure provides a recombinant gene effector (e.g., a recombinant gene repressor, e.g., a transcription repressor) that is not identical to a Kruppel-associated box (KRAB) (e.g., comprising the polypeptide sequence of SEQ ID NO: 64).

[0119] In some embodiments, the size of the recombinant gene effectors disclosed herein is at least about or not more than 500 amino acid residues in length, at least about or not more than 480 amino acid residues in length, at least about or not more than 460 amino acid residues in length, at least about or not more than 450 amino acid residues in length, at least about or not more than 440 amino acid residues in length, at least about or not more than 42 ... at least about 400 amino acid residues in length, at least about 380 amino acid residues in length, at least about 360 amino acid residues in length, at least about 350 amino acid residues in length, at least about 340 amino acid residues in length, at least about 320 amino acid residues in length, at least about 300 amino acid residues in length, at least about 290 amino acid residues in length a length of at least about 290 amino acid residues, at least about 280 amino acid residues, at least about 270 amino acid residues, at least about 260 amino acid residues, at least about 250 amino acid residues, at least about 240 amino acid residues, at least about 230 amino acid residues, at least about 220 amino acid residues 0 amino acid residues or less in length, at least about or less than 210 amino acid residues in length, at least about or less than 200 amino acid residues in length, at least about or less than 190 amino acid residues in length, at least about or less than 180 amino acid residues in length, at least about or less than 170 amino acid residues in length, at least about or less than 160 amino acid residues in length, at least about or less than 150 amino acid residues in length,Lengths of at least about 140 amino acid residues or less, at least about 130 amino acid residues or less, at least about 120 amino acid residues or less, at least about 110 amino acid residues or less, at least about 100 amino acid residues or less, at least about 95 amino acid residues or less, at least about 90 amino acid residues or less, and at least about 85 amino acid residues or less. Alternatively, it may be about 85 amino acid residues or less in length, at least about 80 amino acid residues or less, at least about 70 amino acid residues or less, at least about 60 amino acid residues or less, at least about 50 amino acid residues or less, at least about 40 amino acid residues or less, at least about 30 amino acid residues or less, or at least about 20 amino acid residues or less.

[0120] In some embodiments, the size of the recombinant gene effector disclosed herein may be about 20 to about 200 amino acid residues in length, about 40 to about 180 amino acid residues in length, about 50 to about 150 amino acid residues in length, about 60 to about 140 amino acid residues in length, about 60 to about 130 amino acid residues in length, about 60 to about 120 amino acid residues in length, about 60 to about 110 amino acid residues in length, about 60 to about 100 amino acid residues in length, about 70 to about 120 amino acid residues in length, about 70 to about 110 amino acid residues in length, about 70 to about 100 amino acid residues in length, about 80 to about 120 amino acid residues in length, about 80 to about 110 amino acid residues in length, or about 80 to about 100 amino acid residues in length.

[0121] In some embodiments, the size of the recombinant gene effectors disclosed herein may be less than about 680 amino acid residues in length, less than about 650 amino acid residues in length, less than about 600 amino acid residues in length, less than about 500 amino acid residues in length, less than about 400 amino acid residues in length, less than about 300 amino acid residues in length, less than about 200 amino acid residues in length, less than about 150 amino acid residues in length, less than about 140 amino acid residues in length, less than about 130 amino acid residues in length, less than about 120 amino acid residues in length, less than about 110 amino acid residues in length, or less than about 100 amino acid residues in length.

[0122] In some embodiments, the size of the recombinant gene effectors disclosed herein is about 100 amino acid residues or less in length, about 95 amino acid residues or less in length, about 90 amino acid residues or less in length, about 85 amino acid residues or less in length, about 80 amino acid residues or less in length, about 75 amino acid residues or less in length, about 74 amino acid residues or less in length, about 73 amino acid residues or less in length, about 72 amino acid residues or less in length, about 71 amino acid residues or less in length, about 70 amino acid residues or less in length, about 69 amino acid residues or less in length, about 68 amino acid residues or less in length, about 67 amino acid residues or less in length, about 66 amino acid residues or less in length, about 65 amino acid residues or less in length. The length may be about 64 amino acid residues or less, about 63 amino acid residues or less, about 62 amino acid residues or less, about 61 amino acid residues or less, about 60 amino acid residues or less, about 59 amino acid residues or less, about 58 amino acid residues or less, about 57 amino acid residues or less, about 56 amino acid residues or less, about 55 amino acid residues or less, about 54 amino acid residues or less, about 53 amino acid residues or less, about 52 amino acid residues or less, about 51 amino acid residues or less, about 50 amino acid residues or less, about 45 amino acid residues or less, about 40 amino acid residues or less, or about 35 amino acid residues or less.

[0123] In some embodiments, the recombinant gene effectors disclosed herein may comprise polypeptides.

[0124] In some embodiments, the polypeptide comprised in a recombinant gene effector disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or at least about 78% or less, identical to the polypeptide sequence of SEQ ID NO:1. It may have about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity. For example, the amino acid sequence of a recombinant gene effector of the present disclosure may have about 80% to about 100% identity to the polypeptide sequence of SEQ ID NO:1.

[0125] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein, when aligned to the polypeptide sequence of SEQ ID NO: 1, may comprise C4. In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein, when aligned to the polypeptide sequence of SEQ ID NO: 1, may comprise L5. In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein, when aligned to the polypeptide sequence of SEQ ID NO: 1, may comprise M7. In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein, when aligned to the polypeptide sequence of SEQ ID NO: 1, may comprise L19. In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein, when aligned to the polypeptide sequence of SEQ ID NO: 1, may comprise at least one member, at least two members, at least three members, or at least four members selected from the group consisting of C4, L5, M7, and L19. In some embodiments, the amino acid sequence of the polypeptide comprised in the recombinant gene effector disclosed herein, when aligned with the polypeptide sequence of SEQ ID NO:1, may comprise one or more members selected from the group consisting of C4, L5, M7 and L19.

[0126] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 2. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector of the present disclosure may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 2.

[0127] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 3. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector of the present disclosure may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 3.

[0128] In some embodiments, the size of the polypeptide comprised in the recombinant gene effector disclosed herein is at least or not more than about 250 amino acid residues in length, at least or not more than about 240 amino acid residues in length, at least or not more than about 230 amino acid residues in length, at least or not more than about 220 amino acid residues in length, at least or not more than about 210 amino acid residues in length, at least or not more than about 200 amino acid residues in length, Length of about 200 amino acid residues or less, at least about or less than 190 amino acid residues, at least about or less than 180 amino acid residues, at least about or less than 170 amino acid residues, at least about or less than 160 amino acid residues, at least about or less than 150 amino acid residues, at least about or less than 140 amino acid residues, at least about or less than 130 amino acid residues amino acid residues or less, at least about 120 amino acid residues or less, at least about 110 amino acid residues or less, at least about 100 amino acid residues or less, at least about 95 amino acid residues or less, at least about 90 amino acid residues or less, at least about 85 amino acid residues or less, at least about 80 amino acid residues or less, at least about 70 amino acid residues or less a length of at least about 70 amino acid residues or less, at least about 60 amino acid residues or less, at least about 50 amino acid residues or less, at least about 45 amino acid residues or less, at least about 40 amino acid residues or less, at least about 35 amino acid residues or less, at least about 30 amino acid residues or less, at least about 25 amino acid residues or less,It may be at least about 20 amino acid residues in length or less, at least about 15 amino acid residues in length or less, or at least about 10 amino acid residues in length or less.

[0129] In some embodiments, the size of the polypeptide contained in the recombinant gene effector disclosed herein is from about 10 to about 100 amino acid residues in length, from about 10 to about 90 amino acid residues in length, from about 10 to about 80 amino acid residues in length, from about 10 to about 70 amino acid residues in length, from about 10 to about 60 amino acid residues in length, from about 10 to about 50 amino acid residues in length, from about 10 to about 40 amino acid residues in length, from about 15 to about 100 amino acid residues in length, from about 15 to about 90 amino acid residues in length, or from about 15 to about 80 amino acid residues in length. , about 15 to about 70 amino acid residues in length, about 15 to about 60 amino acid residues in length, about 15 to about 50 amino acid residues in length, about 15 to about 40 amino acid residues in length, about 20 to about 100 amino acid residues in length, about 20 to about 90 amino acid residues in length, about 20 to about 80 amino acid residues in length, about 20 to about 70 amino acid residues in length, about 20 to about 60 amino acid residues in length, about 20 to about 50 amino acid residues in length, about 20 to about 40 amino acid residues in length, or about 25 to about 35 amino acid residues in length.

[0130] In some embodiments, the recombinant gene effector disclosed herein may comprise only one of the polypeptides disclosed herein.

[0131] In some embodiments, the recombinant gene effector disclosed herein may comprise a plurality of such polypeptides (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more) as disclosed herein. Such a plurality of polypeptides may be substantially the same. Additionally or alternatively, such a plurality of polypeptides may be different from one another. In some embodiments, such a plurality of polypeptides may comprise a first polypeptide (e.g., a polypeptide exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:1) and a second polypeptide (e.g., a polypeptide exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:1). The first and second polypeptides may be linked to each other, for example, directly or indirectly (e.g., via a linker). For example, the first and second polypeptides may be fused to each other, for example, directly or indirectly (e.g., via a linker). In another example, the first and second polypeptides may be linked to each other via a non-covalent bond, such as an ionic bond, a hydrogen bond, or an interaction via an oligomerization or dimerization domain.

[0132] In some embodiments of any of the recombinant gene effectors disclosed herein, the central amino acid sequence of the polypeptide comprised in the recombinant gene effector may be closer to the C-terminus than to the N-terminus of the recombinant gene effector, and the degree of proximity to the C-terminus may be at least about 1 amino acid residue or less, at least about 2 amino acid residues or less, at least about 3 amino acid residues or less, at least about 4 amino acid residues or less, at least about 5 amino acid residues or less, at least about 10 amino acid residues or less, at least about 15 amino acid residues or less, at least about 20 amino acid residues or less, at least about 25 amino acid residues or less, at least about 30 amino acid residues or less, or at least about 35 amino acid residues or less. The length may be at least about 40 or less amino acid residues, at least about 45 or less amino acid residues, at least about 50 or less amino acid residues, at least about 60 or less amino acid residues, at least about 70 or less amino acid residues, at least about 80 or less amino acid residues, at least about 90 or less amino acid residues, at least about 100 or less amino acid residues, at least about 110 or less amino acid residues, at least about 120 or less amino acid residues, at least about 130 or less amino acid residues, at least about 140 or less amino acid residues, at least about 150 or less amino acid residues, or at least about 200 amino acid residues.

[0133] In some embodiments of any of the recombinant gene effectors disclosed herein, the central amino acid sequence of the polypeptide comprised in the recombinant gene effector may be closer to the N-terminus than to the C-terminus of the recombinant gene effector, and the degree of proximity to the N-terminus may be at least about 1 amino acid residue or less, at least about 2 amino acid residues or less, at least about 3 amino acid residues or less, at least about 4 amino acid residues or less, at least about 5 amino acid residues or less, at least about 10 amino acid residues or less, at least about 15 amino acid residues or less, at least about 20 amino acid residues or less, at least about 25 amino acid residues or less, at least about 30 amino acid residues or less, or at least about 35 amino acid residues or less. The length may be at least about 40 or less amino acid residues, at least about 45 or less amino acid residues, at least about 50 or less amino acid residues, at least about 60 or less amino acid residues, at least about 70 or less amino acid residues, at least about 80 or less amino acid residues, at least about 90 or less amino acid residues, at least about 100 or less amino acid residues, at least about 110 or less amino acid residues, at least about 120 or less amino acid residues, at least about 130 or less amino acid residues, at least about 140 or less amino acid residues, at least about 150 or less amino acid residues, or at least about 200 amino acid residues.

[0134] In some embodiments, the polypeptide included in a recombinant effector (e.g., a recombinant gene activator) disclosed herein may be heterologous to one or more members selected from the group consisting of VP16, VP64, VP48, VP160, p65, EDLL, TAL, SET1A, SET1B, MLL, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI60, CLOCK, TETDME, DML1, DML2, ROS1, Rta, and VPR (e.g., heterologous to at least one member, at least two members, at least three members, at least four members, at least five members, or more members, or to any one of these members). Such one or more members may be referred to as a "transcriptional activator."

[0135] In some embodiments, the polypeptide included in a recombinant effector (e.g., a recombinant gene activator) disclosed herein may be heterologous to one or more members selected from the group consisting of VP16, VP64, p65, Rta, and VPR (e.g., heterologous to one, two, three, four, or five members). In some cases, the polypeptide included in a recombinant effector disclosed herein may be heterologous to any one (or all) of the members selected from the group consisting of VP16, VP64, p65, Rta, and VPR.

[0136] In some embodiments, a recombinant gene effector (e.g., a recombinant gene activator) disclosed herein can activate the expression level of a target gene in a cell relative to a control (e.g., the recombinant gene effector alone or in combination with a heterologous endonuclease can activate the expression level of a target gene in a cell, e.g., a Cas protein or a dCas protein, e.g., an inactive variant of Un1Cas12f1 comprising the polypeptide sequence of SEQ ID NO: 11). The control can be the expression level (e.g., basal expression level) of a target gene in a control cell that has not been externally manipulated (e.g., the expression level of the target gene in the absence of a recombinant gene effector and / or a heterologous endonuclease). The control can also be the expression level of a target gene in a control cell treated with a heterologous endonuclease (e.g., dCas) alone in the absence of a genetic effector operably linked to the genetic effector disclosed herein. Additionally, the control may be the expression level of the target gene in control cells treated with a control gene effector (e.g., control cells treated with the control gene effector alone or control cells treated with the control gene effector in combination with a heterologous endonuclease). Examples of control gene effectors include, but are not limited to, VP16, VP64, p65, Rta, and VPR.

[0137] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144 The percentage may be at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.

[0138] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144 The percentage may be at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.

[0139] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144 The percentage may be at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.

[0140] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144 The percentage may be at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.

[0141] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144 The percentage may be at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.

[0142] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, or at least about 25% higher than the expression level of a target gene activated by a VPR (e.g., in conjunction with a heterologous endonuclease) in a control cell. Alternatively, the expression level of the target gene activated by VPR (e.g., in combination with a heterologous endonuclease) in a control cell may be at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold, or at least about 100% higher, or at least about 100% higher, or at least about 30% higher, at least about 40% higher, at least about 50% higher, at least about 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 100% higher, or at least about 0.5-fold, at least about 100% higher, or ... at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.It may be 5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0143] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, or at least about 25% higher than the expression level of a target gene activated by Rta (e.g., in conjunction with a heterologous endonuclease) in a control cell. Alternatively, the expression level of the target gene activated by Rta (e.g., in combination with a heterologous endonuclease) in a control cell may be at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 100% higher, at least about 150% higher, at least about 200% higher, at least about 250% higher, at least about 30% higher, at least about 40% higher, at least about 50% higher, at least about 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, at least about 100% higher, at least about 150% higher ... at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.It may be 5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0144] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, or at least about 25% higher than the expression level of a target gene activated by p65 (e.g., in conjunction with a heterologous endonuclease) in a control cell. Alternatively, the expression level of a target gene activated by p65 (e.g., in combination with a heterologous endonuclease) in a control cell may be at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 100% higher, at least about 150% higher, at least about 200% higher, at least about 250% higher, at least about 30% higher, at least about 40% higher, at least about 50% higher, at least about 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, at least about 100% higher, at least about 150% higher ... at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.It may be 5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0145] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, or at least about 25% higher than the expression level of a target gene activated by VP64 (e.g., in conjunction with a heterologous endonuclease) in a control cell. Alternatively, the expression level of the target gene activated by VP64 (e.g., in combination with a heterologous endonuclease) in a control cell may be at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 100% higher, at least about 150% higher, at least about 200% higher, at least about 250% higher, at least about 30% higher, at least about 40% higher, at least about 50% higher, at least about 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold higher, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, at least about 100% higher, at least about 150% higher ... at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.It may be 5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0146] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be at least about 1% higher, at least about 2% higher, at least about 3% higher, at least about 4% higher, at least about 5% higher, at least about 6% higher, at least about 7% higher, at least about 8% higher, at least about 9% higher, at least about 10% higher, at least about 11% higher, at least about 12% higher, at least about 13% higher, at least about 14% higher, at least about 15% higher, at least about 16% higher, at least about 17% higher, at least about 18% higher, at least about 19% higher, at least about 20% higher, or at least about 25% higher than the expression level of a target gene activated by VP16 (e.g., in conjunction with a heterologous endonuclease) in a control cell. Alternatively, the expression level of the target gene activated by VP16 (e.g., in combination with a heterologous endonuclease) in a control cell may be at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold, or at least about 100% higher, or at least about 30% higher, at least about 40% higher, at least about 50% higher, at least about 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 0.5-fold, or at least about 0.6-fold, or at least about 0.7-fold, or at least about 0.8-fold, or at least about 0.9-fold, or at least about 100% higher, or at least about 0.9-fold, or at least about 100% higher, or at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, or at least about 100% higher, or at least about 0.5-fold, at least about 100% higher, or at least about 0.6-fold, at least about 100% higher, or at least about 0.7-fold, at least about 100% higher, or at least about 0.8-fold, at least about 100% higher, or at least about 0.9 ... at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.It may be 5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0147] In some embodiments, the expression level of a target gene activated by a recombinant gene effector disclosed herein (e.g., in conjunction with a heterologous endonuclease) may be substantially sustained (e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of the peak or modulated expression level may be persisted or maintained for a period of at least about 1 hour or less, at least about 2 hours or less, at least about 6 hours or less, at least about 12 hours or less, at least about 18 hours or less, at least about 24 hours or less, or at least about 2 days or less. The period may be at least about 3 days or less, at least about 4 days or less, at least about 5 days or less, at least about 6 days or less, at least about 7 days or less, at least about 8 days or less, at least about 9 days or less, at least about 10 days or less, at least about 11 days or less, at least about 12 days or less, at least about 13 days or less, at least about 14 days or less, at least about 3 weeks or less, at least about 4 weeks or less, at least about 2 months or less, at least about 4 months or less, or at least about 6 months or less.

[0148] In some embodiments, the expression level of a target gene activated by a recombinant genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be substantially sustained at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more, of the expression level of a target gene activated by a control genetic effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc., in combination with a comparable heterologous endonuclease) in a control cell.

[0149] In some embodiments, contacting a cell population with a system provided herein (e.g., at least a recombinant gene effector) may result in a higher percentage of cells in which target gene expression is modulated (e.g., a higher percentage of cells in which the target gene is activated or repressed) compared to a cell population contacted with a system including a control gene effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc.). In some embodiments, expression may be modulated in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the cells in a cell population contacted with a system provided herein (e.g., at least a recombinant gene effector). In some embodiments, contacting a cell population with a system provided herein (e.g., at least a recombinant gene effector) may result in an increased percentage of cells whose expression is modulated, and the percentage of cells whose expression is modulated may be at least about 10% higher, at least about 20% higher, at least about 30% higher, at least about 40% higher, at least about 50% higher, or at least about 60% higher than the percentage of cells whose expression is modulated by contacting a cell population with a system comprising a control gene effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc.). It may be 60% higher, at least about 70% higher, at least about 80% higher, at least about 90% higher, at least about 100% higher, at least about 110% higher, at least about 120% higher, at least about 130% higher, at least about 140% higher, at least about 150% higher, at least about 160% higher, at least about 170% higher, at least about 180% higher, at least about 190% higher, at least about 200% higher, or even higher.

[0150] In some embodiments, the polypeptides comprised in the recombinant effectors (e.g., recombinant gene repressors) disclosed herein include a Kruppel-associated box (KRAB or SKD); a KOX1 repression domain; a Mad mSIN3-interacting domains (SIDs); ERF repressor domains (ERDs) (e.g., for repression in plants), SRDX repression domains, etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARIDlB / PLU-1, JARID1C / SMCX, and JARIDID / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; Hal DNA The repressor may be heterologous to one or more members (e.g., at least one member, at least two members, at least three members, at least four members, at least five members, or more members, or any one of these members) selected from the group consisting of DNA methylases such as m5c methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and peripheral recruitment factors such as lamin A and lamin B. Such one or more members may be referred to as a "transcriptional repressor."

[0151] In some embodiments, the polypeptide included in a recombinant effector (e.g., a recombinant gene repressor) disclosed herein may be heterologous to one or more members selected from the group consisting of KRAB, DNMT3A, and DNMT3L (e.g., may be heterologous to one member, two members, or three members).

[0152] In some embodiments, the polypeptides included in the recombinant effectors (e.g., recombinant gene repressors) disclosed herein may not, and may not need to, include KRAB, DNMT3A, and / or DNMT3L.

[0153] In some embodiments, a recombinant gene effector (e.g., a recombinant gene repressor) disclosed herein can reduce the expression level of a target gene in a cell compared to a control (e.g., the recombinant gene effector alone or in combination with a heterologous endonuclease can reduce the expression level of a target gene in a cell, e.g., a Cas protein or a dCas protein, e.g., an inactive variant of Un1Cas12f1 comprising the polypeptide sequence of SEQ ID NO: 11). The control can be the expression level (e.g., basal expression level) of a target gene in a control cell that has not been externally manipulated (e.g., the expression level of the target gene in the absence of a recombinant gene effector and / or a heterologous endonuclease). Alternatively, the control can be the expression level of a target gene in a control cell treated with a heterologous endonuclease (e.g., dCas) alone in the absence of a genetic effector operably linked to the genetic effector disclosed herein. Furthermore, the control may be the expression level of the target gene in control cells treated with a control gene effector (e.g., control cells treated with the control gene effector alone or control cells treated with the control gene effector in combination with a heterologous endonuclease). Examples of control gene effectors include, but are not limited to, KRAB, DNMT3A, and DNMT3L, and fusion repressors comprising KRAB, DNMT3A, and DNMT3L (KAL).

[0154] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 25% or less, at least about 30% or less, at least about 35% or less, of the expression level of a target gene that is reduced by KRAB (e.g., in combination with a heterologous endonuclease) in a control cell. It may be at least about 40% or less, at least about 45% or less, at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, at least about 90% or less, or at least about 95% or less.

[0155] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be comparable to (e.g., substantially the same as) the expression level of a target gene that is reduced by KRAB (e.g., in combination with a heterologous endonuclease) in a control cell.

[0156] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be lower than the expression level of a target gene that is reduced by KRAB (e.g., in combination with a heterologous endonuclease) in a control cell, by a difference of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 40%. %, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0157] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 25% or less, at least about 30% or less, or at least about 35% or less of the expression level of a target gene that is reduced by DNMT3A (e.g., in combination with a heterologous endonuclease) in a control cell. , at least about 40% or less, at least about 45% or less, at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, at least about 90% or less, or at least about 95% or less.

[0158] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be comparable to (e.g., substantially the same as) the expression level of a target gene that is reduced by DNMT3A (e.g., in combination with a heterologous endonuclease) in a control cell.

[0159] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be lower than the expression level of a target gene that is reduced by DNMT3A (e.g., in combination with a heterologous endonuclease) in a control cell, by a difference of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 3 ... The increase may be 0%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0160] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 25% or less, at least about 30% or less, or at least about 35% or less of the expression level of a target gene that is reduced by DNMT3L (e.g., in combination with a heterologous endonuclease) in a control cell. , at least about 40% or less, at least about 45% or less, at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, at least about 90% or less, or at least about 95% or less.

[0161] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be comparable to (e.g., substantially the same as) the expression level of a target gene that is reduced by DNMT3L (e.g., in combination with a heterologous endonuclease) in a control cell.

[0162] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be lower than the expression level of a target gene that is reduced by DNMT3L (e.g., in combination with a heterologous endonuclease) in a control cell, by a difference of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 3 ... The increase may be 0%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0163] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) is at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 25% or less, at least about 30% or less, at least about 35% or less, or at least about 40% or less of the expression level of a target gene that is reduced by a KAL (e.g., in combination with a heterologous endonuclease) in a control cell. It may be at least about 40% or less, at least about 45% or less, at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, at least about 90% or less, or at least about 95% or less.

[0164] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be comparable to (e.g., substantially the same as) the expression level of a target gene that is reduced by a KAL (e.g., in combination with a heterologous endonuclease) in a control cell.

[0165] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be lower than the expression level of a target gene that is reduced by a KAL (e.g., in combination with a heterologous endonuclease) in a control cell by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%. , at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.7 times, at least about 0.8 times, at least about 0.9 times, at least about 1 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0166] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be substantially sustained by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more, compared to the expression level of a target gene that is reduced by KRAB (e.g., in combination with a heterologous endonuclease) in a control cell.

[0167] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be substantially sustained by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more, compared to the expression level of a target gene that is reduced by DNMT3A (e.g., in combination with a heterologous endonuclease) in a control cell.

[0168] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be substantially sustained by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more, compared to the expression level of a target gene that is reduced by DNMT3L (e.g., in combination with a heterologous endonuclease) in a control cell.

[0169] In some embodiments, the expression level of a target gene that is reduced by a recombinant gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) may be substantially sustained by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200% or more compared to the expression level of a target gene that is reduced by a KAL (e.g., in combination with a heterologous endonuclease) in a control cell.

[0170] In some embodiments, a recombinant gene effector disclosed herein may comprise the polypeptide and an additional polypeptide. The polypeptide and additional polypeptide may be heterologous to each other. The polypeptide and additional polypeptide may be linked to each other, for example, directly or indirectly (e.g., via a linker). For example, the polypeptide and additional polypeptide may be fused to each other, for example, directly or indirectly (e.g., via a linker). In another example, the polypeptide and additional polypeptide may be linked to each other non-covalently, for example, by ionic bonds, hydrogen bonds, or interactions via oligomerization or dimerization domains. In some embodiments, a recombinant gene effector disclosed herein may comprise only one of the additional polypeptides disclosed herein. In some embodiments, a recombinant gene effector disclosed herein may comprise multiple (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) additional polypeptides disclosed herein. Such additional multiple polypeptides may be substantially the same. Additionally or alternatively, such additional multiple polypeptides may be different from one another.

[0171] In some embodiments, the additional polypeptide of the recombinant gene effector may not include the polypeptide sequence of SEQ ID NO:1.

[0172] Without wishing to be bound by any theory, in some embodiments, (1) a first recombinant gene effector may include at least a portion of the polypeptide sequence of SEQ ID NO: 1, and (2) a second recombinant gene effector may include at least a portion of the polypeptide sequence of SEQ ID NO: 1 that is different from that included in the first recombinant gene effector, and the first recombinant gene effector and the second recombinant gene effector may exhibit different gene regulatory activities. In some cases, even if at least a portion of the polypeptide sequence of SEQ ID NO: 1 included in (1) the first recombinant gene effector is substantially identical to at least a portion of the polypeptide sequence of SEQ ID NO: 1 included in (2) the second recombinant gene effector, the first recombinant gene effector and the second recombinant gene effector may exhibit different (e.g., opposite) gene regulatory activities. For example, the first recombinant gene effector may be a gene activator, while the second recombinant gene effector may be a gene repressor. For example, the different activity between a first and second recombinant gene effector may be due to one or more differences between the first and second recombinant gene effector, such as, for example, (i) the location of at least a portion of the polypeptide sequence of SEQ ID NO: 1 within each recombinant gene effector, (ii) the overall size / length of each recombinant gene effector, (iii) the presence or absence, size or amino acid residue composition of an additional polypeptide sequence added to the N-terminal side of at least a portion of the polypeptide sequence of SEQ ID NO: 1, (iv) the presence or absence, size or amino acid residue composition of an additional polypeptide sequence added to the C-terminal side of at least a portion of the polypeptide sequence of SEQ ID NO: 1, and / or (v) the size or amino acid residue composition of at least a portion of the polypeptide sequence of SEQ ID NO: 1.

[0173] In some embodiments, an additional polypeptide of a genetic effector disclosed herein can be positioned adjacent to the N-terminus of the polypeptide of the genetic effector. In some embodiments, an additional polypeptide of a genetic effector disclosed herein can be positioned adjacent to the C-terminus of the polypeptide of the genetic effector. In some embodiments, a first type of additional polypeptide of a genetic effector (e.g., a first additional polypeptide) can be positioned adjacent to the N-terminus of the polypeptide of the genetic effector, and a second type of additional polypeptide of the genetic effector (e.g., a second additional polypeptide) can be positioned adjacent to the C-terminus of the polypeptide of the genetic effector (e.g., a first additional polypeptide-said polypeptide-second additional polypeptide configuration). The polypeptide can be sandwiched between the first additional polypeptide and the second additional polypeptide. The first additional polypeptide and the second additional polypeptide can be substantially the same. Alternatively, the first additional polypeptide and the second additional polypeptide can be different. In some embodiments, a first type of polypeptide (e.g., a first polypeptide) of a genetic effector can be positioned adjacent to the N-terminus of an additional polypeptide of the genetic effector, and a second type of polypeptide (e.g., a second polypeptide) of the genetic effector can be positioned adjacent to the C-terminus of the additional polypeptide of the genetic effector (e.g., a first polypeptide-additional polypeptide-second polypeptide configuration). The additional polypeptide can be sandwiched between the first polypeptide and the second polypeptide. The first polypeptide and the second polypeptide can be substantially the same. Alternatively, the first polypeptide and the second polypeptide can be different.

[0174] In some embodiments, an additional polypeptide of a recombinant gene effector disclosed herein can be positioned (e.g., inserted) within the polypeptide contained in the recombinant gene effector. The additional polypeptide can be inserted at the N-terminus of the polypeptide contained in the recombinant gene effector disclosed herein, adjacent to the N-terminus of the polypeptide. Additionally or alternatively, the additional polypeptide can be inserted at the C-terminus of the polypeptide contained in the recombinant gene effector disclosed herein, adjacent to the N-terminus of the polypeptide.

[0175] In some embodiments, the size of the polypeptide and the additional polypeptide contained in the recombinant genetic effector disclosed herein may be substantially the same, hi some embodiments, the size of the polypeptide and the additional polypeptide contained in the recombinant genetic effector disclosed herein may be different.

[0176] In some embodiments of the recombinant gene effector disclosed herein, the size of the polypeptide comprised in the recombinant gene effector may be longer than the size of the additional polypeptide, for example, the difference may be at least or less than about 1 amino acid residue, at least or less than about 2 amino acid residues, at least or less than about 3 amino acid residues, at least or less than about 4 amino acid residues, at least or less than about 5 amino acid residues, at least or less than about 10 amino acid residues, at least or less than about 15 amino acid residues, at least or less than about 20 amino acid residues, at least or less than about 25 amino acid residues, at least or less than about 30 amino acid residues, at least or less than about 35 amino acid residues, at least or less than about 40 amino acid residues. The amino acid residues may be at least about 40 or less amino acid residues, at least about 45 or less amino acid residues, at least about 50 or less amino acid residues, at least about 60 or less amino acid residues, at least about 70 or less amino acid residues, at least about 80 or less amino acid residues, at least about 90 or less amino acid residues, at least about 100 or less amino acid residues, at least about 110 or less amino acid residues, at least about 120 or less amino acid residues, at least about 130 or less amino acid residues, at least about 140 or less amino acid residues, at least about 150 or less amino acid residues, or at least about 200 or less amino acid residues.

[0177] In some embodiments of the recombinant gene effectors disclosed herein, the size of the additional polypeptide may be longer than the size of the polypeptide comprised in the recombinant gene effector, for example, by at least about 1 amino acid residue or less, at least about 2 amino acid residues or less, at least about 3 amino acid residues or less, at least about 4 amino acid residues or less, at least about 5 amino acid residues or less, at least about 10 amino acid residues or less, at least about 15 amino acid residues or less, at least about 20 amino acid residues or less, at least about 25 amino acid residues or less, at least about 30 amino acid residues or less, at least about 35 amino acid residues or less, or at least about 40 amino acid residues. Alternatively, it may be about 40 amino acid residues or less, at least about 45 amino acid residues or less, at least about 50 amino acid residues or less, at least about 60 amino acid residues or less, at least about 70 amino acid residues or less, at least about 80 amino acid residues or less, at least about 90 amino acid residues or less, at least about 100 amino acid residues or less, at least about 110 amino acid residues or less, at least about 120 amino acid residues or less, at least about 130 amino acid residues or less, at least about 140 amino acid residues or less, at least about 150 amino acid residues or less, at least about 200 amino acid residues or less, or more.

[0178] In some embodiments of the recombinant gene effectors disclosed herein, the size of the polypeptide included in the recombinant gene effector may be at least about 1% longer, at least about 2% longer, at least about 5% longer, at least about 10% longer, at least about 15% longer, at least about 20% longer, at least about 25% longer, at least about 26% longer, at least about 27% longer, at least about 28% longer, at least about 29% longer, at least about 30% longer, at least about 31% longer, at least about 32% longer, at least about 33% longer, at least about 34% longer, at least about 35% longer, at least about 40% longer, or at least about 45% longer than the size of the additional polypeptide. It may be 0% longer, at least about 50% longer, at least about 60% longer, at least about 70% longer, at least about 80% longer, at least about 90% longer, at least about 100% longer, at least about 110% longer, at least about 120% longer, at least about 130% longer, at least about 140% longer, at least about 150% longer, at least about 160% longer, at least about 170% longer, at least about 180% longer, at least about 190% longer, at least about 200% longer, at least about 250% longer, at least about 300% longer, at least about 400% longer, at least about 500% longer, or more.In some embodiments of the recombinant gene effector disclosed herein, the size of the polypeptide comprised in the recombinant gene effector may be up to about 1% longer, up to about 2% longer, up to about 5% longer, up to about 10% longer, up to about 15% longer, up to about 20% longer, up to about 25% longer, up to about 26% longer, up to about 27% longer, up to about 28% longer, up to about 29% longer, up to about 30% longer, up to about 31% longer, up to about 32% longer, up to about 33% longer, up to about 34% longer, or up to about 35% longer than the size of the additional polypeptide. may be up to about 40% longer, may be up to about 50% longer, may be up to about 60% longer, may be up to about 70% longer, may be up to about 80% longer, may be up to about 90% longer, may be up to about 100% longer, may be up to about 110% longer, may be up to about 120% longer, may be up to about 130% longer, may be up to about 140% longer, may be up to about 150% longer, may be up to about 160% longer, may be up to about 170% longer, may be up to about 180% longer, may be up to about 190% longer, may be up to about 200% longer, may be up to about 250% longer, may be up to about 300% longer, may be up to about 400% longer, or may be up to about 500% longer.

[0179] In some embodiments of the recombinant gene effectors disclosed herein, the size of the additional polypeptide may be at least about 1% longer, at least about 2% longer, at least about 5% longer, at least about 10% longer, at least about 15% longer, at least about 20% longer, at least about 25% longer, at least about 26% longer, at least about 27% longer, at least about 28% longer, at least about 29% longer, at least about 30% longer, at least about 31% longer, at least about 32% longer, at least about 33% longer, at least about 34% longer, at least about 35% longer, at least about 40% longer, at least about 45% longer, at least about 46% longer, at least about 47% longer, at least about 48% longer, at least about 49% longer, at least about 50% longer, at least about 51% longer, at least about 52% longer, at least about 53% longer, at least about 54% longer, at least about 55% longer, at least about 56% longer, at least about 57% longer, at least about 58% longer, at least about 59% longer, at least about 60% longer, at least about 61% longer, at least about 62% longer, at least about 63% longer, at least about 64% longer, at least about 65% longer, at least about 66% longer, at least about 67% longer, at least about 68% longer, at least about 69% longer, at least about 70% longer, at least about 71% longer, at least about 72% longer, at least about 73% longer, at least about 74% longer, at least about 75% longer, at least about 76% longer, at least about 77% longer, at least about 78% longer, at least about 79% longer, at least about 80% longer, at least about 81% longer It may be 0% longer, at least about 50% longer, at least about 60% longer, at least about 70% longer, at least about 80% longer, at least about 90% longer, at least about 100% longer, at least about 110% longer, at least about 120% longer, at least about 130% longer, at least about 140% longer, at least about 150% longer, at least about 160% longer, at least about 170% longer, at least about 180% longer, at least about 190% longer, at least about 200% longer, at least about 250% longer, at least about 300% longer, at least about 400% longer, at least about 500% longer, or more.In some embodiments of the recombinant gene effectors disclosed herein, the size of the additional polypeptide may be up to about 1% longer, up to about 2% longer, up to about 5% longer, up to about 10% longer, up to about 15% longer, up to about 20% longer, up to about 25% longer, up to about 26% longer, up to about 27% longer, up to about 28% longer, up to about 29% longer, up to about 30% longer, up to about 31% longer, up to about 32% longer, up to about 33% longer, up to about 34% longer, or up to about 35% longer than the size of the polypeptide comprised in the recombinant gene effector. may be up to about 40% longer, may be up to about 50% longer, may be up to about 60% longer, may be up to about 70% longer, may be up to about 80% longer, may be up to about 90% longer, may be up to about 100% longer, may be up to about 110% longer, may be up to about 120% longer, may be up to about 130% longer, may be up to about 140% longer, may be up to about 150% longer, may be up to about 160% longer, may be up to about 170% longer, may be up to about 180% longer, may be up to about 190% longer, may be up to about 200% longer, may be up to about 250% longer, may be up to about 300% longer, may be up to about 400% longer, or may be up to about 500% longer.

[0180] In some embodiments, the size of the additional polypeptide of the recombinant gene effector disclosed herein is at least or not more than about 500 amino acid residues in length, at least or not more than about 480 amino acid residues in length, at least or not more than about 460 amino acid residues in length, at least or not more than about 450 amino acid residues in length, at least or not more than about 440 amino acid residues in length, at least or not more than about 420 amino acid residues in length, The length of the nucleotide sequence is about 420 amino acid residues or less, at least about 400 amino acid residues or less, at least about 380 amino acid residues or less, at least about 360 amino acid residues or less, at least about 350 amino acid residues or less, at least about 340 amino acid residues or less, at least about 320 amino acid residues or less, at least about 300 amino acid residues or less. amino acid residues or less, at least about 290 amino acid residues or less, at least about 280 amino acid residues or less, at least about 270 amino acid residues or less, at least about 260 amino acid residues or less, at least about 250 amino acid residues or less, at least about 240 amino acid residues or less, at least about 230 amino acid residues or less length or less, at least about 220 amino acid residues or less, at least about 210 amino acid residues or less, at least about 200 amino acid residues or less, at least about 190 amino acid residues or less, at least about 180 amino acid residues or less, at least about 170 amino acid residues or less, at least about 160 amino acid residues or less,at least about 150 amino acid residues in length or less, at least about 140 amino acid residues in length or less, at least about 130 amino acid residues in length or less, at least about 120 amino acid residues in length or less, at least about 110 amino acid residues in length or less, at least about 100 amino acid residues in length or less, at least about 95 amino acid residues in length or less, Length of about 90 amino acid residues or less, at least about 85 amino acid residues or less, at least about 80 amino acid residues or less, at least about 70 amino acid residues or less, at least about 60 amino acid residues or less, at least about 50 amino acid residues or less, at least about 45 amino acid residues or less, at least about 40 amino acid residues or less The length may be at least about 35 amino acid residues or less, at least about 30 amino acid residues or less, at least about 25 amino acid residues or less, at least about 20 amino acid residues or less, at least about 15 amino acid residues or less, at least about 10 amino acid residues or less, at least about 9 amino acid residues or less, at least about 8 amino acid residues or less, at least about 7 amino acid residues or less, at least about 6 amino acid residues or less, at least about 5 amino acid residues or less, at least about 4 amino acid residues or less, at least about 3 amino acid residues or less, at least about 2 amino acid residues or less, or at least about 1 amino acid residue or less.

[0181] In some embodiments, the size of the additional polypeptide of the recombinant genetic effector disclosed herein is from about 2 amino acid residues to about 50 amino acid residues, from about 2 amino acid residues to about 40 amino acid residues, from about 2 amino acid residues to about 30 amino acid residues, from about 2 amino acid residues to about 25 amino acid residues, from about 2 amino acid residues to about 20 amino acid residues, from about 2 amino acid residues to about 15 amino acid residues, from about 2 amino acid residues to about 10 amino acid residues, from about 2 amino acid residues to about 9 amino acid residues, from about 2 amino acid residues to about 8 amino acid residues, from about 2 amino acid residues to about 7 amino acid residues, from about 2 amino acid residues to about 6 amino acid residues, from about 2 amino acid residues to about 5 amino acid residues, from about 2 amino acid residues to about 4 amino acid residues, or from about 3 amino acid residues. The length may be from about 1 to about 15 amino acid residues, from about 3 to about 10 amino acid residues, from about 3 to about 9 amino acid residues, from about 3 to about 8 amino acid residues, from about 3 to about 7 amino acid residues, from about 3 to about 6 amino acid residues, from about 3 to about 5 amino acid residues, from about 1 to about 15 amino acid residues, from about 1 to about 10 amino acid residues, from about 1 to about 9 amino acid residues, from about 1 to about 8 amino acid residues, from about 1 to about 7 amino acid residues, from about 1 to about 6 amino acid residues, from about 1 to about 5 amino acid residues, from about 1 to about 4 amino acid residues, or from about 1 to about 3 amino acid residues.

[0182] In some embodiments, the size of the additional polypeptide of the recombinant gene effector disclosed herein is about 50 amino acid residues or less in length, about 45 amino acid residues or less in length, about 44 amino acid residues or less in length, about 43 amino acid residues or less in length, about 42 amino acid residues or less in length, about 41 amino acid residues or less in length, about 40 amino acid residues or less in length, about 39 amino acid residues or less in length, about 38 amino acid residues or less in length, about 37 amino acid residues or less in length, about 36 amino acid residues or less in length, about 35 amino acid residues or less in length, about 34 amino acid residues or less in length, about 33 amino acid residues or less in length, about 32 amino acid residues or less in length, about The length may be 31 amino acid residues or less, about 30 amino acid residues or less, about 29 amino acid residues or less, about 28 amino acid residues or less, about 27 amino acid residues or less, about 26 amino acid residues or less, about 25 amino acid residues or less, about 24 amino acid residues or less, about 23 amino acid residues or less, about 22 amino acid residues or less, about 21 amino acid residues or less, about 20 amino acid residues or less, about 15 amino acid residues or less, about 10 amino acid residues or less, about 5 amino acid residues or less, about 4 amino acid residues or less, about 3 amino acid residues or less, about 2 amino acid residues or less, or about 1 amino acid residue.

[0183] In some embodiments, an additional polypeptide (of a recombinant gene effector disclosed herein) may be inactive by itself, e.g., may not by itself induce a desired biological activity in a cell. In some embodiments, an additional polypeptide (of a recombinant gene effector disclosed herein) may not by itself encode (or comprise) a genetic effector, and need not by itself encode (or comprise) a genetic effector. Thus, the additional polypeptide may not be able to modulate the expression and / or activity of a target gene in a cell in the absence of the polypeptide contained in the recombinant gene effector disclosed herein.

[0184] In some embodiments, the additional polypeptide of the recombinant gene effector disclosed herein may comprise a filler polypeptide sequence. A filler polypeptide sequence can be used in combination with another polypeptide sequence (e.g., the polypeptide contained in the recombinant gene effector of the present disclosure) to increase the size of the entire polypeptide (e.g., the entire recombinant gene effector of the present disclosure). The filler polypeptide sequence may not be capable of exhibiting a desired biological activity by itself. For example, the filler polypeptide sequence may not be capable of regulating the expression level or activity level of a target gene in a cell (e.g., activating or repressing) in the absence of other portions of the recombinant gene effector of the present disclosure (e.g., in the absence of the polypeptide contained in the recombinant gene effector of the present disclosure). The filler polypeptide sequence may not be found in a naturally occurring protein (e.g., a naturally occurring mammalian protein). In some embodiments, the filler polypeptide sequence may comprise at least one (e.g., one or more) aliphatic amino acids selected from the group consisting of isoleucine (I), leucine (L), valine (V), alanine (A), and glycine (G). In some embodiments, a filler polypeptide sequence may include multiple I residues (e.g., II and III). In some embodiments, a filler polypeptide sequence may include multiple L residues (e.g., LL and LLL). In some embodiments, a filler polypeptide sequence may include multiple V residues (e.g., VV and VVV). In some embodiments, a filler polypeptide sequence may include multiple A residues (e.g., AA and AAA). In some embodiments, a filler polypeptide sequence may include multiple G residues (e.g., GG and GGG).

[0185] In some embodiments, the filler polypeptide sequence disclosed herein may be a linker sequence. Any suitable linker can be used. A flexible linker may have a sequence containing a region consisting of glycine and serine residues. Glycine and serine residues can provide flexibility due to their small size, thereby imparting flexibility to the linked functional domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby suppressing undesirable interactions between the linker and the protein moiety. Flexible linkers may also contain additional amino acids, such as threonine or alanine, to maintain flexibility, and polar amino acids, such as lysine or glutamine, to improve solubility. Rigid linkers may, for example, have an α-helical structure. Rigid linkers with an α-helical structure can function as spacers between protein domains. Examples of such linkers include, but are not limited to, the sequences set forth in Table 4 and repeats thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats thereof. SEQ ID NOs: 65-70 provide flexible linkers or subunits thereof. SEQ ID NOs: 71-74 provide rigid linkers or subunits thereof. [Table 1]

[0186] In some embodiments, the length of the linker sequences disclosed herein can be, for example, 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues, 20 amino acid residues, 21 amino acid residues, 22 amino acid residues, 23 amino acid residues, 24 amino acid residues, The length may be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0187] In some embodiments, the linker sequences disclosed herein may comprise at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 7 amino acids, at least 9 amino acids, at least 11 amino acids, at least 13 amino acids, at least 15 amino acids, or at least 20 amino acids. In some embodiments, the linker sequences may comprise up to 5 amino acids, up to 7 amino acids, up to 9 amino acids, up to 11 amino acids, up to 13 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 30 amino acids, up to 40 amino acids, or up to 50 amino acids.

[0188] In some embodiments, a non-peptide linker is used. The non-peptide linker may be, for example, a chemical linker. The two parts of the conjugate of the present disclosure can be linked by a chemical linker. Each chemical linker of the present disclosure may be an alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, and these linkers may be substituted. In some embodiments, the chemical linker of the present disclosure may be an ester, ether, amide, thioether, or polyethylene glycol (PEG). In some embodiments, the linker can reverse the order of the amino acid sequences in the compound, for example, so that the amino acid sequences connected by the linker are linked head-to-head rather than head-to-tail. Examples of such linkers include, but are not limited to, diesters of dicarboxylic acids, such as oxalyl diesters, malonyl diesters, succinyl diesters, glutaryl diesters, adipyl diesters, dimethyl diesters, fumaryl diesters, maleyl diesters, phthalyl diesters, isophthalyl diesters, and terephthalyl diesters. Examples of such linkers include, but are not limited to, diamides of dicarboxylic acids, such as oxalyl diamides, malonyl diamides, succinyl diamides, glutaryl diamides, adipyl diamides, dimethyl diamides, fumaryl diamides, maleyl diamides, phthalyl diamides, isophthalyl diamides, and terephthalyl diamides. Examples of such linkers include, but are not limited to, diaminodiamide linkers, such as ethylenediamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane, 1,3-di(methylamino)propane, 1,4-di(methylamino)butane, 1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and piperazine.Examples of substituents that may be introduced into a linker include, but are not limited to, a hydroxyl group, a sulfhydryl group, a halogen, an amino group, a nitro group, a nitroso group, a cyano group, an azide group, a sulfoxide group, a sulfone group, a sulfonamide group, a carboxyl group, a carboxaldehyde group, an imine group, an alkyl group, a haloalkyl group, an alkenyl group, a haloalkenyl group, an alkynyl group, a haloalkynyl group, an alkoxy group, an aryl group, an aryloxy group, an aralkyl group, an arylalkoxy group, a heterocyclyl group, an acyl group, an acyloxy group, a carbamate group, an amido group, a ureido group, an epoxy group, and an ester group.

[0189] In some embodiments, the additional polypeptides of the recombinant gene effectors disclosed herein may include up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or about 1 glycine serine (GS) linkers. In some embodiments, the additional polypeptides of a recombinant gene effector (e.g., recombinant gene repressor) disclosed herein may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, or more GS linkers.

[0190] In some embodiments, the additional polypeptides of the recombinant gene effectors disclosed herein may include up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or about 1 glycine (G) linkers. In some embodiments, the additional polypeptides of a recombinant gene effector (e.g., recombinant gene repressor) disclosed herein may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, or more G linkers.

[0191] In some embodiments, the additional polypeptide (of the recombinant gene effector disclosed herein) may solely encode (or comprise) at least one gene effector. Thus, the at least one gene effector encoded by (or comprised in) the additional polypeptide can modulate the expression and / or activity of a target gene in a cell in the absence of the polypeptide comprised in the recombinant gene effector of the present disclosure. In some cases, the at least one gene effector may comprise multiple identical gene effectors (e.g., at least two, at least three, at least four, at least five, or more identical gene effectors). Alternatively, the multiple gene effectors may be different from one another. The multiple gene effectors may comprise two or more gene activators. The multiple gene effectors may comprise two or more gene repressors. The multiple gene effectors may comprise a gene activator and a gene repressor.

[0192] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise at least a portion of one or more members selected from the group consisting of VP16, VP64, VP48, VP160, p65, EDLL, TAL, SET1A, SET1B, MLL, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI60, CLOCK, TETDME, DML1, DML2, ROS1, Rta, and VPR. In some embodiments, the additional polypeptides of the recombinant gene effectors (e.g., recombinant gene activators) disclosed herein may not include two or more members selected from the group consisting of VP16, VP64, VP48, VP160, p65, EDLL, TAL, SET1A, SET1B, MLL, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI60, CLOCK, TETDME, DML1, DML2, ROS1, and Rta.

[0193] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise at least a portion of one or more members selected from the group consisting of VP16, VP64, p65, Rta, and VPR.

[0194] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., a recombinant gene activator) disclosed herein may comprise two or more members selected from the group consisting of VP16, VP64, p65, and Rta. In some embodiments, the additional polypeptide of a recombinant gene effector disclosed herein may comprise multiple VP16 domains. In some embodiments, the additional polypeptide of a recombinant gene effector disclosed herein may comprise at least two VP16 domains, at least three VP16 domains, at least four VP16 domains, or at least five VP16 domains.

[0195] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may not comprise two or more members selected from the group consisting of VP16, VP64, p65, and Rta. The additional polypeptide may include VP16 but may not include VP64, p65, and Rta. The additional polypeptide may include VP64 but may not include VP16, p65, and Rta. The additional polypeptide may include p65 but may not include VP16, VP64, and Rta. The additional polypeptide may include Rta but may not include VP16, VP64, and p65. Alternatively, the additional polypeptide may not include VP16, VP64, p65, Rta, and VPR.

[0196] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or It may have about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0197] In some embodiments, the recombinant gene effectors disclosed herein do not comprise a VPR.

[0198] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or It may have about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0199] In some embodiments, the recombinant gene effectors disclosed herein do not include Rta.

[0200] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or It may have about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0201] In some embodiments, the recombinant gene effectors disclosed herein do not include p65.

[0202] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less of the polypeptide sequence of SEQ ID NO: 6 (VP64). may have about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0203] In some embodiments, the recombinant gene effectors disclosed herein do not include VP64.

[0204] In some embodiments, the additional polypeptide of a recombinant gene effector (e.g., recombinant gene activator) disclosed herein may comprise an amino acid sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or the polypeptide sequence of SEQ ID NO: 5 (VP16). may have about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0205] In some embodiments, the recombinant gene effector disclosed herein does not comprise VP16. In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a polypeptide sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, or at least about or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity. [Table 2] JPEG2025510781000004.jpg232164 JPEG2025510781000005.jpg240163 JPEG2025510781000006.jpg241163 JPEG2025510781000007.jpg242164 JPEG2025510781000008.jpg18164

[0206] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence identity at or below about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, or The amino acid sequence may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0207] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 19 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0208] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:20 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0209] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:23 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0210] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:24 of at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0211] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 30 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0212] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO: 31, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0213] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence identity at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, or at least about 80% or less, of the polypeptide sequence of a member selected from SEQ ID NOs: 79-84, 86-92, 94-103, 116, 119-120, 122, 123, and 125 (e.g., a member selected from SEQ ID NOs: 79-84, 88, 90, 91, and 95). or less, at least about 76% or less, at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity (e.g., contiguous polypeptide sequences).

[0214] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 79 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0215] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 80 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0216] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 81 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0217] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 82 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0218] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 83 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0219] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO: 84, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0220] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 86 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0221] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO: 87, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0222] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO: 88, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0223] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 89 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0224] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 90 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0225] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:91 at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0226] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO:92, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0227] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:94 of at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0228] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:95 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0229] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 96 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0230] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO:97, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0231] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:98 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0232] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or similar to the polypeptide sequence of SEQ ID NO:99, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0233] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 100, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0234] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 101, and is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0235] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 102, and / or a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 102, and / or a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 102, and / or a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 102, and / or a sequence similar to or identical to the polypeptide sequence of SEQ ID NO: 102. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0236] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 103 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0237] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 116 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0238] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 119 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0239] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 120 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0240] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 122 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0241] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 123 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0242] In some embodiments, the recombinant gene effector (e.g., recombinant gene activator) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 125 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80%. Alternatively, it may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0243] In some embodiments, a recombinant gene effector (e.g., a recombinant gene repressor) disclosed herein may comprise at least a portion of the polypeptide sequence of SEQ ID NO: 1. In some embodiments, a recombinant gene effector (e.g., a recombinant gene repressor) disclosed herein may not, and may not need to, comprise the polypeptide sequence of SEQ ID NO: 1.

[0244] In some embodiments, a recombinant gene effector (e.g., a recombinant gene repressor) disclosed herein has a polypeptide sequence that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less identical to the polypeptide sequence of a member selected from Table 3 (e.g., a member selected from SEQ ID NOs: 32-51). The amino acid sequence may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity. [Table 3]

[0245] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a polypeptide sequence identical to or similar to at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, or at least about 78% of the polypeptide sequence of a member selected from SEQ ID NOs: 32-35, 38, and 39. , at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity (e.g., contiguous polypeptide sequence).

[0246] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 32 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO: 32. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0247] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 33 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO: 33. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0248] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 34 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO: 34. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0249] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 35 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO: 35. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0250] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 38 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO: 38. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0251] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:39 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0252] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a polypeptide sequence similar to or different from at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, or at least about 80% of the polypeptide sequence of a member selected from SEQ ID NOs: 44-51. or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity (e.g., contiguous polypeptide sequences).

[0253] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:44 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0254] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:45 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% identical to the polypeptide sequence of SEQ ID NO:45. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0255] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:46 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0256] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:47 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0257] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:48 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0258] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:49 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0259] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 50 at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 80% or more. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0260] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:51 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0261] In some embodiments, a recombinant gene effector (e.g., a recombinant gene repressor) disclosed herein may comprise one or more members selected from the group consisting of: (i) at least a portion of the polypeptide sequence of SEQ ID NO: 52, (ii) at least a portion of the polypeptide sequence of SEQ ID NO: 55, (iii) at least a portion of the polypeptide sequence of SEQ ID NO: 58, and (iv) at least a portion of the polypeptide sequence of SEQ ID NO: 61.

[0262] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:52 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0263] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 53. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 53.

[0264] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 54. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 54.

[0265] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO: 55 at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 80% or more. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0266] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 56. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 56.

[0267] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 57. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 57.

[0268] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:58 that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 78% or less, or at least about 80% or less. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0269] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 59. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 59.

[0270] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 60. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 60.

[0271] In some embodiments, the recombinant gene effector (e.g., recombinant gene repressor) disclosed herein has a homology with the polypeptide sequence of SEQ ID NO:61 at least about 50%, at least about 55%, at least about 60%, at least about 62%, at least about 64%, at least about 65%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 80% or more. or may comprise an amino acid sequence (e.g., a contiguous polypeptide sequence) having about 80% or less, at least about or at 82% or less, at least about or at 84% or less, at least about or at 85% or less, at least about or at 86% or less, at least about or at 88% or less, at least about or at 90% or less, at least about or at 91% or less, at least about or at 92% or less, at least about or at 93% or less, at least about or at 94% or less, at least about or at 95% or less, at least about or at 96% or less, at least about or at 97% or less, at least about or at 98% or less, at least about or at 99% or less, or about 100% sequence identity.

[0272] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 62. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 62.

[0273] In some embodiments, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector disclosed herein may have at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10% identity to the polypeptide sequence of SEQ ID NO: 63. For example, the amino acid sequence of the polypeptide comprised in a recombinant genetic effector may not, and need not, be identical to the polypeptide sequence of SEQ ID NO: 63.

[0274] In some embodiments, recombinant gene effectors of the present disclosure may be enriched in hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, methionine, proline) and / or acidic amino acid residues (e.g., aspartic acid, glutamic acid) relative to other types of amino acid residues, such as aromatic amino acid residues (e.g., phenylalanine, tyrosine, tryptophan), neutral amino acid residues (e.g., asparagine, glutamine, glycine, alanine, etc.), and / or basic amino acid residues (e.g., histidine, lysine, arginine). Alternatively, in some embodiments, recombinant gene effectors of the present disclosure may be enriched in one or more of aromatic amino acid residues, neutral amino acid residues, and / or basic amino acid residues relative to hydrophobic amino acid residues and / or acidic amino acid residues.

[0275] In some embodiments, the polypeptides included in the recombinant genetic effectors of the present disclosure may be enriched in hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, methionine, proline) and / or acidic amino acid residues (e.g., aspartic acid, glutamic acid) relative to other types of amino acid residues, such as aromatic amino acid residues (e.g., phenylalanine, tyrosine, tryptophan), neutral amino acid residues (e.g., asparagine, glutamine, glycine, alanine, etc.), and / or basic amino acid residues (e.g., histidine, lysine, arginine). Alternatively, in some embodiments, the polypeptides included in the recombinant genetic effectors of the present disclosure may be enriched in one or more of aromatic amino acid residues, neutral amino acid residues, and / or basic amino acid residues relative to hydrophobic amino acid residues and / or acidic amino acid residues.

[0276] In some embodiments, the additional polypeptides of the recombinant genetic effector of the present disclosure may be enriched in hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, methionine, proline) and / or acidic amino acid residues (e.g., aspartic acid, glutamic acid) relative to other types of amino acid residues, such as aromatic amino acid residues (e.g., phenylalanine, tyrosine, tryptophan), neutral amino acid residues (e.g., asparagine, glutamine, glycine, alanine, etc.), and / or basic amino acid residues (e.g., histidine, lysine, arginine). Alternatively, in some embodiments, the additional polypeptides of the recombinant genetic effector of the present disclosure may be enriched in one or more of aromatic amino acid residues, neutral amino acid residues, and / or basic amino acid residues relative to hydrophobic amino acid residues and / or acidic amino acid residues.

[0277] array SEQ ID NO: 1 (vIRF2 domain 1) JPEG2025510781000010.jpg6142 SEQ ID NO: 2 (vIRF2 domain 2) JPEG2025510781000011.jpg13147 SEQ ID NO: 3 (vIRF2 domain 3) JPEG2025510781000012.jpg12147 SEQ ID NO: 4 (wild-type viral IRF2 or "vIRF2") JPEG2025510781000013.jpg77147 SEQ ID NO: 5 (VP16) JPEG2025510781000014.jpg7136 SEQ ID NO: 6 (VP64) JPEG2025510781000015.jpg7146 SEQ ID NO: 7 (p65) JPEG2025510781000016.jpg33144 SEQ ID NO: 8 (Rta) JPEG2025510781000017.jpg22146 SEQ ID NO:9 (VP64-p65-Rta or "VPR"; linker sequence underlined) JPEG2025510781000018.jpg59146 SEQ ID NO: 10 (Un1Cas12f1 or "CasMini") JPEG2025510781000019.jpg58142 SEQ ID NO: 11 (Inactive nuclease variant of Un1Cas12f1 or "dCasMini") JPEG2025510781000020.jpg60143 SEQ ID NO: 52 (Bel-1 domain 1) JPEG2025510781000021.jpg7140 SEQ ID NO: 53 (Bel-1 domain 2) JPEG2025510781000022.jpg11142 SEQ ID NO: 54 (wild-type Bel-1) JPEG2025510781000023.jpg33146 SEQ ID NO: 55 (Chikungunya virus domain 1) JPEG2025510781000024.jpg6143 SEQ ID NO: 56 (Chikungunya virus domain 2) JPEG2025510781000025.jpg11148 SEQ ID NO: 57 (wild-type chikungunya virus) JPEG2025510781000026.jpg231154 SEQ ID NO: 58 (Eptv1 domain 1) JPEG2025510781000027.jpg5137 SEQ ID NO: 59 (Eptv1 domain 2) JPEG2025510781000028.jpg11148 SEQ ID NO: 60 (wild type Eptv1) JPEG2025510781000029.jpg112144 SEQ ID NO: 61 (Eptv2 active domain 1) JPEG2025510781000030.jpg6138 SEQ ID NO: 62 (Eptv2 active domain 2) JPEG2025510781000031.jpg11150 SEQ ID NO: 63 (wild type Eptv2) JPEG2025510781000032.jpg21149 SEQ ID NO: 64 (KRAB) JPEG2025510781000033.jpg13145

[0278] Further aspects of recombinant gene effectors

[0279] In some embodiments, the recombinant gene effectors disclosed herein can be used to regulate the expression level and / or activity level of a target gene (e.g., one or more target genes listed in Table 1). In some cases, the expression level and / or activity level of a target gene can be regulated by introducing a heterologous polypeptide comprising at least a recombinant gene effector of the present disclosure into a cell (e.g., a mammalian cell). In some embodiments, the recombinant gene effectors disclosed herein, or proteins comprising the recombinant gene effectors (e.g., proteins comprising a recombinant gene effector linked to a heterologous endonuclease), can also be referred to as "actuator moieties."

[0280] In some embodiments, the recombinant gene effector disclosed herein can be linked to a heterologous endonuclease (e.g., an enzymatically active Cas protein or an enzymatically inactivated Cas protein). The recombinant gene effector and heterologous endonuclease can be linked to each other, for example, directly or indirectly (e.g., via a linker). For example, the recombinant gene effector and heterologous endonuclease can be fused to each other, for example, directly or indirectly (e.g., via a linker). In another example, the recombinant gene effector and heterologous endonuclease can be linked to each other via a non-covalent bond, such as an ionic bond, hydrogen bond, or interaction via an oligomerization or dimerization domain. In some cases, the recombinant gene effector and heterologous endonuclease can be part of a single polypeptide molecule (e.g., part of a chimeric or fusion polypeptide).

[0281] Examples of heterologous endonucleases disclosed herein include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases, such as type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); derivatives thereof; and variants and fragments thereof.

[0282] In some embodiments, the heterologous endonucleases disclosed herein are up to about 1000 amino acids in length, up to about 950 amino acids in length, up to about 900 amino acids in length, up to about 850 amino acids in length, up to about 800 amino acids in length, up to about 750 amino acids in length, up to about 700 amino acids in length, up to about 650 amino acids in length, up to about 640 amino acids in length, up to about 630 amino acids in length, up to about 620 amino acids in length, up to about 610 amino acids in length, up to about 600 amino acids in length, up to about 590 amino acids in length, up to about 580 amino acids in length, up to about 570 amino acids in length, up to about 560 amino acids in length, amino acid length, up to about 550 amino acids in length, up to about 540 amino acids in length, up to about 530 amino acids in length, up to about 520 amino acids in length, up to about 510 amino acids in length, up to about 500 amino acids in length, up to about 490 amino acids in length, up to about 480 amino acids in length, up to about 470 amino acids in length, up to about 460 amino acids in length, up to about 450 amino acids in length, up to about 440 amino acids in length, up to about 430 amino acids in length, up to about 420 amino acids in length, up to about 410 amino acids in length, up to about 400 amino acids in length, up to about 350 amino acids in length, or up to about 300 amino acids in length.

[0283] In some embodiments, the heterologous endonucleases disclosed herein may be nuclease-defective. In some embodiments, the heterologous endonucleases disclosed herein may be nuclease-null DNA-binding proteins that are unable to induce transcriptional activation or repression of a target DNA sequence unless complexed with one or more heterologous genetic effectors of the present disclosure. In some embodiments, the heterologous endonucleases disclosed herein may be nuclease-null DNA-binding proteins that are able to induce transcriptional activation or repression of a target DNA sequence (e.g., transcriptional activation or repression of a target DNA sequence can be altered or enhanced by the presence of a heterologous genetic effector provided herein).

[0284] In some embodiments, a heterologous endonuclease disclosed herein can be an RNA nuclease, such as a recombinant RNA nuclease (e.g., a programmable RNA nuclease or a targetable RNA nuclease). In some embodiments, a heterologous endonuclease disclosed herein can be an RNA binding protein in a nuclease-null form that is incapable of inducing transcriptional activation or repression of a target RNA sequence unless complexed with one or more heterologous genetic effectors of the present disclosure. In some embodiments, a heterologous endonuclease disclosed herein can be an RNA binding protein in a nuclease-null form that is capable of inducing transcriptional activation or repression of a target RNA sequence (e.g., transcriptional activation or repression of a target RNA sequence can be altered or enhanced by the presence of a heterologous genetic effector provided herein).

[0285] In some embodiments, the heterologous endonucleases disclosed herein can be nucleic acid-guided targeting systems. In some embodiments, the heterologous endonucleases disclosed herein can be DNA-guided targeting systems. In some embodiments, the heterologous endonucleases disclosed herein can be RNA-guided targeting systems. The nucleic acid-guided targeting systems can include, for example, a guide nucleic acid sequence that facilitates specific binding of a CRISPR-Cas system (e.g., a nuclease-deficient version thereof, e.g., dCas9 or dCas14) to a target gene (e.g., an endogenous target gene) or a regulatory sequence of the target gene. For example, the target gene can be any one of the genes listed in Table 1, and the regulatory sequence of the target gene can be operably linked to any one of the genes listed in Table 1. Binding specificity can be determined using a guide nucleic acid, such as a single guide RNA (sgRNA) or a portion thereof. In some embodiments, the compositions and methods of the present disclosure can be used against different target genes (e.g., endogenous target genes) or regulatory sequences of target genes (e.g., to target different target genes or their regulatory sequences) by using different types of sgRNAs.

[0286] In some embodiments, the heterologous endonuclease disclosed herein is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, or at least about or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity. In some embodiments, the heterologous endonuclease disclosed herein is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, at least about 85% or less, or at least about 90% or less, with the polypeptide sequence of SEQ ID NO: 11 (e.g., dCasMini). The amino acid sequence may have about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% sequence identity.

[0287] In some embodiments, the amino acid sequences of the heterologous endonucleases disclosed herein can be mutated and / or modified to obtain proteins that are deficient in nuclease activity or that have reduced nuclease activity compared to wild-type Cas proteins. Nuclease-defective proteins can retain the ability to bind to target genes (e.g., DNA) but may lack or have reduced nucleic acid cleavage activity. In some embodiments, the heterologous endonucleases disclosed herein can exhibit reduced nuclease activity (e.g., nuclease-defective or nuclease null) compared to wild-type Un1Cas12f1. The reduced nuclease activity may be up to about 95% nuclease activity, up to about 90% nuclease activity, up to about 80% nuclease activity, up to about 70% nuclease activity, up to about 60% nuclease activity, up to about 50% nuclease activity, up to about 40% nuclease activity, up to about 30% nuclease activity, up to about 20% nuclease activity, up to about 10% nuclease activity, up to about 5% nuclease activity, up to about 1% nuclease activity, up to about 0.5% nuclease activity, up to about 0.1% nuclease activity, or less nuclease activity relative to the nuclease activity of wild-type Un1Cas12f1. In some cases, the heterologous endonucleases disclosed herein may contain substitutions at D326 and / or D510 compared to wild-type Un1Cas12f1. For example, the substitutions at D326 and / or D510 may be to alanine (e.g., D326A and / or D510A).

[0288] In some embodiments, the amino acid sequence of a heterologous endonuclease (e.g., a Cas protein) disclosed herein may contain one or more substitutions in a native amino acid sequence, at least some of which follow one or more specific rules determined to provide surprising advantages for the heterologous endonuclease. In some cases, the specific substitution rules are selected to, for example, generate variants of the heterologous endonuclease that can function in eukaryotic cells. According to these specific rules, all or some of the one or more substitutions in the native amino acid sequence are (1) substitutions within or not exceeding 30 amino acid residues downstream of the (D / E / K / N)X(R / F)(E / K)N motif of the native amino acid sequence, (2) substitutions within or not exceeding 30 amino acid residues upstream or downstream of position 241 of the native amino acid sequence, (3) substitutions within or not exceeding 30 amino acid residues upstream or downstream of position 516 of the native amino acid sequence, and / or (4) substitutions of charged amino acids in the native amino acid sequence.

[0289] In some embodiments, the amino acid sequence of a heterologous endonuclease (e.g., a Cas protein) disclosed herein may comprise one or more substitutions at amino acid positions within or below a threshold length (e.g., 30 amino acid residues) upstream and / or downstream of a (D / E / K / N)X(R / F)(E / K)N motif, e.g., compared to the polypeptide sequence of wild-type Un1Cas12f1. Optionally, at least one of the one or more substitutions may be present, e.g., within 28 or fewer, 26 or fewer, 24 or fewer, 22 or fewer, 20 or fewer, 18 or fewer, 16 or fewer, 14 or fewer, 12 or fewer, or 10 or fewer amino acid residues upstream and / or downstream of the motif. In some cases, at least one of the one or more substitutions may be to an R, A, S, or G amino acid residue. In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of D143, T147, E151, and K154 (e.g., D143R, T147R, E151R, and / or K154R). In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of N504, E507, N516, N519, E527, and E528 (e.g., N504R, E507R, N516R, N519R, E527R, and / or E528R).In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of K11, K73, D143, T147, E151, K154, E241, D318, K330, K457, E425, E462, N504, E507, N516, N519, E527 and E528 (e.g., K11R, K73R, D143R, T147R, E151R, K154R, E241R, D318R, K330R, E425N, K457R, E462R, N504R, E507R, N516R, N519R, E527R and / or E528R).

[0290] In some embodiments, the amino acid sequence of a heterologous endonuclease comprising one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif disclosed herein may exhibit a higher cationic charge (e.g., positive charge) than a wild-type variant of the heterologous endonuclease, the difference being at least about monovalent or less than monovalent cationic charge, at least about divalent or less than divalent cationic charge, at least about trivalent or less than trivalent cationic charge, at least about tetravalent or less than divalent cationic charge, at least about pentavalent or less than divalent cationic charge, at least about hexavalent or less than divalent cationic charge, at least about hepta ... cationic charge of at least about 8 or less, cationic charge of at least about 9 or less, cationic charge of at least about 10 or less, cationic charge of at least about 11 or less, cationic charge of at least about 12 or less, cationic charge of at least about 13 or less, cationic charge of at least about 14 or less, cationic charge of at least about 15 or less, cationic charge of at least about 16 or less, cationic charge of at least about 17 or less, or cationic charge of at least about 18 or less.

[0291] In some embodiments, the heterologous endonuclease (e.g., a recombinant gene effector operably linked to a heterologous endonuclease) can form a complex with a guide nucleic acid, such as a guide RNA or a portion thereof. In some embodiments, the heterologous endonuclease can form a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the heterologous endonuclease can be an RNA-binding protein (RBP) that can form a complex with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), which can form a complex with a Cas protein. In some embodiments, the heterologous endonuclease can be a DNA-binding protein with a nuclease null form that can induce activation or repression of transcription of a target DNA sequence. In some embodiments, the heterologous endonuclease can be an RNA-binding protein with a nuclease null form derived from RNA.

[0292] Guide nucleic acids used in the compositions and methods of the disclosure may be, for example, at least 8 nucleotides in length, at least 9 nucleotides in length, at least 10 nucleotides in length, at least 11 nucleotides in length, at least 12 nucleotides in length, at least 13 nucleotides in length, at least 14 nucleotides in length, at least 15 nucleotides in length, at least 16 nucleotides in length, at least 17 nucleotides in length, at least 18 nucleotides in length, at least 19 nucleotides in length, at least 20 nucleotides in length, at least 21 nucleotides in length, at least 22 nucleotides in length, at least 23 nucleotides in length, at least 24 nucleotides in length, at least 25 nucleotides in length, at least 26 nucleotides in length, at least 27 nucleotides in length, at least 28 nucleotides in length, at least 29 nucleotides in length, at least 30 nucleotides in length, at least 31 nucleotides in length, at least 32 nucleotides in length, at least 33 nucleotides in length, at least 34 nucleotides in length, at least 35 nucleotides in length, at least 36 nucleotides in length, at least 37 nucleotides in length, at least 38 nucleotides in length, at least 39 nucleotides in length, or at least 40 nucleotides in length.

[0293] In some embodiments, a guide nucleic acid used in the compositions and methods of the disclosure is up to 10 nucleotides in length, up to 11 nucleotides in length, up to 12 nucleotides in length, up to 13 nucleotides in length, up to 14 nucleotides in length, up to 15 nucleotides in length, up to 16 nucleotides in length, up to 17 nucleotides in length, up to 18 nucleotides in length, up to 19 nucleotides in length, up to 20 nucleotides in length, up to 21 nucleotides in length, up to 22 nucleotides in length, up to 23 nucleotides in length, up to 24 nucleotides in length, up to 25 nucleotides in length, up to 26 nucleotides in length, up to 27 nucleotides in length, up to 28 nucleotides in length, up to 29 nucleotides in length, up to 30 nucleotides in length, up to 31 nucleotides in length, up to 32 nucleotides in length, up to 33 nucleotides in length, up to 34 nucleotides in length, up to 35 nucleotides in length, up to 36 nucleotides in length, up to 37 nucleotides in length, up to 38 nucleotides in length, up to 39 nucleotides in length, or up to 40 nucleotides in length.

[0294] In some embodiments, the guide nucleic acid used in the compositions and methods of the present disclosure is between about 8 nucleotides and about 40 nucleotides in length, between about 10 nucleotides and about 40 nucleotides in length, between about 11 nucleotides and about 40 nucleotides in length, between about 12 nucleotides and about 40 nucleotides in length, between about 13 nucleotides and about 40 nucleotides in length, between about 14 nucleotides and about 40 nucleotides in length, between about 15 nucleotides and about 40 nucleotides in length, between about 16 nucleotides and about 40 nucleotides in length, between about 17 nucleotides and about 40 nucleotides in length, between about 18 nucleotides in length, between about 20 nucleotides in length, between about 22 nucleotides in length, between about 24 nucleotides in length, between about 26 nucleotides in length, between about 28 nucleotides in length, between about 29 nucleotides in length, between about 30 nucleotides in length, between about 31 nucleotides in length, between about 32 nucleotides in length, between about 33 nucleotides in length, between about 34 nucleotides in length, between about 35 nucleotides in length, between about 36 nucleotides in length, between about 37 nucleotides in length, between about 38 nucleotides in length, between about 39 nucleotides in length, between about 40 nucleotides in length, between about 41 nucleotides in length, between about 42 nucleotides in length, between about 43 nucleotides in length, between about 44 nucleotides in length, between about 45 nucleotides in length, between about 46 nucleotides in length, between about 47 nucleotides in length, between about 48 nucleotides in length, between about 49 nucleotides in length, between about 50 nucleotides in length, between about 51 nucleotides in length, between about nucleotide length to about 40 nucleotides, about 19 nucleotides to about 40 nucleotides, about 20 nucleotides to about 40 nucleotides, about 22 nucleotides to about 40 nucleotides, about 24 nucleotides to about 40 nucleotides, about 26 nucleotides to about 40 nucleotides, about 28 nucleotides to about 40 nucleotides, about 30 nucleotides to about 40 nucleotides, about 8 nucleotides to about 30 nucleotides, about 10 nucleotides to about 30 nucleotides, about 11 nucleotides to about 30 nucleotides, about 12 nucleotides to about 30 nucleotides long, about 13 nucleotides to about 30 nucleotides long, about 14 nucleotides to about 30 nucleotides long, about 15 nucleotides to about 30 nucleotides long, about 16 nucleotides to about 30 nucleotides long, about 17 nucleotides to about 30 nucleotides long, about 18 nucleotides to about 30 nucleotides long, about 19 nucleotides to about 30 nucleotides long, about 20 nucleotides to about 30 nucleotides long, about 22 nucleotides to about 30 nucleotides long, about 24 nucleotides to about 30 nucleotides long, about 26 nucleotides to about 30 nucleotides long nucleotide length, about 28 nucleotides to about 30 nucleotides, about 8 nucleotides to about 25 nucleotides, about 10 nucleotides to about 25 nucleotides, about 11 nucleotides to about 25 nucleotides, about 12 nucleotides to about 25 nucleotides, about 13 nucleotides to about 25 nucleotides, about 14 nucleotides to about 25 nucleotides, about 15 nucleotides to about 25 nucleotides, about 16 nucleotides to about 25 nucleotides, about 17 nucleotides to about 25 nucleotides, about 18 nucleotides to about 25 nucleotides,about 19 nucleotides to about 25 nucleotides in length, about 20 nucleotides to about 25 nucleotides in length, about 22 nucleotides to about 25 nucleotides in length, about 24 nucleotides to about 25 nucleotides in length, about 8 nucleotides to about 20 nucleotides in length, about 10 nucleotides to about 20 nucleotides in length, about 11 nucleotides to about 20 nucleotides in length, about 12 nucleotides to about 20 nucleotides in length, about 13 nucleotides to about 20 nucleotides in length, about 14 nucleotides to about 20 nucleotides in length, about 15 nucleotides to about 20 nucleotides in length, about 16 nucleotides to about 20 nucleotides in length, about 17 nucleotides to about 20 nucleotides in length, about 18 nucleotides to about 20 nucleotides in length, about 19 nucleotides to about 20 nucleotides in length, about 8 nucleotides to about 20 nucleotides in length, about 19 nucleotides to about 20 nucleotides in length, about 20 ... The length of the guide nucleic acid is about 1 to about 18 nucleotides, about 10 to about 18 nucleotides, about 11 to about 18 nucleotides, about 12 to about 18 nucleotides, about 13 to about 18 nucleotides, about 14 to about 18 nucleotides, about 15 to about 18 nucleotides, about 16 to about 18 nucleotides, about 8 to about 16 nucleotides, about 10 to about 16 nucleotides, about 11 to about 16 nucleotides, about 12 to about 16 nucleotides, about 13 to about 16 nucleotides, about 14 to about 16 nucleotides, or about 15 to about 16 nucleotides. In some embodiments, the guide nucleic acid may be a guide RNA or a portion thereof.

[0295] The heterologous endonucleases disclosed herein can be modified to enhance regulation of gene expression using the compositions and methods of the present disclosure, for example, as part of a complex disclosed herein. The heterologous endonucleases of the present disclosure can be modified to have increased or decreased nucleic acid binding affinity, increased or decreased nucleic acid binding specificity, increased or decreased enzymatic activity, and / or increased or decreased binding to and ligand attraction of other factors, such as heterodimerization or oligomerization domains. The heterologous endonucleases of the present disclosure can also be modified to alter other activities or properties (e.g., stability). For example, one or more nuclease domains of the heterologous endonucleases of the present disclosure can be modified, deleted, or inactivated, or at least a portion of the heterologous endonuclease of the present disclosure can be truncated to remove domains that are not essential for the desired function of the heterologous endonuclease or complex thereof. Furthermore, the heterologous endonucleases of the present disclosure can also be modified such that the activity of the heterologous endonuclease to regulate gene expression is modulated (e.g., increased or decreased) by a complex of the present disclosure that includes a heterologous gene effector.

[0296] For example, a heterologous endonuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repression domain). A heterologous endonuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to an oligomerization domain or dimerization domain (e.g., a heterodimerization domain) disclosed herein. A heterologous endonuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a heterologous polypeptide that increases or decreases stability. A heterologous endonuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a sequence that can promote degradation of the heterologous endonuclease or a complex comprising the heterologous endonuclease.

[0297] A heterologous endonuclease of the disclosure can be linked (e.g., fused, covalently linked, or non-covalently linked) to any suitable number of partners, for example, at least 1 partner, at least 2 partners, at least 3 partners, at least 4 partners, at least 5 partners, at least 6 partners, at least 7 partners, or at least 8 partners. In some embodiments, a heterologous endonuclease of the disclosure is linked (e.g., fused, covalently linked, or non-covalently linked) to a maximum of 2 partners, a maximum of 3 partners, a maximum of 4 partners, a maximum of 5 partners, a maximum of 6 partners, a maximum of 7 partners, a maximum of 8 partners, or a maximum of 10 partners. In some embodiments, a heterologous endonuclease of the disclosure is linked to 1 to 5 partners, 1 to 4 partners, 1 to 3 partners, 1 to 2 partners, 2 to 5 partners, 2 to 4 partners, 2 to 3 partners, 3 to 5 partners, 3 to 4 partners, or 4 to 5 partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a heterologous endonuclease of the disclosure is linked to one partner (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a heterologous endonuclease of the disclosure is linked to two partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a heterologous endonuclease of the disclosure is linked to three partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a heterologous endonuclease of the present disclosure is linked (eg, fused, covalently linked, or non-covalently linked) to four partners.In some embodiments, a heterologous endonuclease of the disclosure is linked to five partners (e.g., fused, covalently linked, or non-covalently linked), hi some embodiments, a heterologous endonuclease of the disclosure is linked to six partners (e.g., fused, covalently linked, or non-covalently linked).

[0298] The heterologous endonucleases disclosed herein can be linked (e.g., linked via covalent or non-covalent bonds) to one or more partners disclosed herein, which may include (i) any one of the recombinant gene effectors disclosed herein and (ii) one or more additional partners provided in this disclosure.

[0299] The heterologous endonucleases disclosed herein can be fusion proteins, for example, fusion proteins comprising a heterologous endonuclease disclosed herein and one or more partners disclosed herein, where the fused domains can be located at the N-terminus, C-terminus, or internal to the heterologous endonuclease.

[0300] A partner of a heterologous endonuclease disclosed herein (e.g., a partner covalently or non-covalently linked to a nuclease activity-deficient or nuclease null variant of a heterologous endonuclease disclosed herein) can be a transcriptional effector (e.g., a transcriptional activator or transcriptional repressor). The transcriptional effector can be heterologous to the cells provided herein.

[0301] In some embodiments, the transcriptional effector may be an epigenetic histone modifier (or histone modifier). In some cases, the epigenetic histone modifier may regulate histones by methylation (e.g., a histone methylation modifier, e.g., an amino acid methyltransferase (e.g., KRAB)). In some cases, the epigenetic histone modifier may regulate histones by acetylation. In some cases, the epigenetic histone modifier may regulate histones by phosphorylation. In some cases, the epigenetic histone modifier may regulate histones by ADP-ribosylation. In some cases, the epigenetic histone modifier may regulate histones by glycosylation. In some cases, the epigenetic histone modifier may regulate histones by sumoylation. In some cases, the epigenetic histone modifier may regulate histones by ubiquitination. In some cases, epigenetic histone modifiers can regulate histones by, for example, remodeling histone structure via ATP hydrolysis-dependent processes.

[0302] In some embodiments, the transcriptional effector may be an epigenetic gene modifier (or gene modifier). In some cases, the gene modifier may regulate genes by methylation (e.g., a gene methylation modifier, e.g., a DNA methyltransferase, or DNMT). In some cases, the gene modifier may regulate genes by acetylation.

[0303] In some embodiments, the transcriptional effector is selected from a family of related histone acetyltransferases, including, but not limited to, the GNAT subfamily, the MYST subfamily, the p300 / CBP subfamily, the HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0304] In some embodiments, the transcriptional effector may comprise an epigenetic modifier. In some embodiments, the transcriptional effector comprises an epigenetic histone modifier (e.g., histone lysine methyltransferase, histone lysine demethylase, or DNA methylase). Examples of epigenetic modifiers include the EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, PRDM13, and PRDM14. 14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0305] Examples of proteins (or fragments thereof) that can be used as fusion partners to increase transcription include, but are not limited to, the VP16 subdomain, VP64 subdomain, VP48 subdomain, VP160 subdomain, and p65 subdomain (e.g., from NFkB); the activation domain of EDLL; and / or the TAL activation domain (e.g., for activity in plants); and transcriptional activators such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60, CLOCK, Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.

[0306] Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include Kruppel-associated boxes (KRAB or SKD); KOX1 repression domains; Mad mSIN3-interacting domains (SIDs); ERF repressor domains (ERDs) (e.g., for repression in plants), SRDX repression domains, etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD 1 A / RBP2, JARIDlB / PLU-1, JARID 1C / SMCX, and JARIDID / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; and Hhal DNA These include, but are not limited to, DNA methylases such as m5c methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and transcriptional repressors such as peripheral recruitment factors such as lamin A and lamin B.

[0307] The heterologous endonucleases disclosed herein may be provided in any form. For example, the heterologous endonucleases disclosed herein can be provided in the form of a protein, e.g., the heterologous endonuclease alone or in a complex with a guide nucleic acid as a ribonucleoprotein. The heterologous endonuclease can be provided in the form of a complex, e.g., the heterologous endonuclease can be provided in a complex with a guide nucleic acid and / or one or more heterologous gene effectors of the present disclosure. The heterologous endonuclease can also be provided in the form of a nucleic acid encoding at least the heterologous endonuclease, e.g., RNA (e.g., messenger RNA (mRNA)) or DNA. The nucleic acid encoding at least the heterologous endonuclease can be codon-optimized (e.g., optimized for human codons) for efficient protein translation in a particular cell or organism.

[0308] The nucleic acid encoding at least the heterologous endonuclease disclosed herein, or a fragment or derivative thereof, can be stably integrated into the genome of a cell. The nucleic acid encoding at least the heterologous endonuclease can be operably linked to a promoter, such as a promoter that is constitutively active in the cell or a promoter that is activated upon induction. The nucleic acid encoding at least the heterologous endonuclease can be operably linked to a promoter in an expression construct. The expression construct can include a nucleic acid construct capable of inducing expression of a gene or other nucleic acid sequence of interest (e.g., at least the heterologous endonuclease), and such a nucleic acid sequence of interest can be transferred into a target cell.

[0309] In some embodiments, the heterologous endonucleases disclosed herein can bind to a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., an endogenous target gene), for example, by cooperating with a heterologous gene effector disclosed herein. The sgRNA can also be introduced into cells expressing the heterologous endonucleases provided herein or variants thereof. In some cases, such cells may contain one or more sgRNAs that target the same target gene (e.g., an endogenous target gene) or the same regulatory sequence of a target gene. In other cases, the sgRNAs target different nucleic acids within the cell (e.g., targeting different target genes, different regulatory sequences of a target gene, or different sequences within the same target gene or the same regulatory sequence of a target gene).

[0310] "Enzymatically inactive" (e.g., as used with respect to a defective nuclease) refers to a nuclease that can bind sequence-specifically to a nucleic acid sequence in a polynucleotide but that may not cleave the target polynucleotide or that cleaves the target polynucleotide significantly less frequently. An enzymatically inactive guide portion may comprise an enzymatically inactive domain (e.g., an enzymatically inactive nuclease domain). "Enzymatically inactive" may mean no activity. "Enzymatically inactive" may mean substantially no activity. "Enzymatically inactive" may mean essentially no activity. "Enzymatically inactive" may mean 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, or 10% or less of the activity of a comparable wild-type activity (e.g., nucleic acid cleavage activity or wild-type Cas activity).

[0311] In some embodiments, the target nucleic acid of the heterologous endonuclease disclosed herein may be dsDNA. In such embodiments, target specificity for dsDNA is determined at least in part by two parameters: the gRNA spacer that targets the protospacer in the target dsDNA (the sequence in the target dsDNA that corresponds to the gRNA spacer on the non-complementary DNA strand), and a short sequence called the protospacer adjacent motif (PAM) located immediately 5' (upstream) of the protospacer on the non-complementary DNA strand. In some embodiments, the PAM is 5'-TTTG-3' or 5'-TTTA-3'. In some embodiments, the PAM is 5'-TTTG-3'. In some embodiments, the PAM is 5'-TTTA-3'.

[0312] In some embodiments, the target nucleic acid of the heterologous endonuclease disclosed herein may be RNA. In such embodiments, target specificity for RNA is determined at least in part by the gRNA spacer targeting a protospacer-like sequence (a sequence in the target RNA that is complementary to the gRNA spacer) in the target RNA, and is independent of the sequence located immediately 5' (upstream) of the protospacer-like sequence. In some embodiments, the heterologous endonuclease can also target a dsDNA molecule, and the gRNA spacer is selected to target a protospacer in a target dsDNA molecule having a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In another embodiment, the heterologous endonuclease does not target a dsDNA molecule, and the gRNA spacer is selected to target a protospacer in a dsDNA molecule that does not have a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'.

[0313] In some embodiments, a heterologous polypeptide (e.g., and / or a complex comprising a heterologous polypeptide) comprising a recombinant gene effector and a heterologous endonuclease disclosed herein can regulate the expression and / or activity of a target gene (e.g., an endogenous target gene). In some embodiments, the heterologous polypeptide and / or complex thereof can edit the sequence of a nucleic acid (e.g., a gene and / or gene product). A nuclease-active variant of the heterologous endonuclease can edit a nucleic acid sequence by introducing a double-stranded or single-stranded break in a target polynucleotide.

[0314] In some embodiments, a heterologous polypeptide (e.g., and / or a complex comprising a heterologous polypeptide) comprising a recombinant gene effector and a heterologous endonuclease disclosed herein can introduce a double-strand break into a target polynucleotide, such as DNA. The introduction of a double-strand break into DNA allows the DNA break to be repaired, allowing for the introduction of a genetic modification (e.g., nucleic acid editing). In some embodiments, the nuclease induces a site-specific single-strand DNA break, or nick, allowing homology-directed repair to occur.

[0315] By introducing double-strand breaks into DNA, DNA breaks can be repaired and genetic modifications (e.g., nucleic acid editing) can be introduced. DNA break repair can occur by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In homology-directed repair, a donor DNA repair template or template polynucleotide can be provided that includes homologous arms that flank the target DNA site.

[0316] In some embodiments, a heterologous polypeptide comprising a recombinant gene effector and a heterologous endonuclease disclosed herein (e.g., and / or a complex comprising the heterologous polypeptide) does not introduce a double-strand break in a target polynucleotide such as DNA. The heterologous polypeptide or a complex comprising the heterologous polypeptide (e.g., a complex comprising a nuclease-deficient variant of a heterologous endonuclease and a guide RNA) can bind to a target polynucleotide without cleaving the nucleic acid, thereby sufficiently modulating (e.g., enhancing or suppressing) expression of a target gene (e.g., an endogenous target gene).

[0317] Target gene

[0318] The present disclosure provides compositions, methods, and systems for regulating the expression of one or more target genes. The target genes may be one or more heterologous target genes. The target genes may be one or more endogenous target genes, for example, (i) disease-causing alleles (e.g., mutant alleles) and / or (ii) non-disease-causing alleles (e.g., wild-type alleles). For example, disclosed herein are complexes comprising one or more heterologous polypeptides, including a recombinant gene effector and a heterologous endonuclease disclosed herein, and a guide moiety, that can regulate (e.g., increase or decrease) the activity level or expression amount of a target gene (e.g., in a cell).

[0319] In some embodiments, the target gene or its regulatory sequence is endogenous to the cell, e.g., present in the genome of the cell, or endogenous to the subject, e.g., present in the genome of the subject. In some embodiments, the target gene or its regulatory sequence is not part of an engineered reporter system.

[0320] In some embodiments, the target gene is exogenous to the host subject, e.g., a pathogen-targeting gene or foreign gene is expressed as a result of therapeutic intervention, such as gene therapy and / or cell therapy. In some embodiments, the target gene is an exogenous reporter gene. In some embodiments, the target gene is an exogenous synthetic gene.

[0321] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., by introducing into a cell or cell population a complex comprising a recombinant gene effector and a heterologous polypeptide). In some embodiments, the expression level is RNA expression level, which can be measured, for example, by RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, the expression level is protein expression level, which can be measured, for example, by Western blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, etc.

[0322] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., by introducing into a cell or cell population a complex comprising a recombinant gene effector and a heterologous polypeptide), the degree of modulation can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45 The increase may be about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, or at least about 3000-fold.

[0323] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., by introducing into a cell or cell population a complex comprising a recombinant gene effector and a heterologous polypeptide) by a degree of modulation of up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 2-fold, up to about 3-fold, up to about 4-fold, up to about 5-fold, up to about 6-fold, up to about 7-fold, up to about 8-fold, up to about 9-fold, up to about 10-fold, up to about 11-fold, up to about 12-fold, up to about 14-fold, up to about 16-fold, up to about 18-fold, up to about 20-fold, up to about 22-fold, up to about 24-fold, up to about 26-fold, up to about 28-fold, up to about 29-fold, up to about 30-fold, up to about 31-fold, up to about 32-fold, up to about 33-fold, up to about 34-fold, up to about 35-fold, up to about 36-fold, up to about 37-fold, up to about 38-fold, up to about 39-fold, up to about 40-fold, up to about 41-fold, up to about 42-fold, up to about 43-fold, up to about 44-fold, up to about 45-fold, up to about 46-fold, up to about 47-fold, up to about 48-fold, up to about 49-fold, up to about 50-fold, up to about 51-fold, up to about 52-fold, up to about 53-fold, up to about 54-fold, up to about 55-fold, up to about 56- The amplification may be 13-fold, up to about 14-fold, up to about 15-fold, up to about 20-fold, up to about 30-fold, up to about 40-fold, up to about 50-fold, up to about 60-fold, up to about 70-fold, up to about 80-fold, up to about 90-fold, up to about 100-fold, up to about 150-fold, up to about 200-fold, up to about 250-fold, up to about 300-fold, up to about 350-fold, up to about 400-fold, up to about 500-fold, up to about 600-fold, up to about 700-fold, up to about 800-fold, up to about 900-fold, up to about 1000-fold, up to about 1500-fold, up to about 2000-fold, up to about 3000-fold, up to about 5000-fold, or up to about 10,000-fold.

[0324] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., by introducing into a cell or cell population a complex comprising a recombinant gene effector and a heterologous polypeptide), and the degree of modulation can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10 ... fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1500-fold, about 2000-fold, about 3000-fold, about 5000-fold, or about 10000-fold.

[0325] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) expression of a target gene from below the limit of detection to detectable levels (e.g., by introducing into a cell or cell population a complex comprising a recombinant gene effector and a heterologous polypeptide).

[0326] In some embodiments, the degree of change in expression is relative to the state before introduction of a system of the present disclosure (e.g., a complex comprising a recombinant gene effector and a heterologous polypeptide) into the cell or cell population. In some embodiments, the degree of change in expression is relative to a corresponding control cell or control cell population that has not been treated with a system of the present disclosure. In some embodiments, the degree of change in expression is relative to a corresponding control cell or control cell population that has been treated with another system of the present disclosure.

[0327] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the activity level of a target gene (e.g., by introducing into a cell or cell population a complex comprising a heterologous polypeptide, including a heterologous endonuclease disclosed herein, and a recombinant gene effector). Depending on the functional nature of the target gene of interest, the activity level can be measured by a functional assay appropriate for the target gene. For example, the activity level of a mitogenic target gene can be measured by measuring cell proliferation; the activity level of a target gene that induces apoptosis can be measured by an Annexin V assay or other appropriate cell death assay; and the activity level of an anti-inflammatory cytokine can be measured by an LPS-induced cytokine release assay.

[0328] In some cases, the disclosed systems and methods can induce changes in the expression and / or activity levels of a target gene (e.g., an endogenous target gene) for a longer duration than can be achieved by alternative compositions and methods (e.g., RNAi-mediated suppression, e.g., suppression using siRNA). In some embodiments, sustained modulation of gene expression (e.g., durable gene activation or durable gene suppression) is advantageous compared to transient modulation.

[0329] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity levels of a target gene over a period of time, wherein the duration of modulation is at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, The period may be at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months or longer.

[0330] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity level of a target gene (e.g., an endogenous target gene) above a particular threshold for a period of time, the duration of modulation being up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 5 hours, up to about 6 hours, up to about 7 hours, up to about 8 hours, up to about 9 hours, up to about 10 hours, up to about 12 hours, up to about 14 hours, up to about 18 hours, up to about 20 hours, The period may be up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 14 days, up to about 21 days, up to about 28 days, up to about 5 weeks, up to about 6 weeks, up to about 7 weeks, up to about 8 weeks, up to about 9 weeks, up to about 10 weeks, up to about 12 weeks, up to about 14 weeks, up to about 18 weeks, up to about 20 weeks, up to about 26 weeks, up to about 5 months, up to about 6 months, up to about 9 months, up to about 12 months, or longer.

[0331] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity level of a target gene (e.g., an endogenous target gene) above a particular threshold over a period of time, where the duration of modulation can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.

[0332] In some embodiments, the target gene (e.g., an endogenous target gene) may be a disease-causing allele, e.g., a mutant variant of a wild-type allele. The disease may be a genetic disease (e.g., an inherited disease). Examples of genetic diseases include, but are not limited to, Duchenne muscular dystrophy (DMD), hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease. In some cases, the target gene may be a protein-coding gene. In some cases, the target gene may be a gene regulatory sequence (e.g., a promoter, enhancer, repressor, silencer, insulator, cis-regulatory element, trans-regulatory element, epigenetic modification site (e.g., DNA methylation site), etc.) that can affect the expression of a gene encoding a protein of interest provided herein. For example, the regulatory sequence of the target gene may be physically located outside the transcription unit or open reading frame that encodes the target gene product.

[0333] In some embodiments, the target gene regulatory sequence does not comprise a nucleotide sequence exogenous to the subject or host cell, hi some embodiments, the target gene regulatory sequence does not comprise a recombinant, artificially created, or introduced nucleotide sequence.

[0334] In some embodiments, the target gene (e.g., an endogenous target gene) is a gene that is overexpressed or underexpressed in a disease or condition. In some embodiments, the target gene is a gene that is overexpressed or underexpressed in an inherited genetic disease.

[0335] In some embodiments, the target gene (e.g., endogenous target gene) is a gene that is overexpressed or underexpressed in cancer, such as acute leukemia, astrocytoma, biliary tract cancer (cholangiocarcinoma), bone cancer, breast cancer, brainstem glioma, bronchioloalveolar cell lung cancer, adrenal gland cancer, anal region cancer, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureter cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, cutaneous melanoma, ependymoma, epidermoid tumor, Ewing's disease, sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, glioma, hematologic malignancies, hepatocellular (liver) carcinoma, hepatoma, Hodgkin's disease, intraocular melanoma, Kaposi's sarcoma, lung cancer, lymphoma, medulloblastoma, malignant melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, central nervous system (CNS) neoplasms, neuronal carcinoma, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma, skin cancer, spinal axis tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, uterine cancer, and tumors, as well as metastases and refractory forms of said cancers, and combinations thereof.

[0336] Non-limiting examples of target genes or genes encoding proteins of interest disclosed herein are shown in Table 1 below. table Table 1: List of examples of target genes (e.g., target genes encoding proteins of interest). List of target genes / proteins of interest A1BG, CCND3, FAM163A, KCNK10, NRG4, REM1, TECTB, A1CF, CCNDBP1, FAM163B, KCNK12, NRGN, REM2, TEDDM1, A2M, CCNE1, FAM166A, KCNK13, NRIP1, REN, TEF, A2ML1, CCNE2, FAM166B, KCNK15, NRIP2, RENBP, TEFM, A3GALT2, CCNF, FAM167A, KCNK16, NRIP3, REP15, TEK, A4GALT, CCNG1, FAM167B, KCNK17, NRK, REPIN1, TEKT1, A4GNT, CCNG2, FAM168A, KCNK18, NRL, REPS1, TEKT2, AAAS, CCNH, FAM168B, KCNK2, NRM, REPS2, TEKT3, AACS, CCNI, FAM169A, KCNK3, NRN1, RER1, TEKT4, AADAC, CCNI2, FAM169B, KCNK4, NRN1L, RERE, TEKT5, AADACL2, CCNJ, FAM170A, KCNK5, NRP1, RERG, TELO2, AADACL3, CCNJL, FAM170B, KCNK6, NRP2, RERGL, TEN1, AADACL4, CCNK, FAM171A1, KCNK7, NRROS, RESP18, TENC1, AADAT, CCNL1, FAM171A2, KCNK9, NRSN1,REST, TENM1, AAED1, CCNL2, FAM171B, KCNMA1, NRSN2, RET, TENM2, AAGAB, CCNO, FAM172A, KCNMB1, NRTN, RETN, TENM3, AAK1, CCNT1, FAM173A, KCNMB2, NRXN1, RETNLB, TENM4, AAMDC, CCNT2, FAM173B, KCNMB3, NRXN2, RETSAT, TEP1, AAMP, CCNY, FAM174A, KCNMB4, NRXN3, REV1, TEPP, AANAT, CCNYL1, FAM174B, KCNN1, NSA2, REV3L,TERF1, AAR2, CCP110, FAM175A, KCNN2, NSD1, REXO1, TERF2, AARD, CCPG1, FAM175B, KCNN3, NSDHL, REXO2, TERF2IP, AARS, CCR1, FAM177A1, KCNN4, NSF, REXO4, TERT, AARS2, CCR10, FAM177B, KCNQ1, NSFL1C, RFC1, TES, AARSD1, CCR2, FAM178A, KCNQ2, NSG1, RFC2, TESC, AASDH, CCR3, FAM178B, KCNQ3, NSL1, RFC3, TESK1, AASDHPPT, CCR4, FAM179A, KCNQ4, NSMAF, RFC4, TESK2, AASS, CCR5, FAM179B, KCNQ5, NSMCE1, RFC5, TESPA1, AATF, CCR6, FAM180A, KCNRG, NSMCE2, RFESD, TET1, AATK, CCR7, FAM180B, KCNS1, NSMCE4A, RFFL, TET2, ABAT, CCR8, FAM181A, KCNS2, NSMF, RFK, TET3, ABCA1, CCR9, FAM181B, KCNS3, NSRP1, RFNG, TEX10, ABCA10, CCRL2, FAM183A, KCNT1, NSUN2, RFPL1, TEX101, ABCA12, CCRN4L, FAM184A, KCNT2, NSUN3, RFPL2, TEX11, ABCA13, CCS, FAM184B, KCNU1, NSUN4, RFPL3, TEX12, ABCA2, CCSAP, FAM185A, KCNV1, NSUN5, RFPL4A, TEX13A, ABCA3, CCSER1, FAM186A, KCNV2, NSUN6, RFPL4AL1, TEX13B, ABCA4, CCSER2, FAM186B, KCP, NSUN7, RFPL4B, TEX14, ABCA5, CCT2, FAM187B, KCTD1, NT5C, RFT1, TEX15, ABCA6, CCT3, FAM188A, KCTD10, NT5C1A,RFTN1, TEX19, ABCA7, CCT4, FAM188B, KCTD11, NT5C1B, RFTN2, TEX2, ABCA8, CCT5, FAM189A1, KCTD12, NT5C1B-RDH14, RFWD2, TEX22, ABCA9, CCT6A, FAM189A2, KCTD13, NT5C2, RFWD3, TEX26, ABCB1, CCT6B, FAM189B, KCTD14, NT5C3A, RFX1, TEX261, ABCB10, CCT7, FAM192A, KCTD15, NT5C3B, RFX2, TEX264, ABCB11, CCT8, FAM193A, KCTD16, NT5DC1, RFX3, TEX28, ABCB4, CCT8L2, FAM193B, KCTD17, NT5DC2, RFX4, TEX29, ABCB5, CCZ1, FAM194A, KCTD18, NT5DC3, RFX5, TEX30, ABCB6, CCZ1B, FAM194B, KCTD19, NT5E, RFX6, TEX33, ABCB7, CD101, FAM195A, KCTD2, NT5M, RFX7, TEX35, ABCB8, CD109, FAM195B, KCTD20, NTAN1, RFX8, TEX36, ABCB9, CD14, FAM196A, KCTD21, NTF3, RFXANK, TEX37, ABCC1, CD151, FAM196B, KCTD3, NTF4, RFXAP, TEX38, ABCC10, CD160, FAM198A, KCTD4, NTHL1, RGAG1, TEX40, ABCC11, CD163, FAM198B, KCTD5, NTM, RGAG4, TEX9, ABCC12, CD163L1, FAM199X, KCTD6, NTMT1, RGCC, TF, ABCC2, CD164, FAM19A1, KCTD7, NTN1, RGL1, TFAM, ABCC3, CD164L2, FAM19A2, KCTD8, NTN3, RGL2, TFAP2A, ABCC4, CD177, FAM19A3, KCTD9, NTN4, RGL3, TFAP2B, ABCC5,CD180, FAM19A4, KDELC1, NTN5, RGL4, TFAP2C, ABCC6, CD19, FAM19A5, KDELC2, NTNG1, RGMA, TFAP2D, ABCC8, CD1A, FAM200A, KDELR1, NTNG2, RGMB, TFAP2E, ABCC9, CD1B, FAM203A, KDELR2, NTPCR, RGN, TFAP4, ABCD1, CD1C, FAM203B, KDELR3, NTRK1, RGP1, TFB1M, ABCD2, CD1D, FAM204A, KDM1A, NTRK2, RGPD1, TFB2M, ABCD3, CD1E, FAM205A, KDM1B, NTRK3, RGPD2, TFCP2, ABCD4, CD2, FAM206A, KDM2A, NTS, RGPD3, TFCP2L1, ABCE1, CD200, FAM207A, KDM2B, NTSR1, RGPD4, TFDP1, ABCF1, CD200R1, FAM208A, KDM3A, NTSR2, RGPD5, TFDP2, ABCF2, CD200R1L, FAM208B, KDM3B, NUAK1, RGPD6, TFDP3, ABCF3, CD207, FAM209A, KDM4A, NUAK2, RGPD8, TFE3, ABCG1, CD209, FAM209B, KDM4B, NUB1, RGR, TFEB, ABCG2, CD22, FAM20A, KDM4C, NUBP1, RGS1, TFEC, ABCG4, CD226, FAM20B, KDM4D, NUBP2, RGS10, TFF1, ABCG5, CD24, FAM20C, KDM4E, NUBPL, RGS11, TFF2, ABCG8, CD244, FAM210A, KDM5A, NUCB1, RGS12, TFF3, ABHD1, CD247, FAM210B, KDM5B, NUCB2, RGS13, TFG, ABHD10, CD248, FAM211A, KDM5C, NUCKS1, RGS14, TFIP11, ABHD11, CD27, FAM211B, KDM5D, NUDC, RGS16, TFPI,ABHD12, CD274, FAM212A, KDM6A, NUDCD1, RGS17, TFPI2, ABHD12B, CD276, FAM212B, KDM6B, NUDCD2, RGS18, TFPT, ABHD13, CD28, FAM213A, KDM8, NUDCD3, RGS19, TFR2, ABHD14A, CD2AP, FAM213B, KDR, NUDT1, RGS2, TFRC, ABHD14B, CD2BP2, FAM214A, KDSR, NUDT10, RGS20, TG, ABHD15, CD300A, FAM214B, KEAP1, NUDT11, RGS21, TGDS, ABHD16A, CD300C, FAM216A, KEL, NUDT12, RGS22, TGFA, ABHD16B, CD300E, FAM216B, KERA, NUDT13, RGS3, TGFB1, ABHD17A, CD300LB, FAM217A, KHDC1, NUDT14, RGS4, TGFB1I1, ABHD17B, CD300LD, FAM217B, KHDC1L, NUDT15, RGS5, TGFB2, ABHD17C, CD300LF, FAM218A, KHDC3L, NUDT16, RGS6, TGFB3, ABHD2, CD300LG, FAM219A, KHDRBS1, NUDT16L1, RGS7, TGFBI, ABHD3, CD302, FAM219B, KHDRBS2, NUDT17, RGS7BP, TGFBR1, ABHD4, CD320, FAM21A, KHDRBS3, NUDT18, RGS8, TGFBR2, ABHD5, CD33, FAM21B, KHK, NUDT19, RGS9, TGFBR3, ABHD6, CD34, FAM21C, KHNYN, NUDT2, RGS9BP, TGFBR3L, ABHD8, CD36, FAM220A, KHSRP, NUDT21, RGSL1, TGFBRAP1, ABI1, CD37, FAM221A, KIAA0020, NUDT22, RHAG, TGIF1, ABI2, CD38, FAM221B, KIAA0040, NUDT3, RHBDD1,TGIF2, ABI3, CD3D, FAM222A, KIAA0100, NUDT4, RHBDD2, TGIF2-C20orf24, ABI3BP, CD3E, FAM222B, KIAA0101, NUDT5, RHBDD3, TGIF2LX, ABL1, CD3EAP, FAM227A, KIAA0141, NUDT6, RHBDF1, TGIF2LY, ABL2, CD3G, FAM227B, KIAA0195, NUDT7, RHBDF2, TGM1, ABLIM1, CD4, FAM228A, KIAA0196, NUDT8, RHBDL1, TGM2, ABLIM2, CD40, FAM228B, KIAA0226, NUDT9, RHBDL2, TGM3, ABLIM3, CD40LG, FAM229A, KIAA0226L, NUF2, RHBDL3, TGM4, ABO, CD44, FAM229B, KIAA0232, NUFIP1, RHBG, TGM5, ABR, CD46, FAM230A, KIAA0247, NUFIP2, RHCE, TGM6, ABRA, CD47, FAM24A, KIAA0319, NUGGC, RHCG, TGM7, ABRACL, CD48, FAM24B, KIAA0319L, NUMA1, RHD, TGOLN2, ABT1, CD5, FAM25A, KIAA0355, NUMB, RHEB, TGS1, ABTB1, CD52, FAM25C, KIAA0368, NUMBL, RHEBL1, TH, ABTB2, CD53, FAM25G, KIAA0391, NUP107, RHNO1, THADA, ACAA1, CD55, FAM26D, KIAA0408, NUP133, RHO, THAP1, ACAA2, CD58, FAM26E, KIAA0430, NUP153, RHOA, THAP10, ACACA, CD59, FAM26F, KIAA0513, NUP155, RHOB, THAP11, ACACB, CD5L, FAM32A, KIAA0556, NUP160, RHOBTB1, THAP2, ACAD10, CD6, FAM35A, KIAA0586, NUP188,RHOBTB2, THAP3, ACAD11, CD63, FAM3A, KIAA0753, NUP205, RHOBTB3, THAP4, ACAD8, CD68, FAM3B, KIAA0754, NUP210, RHOC, THAP5, ACAD9, CD69, FAM3C, KIAA0825, NUP210L, RHOD, THAP6, ACADL, CD7, FAM3D, KIAA0895, NUP214, RHOF, THAP7, ACADM, CD70, FAM43A, KIAA0895L, NUP35, RHOG, THAP8, ACADS, CD72, FAM43B, KIAA0907, NUP37, RHOH, THAP9, ACADSB, CD74, FAM45A, KIAA0922, NUP43, RHOJ, THBD, ACADVL, CD79A, FAM46A, KIAA0930, NUP50, RHOQ, THBS1, ACAN, CD79B, FAM46B, KIAA0947, NUP54, RHOT1, THBS2, ACAP1, CD80, FAM46C, KIAA1009, NUP62, RHOT2, THBS3, ACAP2, CD81, FAM46D, KIAA1024, NUP62CL, RHOU, THBS4, ACAP3, CD82, FAM47A, KIAA1024L, NUP85, RHOV, THEG, ACAT1, CD83, FAM47B, KIAA1033, NUP88, RHOXF1, THEG5, ACAT2, CD84, FAM47C, KIAA1045, NUP93, RHOXF2. THEGL, ACBD3, CD86, FAM47E, KIAA1107, NUP98, RHOXF2B, THEM4, ACBD4, CD8A, FAM47E-STBD1, KIAA1109, NUPL1, RHPN1, THEM5, ACBD5, CD8B, FAM49A, KIAA1143, NUPL2, RHPN2, THEM6, ACBD6, CD9, FAM49B, KIAA1147, NUPR1, RIBC1, THEMIS, ACBD7, CD93, FAM50A, KIAA1161, NUPR1L,RIBC2, THEMIS2, ACCS, CD96, FAM50B, KIAA1191, NUS1, RIC3, ​​THG1L, ACCSL, CD97, FAM53A, KIAA1199, NUSAP1, RIC8A, THNSL1, ACD, CD99, FAM53B, KIAA1210, NUTF2, RIC8B, THNSL2, ACE, CD99L2, FAM53C, KIAA1211, NUTM1, RICTOR, THOC1, ACE2, CDA, FAM57A, KIAA1211L, NUTM2A, RIF1, THOC2, ACER1, CDADC1, FAM57B, KIAA1217, NUTM2B, RIIAD1, THOC3, ACER2, CDAN1, FAM58A, KIAA1239, NUTM2F, RILP, THOC5, ACER3, CDC123, FAM60A, KIAA1244, NUTM2G, RILPL1, THOC6, ACHE, CDC14A, FAM63A, KIAA1257, NVL, RILPL2, THOC7, ACIN1, CDC14B, FAM63B, KIAA1279, NWD1, RIMBP2, THOP1, ACKR1, CDC16, FAM64A, KIAA1324, NXF1, RIMBP3, THPO, ACKR2, CDC20, FAM65A, KIAA1324L, NXF2, RIMBP3B, THRA, ACKR3, CDC20B, FAM65B, KIAA1328, NXF2B, RIMBP3C, THRAP3, ACKR4, CDC23, FAM65C, KIAA1377, NXF3, RIMKLA, THRB, ACLY, CDC25A, FAM69A, KIAA1407, NXF5, RIMKLB, THRSP, ACMSD, CDC25B, FAM69B, KIAA1429, NXN, RIMS1, THSD1, ACN9, CDC25C, FAM69C, KIAA1430, NXNL1, RIMS2, THSD4, ACO1, CDC26, FAM71A, KIAA1432, NXNL2, RIMS3, THSD7A, ACO2, CDC27, FAM71B,KIAA1456, NXPE1, RIMS4, THSD7B, ACOT1, CDC34, FAM71C, KIAA1462, NXPE2, RIN1, THTPA, ACOT11, CDC37, FAM71D, KIAA1467, NXPE3, RIN2, THUMPD1, ACOT12, CDC37L1, FAM71E1, KIAA1468, NXPE4, RIN3, THUMPD2, ACOT13, CDC40, FAM71E2, KIAA1522, NXPH1, RING1, THUMPD3, ACOT2, CDC42, FAM71F1, KIAA1524, NXPH2, RINL, THY1, ACOT4, CDC42BPA, FAM71F2, KIAA1549, NXPH3, RINT1, THYN1, ACOT6, CDC42BPB, FAM72A, KIAA1549L, NXPH4, RIOK1, TIA1, ACOT7, CDC42BPG, FAM72B, KIAA1551, NXT1, RIOK2, TIAF1, ACOT8, CDC42EP1, FAM72D, KIAA1586, NXT2, RIOK3, TIAL1, ACOT9, CDC42EP2, FAM73A, KIAA1598, NYAP1, RIPK1, TIAM1, ACOX1, CDC42EP3, FAM73B, KIAA1614, NYAP2, RIPK2, TIAM2, ACOX2, CDC42EP4, FAM76A, KIAA1644, NYNRIN, RIPK3, TICAM1, ACOX3, CDC42EP5, FAM76B, KIAA1671, NYX, RIPK4, TICAM2, ACOXL, CDC42SE1, FAM78A, KIAA1683, OAF, RIPPLY1, TICRR, ACP1, CDC42SE2, FAM78B, KIAA1715, OARD1, RIPPLY2, TIE1, ACP2, CDC45, FAM81A, KIAA1731, OAS1, RIPPLY3, TIFA, ACP5, CDC5L, FAM81B, KIAA1737, OAS2, RIT1, TIFAB, ACP6, CDC6, FAM83A, KIAA1751,OAS3, RIT2, TIGD2, ACPL2, CDC7, FAM83B, KIAA1755, OASL, RLBP1, TIGD3, ACPP, CDC73, FAM83C, KIAA1804, OAT, RLF, TIGD4, ACPT, CDCA2, FAM83D, KIAA1841, OAZ1, RLIM, TIGD5, ACR, CDCA3, FAM83E, KIAA1919, OAZ2, RLN1, TIGD6, ACRBP, CDCA4, FAM83F, KIAA1958, OAZ3, RLN2, TIGD7, ACRC, CDCA5, FAM83G, KIAA1984, OBFC1, RLN3, TIGIT, ACRV1, CDCA7, FAM83H, KIAA2013, OBP2A, RLTPR, TIMD4, ACSBG1, CDCA7L, FAM84A, KIAA2018, OBP2B, RMDN1, TIMELESS, ACSBG2, CDCA8, FAM84B, KIAA2022, OBSCN, RMDN2, TIMM10, ACSF2, CDCP1, FAM86A, KIAA2026, OBSL1, RMDN3, TIMM10B, ACSF3, CDCP2, FAM86B1, KIDINS220, OC90, RMI1, TIMM13, ACSL1, CDH1, FAM86B2, KIF11, OCA2, RMI2, TIMM17A, ACSL3, CDH10, FAM86C1, KIF12, OCEL1, RMND1, TIMM17B, ACSL4, CDH11, FAM86KP, KIF13A, OCIAD1, RMND5A, TIMM21, ACSL5, CDH12, FAM89A, KIF13B, OCIAD2, RMND5B, TIMM22, ACSL6, CDH13, FAM89B, KIF14, OCLM, RNASE1, TIMM23, ACSM1, CDH15, FAM8A1, KIF15, OCLN, RNASE10, TIMM23B, ACSM2A, CDH16, FAM90A1, KIF16B, OCM, RNASE11, TIMM44, ACSM2B, CDH17, FAM91A1, KIF17, OCM2,RNASE12, TIMM50, ACSM3, CDH18, FAM92A1, KIF18A, OCRL, RNASE13, TIMM8A, ACSM4, CDH19, FAM92B, KIF18B, OCSTAMP, RNASE2, TIMM8B, ACSM5, CDH2, FAM96A, KIF19, ADAM, RNASE3, TIMM9, ACSS1, CDH20, FAM96B, KIF1A, ODC1, RNASE4, TIMMDC1, ACSS2, CDH22, FAM98A, KIF1B, ODF1, RNASE6, TIMP1, ACSS3, CDH23, FAM98B, KIF1C, ODF2, RNASE7, TIMP2, ACTA1, CDH24, FAM98C, KIF20A, ODF2L, RNASE8, TIMP3, ACTA2, CDH26, FAM9A, KIF20B, ODF3, RNASE9, TIMP4, ACTB, CDH3, FAM9B, KIF21A, ODF3B, RNASEH1, TINAG, ACTBL2, CDH4, FAM9C, KIF21B, ODF3L1, RNASEH2A, TINAGL1, ACTC1, CDH5, FAN1, KIF22, ODF3L2, RNASEH2B, TINF2, ACTG1, CDH6, FANCA, KIF23, ODF4, RNASEH2C, TIPARP, ACTG2, CDH7, FANCB, KIF24, OFCC1, RNASEK, TIPIN, ACTL10, CDH8, FANCC, KIF25, OFD1, RNASEL, TIPRL, ​​ACTL6A, CDH9, FANCD2, KIF26A, OGDH, RNASET2, TIRAP, ACTL6B, CDHR1, FANCD2OS, KIF26B, OGDHL, RND1, TJAP1, ACTL7A, CDHR2, FANCE, KIF27, OGFOD1, RND2, TJP1, ACTL7B, CDHR3, FANCF, KIF28P, OGFOD2, RND3, TJP2, ACTL8, CDHR4, FANCG, KIF2A, OGFOD3, RNF10, TJP3, ACTL9, CDHR5, FANCI, KIF2B, OGFR, RNF103, TK1, ACTN1, CDIP1, FANCL, KIF2C, OGFRL1, RNF103-CHMP3, TK2, ACTN2, CDIPT, FANCM, KIF3A, OGG1, RNF11, TKT, ACTN3, CDK1, FANK1, KIF3B, OGN, RNF111, TKTL1,ACTN4, CDK10, FAP, KIF3C, OGT, RNF112, TKTL2, ACTR10, CDK11A, FAR1, KIF4A, OIP5, RNF113A, TLCD1, ACTR1A, CDK11B, FAR2, KIF4B, OIT3, RNF113B, TLCD2, ACTR1B, CDK12, FARP1, KIF5A, OLA1, RNF114, TLDC1, ACTR2, CDK13, FARP2, KIF5B, OLAH, RNF115, TLDC2, ACTR3, CDK14, FARS2, KIF5C, OLFM1, RNF121, TLE1, ACTR3B, CDK15, FARSA, KIF6, OLFM2, RNF122, TLE2, ACTR3C, CDK16, FARSB, KIF7, OLFM3, RNF123, TLE3, ACTR5, CDK17, FAS, KIF9, OLFM4, RNF125, TLE4, ACTR6, CDK18, FASLG, KIFAP3, OLFML1, RNF126, TLE6, ACTR8, CDK19, FASN, KIFC1, OLFML2A, RNF128, TLK1, ACTRT1, CDK2, FASTK, KIFC2, OLFML2B, RNF13, TLK2, ACTRT2, CDK20, FASTKD1, KIFC3, OLFML3, RNF130, TLL1, ACTRT3, CDK2AP1, FASTKD2, KIN, OLIG1, RNF133, TLL2, ACVR1, CDK2AP2, FASTKD3, KIR2DL1, OLIG2, RNF135, TLN1, ACVR1B, CDK3, FASTKD5, KIR2DL3, OLIG3, RNF138, TLN2, ACVR1C, CDK4, FAT1, KIR2DL4, OLR1, RNF139, TLR1, ACVR2A, CDK5, FAT2, KIR2DS4, OMA1, RNF14, TLR10, ACVR2B, CDK5R1, FAT3, KIR3DL1, OMD, RNF141, TLR2, ACVRL1, CDK5R2, FAT4, KIR3DL2, OMG, RNF144A,TLR3, ACY1, CDK5RAP1, FATE1, KIR3DL3, OMP,...

Claims

1. A recombinant gene effector containing a polypeptide, comprising an amino acid sequence having at least 90% sequence identity with the polypeptide sequence shown in SEQ ID NO: 1, The polypeptide is heterogeneous to any member selected from the group consisting of VP16, VP64, p65, and Rta; The size of the recombinant gene effector is up to 500 amino acid residues in length; The recombinant gene effector can activate the expression level of a target gene in cells, and the expression level of the target gene activated via the recombinant gene effector is at least 80% of the expression level of the target gene activated by the VP64-p65-Rta fusion polypeptide (VPR) in control cells. Recombinant gene effectors.

2. The recombinant gene effector according to claim 1, wherein the expression level of the target gene activated via the recombinant gene effector is sustained for at least 9 days.

3. The recombinant gene effector according to claim 1, wherein the size is (a) 400 amino acid residues or less in length, (b) 300 amino acid residues or less in length, or (c) 250 amino acid residues or less in length.

4. The recombinant gene effector according to claim 1, wherein the target gene is endogenous to the cell.

5. The recombinant gene effector according to claim 1, wherein the amino acid sequence of the polypeptide, when aligned with the polypeptide sequence shown in SEQ ID NO: 1, includes one or more members selected from the group consisting of C4, L5, M7, and L19.

6. The recombinant gene effector according to claim 1, wherein the amino acid sequence of the recombinant gene effector is not identical to any of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

7. The polypeptide is linked to an additional polypeptide, The additional polypeptide comprises at least a portion of one or more members selected from the group consisting of VP16, VP64, p65, and Rta; The recombinant gene effector has a size of 250 amino acid residues or less. A recombinant gene effector according to any one of claims 1 to 6.

8. The recombinant gene effector according to claim 7, wherein the additional polypeptide comprises VP16 or an amino acid sequence having at least 90% sequence identity with the polypeptide sequence shown in SEQ ID NO:

5.

9. The recombinant gene effector according to claim 7, wherein the additional polypeptide comprises VP64 or an amino acid sequence having at least 90% sequence identity with the polypeptide sequence shown in SEQ ID NO:

6.

10. The recombinant gene effector according to claim 7, wherein the polypeptide is fused to the additional polypeptide.

11. The recombinant gene effector according to claim 7, wherein the amino acid sequence of the polypeptide is not identical to either of the sequences shown in SEQ ID NO: 2 and SEQ ID NO:

3.

12. The recombinant gene effector according to claim 7, wherein the additional polypeptide is located adjacent to the C-terminus of the polypeptide.

13. The recombinant gene effector according to claim 7, wherein the additional polypeptide is positioned adjacent to the N-terminus of the polypeptide.

14. The recombinant gene effector according to claim 7, wherein the amino acid sequence of the polypeptide includes one or more members selected from the group consisting of C4, L5, M7, and L19 when aligned with the polypeptide sequence shown in SEQ ID NO:

1.

15. Recombinant gene effector according to any one of claims 1 to 6; and Heterogeneous endonuclease linked to the recombinant gene effector A system that includes this.

16. The system according to claim 15, wherein the heterologous endonuclease is a Cas protein.

17. The system according to claim 15, further comprising a guide nucleic acid capable of forming a complex comprising the recombinant gene effector and the heterologous endonuclease, wherein the complex exhibits specific binding to a target gene.

18. One or more polynucleotides that code for the system described in Claim 15.

19. A cell comprising the system described in Claim 15.

20. A method for controlling a target gene in a cell in vitro or ex vivo, A method comprising the step of bringing cells into contact with the system described in claim 15.

21. The method according to claim 20, wherein the control includes activating the expression level of the target gene.

22. The method according to claim 20, wherein the target gene is endogenous to the cell.

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